Data are the pooled means of three estimates each over two years ±standard deviation. 'p-value' denotes the significance of difference between the means by one way ANOVA statistics. a The values

**\* Cul**: *Cassia uniflora* Mill.non Spreng ; **Snl**: *Synedrella nodiflora*(L) Gaertn; **Alt**: *Alternanthera tenella* Colla; **Eug**: *Euphorbia geniculata* Orteg.; **Ach**: *Achyranthes aspera* L.; **Bod**: *Boerhaavia erecta* L.; **Bln**: *Blainvillea acmella* L.; **Aca**: *Acalypha ciliata* Forsk.; **Tum:** *Triumfetta rhomboidea* Jacq.; **Cab**: *Cassia absus* L.; **Cfl:** *Cassia obtusifolia* L.; **Bdn**: *Bidens biternata* Lour.; **Raw:** *Rauwolfia tetraphylla* L.; **Opl:** *Oplismenus compositus* P.Beauv.

The alien species like *Cassia uniflora, Synedrella* and *Alternanthera* have higher contents of different types of antioxidants as compared to the co- occurring invasive and native weeds at the selected sites of Pune University campus (Table 5). Along with this lower values of lipid peroxidation and higher MSI and RWC might be offering them additional mechanisms for abiotic stress tolerance. As a result of this the selected invasive weeds might have succeeded to invade and encroach over the native plants of Pune University campus even

followed by different letters differ significantly by Duncan's multiple range test at p=0.05.

<0.001 0.024 <0.001 <0.001 <0.001 <0.001

Weed species

Proline m moles/g DW

± 4.68

± 2.63

± 1.02

± 0.35

± 0.74

± 0.32

± 2.87

± 0.43

± 0.58

± 0.21

± 0.29

± 0.379

± 0.31

± 0.22

during the harsh environmental conditions.

Cul 38.28 a

Snl 34.48 b

Alt 25.57 c

Eug 11.82 de

Ach 12.44 d

Bod 6.54 gh

Bln 9.264ef

Aca 6.27 h

Tum 8.37 efg

Cab 12.41 d

Cfl 20.84 c

Bdn 9.486 def

Raw 15.24 cd

Opl 7.54 fgh

Glycine Betaine mg/g

> 0.42 a ± 0.01

> 0.37 a ± 0.01

> 0.33 a ± 0.21

> 0.30 a ± 0.18

0.334 a ± 0.023

0.27 a ± 0.01

0.303 a ± 0.009

0.26 a ± 0.19

0.28 a ± 0.01

0.24 ab ± 0.017

0.26 a ± 0.18

0.305 a ± 0.015

0.059 b ± 0.002

0.054 b ± 0.003

Table 5. Osmolytes and antioxidants in invasive and native weeds

The enhancement in various antioxidants was reported by many allelopathy workers like Tambussi et al. (2000), Horling et al. (2003), Guha et al*.* (2004), Yang and Lu (2005) in different types of invasive plants growing in terrestrial and boreal forest communities of North America. The role(s) of different antioxidants and osmolytes existing in the invasive and native weeds of forest and cropland ecosystems are very much important. They have explained that the antioxidants were helpful for these weeds to become dominant over cooccuring plant species.

The free radicals are constantly generated under stress conditions which are quenched by an efficient antioxidant network in the plant body. The complex network of such adaptive mechanisms at physiological and molecular levels cause changes in the synthesis and accumulation of various osmolytes, antioxidants and antioxidant enzymes, which provide stress tolerance to the plants (Bagul et al. 2005, Bhattacharya et al. 2009).

Proline is a major organic osmolyte accumulating in a variety of plant species in response to biotic and abiotic stresses, though its actual role in plant osmo-tolerance remains controversial. It is also thought to help in stabilization of sub-cellular structures (e.g. membranes and proteins), and to scavenge free radicals under stress conditions. Proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental stresses (Kavi Kishore et al. 2005).

The rapid breakdown of proline upon relief of stress may provide sufficient reducing agents that support mitochondrial oxidative phosphorylation and generation of ATP for recovery from stress and repairing of stress induced damages (Zhu 2002). In response to drought or salinity stress in plants proline also helps for cytoplasmic osmotic adjustment. Accumulation of proline under stress in many plant species has been correlated with stress tolerance, and its concentration has been shown to be generally higher in stress - tolerant than in stress - sensitive plants (Ashraf and Harris 2004, Ashraf and Foolad 2007). Comparatively higher amount of proline accumulation in *Cassia* and *Synedrella* might be functioning as mentioned above providing stress tolerance to these weeds, as a result of which both the weeds were able to survive throughout the year and producing large no. of seeds even under unfavourable stress conditions. These outnumbering seeds of both the invasive weeds when germinate during favourable season, naturally establish the monothickets or pure stands which caused substitution of many natives resulting into loss of phytodiversity of Pune University campus.

Malondialdehyde (MDA) is a product of lipid peroxidation by a thiobarbituric acid reaction. During drought conditions high activities of antioxidant enzymes are associated with lower concentration of MDA, being linked to drought tolerance (Gao et al. 2008). Like proline lowest values of MDA in *Cassia* and *Synedrella* can be linked with drought tolerance and better survival in extremely adverse environmental conditions.

One of the most common responses in plants to abiotic stresses is overproduction of different types of compatible organic solutes (Serraj and Sinclair 2002), which protect the plants from stress injuries by cellular osmotic adjustment, detoxification of ROS, protection of membrane integrity and stabilization of enzymes/ proteins. The antioxidants also protect cellular components from dehydration injury. These solutes include proline, sucrose, polyols, trehalose and quaternary ammonium compounds (QACs) such as glycine-betaine, alanine-betaine, proline-betaine, choline *O*-sulfate, hydroxyproline-betaine and pipecolatebetaine (Rhodes and Hanson 1993).

Morphophysiological Investigations in Some Dominant Alien Invasive Weeds 37

high stimulation in the activities of above mentioned enzymes in response to stress conditions, which might be responsible for survival of these weeds in extreme ecological conditions existing in the campus of Pune University. Ping Lu et al. (2007) supported the

**efg def g fg def h h**

**Cfl**

**cde cde**

**Bdn**

 **c**

 **e**

**Raw**

**f**

**Opl**

**0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45**

**PPO POX**

**e**

**Opl**

**e**

**Raw**

**bcd**

**Bdn**

**de**

**Cfl**

**e**

**Cab**

**<sup>e</sup><sup>e</sup>**

above view.

**Δ OD µg protein-1 min-1**

 **a**

**Cul**

**0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09**  **a a**

**b**

**Snl**

**Alt**

**Eug**

 **abc**

 **ab**

 **a**

**0 0.005 0.01 0.015 0.02 0.025 0.03 0.035**

**units g-1min-1**

at p<0.05.

**Cul**

**Snl**

**Alt**

**Eug**

**Ach**

Fig. 5. Activity of superoxide dismutase in the invasive and native weeds

 **b**

 **bc**

**c de d def def**

**Ach**

 **e**

**Bod**

**abc abc**

**Bln**

 **d**

 **e de**

**Aca**

Fig. 4. Activity of polyphenol oxidase and peroxidase in the invasive and native weeds

**Weeds**

**Tum**

**bcd**

**Bln**

**Weeds**

#Data points are the pooled means of three replicates over two years with standard deviation as error bars. a Different letters at the data points denote significant difference by Duncan's multiple range test

**Aca**

**Tum**

**cde**

**Bod**

**Cab**

**b**

Amongst the many quaternary ammonium compounds known in plants, glycine betaine occurs most abundantly in response to dehydration stress (Venkatesan and Chellappan 1998, Mansour 2000). GB is abundant mainly in chloroplast where it plays a vital role in adjustment and protection of thylakoid membrane, thereby maintaining photosynthetic efficiency. The results of present investigation on antioxidants indicated more accumulation of GB in *Cassia* and *Synedrella* as compared to the native weeds at selected sites of Pune University campus. Along with high proline and low MDA, GB might also be contributing for stress tolerance and thereby maintaining the photosynthetic efficiency of both the dominant invasive weeds as suggested by Genard et al. (1991).

RWC has special significance in physiological activities of plants. The results of the present investigation indicated that *Cassia* and *Synedrella* had significantly higher RWC as compared to other invasive and native weeds. It may be the additional physiological adaptation for drought tolerance along with above mentioned antioxidants.

Likewise MSI decides the extent of membrane perturbations in structure and dysfunctioning in the cellular activities during the stress conditions. The membrane stability index (MSI) is very important parameter that gives idea about the stress tolerance ability of invasive and native weeds. The MSI of weeds under investigations agree with this. Increase in allelochemicals in these weeds might also be helping the weeds to get more stress tolerance. Membranes are barriers isolating aqueous compartments of the cells and the membrane proteins participate in signal reception and in transport of specific solutes giving them stability and thereby afford stress tolerance to the plants (Ramadevi et al. 1997). The higher values of MSI in both the invasive weeds recorded in the present investigation may be having similar role as mentioned above, because of which these weeds are tolerating extreme environmental conditions, survive comfortably and invade successfully in the new habitats. On the contrary the native weeds are not able to tolerate the stress conditions and hence make the place for highly tolerant invasive weeds. This results in to loss of native phytodiversity in that particular ecosystem.

### **4.10 Antioxidant enzymes in invasive and native weeds**

The activities of antioxidant enzymes like PPO (Polyphenol oxidase), POX (Peroxidase) and SOD (Superoxide dismutase) (Figure 4, 5), were stimulated in *Cassia uniflora* followed by *Synedrella* and *Alternanthera.* The remaining invasive and native weeds followed the above mentioned weed species. The difference in stimulation of these enzymes might be due to the difference in stress tolerance ability of these weeds. More accumulation of antioxidants and stimulated activities of antioxidant enzymes might be becoming helpful for stress tolerance to these weeds. The results of the present investigations are in agreement with the findings of Bhalerao (2003), Jadhav (2006), Vaidya (2009) and Ghayal et al. (2009). They have also reported comparatively higher stimulation of antioxidant enzymes like PPO, POX and SOD in some invasive as well as native weeds of forest, aquatic and terrestrial ecosystems. They further concluded that more accumulation of antioxidants and stimulated activities of antioxidant enzymes were helpful for stress tolerance to these weeds.

Antioxidative enzymes, such as superoxide dismutase (SOD), peroxidases (POD) and polyphenol oxidase (PPO), are the most important components in the ROS scavenging system. SOD dismutates O2 to H2O2, POD and PPO subsequently scavenge the H2O2. The activities of antioxidant enzymes are usually stimulated on exposure to oxidative stress, for protecting the plants, because these enzymes scavenge the reactive oxygen species (Tanaka 1994). The invasive and native weeds studied in the present investigation had shown very

Amongst the many quaternary ammonium compounds known in plants, glycine betaine occurs most abundantly in response to dehydration stress (Venkatesan and Chellappan 1998, Mansour 2000). GB is abundant mainly in chloroplast where it plays a vital role in adjustment and protection of thylakoid membrane, thereby maintaining photosynthetic efficiency. The results of present investigation on antioxidants indicated more accumulation of GB in *Cassia* and *Synedrella* as compared to the native weeds at selected sites of Pune University campus. Along with high proline and low MDA, GB might also be contributing for stress tolerance and thereby maintaining the photosynthetic efficiency of both the

RWC has special significance in physiological activities of plants. The results of the present investigation indicated that *Cassia* and *Synedrella* had significantly higher RWC as compared to other invasive and native weeds. It may be the additional physiological adaptation for

Likewise MSI decides the extent of membrane perturbations in structure and dysfunctioning in the cellular activities during the stress conditions. The membrane stability index (MSI) is very important parameter that gives idea about the stress tolerance ability of invasive and native weeds. The MSI of weeds under investigations agree with this. Increase in allelochemicals in these weeds might also be helping the weeds to get more stress tolerance. Membranes are barriers isolating aqueous compartments of the cells and the membrane proteins participate in signal reception and in transport of specific solutes giving them stability and thereby afford stress tolerance to the plants (Ramadevi et al. 1997). The higher values of MSI in both the invasive weeds recorded in the present investigation may be having similar role as mentioned above, because of which these weeds are tolerating extreme environmental conditions, survive comfortably and invade successfully in the new habitats. On the contrary the native weeds are not able to tolerate the stress conditions and hence make the place for highly tolerant invasive weeds. This results in to loss of native

The activities of antioxidant enzymes like PPO (Polyphenol oxidase), POX (Peroxidase) and SOD (Superoxide dismutase) (Figure 4, 5), were stimulated in *Cassia uniflora* followed by *Synedrella* and *Alternanthera.* The remaining invasive and native weeds followed the above mentioned weed species. The difference in stimulation of these enzymes might be due to the difference in stress tolerance ability of these weeds. More accumulation of antioxidants and stimulated activities of antioxidant enzymes might be becoming helpful for stress tolerance to these weeds. The results of the present investigations are in agreement with the findings of Bhalerao (2003), Jadhav (2006), Vaidya (2009) and Ghayal et al. (2009). They have also reported comparatively higher stimulation of antioxidant enzymes like PPO, POX and SOD in some invasive as well as native weeds of forest, aquatic and terrestrial ecosystems. They further concluded that more accumulation of antioxidants and stimulated activities of

Antioxidative enzymes, such as superoxide dismutase (SOD), peroxidases (POD) and polyphenol oxidase (PPO), are the most important components in the ROS scavenging system. SOD dismutates O2- to H2O2, POD and PPO subsequently scavenge the H2O2. The activities of antioxidant enzymes are usually stimulated on exposure to oxidative stress, for protecting the plants, because these enzymes scavenge the reactive oxygen species (Tanaka 1994). The invasive and native weeds studied in the present investigation had shown very

dominant invasive weeds as suggested by Genard et al. (1991).

drought tolerance along with above mentioned antioxidants.

phytodiversity in that particular ecosystem.

**4.10 Antioxidant enzymes in invasive and native weeds** 

antioxidant enzymes were helpful for stress tolerance to these weeds.

high stimulation in the activities of above mentioned enzymes in response to stress conditions, which might be responsible for survival of these weeds in extreme ecological conditions existing in the campus of Pune University. Ping Lu et al. (2007) supported the above view.

Fig. 4. Activity of polyphenol oxidase and peroxidase in the invasive and native weeds

#Data points are the pooled means of three replicates over two years with standard deviation as error bars. a Different letters at the data points denote significant difference by Duncan's multiple range test at p<0.05.

Fig. 5. Activity of superoxide dismutase in the invasive and native weeds

Morphophysiological Investigations in Some Dominant Alien Invasive Weeds 39

in weed species like Shepherd's purse (*Capsella bursa-pastoris*) by GC-MS and studied its

The dominance, negative weed-weed interaction, encroachment over native weeds as well as successful invasion of *Cassia uniflora* and *Synedrella* recorded at all the four selected sites in the campus of Pune University can be attributed to the existence of different types of allelochemicals in the leaves of both the weeds such as 2(4H)- Benzofuranone,5,6,7,7atetrahydro-4,4,7a-trimethyl (Dihydroactinidiolide), 2-pentadecanone, Isobutyl phthalate, 4,4,8-trimethyltricyclo [6.3.1.0(1,5)]dodecane-2,9-diol, Hexadecanoic acid, Phytol, Dioctyl phthalate, Neophytidiene, Caryophylene oxide and Di-isooctyl phthalate. The presence of above allelochemicals in both the invasive weeds was well documented by Ghayal et al. (2007). The presence of allelochemicals such as Dodecane-4- yl butyrate in *Cassia* leaves and 3-(5-(1-(3-methylpentyloxy) propyl)-tetrahydro-2-oxofuran-3-yl)-dihydrofuran-2(3*H*)-one in

Many allelopathy researchers such as An et al. (2000), Orr et al. (2005) and Santos et al. (2007) separated, identified and quantified the allelochemicals from different weeds and tested their phytotoxicity*.* Yang et al. (2006) also have separated and identified allelochemicals by NMR in *Ageratina adenophora* and studied their allelopathic activity on rice seedlings. Isolation and bioactivity of withaferin A from *Withania somnifera* roots was done by Kannan and Kulandaivelu (2007). Leicach et al. (2007, 2009) have used different methods of chromatography and spectroscopy for alkaloid separation and identification from different plants and elaborately discussed the importance of extraction, separation and identification of allelochemicals from different plants having allelopathic activity. Ma et al. (2009) and Li et al. (2009) have attempted the isolation and identification of allelochemicals by NMR and Mass in invasive plants *Ipomoea cairica* and *Polygonatum odoratum* respectively. The results of the present investigation are in conformity with the above findings.These allelochemicals usually had greater adverse impact on the physiological as well as biochemical processes, enzymological activities, nutrient uptake and assimilation, reproductive abilities, growth and development of recipient plant species. The changes induced by allelochemicals at molecular level are also expressed in phenotypes. The antimicrobial activity in leaf leachates and extracts of *Cassia* and *Synedrella* might be due to the presence of allelochemicals such as terpenoids, steroids, flavonoids, pungent and bitter

The present investigations attempted on phytosociology, physiology, biochemistry and enzymology of selected weeds, phytochemicals and allelochemicals existing in them, their allelopathic potential tested through seed germination bioassays, seedling growth and physiological, biochemical and enzymological changes including treated seedlings of testcrops due to leachates of selected invasive weeds, clearly revealed that the basis for all such

The weed – crop interactions at molecular, cellular and whole plant level were also attempted with special emphasis, as these weeds in future are likely to invade agroecosystems and croplands. At present slowly they are spreading from wastelands to agricultural lands and competing with the crops of interest and cause significant yield loss.

*Synedrella* leaves were detected for the first time in the present investigation.

essential oils and various types of phenols present in them.

events was allelopathic nature of *Cassia* and *Synedrella.*

**6. Conclusions** 

phytotoxicity.

**5.2 IR, NMR, and MASS spectra studies** 

### **5. Allelobiogenesis of invasive weeds**

To understand the allelopathic nature of any plant, extraction, identification and characterization of allelochemicals in its roots, stems and leaves has predominant role. In fact, all the interpretations in allelopathy are mostly based on such investigations. However, collection, isolation and complete identification, characterization and quantification of allelochemicals is difficult and a challenge to the allelopathy scientists. The allelochemicals like terpenes, steroids, flavonoids, alkaloids etc. have major impact on physiology of recipient plants, right from gene to organism level e.g. the monoterpenes which are the main constituents of the essential oils from many higher plants, interfere with basic biological processes like DNA replication, respiration, enzyme activities, seed germination and plant growth. These monoterpenes have allelopathic action. Triterpenes from many different weeds like *Cassia*, *Lantana, Mikania* are known for their allelopathic responses and great ecological significance with respect to invasion Ghayal et al. (2007a).

The allelochemicals like terpenoids, steroids, phenols and bitter essential oils present in roots, stems and leaves of *Cassia* and *Synedrella* might be released in to their environment, through various processes in the form of extracts, leachates, root exudates and even residues of all above plant parts which in due course of time become allelopathic to associated invasive and native weeds as a result of which they were suppressed slowly and substituted by *Cassia* and *Synedrella*. This phenomenon was observed at all the four sites of Pune University campus.

#### **5.1 GC-MS study**

The phytosociological dominance of *Cassia uniflora* and *Synedrella nodiflora* at the four selected sites in Pune University campus recorded previously and the inhibitory weed-weed interaction between these invasive weeds and co-occuring native weeds can be attributed to the different types of allelochemicals existing in them which are detected with GC-MS. The allelopathic potential exhibited by both the weeds might be due to different types of allelochemicals existing in them.

The distribution, quantity and type of allelochemicals depends on various factors such as age of the plant, growing season, vegetative or reproductive phase, environmental conditions and habitat. The allelopathic influence of extracts, leachates or residues of such plants is due to the different types of allelochemicals such as salts, esters, fatty acids, alkaloids, glycosides, terpenoids, flavonoids and steroids present in them. Their solubility in different solvents and mechanism of actions of such allelochemicals mostly depend on their chemical nature. These allelochemicals might be exuded, excreted or released from the plants. The chemical nature of such allelopathic compounds governs the process of invasion, dominance, distribution and encroachment over co-occurring species in any ecosystem.

Many researchers have isolated more than ten thousand low molecular weight secondary metabolites from higher plants and fungi. These compounds or their analogs are new sources of allelochemicals. Drager (2002), Mashhadi and Rodosevich (2003), Bhalerao (2003), Haig (2004), Elzaawely et al. (2005), and Alonso-Amelot (2006) had detected different types of allelochemicals from various weeds and fern species with GC-MS technique and studied their allelopathic activity. Ru Bai et al. (2009) also reported many allelopathic compounds by GC-MS in root exudates of *Malus prunifolia*. Qiaoying Zhang et al. (2009) detected allelopathic potential of flowers and fruits of *Lantana camara* which was ascribed to the allelochemicals by GC-MS. Seal et al. (2009) had also identified different allelochemicals in weed species like Shepherd's purse (*Capsella bursa-pastoris*) by GC-MS and studied its phytotoxicity.

### **5.2 IR, NMR, and MASS spectra studies**

38 Plants and Environment

To understand the allelopathic nature of any plant, extraction, identification and characterization of allelochemicals in its roots, stems and leaves has predominant role. In fact, all the interpretations in allelopathy are mostly based on such investigations. However, collection, isolation and complete identification, characterization and quantification of allelochemicals is difficult and a challenge to the allelopathy scientists. The allelochemicals like terpenes, steroids, flavonoids, alkaloids etc. have major impact on physiology of recipient plants, right from gene to organism level e.g. the monoterpenes which are the main constituents of the essential oils from many higher plants, interfere with basic biological processes like DNA replication, respiration, enzyme activities, seed germination and plant growth. These monoterpenes have allelopathic action. Triterpenes from many different weeds like *Cassia*, *Lantana, Mikania* are known for their allelopathic responses and great

The allelochemicals like terpenoids, steroids, phenols and bitter essential oils present in roots, stems and leaves of *Cassia* and *Synedrella* might be released in to their environment, through various processes in the form of extracts, leachates, root exudates and even residues of all above plant parts which in due course of time become allelopathic to associated invasive and native weeds as a result of which they were suppressed slowly and substituted by *Cassia* and *Synedrella*. This phenomenon was observed at all the four sites of Pune

The phytosociological dominance of *Cassia uniflora* and *Synedrella nodiflora* at the four selected sites in Pune University campus recorded previously and the inhibitory weed-weed interaction between these invasive weeds and co-occuring native weeds can be attributed to the different types of allelochemicals existing in them which are detected with GC-MS. The allelopathic potential exhibited by both the weeds might be due to different types of

The distribution, quantity and type of allelochemicals depends on various factors such as age of the plant, growing season, vegetative or reproductive phase, environmental conditions and habitat. The allelopathic influence of extracts, leachates or residues of such plants is due to the different types of allelochemicals such as salts, esters, fatty acids, alkaloids, glycosides, terpenoids, flavonoids and steroids present in them. Their solubility in different solvents and mechanism of actions of such allelochemicals mostly depend on their chemical nature. These allelochemicals might be exuded, excreted or released from the plants. The chemical nature of such allelopathic compounds governs the process of invasion, dominance, distribution and encroachment over co-occurring species in any ecosystem. Many researchers have isolated more than ten thousand low molecular weight secondary metabolites from higher plants and fungi. These compounds or their analogs are new sources of allelochemicals. Drager (2002), Mashhadi and Rodosevich (2003), Bhalerao (2003), Haig (2004), Elzaawely et al. (2005), and Alonso-Amelot (2006) had detected different types of allelochemicals from various weeds and fern species with GC-MS technique and studied their allelopathic activity. Ru Bai et al. (2009) also reported many allelopathic compounds by GC-MS in root exudates of *Malus prunifolia*. Qiaoying Zhang et al. (2009) detected allelopathic potential of flowers and fruits of *Lantana camara* which was ascribed to the allelochemicals by GC-MS. Seal et al. (2009) had also identified different allelochemicals

ecological significance with respect to invasion Ghayal et al. (2007a).

**5. Allelobiogenesis of invasive weeds** 

University campus.

**5.1 GC-MS study** 

allelochemicals existing in them.

The dominance, negative weed-weed interaction, encroachment over native weeds as well as successful invasion of *Cassia uniflora* and *Synedrella* recorded at all the four selected sites in the campus of Pune University can be attributed to the existence of different types of allelochemicals in the leaves of both the weeds such as 2(4H)- Benzofuranone,5,6,7,7atetrahydro-4,4,7a-trimethyl (Dihydroactinidiolide), 2-pentadecanone, Isobutyl phthalate, 4,4,8-trimethyltricyclo [6.3.1.0(1,5)]dodecane-2,9-diol, Hexadecanoic acid, Phytol, Dioctyl phthalate, Neophytidiene, Caryophylene oxide and Di-isooctyl phthalate. The presence of above allelochemicals in both the invasive weeds was well documented by Ghayal et al. (2007). The presence of allelochemicals such as Dodecane-4- yl butyrate in *Cassia* leaves and 3-(5-(1-(3-methylpentyloxy) propyl)-tetrahydro-2-oxofuran-3-yl)-dihydrofuran-2(3*H*)-one in *Synedrella* leaves were detected for the first time in the present investigation.

Many allelopathy researchers such as An et al. (2000), Orr et al. (2005) and Santos et al. (2007) separated, identified and quantified the allelochemicals from different weeds and tested their phytotoxicity*.* Yang et al. (2006) also have separated and identified allelochemicals by NMR in *Ageratina adenophora* and studied their allelopathic activity on rice seedlings. Isolation and bioactivity of withaferin A from *Withania somnifera* roots was done by Kannan and Kulandaivelu (2007). Leicach et al. (2007, 2009) have used different methods of chromatography and spectroscopy for alkaloid separation and identification from different plants and elaborately discussed the importance of extraction, separation and identification of allelochemicals from different plants having allelopathic activity. Ma et al. (2009) and Li et al. (2009) have attempted the isolation and identification of allelochemicals by NMR and Mass in invasive plants *Ipomoea cairica* and *Polygonatum odoratum* respectively.

The results of the present investigation are in conformity with the above findings.These allelochemicals usually had greater adverse impact on the physiological as well as biochemical processes, enzymological activities, nutrient uptake and assimilation, reproductive abilities, growth and development of recipient plant species. The changes induced by allelochemicals at molecular level are also expressed in phenotypes. The antimicrobial activity in leaf leachates and extracts of *Cassia* and *Synedrella* might be due to the presence of allelochemicals such as terpenoids, steroids, flavonoids, pungent and bitter essential oils and various types of phenols present in them.

### **6. Conclusions**

The present investigations attempted on phytosociology, physiology, biochemistry and enzymology of selected weeds, phytochemicals and allelochemicals existing in them, their allelopathic potential tested through seed germination bioassays, seedling growth and physiological, biochemical and enzymological changes including treated seedlings of testcrops due to leachates of selected invasive weeds, clearly revealed that the basis for all such events was allelopathic nature of *Cassia* and *Synedrella.*

The weed – crop interactions at molecular, cellular and whole plant level were also attempted with special emphasis, as these weeds in future are likely to invade agroecosystems and croplands. At present slowly they are spreading from wastelands to agricultural lands and competing with the crops of interest and cause significant yield loss.

Morphophysiological Investigations in Some Dominant Alien Invasive Weeds 41

An, M., Haig, T. and Pratley, J. E., (2000). Phytotoxicity of *Vulpia* : II Separation,

Ashraf, M. and Harris, P. J. (2004). Potential biochemical indicators of salinity tolerance in

Ashraf, M. and Foolad, M.R. (2007). Roles of glycine betaine and proline in improving plant

Azania, A.A.P.M., Azina, C.A.M., Alves, P.L.C.A., Palaniraj, R., Kadian, H.S., Sati, S.C.,

Bagul, M.S., Kanaki, S.N. and Rajani, M. (2005). Evaluation of free radical scavenging properties of two classical polyhedral formulations. *Ind. J. Exp. Biol*. 43: 732 – 736. Baker, H. G. (1965). Charactristics and modes of origin of weeds. Pp. 147 – 172, in Genetics

Bartariya, G., Saxena, A., Srivastva, J. N., and Satsangi, G. P. (2005). Allelopathic potential of

Batish, D.R., Singh, H.P., Pandher, J.K., Arora, V. and Kohli, R.K. (2002). Phytotoxic effect of

Bhakat, R.K., Bhattacharjee, A., Maiti, P.P., Das, R.K. and Kanp, U.K. (2006). Effect of *Eupatorium odoratum* L. on *Mimosa pudica* L. *Allelopathy Journal* 17(1): 113 – 116. Bhalerao, E.B., Laware, S.L., Vaidya, R.R.and Dhumal, K.N. (2000a). Influence of leaf

Bhalerao, E.B., Laware, S.L., Vaidya, R.R.and Dhumal, K.N. (2000b). Effect of *Aspidium* 

Bhalerao, E. B. (2003). Physiological studies in some crop plants with reference to

Bhan, V.M., D.B. Bhaskar Choudhary (1976). Germination, growth and reproductive

Bhatt, B.P. and Chauhan, D.S. (2000). Allelopathic effects of *Quercus* spp. on crops of

Bhattacharya Malay, Mandal Palash and Sen Arnab (2009). *In vitro* detection of antioxidants

Blicker P.S., B.E. Olson and J.M. Wraith (2003). Water use and water-use efficiency of the invasive *Centaurea maculosa* and three native grasses. *Plant and Soil.* 254: 371–381. Blum, U. (1996). Allelopathic interactions involving phenolic acids. *J. Nematol.,* 28(3): 259-

(*Helianthus annus* L.). *Allelopathy Journal* 11(1): 1 - 20.

G. Baker and G. L. Stebbins, ed. Academic Press, New York.

bajra. *J. Med. Arom. Plant Sci.*, 22 (4A) and 23(1A): 499-501.

Garlwal Himalaya. *Allelopathy Journal* 7: 265-273.

*Ecol.* 26: 1465 – 1476.

2005.12.006

plants. *Plant Sci.* 166: 3 – 16.

*Allelopathy Journal.* 16(2): 353-358.

*Sci.,* 22/4A & 23/1A: 502-504.

Pune.

126 -130.

267.

14(1) N.S.: 23 – 27.

radish. *Weed Biol. Management* 2: 73-78.

identification and quantification of the allelochemicals from *Vulpia myuros. J. Chem.* 

abiotic stress resistance. *Environ. Exp. Bot.* 59: 206 – 216. DOI: 10.1016/j.envexpbot.

Rawat, L.S, Dahiya, D.S. and Narwal, S.S. (2003). Allelopathic plants 7. sunflower

of colonizing species. Proc. First Int. Union of Biological Sci. Symp. On Gen. Biol. H.

*Cyperus rotundus* L. on germination and seedling growth of *Oryza sativa* L.

*Parthenium* residues on the selected soil properties and growth of chickpea and

leachates of *Pteridium* and *Aspidium* on physiology of *Mentha. J. Med. Arom. Plant* 

*cicutarium* rhizome extracts on seed germination and seedling growth of jowar and

application of fern frond extracts, Ph.D thesis, Department of Botany University of

behaviour of *Phalaris minor* Retz. As affected by date of planting. *Ind. J. Weed Sci*. 18:

in different solvent fractions of Ginger (*Zingiber officinale* Rosc.). *Ind. J. Plt. Physiol.* 

The studies on impact of leachates, extracts and even residues of *Cassia* and *Synedrella*  revealed that higher concentrations had severe and very dreadful influence on physiology, biochemistry and enzymology of test crops and such negative changes were also manifested on growth and yield attributes of crops interacting with weeds.

These investigations have also thrown a light on successful invasion of both the weeds in the campus of Pune University, their dominance, aggressiveness and encroachement over native and even other invasive weeds. The ecological and morphological superiority enabled them to do so very efficiently and effectively. The exclusive dominant nature of selected invasive weeds and their allelopathic potential resulted into the loss of native phytodiversity, which is the major threat of such invasion to any ecosystem. Such investigations may become the basis for exploring environmental and ecological degradations in nature.

### **7. Future research**

The need for research and development in allelopathy for the improvement of agriculture, forestry and different types of ecosystems, community structures and functioning is of extreme urgency, because the understanding of allelopathy has major role(s) in the interactions between invasive/ exotic and native weeds, weeds-crops, crops-crops etc. These studies are of utmost importance in agriculture, forestry and environmental degradation. Many of these weeds cause damages to agroecosystems and disturb natural phytodiversity. Their dominance, luxuriant growth, persistence throughout the year, tolerance to biotic and abiotic stress conditions and allelopathic potential might be the probable factors of successful invasion in new habitats.

The use of naturally produced huge weed biomass for weedicidal, cytotoxic, larvicidal, insecticidal and antimicrobial activity is gaining ground in sustainable agriculture. With this view many research workers have reported the antimicrobial activity in different plant parts and extracts, leachates or residues of large number of plant species available in plenty.

The richness of bioactive compounds, secondary metabolites and variety of allelochemicals present in these weeds and other co-dominant weeds can give enticement to screen their cytotoxic, genotoxic, larvicidal, antimicrobial activities etc. The results of such experiments could be positive, if the analyses of their bioactive compounds, antioxidants and antioxidant enzymes is given due importance. The studies on genomics and proteomics of different weeds, having biotic and abiotic stress tolerance can be exploited with the aid of biotechnological tools, to have such type of agronomic traits in various crops. Only the coupling of all the aspects of studies can give an applied touch to the entire field of allelopathy.

### **8. References**


The studies on impact of leachates, extracts and even residues of *Cassia* and *Synedrella*  revealed that higher concentrations had severe and very dreadful influence on physiology, biochemistry and enzymology of test crops and such negative changes were also manifested

These investigations have also thrown a light on successful invasion of both the weeds in the campus of Pune University, their dominance, aggressiveness and encroachement over native and even other invasive weeds. The ecological and morphological superiority enabled them to do so very efficiently and effectively. The exclusive dominant nature of selected invasive weeds and their allelopathic potential resulted into the loss of native phytodiversity, which is the major threat of such invasion to any ecosystem. Such investigations may become the basis for exploring environmental and ecological

The need for research and development in allelopathy for the improvement of agriculture, forestry and different types of ecosystems, community structures and functioning is of extreme urgency, because the understanding of allelopathy has major role(s) in the interactions between invasive/ exotic and native weeds, weeds-crops, crops-crops etc. These studies are of utmost importance in agriculture, forestry and environmental degradation. Many of these weeds cause damages to agroecosystems and disturb natural phytodiversity. Their dominance, luxuriant growth, persistence throughout the year, tolerance to biotic and abiotic stress conditions and allelopathic potential might be the probable factors of

The use of naturally produced huge weed biomass for weedicidal, cytotoxic, larvicidal, insecticidal and antimicrobial activity is gaining ground in sustainable agriculture. With this view many research workers have reported the antimicrobial activity in different plant parts and extracts, leachates or residues of large number of plant species available in plenty. The richness of bioactive compounds, secondary metabolites and variety of allelochemicals present in these weeds and other co-dominant weeds can give enticement to screen their cytotoxic, genotoxic, larvicidal, antimicrobial activities etc. The results of such experiments could be positive, if the analyses of their bioactive compounds, antioxidants and antioxidant enzymes is given due importance. The studies on genomics and proteomics of different weeds, having biotic and abiotic stress tolerance can be exploited with the aid of biotechnological tools, to have such type of agronomic traits in various crops. Only the coupling of all the aspects of studies can give an applied touch to the entire field of

Aldrich, R.J. (1984). *Weed – crop ecology: Principles in weed management*. Breton Pub., N.

Alonso-Amelot, M.E., Usubillaga, A., Avila-Nunez, J.L., Oliveros-Bastidas, A. and

Avendano, M. (2006). Effects of *Minthostachys mollis* essential oil and volatiles on seedlings of lettuce, tomato, cucumber and *Bidens pilosa*. *Allelopathy Journal* 18(2):

on growth and yield attributes of crops interacting with weeds.

degradations in nature.

**7. Future research** 

allelopathy.

**8. References** 

successful invasion in new habitats.

Scituate, MA. pp. 5 – 6.

267 – 276.


Morphophysiological Investigations in Some Dominant Alien Invasive Weeds 43

Ehrenfeld, J.G. (2003). Effects of exotic plant invasions on soil nutrient cycling processes.

Einhellig, F.A. (1987). Interactions among allelochemicals and other stress factors of the

Elzaawely, A.A., Xuan T.D. and Tawata S. (2005). Allelopathic activity and identification of allelochemicals from *Rumex japonicas* Houtt. *Allelopathy Journal* 16(2): 209 – 216. Endress, B.A. and Parks, C.G. (2004). Element *Potentilla recta*. Vol. 2004. The Nature

Ewe, S.M.L., Sternberg, L.S.L. (2003) Seasonal exchange characteristics of *Schinus* 

Friedman, J. and Waller, G.R. (1983). Seeds as allelopathic agents. *J Chem Ecol.* 9: 1107 –

Fujii, Y., Hirdate S. and Hrya, H. (2002). In: Abstr. 3rd World cong. on Allelopathy Challenge

Funk J.L. and Vitousek P.M. (2007). Resource use efficiency and plant invasion in low –

Gao Jianming, Qiang Xiao, Liping Ding, Mingjie Chen, Liang Yin, Jinzhi Li, Shiyi Zhou,

Ghayal, N.A., Dhumal, K.N., Deshpande, N.R., Kulkarni, A.M., Phadke, A.U. and Shah, S.M.

Ghayal N.A., Dhumal K.N., Deshpande N.R., Shah, S.M. and Ruikar A.D. (2007b). Studies

Ghayal N.A., Dhumal, K.N., Deshpande, N.R., Shah, S.M. and Tambe, A. (2007c). Steam

Ghayal, N. A., Dhumal, K. N., Gupta, S. G., Parange S. and Phadke M. (2009).

*of Plant interactions.* 4(1): 33 – 39. DOI- 10.1080 / 17429140802385964 Gill, D.S. and Sandhu, K.S. (1996). Growth stages of sunflower (*Helianthus annus* L.) in relation to allelopathic effects on pearlmillet. *Indian J. Ecol.* 23: 54 -56. Guha, P., Mukhopadhyay, R., Pal, P.K. and Gupta, K. (2004). Antimicrobial activity of crude

*Adiantum capillus- veneris* L. *Allelopathy Journal* 13(1): 57-66.

stress. *Plant Growth Regul*. 56: 89–95. DOI 10.1007/s10725-008-9291-6. Genard, H., Le Saos, J., Hillard, J., Tremolieres, A., Boucand, J. (1991). Effect of salinity on

chloroplasts of *Snaeda maritime*. *Plant Physiol. Biochem*. 29: 421 – 427.

(*Brassica juncea*) – *Asian Journal of Chemistry* 20 (8): 6114 – 6120.

Guangyuan He. (2008). Differential responses of lipid peroxidation and antioxidants in *Alternanthera philoxeroides* and *Oryza sativa* subjected to drought

lipid composition, glycine betaine content and photosynthetic activity in

(2007a). Phytotoxic effects of *Cassia uniflora* leaf leachates on gerrminartion and seedling growth of radish (*Raphanus sativus*) and mustard (*Brassica juncea)*

on Allelochemicals in *Synedrella nodiflora* and Impact of its Leaf Leachates on Germination and Seedling growth of Radish (*Raphanus sativus*) and Mustard

Volatile Components from *Cassia uniflora* and *Synedrella nodiflora* by Gas Liquid Chromatography – Mass Spectroscopy *– Journal of Indian Council of Chemists* 24(2):

Mophophysiological studies in some invasive weeds from deccan pleateau. *Journal* 

extracts and extracted phenols from gametophytic and sporophytic plant parts of

*terebinthifolius* in a native and disturbed upland community in Everglade National

plant environment. Am. Chem. Soc. Symp. Ser. 330: 343–357. Elton, C.S. (1958). *The ecology of invasions by animals and plants.* Methuen, London, UK.

*Ecosystems* 6: 503 – 523.

Conservancy, Arlington, VA.

1117.

60 -62.

Park, Florida. *Forest Ecol. Manag.* 179: 27-36.

for the new millennium Ed.: Tsukuba, Japan.

resource systems. *Nature.* 446: 1079 – 1081.

*Allelopathy Journal* 19(2): 361-372.


Blum, U. (1997). Benefits of citrate over EDTA for extracting phenolic acids from soils and

Callaway, R.M., Ridenour, E.T. (2004). Novel weapons: invasive success and the evolution

Carballeira, A. and Reigosa, M.J. (1999). Effects of natural leachates of *Acacia dealbata* link in

Carlton, J. T. (2001). Introduced species in U.S. Coastal Waters: Pew Oceans Commissions

Carpenter, D., Cappuccino, N. (2005). Herbivory, time since introduction and the

Castillo Jesu´ s M., Pablo Leira-Doce, Jorge Carrio´ n-Tacuri, Edison Mun˜ oz-Guacho, A´da

Chapin, F.S. III, Zavaleta, E.S., Eviner, V.T., Naylor, R.L., Vitousek, P.M., Reynolds, H.L.,

Chase, J.M. and Leibold, M.A. (2003). *Ecological niches*. The Univesity of Chicago Press,

Cheema, Z.A., Iqbal, M.and Ahmad R., (2002) Response of wheat varieties and some winter

Chon, S.U., Kim,Y.M. Lee, J.C. (2003). Herbicidal potential and quantifications of causative allelochemicals from compositae weeds. *Weed Research* 43: 444 – 450. Chou, C. H. (1999). Roles of allelopathy in plant biodiversity and sustainable agriculture.

Colautti, R.I. and MacIsaac, H.J. (2004). A neutral terminology to define 'invasive' species.

Daehler, C.C. (2003). Performance comparisons of co-occurring native and alien invasive

Davis, M.A., Grime J.P., Thompson, K. (2000). Fluctuating resources in plant communities: a

Dekker, J. (2005). Biology and anthropology of plant invasions. In: *Invasive Plants: Ecological* 

Devi, S.R., Pellissier, F. and Prasad, M.N.V. (1997). Allelochemicals. In: *Plant Ecophysiology* 

DeWalt, S.J., Denslow, J.S., Hamrick, J.L. (2004) Biomass allocation, growth, and

(Ed. M.N.V. Prasad, pub. John Wieley, New York). pp 253-303.

Drager, B. (2002). Analysis of tropane and related alkaloids. *J Chromato*. A. 978: 1 – 35. Durand, L.A., Goldstein, G. (2001). Photosynthesis, photoinhibition, and nitrogen use efficiency in native and invasive tree ferns in Hawaii. – *Oecologia* 126: 345-354.

plants: implications for conservation and restoration. *Ann. Rev. Ecol. Evol.* 

*and agricultural aspects* (Ed. Inderjit) pp. 235 – 250. Birkhauser Verlag / Switzerland.

photosynthesis of genotypes from native and introduced ranges of the tropical

Consequences of changing biodiversity. *Nature* 405: 234–242.

Arroyo-Sol´s, Guillermo Curado, David Doblas, Alfredo E. Rubio-Casal, Antonio A. A´ lvarez-Lo´ pez, Susana Redondo-Go´mez, Regina Berjano, Giovanny Guerrero, Alfonso De Cires, Enrique Figueroa, Alan Tye. (2007). Contrasting strategies to cope with drought by invasive and endemic species of *Lantana* in

Hooper, D.U., Lavorel, S., Sala, O.E., Hobbie, S.E., Mack, M.C. and Diaz, S. (2000).

weeds to allelopathic effects of sorghum water extract. *Int. J. Agri. Biol*. 4: 52 -

of increased competitive ability. *Front Ecol Environ*. 2: 436 - 443.

plant debris. *J. Chem. Ecol.,* 223: 347- 362.

Galicia (NW Spain). *Bot. Bull. Acad. Sin.,* 40: 87-92.

invasiveness of exotic plants. J Ecol. 93: 315 - 321.

Galapagos. *Biodivers Conserv.* 16: 2123–2136.

*Critical reviews in Plant Sciences.* 18: 609 – 636.

general theory of invisibility. *J. Ecol.* 88: 528 -534.

shrub *Clidemia hirta*. *Oecologia* 138: 521-531.

*Diversity and Distributions* 10: 134 – 141.

*Systematics*. 34: 183 - 211.

Chicago.

55.

Report. Pew Oceans Commissions: Washington, DC.


Morphophysiological Investigations in Some Dominant Alien Invasive Weeds 45

Lee, K.A. and Klasing, K.C. (2004). A role for immunology in invasion biology. *Trends in* 

Leicach, S.R., Chludil, H. and Yaber Grass (2007). Chromatographic and spectroscopic

Leicach, S.R., Sampietro, D.A. and Narwal, S.S. (2009). *Separation and identification of* 

Li, D.W., Wang, D.M., Li, J.L. and Chen, C. (2009). Allelopathic effects of *Polygonatum* 

Lin, S.Z., Du, L. and Cao, G.Q. (2002). Advance and application prospects on allelopathy

Lonsdale, W. M. (1999). Global patterns of plant invasions and the concept of invasibility.

Ma, R.J., Wang, N.L., Zhu, H., Guo, S.J. and Chen, D.S. (2009). Isolation and identification of

MacDougall, A.S., Turkington, R. (2005). Are invasive species the drivers or passengers of

Mack, R. N., Simberloff, D., Lonsdale, W. M., Evans, H., Clout, M. and Bazzaz, F. A. (2000).

Mansour, M. M. F. (2000). Nitrogen containing compounds and adaptation of plants to

Maron, J.L. and Vila, M. (2001). When do herbivores affect plant invasion?Evidence for the natural enemies and biotic resistance hypothesis. *Oikos* 95: 361 – 373. Mashhadi, H.R. and Steve R. Rodosevich (2003). *Weed Biology and Management* (Ed. Inderjit).

McDowell, S.C.L. (2002). Photosynthetic characteristics of invasive and noninvasive species

Meekins, J.F. and McCarthy, B.C. (2001). Effect of environmental variations on the invasive success of a nonindegenous forest herb. *Ecolog Applic.* 11:1136 - 1348. Millenium Ecosystem Assessment (2005). Ecosystems and human well-being: Synthesis.

Mishra, D., Mishra, T.K. and Banerjee, S.K. (1997). Comparative phytosociological and soil

Molisch, H. (1937). Der Enfusslinear pflanze andre: Allelopathie. Gustav Fischer, Jena. Monaco, T.J., Weller, S.C. and Ashton, F.M. (2002). Weed Biology and Ecology. In: *Weed Science – Principles and Practices.* 4th Ed. Pp. 13 – 43. John Wiley and Sons, Inc. Narwal, S.S., Palaniraj, R., Sati, S.C., Kadian, H.S. and Dahiya, D.S. (2003a). Allelopathic plants: 8. *Parthenium hysterophorus* L. *Allelopathy Journal* 11(2): 151-170.

physic-chemical aspect between managed and un-managed lateritic land. *Ann* 

research in forestry. *J. Fujian College Fores.*, 22: 184-188.

change in degraded ecosystems? *Ecology*. 86: 42 - 55.

salinity stress. *Biol. Plant.* 43: 491 – 500.

Pp. 1 – 28. Kluwer Academic Publishers.

Island Press, Washington, DC, USA.

*Forestry*. 5(1): 16 -25.

of *Rubus* (Rosaceae). – *Amer. J. Bot*. 89: 1431-1438.

techniques applied to alkaloid separation and identification. In: *Isolation, identification and characterization of allelochemicals/ Natural products* (Eds. S.S. Narwal, D.A. Sampietro, C.A.N. Catalan and M.A. Vattuone) USA: Science Publishers. (In

*allelochemicals. In: Allelochemicals: Role in Plant – Environmental Interactions.* pp. 149 –

*odoratum* rhizome extracts and its isolated allelochemicals. *Allelopathy Journal* 23(1):

allelochemicals from invasive plant *Ipomoea cairica. Allelopathy Journal* 24(1): 77 – 84.

Biotic invasions: Causes, epidemiology, global consequences and control. *Ecol.* 

*Ecology and Evolution.* 19: 523-529.

162. Studium Press, LLC, Texas, USA.

press).

119 – 128.

*Ecology* 80: 1522 - 1536.

*Appl.* 10: 689–710.


Haig, T. (2004). *Allelopathy, Chemistry and mode of action of allelochemicals.* Ed. Macias, F.A., Galindo, J.C.G., Molinillo, J.M.G., Cutler, H.G. pp. 149 – 160.CRC Press. London. Hase, C.P. (2008). Allelopathic and physiological studies in sugarcane under monoculturing from different recovery zones of Maharashtra. Ph.D. thesis - University of Pune. Hierro and Callaway R. (2003). Allelopathy and exotic plant invasion, *Plant and soil*. 256: 1,

Horling, F., Lamkemeyer, P., Konnig, J., Finkemeir, I., Kandlbinder, A., Baier, M. (2003).

Hu, Y.J. and Wang, Y.F. (2001). A study on the vegetation and reproduction of two weedy herbaceous vines. *Acta Scientiarum Naturalium Universitatis Sunyatsen*. 40: 93 – 96. Huang Hua, Guo Shuiliang, Chen Guoqi (2007). Reproductive biology in an invasive plant

Inderjit and Nilsen, E.T. (2003). Bioassays and field studies for allelopathy in terrestrial

Inderjit, Marc W. Cadotte and Robert I. Colautti (2005a). The ecology of biological

Inderjit, Weston, L.A. and Duke, S.O. (2005b). Challenges, achievements and opportunities

Jadhav, S.S. (2006). Allelopathic potential of some dominant aquativ weeds of Mula and Mutha River and their bioprospecting. Ph.D. Thesis, University of Pune. Jeschke, J.M. and Strayer, D.L. (2005). Invasion success of vertebrates in Europe and North America. Proceedings of the National Academy of Sciences. 102(20): 7198 – 7202. Kanchan, S.D. and Jayachandra (1977). *Parthenium* weed menace in India and its control.

Kannan, N.D. and Kulandaivelu, G. (2007). Novel method to isolate Withaferin A from *Withania somnifera* roots and its bioactivity. *Allelopathy Journal* 20(1): 213 – 220. Kavi Kishore, P.B., Sangam, S., Amrutha, R. N., Laxmi, P.S., Naidu, K.R., Rao, K.R.S.S., Rao,

Khanh, T.D., Cong, L.C., Xuan, T.D., Uezato, Y. Deba, F., Toyama, T. and Tawata, S. (2009).

Kong, C.H. and Hu, F. (2001). Plant Allelopathy and its application. Pp. 169 – 201. Chinese

Kulvinder, K., Kapoor, K.K. and Kaur, K. (1999). Effect of incorporation of sunflower

Kumar, M., Sharma, C.M. and Rajwar, G.S. (2004). A study on community structure and

Leather, G.R. and Einhellig, F.A. (2005). Bioassay of naturally occurring allelochemicals for

plant growth and abiotic stress tolerance. *Curr. Sci.* 88: 424 – 438.

S., Reddy K.J., Theriappan, P., Sreenivasulu, N. (2005). Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in

Allelopathic Plants: 20. Hairy Beggarticks (*Bidens pilosa* L.) *Allelopathy Journal* 24(2):

residues in soil on germination of mung bean and pearlmillet. *Environ. and Ecol.,* 17:

diversity of a sub-tropical forest of Garhwal Himalayas. *Indian Forester* 130(2): 207 –

plants: Progress and problems. *Crit. Rev. Plant Sci.,* 22: 1-18.

in allelopathy research. *J. Plant Interactions*, 1(2): 69-81.

Weed Sci. Conf. workshop in India. Abstract No. 162.

*aspects* (Ed. Inderjit) pp. 19 – 44. Birkhauser Verlag / Switzerland.

Divergent light-, ascorbate-, and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Aarabidopsis. *Plant Physiol*. 131:

*Solidago canadensis. Front. Biol.China*. 2(2): 196–204. DOI 10.1007/s11515-007-0030-6.

invasions: past, present and future. In: *Invasive Plants: Ecological and agricultural* 

29-39.

243 – 254.

693-695.

214.

Agricultural Press, Beijing, China.

phytotoxicity. *J. Chem. Ecol.,* 14(10): 1821-1828.

317–325. DOI: 10.1104/pp. 010017.


Morphophysiological Investigations in Some Dominant Alien Invasive Weeds 47

Ru Bai, Xin Zhao, Fengwang Ma and Cuiying Li (2009). Identification and bioassay of

Sampietro, D.A., Sgariglia, M.A., Soberon, J.R., Quiroga, E.N.and Vattuone, M.A. (2007).

Santos, C.C., Souzaand, I.F., Alves, L.W.R. (2003). Effect of corn residues on coffee (*Coffea* 

Saswade, R.R. (2007). Allelopathic potential of some dominant weeds of semi arid crop

Seal, A.N., Pratley, J.E., Haig, T.J., Min An and Hanwen Wu (2009). Phytotoxic potential of Shepherd's purse on annual rye and wild radish. *Allelopathy Journal* 24(1): 55 -66. Sen, D.N. (1977). Ecophysiological studies on weeds of cultivated fields with special

3rd US PL - 480 Project report, Jodhpur University, Jodhpur, India. 91pp. Sen, D.N. (1981). Ecological approaches to Indian weeds. Geobios International, Jodhpur,

Serraj, R. and Sinclair, T. R. (2002). Osmolyte accumulation: can it really help increase crop

Singh, D. and NarsingRao, Y.B. (2003). Allelopathic evaluation of *Andrographis paniculata*  aqueous leachates on rice (*Oryza sativa* L.). *Allelopathy Journal* 11(1): 71-76. Singh, N.B. and Singh, R. (2003). Effect of leaf leachate of *Eucalyptus* on germination, growth

Sutherland, S. (2004). What makes a weed a weed: life history traits of native and exotic

Takeuchi, Y., Kawaguchi, S. and Yoneyama, K.F. (2001). Inhibitory and promontory allelopathy in rice (*Oryza sativa* L.) *Weed Biology and Management* 1: 147 – 156. Tambussi, E.A., Bartoli, C.G., Beltrano, J., Guiamet, J.J., Araus, J.L. (2000). Oxidative damage

Tanaka, K. (1994). Tolerance to herbicides and air pollutants. In: *Causes of Photooxidative* 

Thakur, A.S. and Khare, P.K. (2009). Composition of forest vegetation and floristics of Sagar

Vaidya, R.R. (2009). Allelopathic studies in some dominant plants from Mahabaleshwar

Vilai-Santisopasri (2003). Kasetsart University research and development Institute, Bangkok,

Venkatesan, A. and Chellappan, K.P. (1998). Accumulation of praline and glycine betaine in

*Ipomoea pescaprae* induced by NaCl. *Biol Planta* 41: 271–276. DOI : 10.1023/

yield under drought conditions? *Plant Cell Environ.* 25: 333 – 341.

Stevens, O.A. (1957). Weights of seeds and numbers per plant. *Weeds* 5: 46 – 55.

*Planta.* 108: 398–404. DOI : 10.1034/j.1399-3054.2000.108004398.

district, central India. *J. Ind. Bot. Soc*. 88(1 & 2): 11 - 17.

Varadpande, D.G. (1972). *Flora of Ganeshkhind*. University of Pune, Pune, India.

plants in the USA. *Population Ecology.* 141: 24 – 39.

eds.) pp. 365– 378. CRC Press, Boca Raton.

area. Ph.D. Thesis, University of Pune.

*Journal* 23(2): 477 – 484.

Pune.

52.

Thailand.

A:1001839302627

India. pp. 301.

sida. *Environ. Exp. Bot.* 60: 495–503.

*arabica* L.) growth. *Cienciae Agrotochnol,* 21: 991-1101.

allelopathic substances from the root exudates of *Malus prunifolia. Allelopathy* 

Role of sugarcane straw allelochemicals in the growth suppression of arrowleaf

ecosystem in Newasa Tahsil, Dist. Ahmednagar (M.S.). Ph.D. Thesis, University of

reference to bajra (*Pennisetum typhoides* Rich.) and til (*Sesamum indicum* L.) crops.

and metabolism of greengram, blackgram and peanut. *Allelopathy Journal* 11(1): 43-

to thylakoid proteins in water stressed leaves of wheat (*Triticum aestivum*). *Physiol* 

*Stress and Amelioration of Defense Systems in Plants*. (Foyer C.H., Mullineaux P.M.


Narwal, S.S., Palaniraj, R, Sati, S.C. and Rawat, L.S. (2003b). Effect of different parts of

Navaz, M.I., Sansamma, G. and Geethakumari, V.L. (2003). Influence of *Eupatorium*

Nilsen, E.I. (2002). In: Inderjit and Mallik A.U. (Eds.) Birkhauser Verlag, Basel, 109-129. Norris, R.F. (1992). Case history for weed competition/ population ecology: Barnyardgrass (*Echinochloa crus-galli*) in sugarbeets (*Beta vulgaris*). *Weed Technol.* 6: 220 – 227. Orr, S.P., Rudgers, J.A. and Clay, K. (2005). Invasive plants can inhibit native tree seedlings:

testing potential allelopathic mechanisms. *Plant Ecol.,* 181: 153–165.

Pathipati Usha Rani (2008). Allelochemical stress induced biochemical changes in leaves and plant growth regulation in *Ricinus communis*. *Allelopathy Journal* 22(1): 79 -92. Patil, T.M. and Hegde, B.A. (1988). Isolation and purification of a sequiterpene lactone from

Pattison, R.R., Goldstein, G., Ares, A. (1998). Growth, biomass allocation and photosynthesis of invasive and native Hawaiian rainforest species. *Oecologia* 117: 449-459. Pawar, K.B. and Chavan, P.D. (1999). Influence of leaf leachates of plant species on mineral nutrition of *Sorghum bicolor* (L.) Moench. *Allelopathy Journal* 6(1): 87 -92. Pawar, K.B. (2004). Seed germination studies in Sorghum *bicolor* (L.) Moench. with special

Ping Lu, Wei-Guo Sang and Ke-Ping Ma. (2007) Activity of Stress-related antioxidative

Podolska, G., Bialy, Z., Jurzysta, M. and Waller, G.R. (2003). Effect of application of alfalfa

Putnam, A.R. (1985). Weed Allelopathy. In: *Weed Physiol., Reproduction and Ecophysiol.* (Ed.,

Qiaoying Zhang, Shaolin Peng and Yunchun Zhang (2009). Allelopathic potential of

Ramadevi, S., Pellissier, F. and Prasad, M.N.V. (1997). Allelochemicals. In: *Plant* 

Rao, V.S. (2000). Weed Biology and Ecology. In: *Principles of Weed Science.* Pp. 7 -35. Oxford

Reigosa, M.J., Sánchez-Moreiras, A., and González, L. (1999). Ecophysiological approach in

Reigosa, M. and Pedrol, N. (2002). In: *Allelopathy: from Molecules to Ecosystems*. Science

Rizvi, S.J.H. and Rizvi, V. (1992). In: *Allelopathy: Basic and Applied Aspects*. Chapman and

S.O. Duke, CRC press, Boca Raton, Florida, U.S.A.), 1:131-155.

371-376.

cowpea. *Allelopathy J.,* 11(2): 235-240.

*Curr. Sci.,* 57: 1178-1181.

Kolhapur, pages-253.

220.

*Integrative Plant Biology.* 49 (11): 1555–1564.

wheat. *Allelopahty Journal* 11(2): 171-184.

and IBH Publishing Co. Pvt.Ltd.

Publishers Inc., Enfield, NH, 316p.

Hall, New York, NY, 480p.

allelopathy. *Crit. Rev. Plant Sci.,* 18(5): 577–608.

Rice, E.L. (1979). Allelopahty -An update. *Bot. Rev.,* 45: 1 – 93.

Rice, E.L. (1984). "Allelopathy" 2nd Ed. Academic Press, New York, London.

sunflower (*Helianthus annus)* biomass on wheat (*Triticum aestivum*). *J. Ecobiol.,* 15:

(*Chromoleana odorata* L.) leachate on germination and seedling growth of rice and

the leaves of *Parthenium hysterophorus* L. Its allelopathic and phytotoxic effects.

reference to allelopathic effects, Ph.D. thesis submitted to Shivaji University,

enzymes in the Invasive Plant Crofton Weed (*Eupatorium adenophorum*). *Journal of* 

root saponins aqueous solution on the plant structure, yield and quality of winter

reproductive organs of exotic weed *Lantana camara*. *Allelopathy Journal* 23(1): 213 –

*Ecophysiology.* (Ed. M.N.V. Prasad) ISBN 0-471-13157-1 © John Wiley and Sons, Inc.


**3** 

*Malaysia* 

**Alteration of Abiotic Stress Responsive** 

Ismanizan Ismail, Mian-Chee Gor, Zeti-Azura Mohamed-Hussein,

Plants are continuously exposed to both biotic and abiotic stress in their natural environment. Unlike animals, plants are immobilized organisms which tend to be vulnerable to various environmental stresses. In order to survive, plants have evolved a wide range of defense mechanism to cope with these stresses. Both biotic and abiotic stresses might share some common signaling pathway in triggering the defense system in plants. Recent researches have revealed that phytohormones such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are intermediate molecules which play key roles in the crosstalk between biotic and abiotic signaling network (Fujita et al. 2006). In this chapter, we highlight the effects of exogenous applied jasmonic acid in triggering the synthesis of some molecules and activating their respective biosynthetic genes

Abiotic stress is defined as non-living external factors, usually environment conditions, which could reduce plant growth and cause huge devastation on agricultural productivity. Some of these major adverse environmental factors are drought, salinity, heavy metals, extreme temperatures, nutrient poor soils and other source of natural disasters. To our knowledge, these abiotic stresses have account for major crops lost worldwide where more than 50% of their average yields were decreased yearly (Rodriguez et al. 2005). However, not all effects are detrimental. Plants are able to exhibit various molecular mechanisms as a defense system and these responses could be generally divided into three main groups. Firstly, signalling of stress-activated molecules leading to changes of osmotic and ionic homeostasis as well as detoxification mechanism. Secondly, up-regulation of different gene expression leading to synthesis of specific proteins (e.g. heat-shock proteins and LEA proteins) and some protective molecules (e.g. sugars, polyalcohols and amino acids). Thirdly, generation of reactive oxygen species (ROS) and activation of antioxidant systems by synthesizing secondary metabolites such as flavonoids and phenolic compounds (Boscaiu et al. 2008). Among these changes, synthesis of secondary metabolites is at the highest interest because it has a wide range of functions, ranging from plant defense against

Plants have the ability to produce vast variety of secondary metabolites naturally. Secondary metabolites have been defined as compounds that did not play a vital role in

**1. Introduction** 

in plants as a response towards abiotic stresses.

abiotic stresses to human benefits.

**Genes in** *Polygonum minus* **Roots** 

**by Jasmonic Acid Elicitation** 

Zamri Zainal and Normah Mohd Noor

*Universiti Kebangsaan Malaysia* 


## **Alteration of Abiotic Stress Responsive Genes in** *Polygonum minus* **Roots by Jasmonic Acid Elicitation**

Ismanizan Ismail, Mian-Chee Gor, Zeti-Azura Mohamed-Hussein, Zamri Zainal and Normah Mohd Noor *Universiti Kebangsaan Malaysia Malaysia* 

### **1. Introduction**

48 Plants and Environment

Wang, P., Qin, Z.Q., Wang, P. and Zhao, Z.Q. (2001). Effect of allelochemical on cotton seed

Wang, J., Feng, Y. and Liang, H. (2004). Adaptation of *Eupatorium adenophorum*

Weaver, S.E., McWilliams, E.L. (1980). The biology of Canadian weeds. 44. *Amaranthus retroflexus* L. and *A. powellii* S. and *A. hybridus* L. *Can J Plant Sci.* 60:1215 - 1234. Werner, P.A. and Soule, J.D. (1976). The biology of Canadian weeds. 18. *Potentilla recta* L., *P.* 

Wilson, R. G. (1988). Biology of weeds in the soil. In: Altieri MA., Liebman M, (eds.) Weed

Yang, X. and Lu, C. (2005). Photosynthesis is improved by exogenous glycine betaine in

Yang, G.Q., Liu, W.X., Zhang, X.W. (2006). Physiological effects of allelochemicals from

Zhu, J. K. (2002). Salt and drought stress signal transduction in plants. *Ann. Rev.Plant physiol.* 

Zouhar, K. (2003). *Potentilla recta* Vol. 2003. U.S. Department of Agriculture, Forest service, Rocky Mountain Research Station, Fire Sciences Laboratory, Missoula, MT.

photosynthetic characteristics to light intensity. *Yingy Sheng. Xueb.,* 15(8):1373-1377.

management in agroecosystems: ecological approaches. pp. 25 - 39.Boca Raton, FL:

salt-stressed maize plants. *Physiol Planta*. 124: 343–352. DOI : 10.1111/j.1399-

leachates of *Ageratina adenophora* (Spreng.) on rice seedlings. *Allelopathy Journal* 

germination and seedling growth. *J. China Agri. Univ.,* 6: 26-31.

*norvegica* L. and *P. argena* L. *Can J Plant Sci* 56: 591 – 603.

CRC Press.

3054.2005.00518.

18(2): 237-246.

Young, J.A. (1991). Tumbleweed. *Sci. Am*. March: 82 – 87.

*Plant Mol. Biol.* 53: 247 – 273.

Plants are continuously exposed to both biotic and abiotic stress in their natural environment. Unlike animals, plants are immobilized organisms which tend to be vulnerable to various environmental stresses. In order to survive, plants have evolved a wide range of defense mechanism to cope with these stresses. Both biotic and abiotic stresses might share some common signaling pathway in triggering the defense system in plants. Recent researches have revealed that phytohormones such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are intermediate molecules which play key roles in the crosstalk between biotic and abiotic signaling network (Fujita et al. 2006). In this chapter, we highlight the effects of exogenous applied jasmonic acid in triggering the synthesis of some molecules and activating their respective biosynthetic genes in plants as a response towards abiotic stresses.

Abiotic stress is defined as non-living external factors, usually environment conditions, which could reduce plant growth and cause huge devastation on agricultural productivity. Some of these major adverse environmental factors are drought, salinity, heavy metals, extreme temperatures, nutrient poor soils and other source of natural disasters. To our knowledge, these abiotic stresses have account for major crops lost worldwide where more than 50% of their average yields were decreased yearly (Rodriguez et al. 2005). However, not all effects are detrimental. Plants are able to exhibit various molecular mechanisms as a defense system and these responses could be generally divided into three main groups. Firstly, signalling of stress-activated molecules leading to changes of osmotic and ionic homeostasis as well as detoxification mechanism. Secondly, up-regulation of different gene expression leading to synthesis of specific proteins (e.g. heat-shock proteins and LEA proteins) and some protective molecules (e.g. sugars, polyalcohols and amino acids). Thirdly, generation of reactive oxygen species (ROS) and activation of antioxidant systems by synthesizing secondary metabolites such as flavonoids and phenolic compounds (Boscaiu et al. 2008). Among these changes, synthesis of secondary metabolites is at the highest interest because it has a wide range of functions, ranging from plant defense against abiotic stresses to human benefits.

Plants have the ability to produce vast variety of secondary metabolites naturally. Secondary metabolites have been defined as compounds that did not play a vital role in

Alteration of Abiotic Stress Responsive Genes

(Vanisree et al. 2004).

(Gundlach et al. 1992).

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 51

production from *Catharanthus roseus* and anthraquinone synthesize from *Morinda citrifolia*

Up to now, many approaches have been done to increase the yield of secondary metabolites from plants. Strategies that have been accepted for used are manipulation of culturing media such as glucose, nitrate, phosphate and plant growth regulators concentration; screening and selection of cell lines which have the ability to produce better yield; optimization of culturing conditions such as temperature, light, pH and aeration; and addition of precursor or elicitor (Ramachandra Rao & Ravishankar 2002; Vanisree & Tsay 2004). Since the main roles of plant secondary metabolites are to increase plant adaptation to abiotic stresses and enhance plant defense system against pathogen attack, it is better to investigate some strategies to alter the production of metabolites based on this principle (Sekar & Kandavel 2010). In fact, elicitation by using molecules that involve in plant defense mechanism is the most efficient strategy to increase the productivity of secondary metabolites in plants (Roberts & Shuler 1997). Many biotic and abiotic elicitors have been employed to increase the production of desired compounds in plants (Barz et al. 1988), namely methyl jasmonate (MeJA), jasmonic acid (JA), salicylic acid (SA), fungus polysaccharide, yeast extract, heavy metal etc. Extensive researches have been done to study the roles of JA as the key signaling molecules in signal transduction system regulating the alteration of plant defense genes against environmental stresses. For instance, the expression of *pin* genes was activated by JA or its derivative, MeJA, in mechanically wounded tomato and potato (Farmer & Ryan 1992). The expression of *vsp* genes was also activated by MeJA as a defense mechanism towards wounded cells of soy bean (Creelman et al. 1992). Besides, exposure of *Hypericum perforatum* L. suspension culture to JA had activated the genes that expressed phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) enzymes. Activation of these genes had increased the production of phenylpropanoids such as phenolic, flavanol and flavonol a 6-fold in cells treated with JA (Gadzovska et al. 2007). JA also altered the synthesis of ajmalicine and catharantine in *Catharanthus roseus* (Vazquez-Flota & De Luca 1998), rosmarinic acid and shikonin in *Lithospermum erythrorhizon* (Yazaki et al. 1997), scopoletin dan scopolin in *Nicotiana tabacum* (Sharon et al. 1998) and taxol dan paclitaxel in *Taxus* spp. (Palazon et al. 2003). Thus, jasmonate has been shown to be key molecules in the elicitation process leading to de novo transcription and translation that resulted in the enhancement of secondary metabolites biosynthesis in *in vitro* plants

It is clear that harsh environmental conditions would activate the expression of certain abiotic stress related genes which involve in the biosynthesis pathway of secondary metabolites in plants. Therefore it becomes a crucial need to identify the stress responsive genes and study their signal transduction pathways not only for a better understanding of plants response and adaptation towards abiotic stress, but also for further development of strategies for commercial production of valuable compounds by manipulating certain metabolic pathways based on gene expression (Shilpa et al. 2010). A significant amount of studies have been done on application of elicitation to plant cultures, either to enhance secondary metabolites production or to discover novel compounds. Though many of these showed positive and encouraging results, the stress responsive genes which involved in the reactions of defense-related pathway and secondary metabolites biosynthesis pathway remain largely unexploited. Many molecular approaches such as mRNA differential display, representation difference analysis, RNA fingerprinting, cDNA microarray and suppression subtractive hybridization (SSH) technique have been applied to identify and characterize the

plant growth and development but are important in the interaction between plant and its environment (Namdeo 2007). These molecules function primarily in plants adaptation towards their environment such as biotic and abiotic stress and also serve as a major source for pharmaceutical products (Ramachandra Rao & Ravishankar 2002). Secondary metabolites are usually release by plants as a type of defense system against insects feeding; herbivory effects and pathogens attack such as virus, bacteria and fungi. They also protect plants against abiotic stress such as draught, salinity, UV light, heavy metals, extreme temperatures, nutrient poor soils and other environmental factors. Several other functions of secondary metabolites include attracting pollinators for plants reproduction and serving as signaling molecules and hormones in plant cells secondary metabolism (Korkina 2007). Up to date, thousands of different secondary metabolites structures have been identified in plants. The bioactive compounds extracted from various plant parts were usually used in the pharmaceutical, agrochemical, cosmetic, perfumery, food flavouring and pesticide industries (Balandrin & Klocke 1988). For instance, morphine and codeine extracted from the latex of opium poppy are the commercial anesthesia available in market today whereas ginsenosides isolated from ginseng roots have been proven to be the stimulant for health and longetivity (Sticher 1998). Apart from that, alcohols, aldehydes, ester, free fatty acids, ketones and phenolic compounds purified from plants are also being used in the foods and beverages industries. The food flavouring that are succesfully marketed are apple (Drawert et al. 1984), cocoa (Townsley 1972), caryophylene (Longo & Sanroman 2006), flavanol (Nakao et al. 1999) and vanillin (Dornenburg & Knorr 1996). Many of these phytochemicals, especially the volatile compounds are secreted by plants as an indirect defense mechanism against herbivory and some other abiotic stress (Yuan et al. 2008).

The exploitation of novel secondary metabolites and their functions have gained the interest of many scientists worldwide and extensive studies have been done since the past 50 years. More than 80% of 30,000 known natural products were originated from plants (Fowler & Scragg 1988; Phillipson 1990). Although the advancement of computational biology has shedded light to medical field as new drugs could be designed base on predicted chemical structure, plants-derived compounds still serve as the model for drugs synthesis due to the complexity of their chemical structures (Pezzuto 1995). In fact, the world market for plantderived drugs will be expected to achieve more than \$26 billion in year 2011 (Saklani & Kutty 2008). However, there are some major drawbacks associated with phytochemicals production. Naturally these phytochemicals present at a much lower concentration compare to primary metabolites. The production of secondary metabolites is approximately 1% of the plant dry weight. Depending on the type of environmental stresses surrounding the plants, the type and level of secondary metabolites produce in plants changing from time to time, and from one place to another (Dixon 2001; Oksman-Caldenteyl & Inze 2004). Besides, the widespread of deforestation and instability of geopolitics make it difficult to extract pure secondary metabolites in whole plants (Shilpa et al. 2010). Fortunately, the advancement of biotechnology has made it possible to alter the production of secondary metabolites by means of plant cell cultures technology. The major advantages of plant cell cultures are that it could produce a continuous and more reliable source of plant pharmaceuticals (Vijaya et al. 2010). Though many efforts have been done since four decades ago, little success was achieved. Only the production of Shikonin from *Lithospermum erythrorhizon* cell culture and Taxol or Paclitaxel from *Taxus* cell cultures meeting the satisfactory yields for commercialization (Sekar & Kandavel 2010). Some other successful cases are such as rosmarinic acid production from *Anchusa officinallis*, indole alkaloids and catharantine

plant growth and development but are important in the interaction between plant and its environment (Namdeo 2007). These molecules function primarily in plants adaptation towards their environment such as biotic and abiotic stress and also serve as a major source for pharmaceutical products (Ramachandra Rao & Ravishankar 2002). Secondary metabolites are usually release by plants as a type of defense system against insects feeding; herbivory effects and pathogens attack such as virus, bacteria and fungi. They also protect plants against abiotic stress such as draught, salinity, UV light, heavy metals, extreme temperatures, nutrient poor soils and other environmental factors. Several other functions of secondary metabolites include attracting pollinators for plants reproduction and serving as signaling molecules and hormones in plant cells secondary metabolism (Korkina 2007). Up to date, thousands of different secondary metabolites structures have been identified in plants. The bioactive compounds extracted from various plant parts were usually used in the pharmaceutical, agrochemical, cosmetic, perfumery, food flavouring and pesticide industries (Balandrin & Klocke 1988). For instance, morphine and codeine extracted from the latex of opium poppy are the commercial anesthesia available in market today whereas ginsenosides isolated from ginseng roots have been proven to be the stimulant for health and longetivity (Sticher 1998). Apart from that, alcohols, aldehydes, ester, free fatty acids, ketones and phenolic compounds purified from plants are also being used in the foods and beverages industries. The food flavouring that are succesfully marketed are apple (Drawert et al. 1984), cocoa (Townsley 1972), caryophylene (Longo & Sanroman 2006), flavanol (Nakao et al. 1999) and vanillin (Dornenburg & Knorr 1996). Many of these phytochemicals, especially the volatile compounds are secreted by plants as an indirect defense mechanism

The exploitation of novel secondary metabolites and their functions have gained the interest of many scientists worldwide and extensive studies have been done since the past 50 years. More than 80% of 30,000 known natural products were originated from plants (Fowler & Scragg 1988; Phillipson 1990). Although the advancement of computational biology has shedded light to medical field as new drugs could be designed base on predicted chemical structure, plants-derived compounds still serve as the model for drugs synthesis due to the complexity of their chemical structures (Pezzuto 1995). In fact, the world market for plantderived drugs will be expected to achieve more than \$26 billion in year 2011 (Saklani & Kutty 2008). However, there are some major drawbacks associated with phytochemicals production. Naturally these phytochemicals present at a much lower concentration compare to primary metabolites. The production of secondary metabolites is approximately 1% of the plant dry weight. Depending on the type of environmental stresses surrounding the plants, the type and level of secondary metabolites produce in plants changing from time to time, and from one place to another (Dixon 2001; Oksman-Caldenteyl & Inze 2004). Besides, the widespread of deforestation and instability of geopolitics make it difficult to extract pure secondary metabolites in whole plants (Shilpa et al. 2010). Fortunately, the advancement of biotechnology has made it possible to alter the production of secondary metabolites by means of plant cell cultures technology. The major advantages of plant cell cultures are that it could produce a continuous and more reliable source of plant pharmaceuticals (Vijaya et al. 2010). Though many efforts have been done since four decades ago, little success was achieved. Only the production of Shikonin from *Lithospermum erythrorhizon* cell culture and Taxol or Paclitaxel from *Taxus* cell cultures meeting the satisfactory yields for commercialization (Sekar & Kandavel 2010). Some other successful cases are such as rosmarinic acid production from *Anchusa officinallis*, indole alkaloids and catharantine

against herbivory and some other abiotic stress (Yuan et al. 2008).

production from *Catharanthus roseus* and anthraquinone synthesize from *Morinda citrifolia* (Vanisree et al. 2004).

Up to now, many approaches have been done to increase the yield of secondary metabolites from plants. Strategies that have been accepted for used are manipulation of culturing media such as glucose, nitrate, phosphate and plant growth regulators concentration; screening and selection of cell lines which have the ability to produce better yield; optimization of culturing conditions such as temperature, light, pH and aeration; and addition of precursor or elicitor (Ramachandra Rao & Ravishankar 2002; Vanisree & Tsay 2004). Since the main roles of plant secondary metabolites are to increase plant adaptation to abiotic stresses and enhance plant defense system against pathogen attack, it is better to investigate some strategies to alter the production of metabolites based on this principle (Sekar & Kandavel 2010). In fact, elicitation by using molecules that involve in plant defense mechanism is the most efficient strategy to increase the productivity of secondary metabolites in plants (Roberts & Shuler 1997). Many biotic and abiotic elicitors have been employed to increase the production of desired compounds in plants (Barz et al. 1988), namely methyl jasmonate (MeJA), jasmonic acid (JA), salicylic acid (SA), fungus polysaccharide, yeast extract, heavy metal etc. Extensive researches have been done to study the roles of JA as the key signaling molecules in signal transduction system regulating the alteration of plant defense genes against environmental stresses. For instance, the expression of *pin* genes was activated by JA or its derivative, MeJA, in mechanically wounded tomato and potato (Farmer & Ryan 1992). The expression of *vsp* genes was also activated by MeJA as a defense mechanism towards wounded cells of soy bean (Creelman et al. 1992). Besides, exposure of *Hypericum perforatum* L. suspension culture to JA had activated the genes that expressed phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) enzymes. Activation of these genes had increased the production of phenylpropanoids such as phenolic, flavanol and flavonol a 6-fold in cells treated with JA (Gadzovska et al. 2007). JA also altered the synthesis of ajmalicine and catharantine in *Catharanthus roseus* (Vazquez-Flota & De Luca 1998), rosmarinic acid and shikonin in *Lithospermum erythrorhizon* (Yazaki et al. 1997), scopoletin dan scopolin in *Nicotiana tabacum* (Sharon et al. 1998) and taxol dan paclitaxel in *Taxus* spp. (Palazon et al. 2003). Thus, jasmonate has been shown to be key molecules in the elicitation process leading to de novo transcription and translation that resulted in the enhancement of secondary metabolites biosynthesis in *in vitro* plants (Gundlach et al. 1992).

It is clear that harsh environmental conditions would activate the expression of certain abiotic stress related genes which involve in the biosynthesis pathway of secondary metabolites in plants. Therefore it becomes a crucial need to identify the stress responsive genes and study their signal transduction pathways not only for a better understanding of plants response and adaptation towards abiotic stress, but also for further development of strategies for commercial production of valuable compounds by manipulating certain metabolic pathways based on gene expression (Shilpa et al. 2010). A significant amount of studies have been done on application of elicitation to plant cultures, either to enhance secondary metabolites production or to discover novel compounds. Though many of these showed positive and encouraging results, the stress responsive genes which involved in the reactions of defense-related pathway and secondary metabolites biosynthesis pathway remain largely unexploited. Many molecular approaches such as mRNA differential display, representation difference analysis, RNA fingerprinting, cDNA microarray and suppression subtractive hybridization (SSH) technique have been applied to identify and characterize the

Alteration of Abiotic Stress Responsive Genes

Enzyme, cell wall pieces, pectin, chitosan, glucan.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 53

now, a few hypotheses have been proposed as the biochemical responses of plants towards the challenges of elicitors. For examples, ion Ca2+ influx changes in the cytoplasm (Gelli et al. 1997) and significant changes in protein phosphorylation and kinase proteins activation (Romeis 2001). Besides, some scientists also hypothesized that deactivation of H+-ATPase will result in acidic cytoplasm (Armero & Tena 2001). Generation of reactive oxygen species (ROS) such as superoxide anion and H2O2 may have direct antimicrobial effect against pathogen attack, in the case of biotic elicitors. These ROS could trigger the formation of bioactive fatty acids derivatives in plants (Apostol et al. 1989). Furthermore, ROS could act as secondary signals in the activation of plant defence genes expression (Low & Merida 1996). Thus, it could be concluded that the mechanism of elicitors is almost the same according to the origin, specificity, concentration, physio-chemical environment, stages in

**Nature-based elicitors Origin-based elicitors Biotic elicitors Abiotic elicitors Exogenous elicitors Endogenous** 

> Glucomannose, glucan, chitosan, poly-L-lysin,

polyamine, glycoprotein, polygalacturonase,

selulase.

Jasmonic acid (JA) or its methyl ester, Methyl jasmonate (MeJA) are endogenous signalling molecules derived from lipid and distributed widely in plants. They function primarily in plant response towards biotic and abiotic stress and a variety of plant growth and development processes, including flowering, fruit ripening, and root growth. It has also been recognised as promoter for senescence, growth inhibitor and elicitor for secondary metabolism in many plant species. It is synthesized from oxylipin by lipoxygenase pathway in plant cells. The signalling of oxilipin molecules which include JA, MeJA, JA and amino acid conjugate and other JA derivatives, are regulated by the fatty acid residues that form plant membranes (Wasternack 2007). Oxilipins are originated from either α-linolenic acid (α-LeA, 18:3) or linoleic acid (18:2) released by chloroplast membrane. JA biosynthesis is initiated with the formation of (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) and (9S) hydroperoxyoctadecatrienoic acid (9-HPOT) from α-LeA. Product formed from linolenic acid is (13S)-hydroperoxyoctadecadienoic acid (13-HPOD) whereas linoleic acid will produce (9S)-hydroperoxyoctadecadienoic acid (9-HPOD). This reaction is catalysed by lipoxygenase (LOX) enzyme (Feussner & Wasternack 2002). 13-HPOT will then be converted by allene oxide synthase (AOS) to 12-13-epoxy-octadecatrienoic acid (12, 13-EOT) which is very instable (Song et al. 1993). 12, 13-EOT will be converted to *cis*(+)-12-oxophytodienoic acid (OPDA) by allene oxide cyclase (AOC), and consequently to 3-oxo-2(2'-pentenyl) cyclopentane-1-heptanoic acid (OPC) by 12-oxophytodienoic reductase acid (OPR). For the final step in JA biosynthesis, OPC will undergo three β-oxidation cycles in peroxysomes (Miersch & Wasternack 2000). Every gene that coded for JA biosynthesis enzyme could be

**elicitors** 

Dodeca-β-1,4-Dgalacturonide, heptaβ-glucoside, alginate

oligomer

plant's life cycle and nutrient assimilation of plant (Namdeo 2007).

UV light, denatured protein, heavy metal, chemical.

Table 1. Classification of elicitors according to their nature or origin

**2.2 JA biosynthesis and its roles in plant secondary metabolism** 

genes which were expressed differentially during stress condition. Of all, SSH is a more effective and efficient method compared to others especially when non-model organism is of concern because genome sequences information is not required (Huang et al. 2007). By combining normalization and suppression PCR in a single step, SSH technique could reduce the excessive target cDNA sequences; at the same time enriched the low amount differential expressed transcripts up to 1000 – 5000 times in the sample population (Diatchenko et al. 1996). Hence, it is rational to identify the JA responsive genes by SSH technique because these genes may be involved in the synthesis pathways of valuable metabolites and/or abiotic stress tolerance. In order to support this hypothesis, we had demonstrated the effects of jasmonic acid (JA) as the elicitor in altering secondary metabolites synthesis in a type of local herb called *Polygonum minus* and the expression of JA-responsive genes have been identified by subtractive cDNA library construction.

### **2. Elicitation**

#### **2.1 The concept of elicitation**

In general, plants respond towards abiotic stress stimuli by regulating signaling cascade followed by modulating gene expression machinery which could lead to the synthesis of stress responsive protein or valuable bioactive compounds. When stress signal received by the receptor on plant membrane, the small endogenous signaling molecules such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) will regulate defense system, both synergistically and antagonistically. It has been proven scientifically that exogenous application of these signaling molecules on plant cultures could alter the expression of genes involved in biosynthesis of different classes of secondary metabolites. This approach is known as elicitation, a process that involves the application of chemicals or addition of physical stress to plant cultures or whole plants as a way to produce secondary metabolites which are normally absent in the plants (Bourgard et al. 2001; Roberts & Shuler 1997). Elicitors are defined as chemicals from various sources which could trigger the physiological and morphological changes or phytoalexin accumulation in plants as a defense mechanism against stresses (Sekar & Kandavel 2010). They could mimic the mode of action of natural stress stimuli and thus create a stress environment for plants growth and development. They are able to trigger the normal metabolism in plant cells to synthesize enzymes that catalyze the defense-related pathways which ultimately leading to secondary metabolites production.There are a few ways to classify type of elicitors. It could be categorized according to its nature, which is known as biotic and abiotic elicitors or based on its origin, which is known as exogenous and endogenous elicitors. Abiotic elicitors are substances that derived from non-living thing such as inorganic salts, heavy metal ion, UV radiation, high pH level and so on whereas biotic elicitors are derived from living organims, for instance, pectin and selulose from plant cell walls, and chitin and glucan from microorganism. On the other hand, as defined by the prefix "exo-" and "endo-", exogenous elicitors are substances derived from outer cellular compartment such as polisaccharide, polyamine and fatty acids whereas endogenous elicitors are molecules present in the inner cellular compartment, such as glucuronide or hepta-β-glucoside (Namdeo 2007). Table 1 summarizes the classification of elicitors.

A handful of experiments have been done extensively to study the effects of abiotic elicitors in enhancing the production of secondary metabolites in whole plants or plant cell cultures. However, the mechanisms that actually take place in the elicited cells remain unclear. Till

genes which were expressed differentially during stress condition. Of all, SSH is a more effective and efficient method compared to others especially when non-model organism is of concern because genome sequences information is not required (Huang et al. 2007). By combining normalization and suppression PCR in a single step, SSH technique could reduce the excessive target cDNA sequences; at the same time enriched the low amount differential expressed transcripts up to 1000 – 5000 times in the sample population (Diatchenko et al. 1996). Hence, it is rational to identify the JA responsive genes by SSH technique because these genes may be involved in the synthesis pathways of valuable metabolites and/or abiotic stress tolerance. In order to support this hypothesis, we had demonstrated the effects of jasmonic acid (JA) as the elicitor in altering secondary metabolites synthesis in a type of local herb called *Polygonum minus* and the expression of JA-responsive genes have been

In general, plants respond towards abiotic stress stimuli by regulating signaling cascade followed by modulating gene expression machinery which could lead to the synthesis of stress responsive protein or valuable bioactive compounds. When stress signal received by the receptor on plant membrane, the small endogenous signaling molecules such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) will regulate defense system, both synergistically and antagonistically. It has been proven scientifically that exogenous application of these signaling molecules on plant cultures could alter the expression of genes involved in biosynthesis of different classes of secondary metabolites. This approach is known as elicitation, a process that involves the application of chemicals or addition of physical stress to plant cultures or whole plants as a way to produce secondary metabolites which are normally absent in the plants (Bourgard et al. 2001; Roberts & Shuler 1997). Elicitors are defined as chemicals from various sources which could trigger the physiological and morphological changes or phytoalexin accumulation in plants as a defense mechanism against stresses (Sekar & Kandavel 2010). They could mimic the mode of action of natural stress stimuli and thus create a stress environment for plants growth and development. They are able to trigger the normal metabolism in plant cells to synthesize enzymes that catalyze the defense-related pathways which ultimately leading to secondary metabolites production.There are a few ways to classify type of elicitors. It could be categorized according to its nature, which is known as biotic and abiotic elicitors or based on its origin, which is known as exogenous and endogenous elicitors. Abiotic elicitors are substances that derived from non-living thing such as inorganic salts, heavy metal ion, UV radiation, high pH level and so on whereas biotic elicitors are derived from living organims, for instance, pectin and selulose from plant cell walls, and chitin and glucan from microorganism. On the other hand, as defined by the prefix "exo-" and "endo-", exogenous elicitors are substances derived from outer cellular compartment such as polisaccharide, polyamine and fatty acids whereas endogenous elicitors are molecules present in the inner cellular compartment, such as glucuronide or hepta-β-glucoside (Namdeo 2007). Table 1

A handful of experiments have been done extensively to study the effects of abiotic elicitors in enhancing the production of secondary metabolites in whole plants or plant cell cultures. However, the mechanisms that actually take place in the elicited cells remain unclear. Till

identified by subtractive cDNA library construction.

**2. Elicitation** 

**2.1 The concept of elicitation** 

summarizes the classification of elicitors.

now, a few hypotheses have been proposed as the biochemical responses of plants towards the challenges of elicitors. For examples, ion Ca2+ influx changes in the cytoplasm (Gelli et al. 1997) and significant changes in protein phosphorylation and kinase proteins activation (Romeis 2001). Besides, some scientists also hypothesized that deactivation of H+-ATPase will result in acidic cytoplasm (Armero & Tena 2001). Generation of reactive oxygen species (ROS) such as superoxide anion and H2O2 may have direct antimicrobial effect against pathogen attack, in the case of biotic elicitors. These ROS could trigger the formation of bioactive fatty acids derivatives in plants (Apostol et al. 1989). Furthermore, ROS could act as secondary signals in the activation of plant defence genes expression (Low & Merida 1996). Thus, it could be concluded that the mechanism of elicitors is almost the same according to the origin, specificity, concentration, physio-chemical environment, stages in plant's life cycle and nutrient assimilation of plant (Namdeo 2007).


Table 1. Classification of elicitors according to their nature or origin

### **2.2 JA biosynthesis and its roles in plant secondary metabolism**

Jasmonic acid (JA) or its methyl ester, Methyl jasmonate (MeJA) are endogenous signalling molecules derived from lipid and distributed widely in plants. They function primarily in plant response towards biotic and abiotic stress and a variety of plant growth and development processes, including flowering, fruit ripening, and root growth. It has also been recognised as promoter for senescence, growth inhibitor and elicitor for secondary metabolism in many plant species. It is synthesized from oxylipin by lipoxygenase pathway in plant cells. The signalling of oxilipin molecules which include JA, MeJA, JA and amino acid conjugate and other JA derivatives, are regulated by the fatty acid residues that form plant membranes (Wasternack 2007). Oxilipins are originated from either α-linolenic acid (α-LeA, 18:3) or linoleic acid (18:2) released by chloroplast membrane. JA biosynthesis is initiated with the formation of (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) and (9S) hydroperoxyoctadecatrienoic acid (9-HPOT) from α-LeA. Product formed from linolenic acid is (13S)-hydroperoxyoctadecadienoic acid (13-HPOD) whereas linoleic acid will produce (9S)-hydroperoxyoctadecadienoic acid (9-HPOD). This reaction is catalysed by lipoxygenase (LOX) enzyme (Feussner & Wasternack 2002). 13-HPOT will then be converted by allene oxide synthase (AOS) to 12-13-epoxy-octadecatrienoic acid (12, 13-EOT) which is very instable (Song et al. 1993). 12, 13-EOT will be converted to *cis*(+)-12-oxophytodienoic acid (OPDA) by allene oxide cyclase (AOC), and consequently to 3-oxo-2(2'-pentenyl) cyclopentane-1-heptanoic acid (OPC) by 12-oxophytodienoic reductase acid (OPR). For the final step in JA biosynthesis, OPC will undergo three β-oxidation cycles in peroxysomes (Miersch & Wasternack 2000). Every gene that coded for JA biosynthesis enzyme could be

Alteration of Abiotic Stress Responsive Genes

appropriate data obtained.

**2.3.1 Phytochemical analysis of** *P. minus* **roots** 

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 55

properties. For example, researchers have found phytoestrogens from *P. cuspidatum* and *P. hydropiper* roots (Matsuda et al. 2001), phytohormones, and anthraquinones from *P. multiflorum* roots (Yu et al. 2006) and indigo from *P. tinctorium* roots (Chae et al. 2000). We decided to target roots as the organ for elicitation process because of their ability to import and export molecules between root cells and the rhizosphere (Gleba et al. 1999). In addition, roots also possess some advantages against other plant parts. As they are physically unprotected in the soil environment, they are surrounded by many types of microorganisms. Hence, they may produce a vast amount of metabolites that possess antimicrobial or aromatic properties to ensure plant survival (Poulev et al. 2003). The application of elicitors on plant shoots has serious limitations because the hydrophobic surfaces and impermeable characteristics of leaves result in low uptake of chemical elicitors. On the contrary, elicitors can be easily added into growth media and absorbed by roots and can easily harvest and screen for bioactive compounds. Roots also contain low levels of pigments and other compounds found in leaves, such as tannins, which may interfere in

In our experiment, *P. minus* roots harvested from *in vitro* plantlets were used for elicitation process and subtractive cDNA library construction. *P. minus* were micropropagated by internode culture and subculture in MS solid medium at every 2 months interval. The cultures were incubated at 26 + 2 ºC with 16 hours photoperiod and 20 μM/m2/s light intensity. Prior to adding JA solution, *P. minus* were transferred to MS liquid medium supplemented with different concentrations of JA (50µM, 100 µM, 150 µM and 200 µM). The non-treated plantlets were used as control. *P. minus* roots were harvested from JA-treated plantlets at day-1, day-3 and day-6 for GC-MS analysis. Each experiment was performed in triplicate where each treatment contained 10 plantlets. The experimental designed was done based on factorial 5 x 3 (JA concentration x treatment period). The volatile compounds and other secondary metabolites induced by JA stress were extracted from 2g of roots by Solid Phase Micro Extraction (SPME). The extracted compounds were then purified and separated by gas chromatography in Shimadzu AQ5050P with HP-5MS non-polar column (30m x 0.25mm x 0.25µM) and detected by quadrupole mass spectrometry. The parameters for GC-MS analysis were as follow: injection temperature 220ºC; detector temperature 280ºC; column temperature 50ºC – 3 min, 20ºC/min - 100ºC, hold 3 min, 30ºC/min - 250ºC, hold 3 min; flow rate 1.3ml/min; injection volume 1µl; injection method – split ration and mass spectrometry was operated in scan mode. Finally the compounds detected by MS were compared against the GC-MS Nist. 147 library according to similarity index (SI) and retention time (RT). Only compounds with SI unit more than 80 and present consistently in two or more replicate were considered for further analysis. The results from qualitative analysis showed β-caryophyllene present abundantly and consistently in the sample. Hence, it was selected as a marker compounds which is known as single point external standard for quantitative analysis. The data obtained were analysed with SAS (Statistical Analysis Systems) statistic program version 9.0 at significant level p < 0.05. General Linear Modelling (GLM) and Duncan analysis were performed according to experimental design and the

The chemical compounds from some species in the Polygonum family have been studied decades ago, for example *P. minus* (Karim 1987) and *P. odoratum* (Dung et al. 1995; Hunter et al. 1997). From our experiment, 30 compounds were successfully detected by GC-MS from

chemical screening and extraction (Gleba et al. 1999; Poulev et al.2003).

alter by JA itself (Wasternack 2006). Till now, many promoters have been tested and it was found that the activities of promoters have been increase upon exposure to JA (Kubigsteltig et al. 1999). This observation suggested that the biosynthesis of JA is regulated by positive feedback reaction.

JA is recognised as the signalling molecule in elicitation process that leads to *de novo* transcription and translation and eventually activates the secondary metabolites in plant cell cultures (Gundlach et al. 1992). It has also been known as molecule involves in the signal transduction pathway that leads to the activation of plant defense against pathogen, insect and herbivore attack (Menke et al. 1999). There are also some studies which shown that JA activities are not restricted to one type of secondary metabolite only but it is able to alter the production pathways of various classes of secondary metabolites such as phenylpropanoids, alkaloids and terpenoids (Zhao et al. 2005; Pauwels et al. 2009). Many studies were done using JA as the elicitor to induce the production of important metabolites. For example, the *Hypericum perforatum* (St. John's wort) cell cultures showed significant increase in the production of hypericin after treated with JA (Walker et al. 2002). This result was supported by another group of scientists where they have successfully increase 6-8 times the production of phenylpropanoids like phenolic compounds, flavanol, flavonol; and naphtodianthrones like hypericin in *H. perofatum* cell culture after elicited by JA (Gadzovska et al. 2007). Besides, the production of anthraquinone in *Morinda elliptica* cell cultures was also enhanced by JA treatment (Chong et al. 2005), so as to antioxidant like carotenoids, vitamin C and vitamin E (Chong et al. 2005). Furthermore, MeJA was showed to increase the accumulation of silymarin products in *Silybum marianum* (L.) Gaertn cell cultures (Sanchez-Sampedro et al. 2005) and shikonin in *Lithospermum* cell cultures (Yazaki et al. 1997).

In a recent study, we are interested in finding the genes involved in the biosynthesis of aromatic compounds or other valuable metabolites found in *P. minus* roots using the elicitation strategy. Since most of the secondary metabolites induced by elicitor present *de novo* in plant cells (Pare & Tumlinson 1997) and involved certain enzymes activities induction (Bouwmeester et al. 1999; Degenhardt & Gershenzon 2000), the production of volatile compounds triggered by JA in kesum roots will reflect the enzyme activities which catalysed the biosynthesis of those compounds. The enzyme activity is proportional with the mRNA transcripts related to the compounds production. By combining the analysis of metabolomics and differentially expressed genes dataset, we hope to have a better understanding in the correlation between plant defence system against abiotic stress and secondary metabolites production.

### **2.3 Case study: Elicitation of** *Polygonum minus* **roots with JA**

We have recently conducted an experiment to evaluate the effect of JA elicitation on the production of volatile compounds in *Polygonum minus* roots by gas chromatography coupled with mass spectrometry, as well as using SSH technique to identify the transcripts responsive to JA stress. *Polygonum minus* Huds., or commonly known as kesum, is a type of local herbs rich in essential oils. It has been recognized by the Malaysian government in the Herbal Product Blueprint as an essential oil-producing crop (Wan Hassan 2007). Previous studies reported that the essential oil from kesum leaves was found to contain about 76.59% aliphatic aldehydes that consisted of two dominant compounds, decanal and dodecanal, and contained 0.18% b-caryophyllene, a sesquiterpene (Karim 1987). Other studies have successfully identified valuable compounds from *Polygonum* roots that possessed medicinal

alter by JA itself (Wasternack 2006). Till now, many promoters have been tested and it was found that the activities of promoters have been increase upon exposure to JA (Kubigsteltig et al. 1999). This observation suggested that the biosynthesis of JA is regulated by positive

JA is recognised as the signalling molecule in elicitation process that leads to *de novo* transcription and translation and eventually activates the secondary metabolites in plant cell cultures (Gundlach et al. 1992). It has also been known as molecule involves in the signal transduction pathway that leads to the activation of plant defense against pathogen, insect and herbivore attack (Menke et al. 1999). There are also some studies which shown that JA activities are not restricted to one type of secondary metabolite only but it is able to alter the production pathways of various classes of secondary metabolites such as phenylpropanoids, alkaloids and terpenoids (Zhao et al. 2005; Pauwels et al. 2009). Many studies were done using JA as the elicitor to induce the production of important metabolites. For example, the *Hypericum perforatum* (St. John's wort) cell cultures showed significant increase in the production of hypericin after treated with JA (Walker et al. 2002). This result was supported by another group of scientists where they have successfully increase 6-8 times the production of phenylpropanoids like phenolic compounds, flavanol, flavonol; and naphtodianthrones like hypericin in *H. perofatum* cell culture after elicited by JA (Gadzovska et al. 2007). Besides, the production of anthraquinone in *Morinda elliptica* cell cultures was also enhanced by JA treatment (Chong et al. 2005), so as to antioxidant like carotenoids, vitamin C and vitamin E (Chong et al. 2005). Furthermore, MeJA was showed to increase the accumulation of silymarin products in *Silybum marianum* (L.) Gaertn cell cultures (Sanchez-

Sampedro et al. 2005) and shikonin in *Lithospermum* cell cultures (Yazaki et al. 1997).

**2.3 Case study: Elicitation of** *Polygonum minus* **roots with JA** 

In a recent study, we are interested in finding the genes involved in the biosynthesis of aromatic compounds or other valuable metabolites found in *P. minus* roots using the elicitation strategy. Since most of the secondary metabolites induced by elicitor present *de novo* in plant cells (Pare & Tumlinson 1997) and involved certain enzymes activities induction (Bouwmeester et al. 1999; Degenhardt & Gershenzon 2000), the production of volatile compounds triggered by JA in kesum roots will reflect the enzyme activities which catalysed the biosynthesis of those compounds. The enzyme activity is proportional with the mRNA transcripts related to the compounds production. By combining the analysis of metabolomics and differentially expressed genes dataset, we hope to have a better understanding in the correlation between plant defence system against abiotic stress and

We have recently conducted an experiment to evaluate the effect of JA elicitation on the production of volatile compounds in *Polygonum minus* roots by gas chromatography coupled with mass spectrometry, as well as using SSH technique to identify the transcripts responsive to JA stress. *Polygonum minus* Huds., or commonly known as kesum, is a type of local herbs rich in essential oils. It has been recognized by the Malaysian government in the Herbal Product Blueprint as an essential oil-producing crop (Wan Hassan 2007). Previous studies reported that the essential oil from kesum leaves was found to contain about 76.59% aliphatic aldehydes that consisted of two dominant compounds, decanal and dodecanal, and contained 0.18% b-caryophyllene, a sesquiterpene (Karim 1987). Other studies have successfully identified valuable compounds from *Polygonum* roots that possessed medicinal

feedback reaction.

secondary metabolites production.

properties. For example, researchers have found phytoestrogens from *P. cuspidatum* and *P. hydropiper* roots (Matsuda et al. 2001), phytohormones, and anthraquinones from *P. multiflorum* roots (Yu et al. 2006) and indigo from *P. tinctorium* roots (Chae et al. 2000). We decided to target roots as the organ for elicitation process because of their ability to import and export molecules between root cells and the rhizosphere (Gleba et al. 1999). In addition, roots also possess some advantages against other plant parts. As they are physically unprotected in the soil environment, they are surrounded by many types of microorganisms. Hence, they may produce a vast amount of metabolites that possess antimicrobial or aromatic properties to ensure plant survival (Poulev et al. 2003). The application of elicitors on plant shoots has serious limitations because the hydrophobic surfaces and impermeable characteristics of leaves result in low uptake of chemical elicitors. On the contrary, elicitors can be easily added into growth media and absorbed by roots and can easily harvest and screen for bioactive compounds. Roots also contain low levels of pigments and other compounds found in leaves, such as tannins, which may interfere in chemical screening and extraction (Gleba et al. 1999; Poulev et al.2003).

In our experiment, *P. minus* roots harvested from *in vitro* plantlets were used for elicitation process and subtractive cDNA library construction. *P. minus* were micropropagated by internode culture and subculture in MS solid medium at every 2 months interval. The cultures were incubated at 26 + 2 ºC with 16 hours photoperiod and 20 μM/m2/s light intensity. Prior to adding JA solution, *P. minus* were transferred to MS liquid medium supplemented with different concentrations of JA (50µM, 100 µM, 150 µM and 200 µM). The non-treated plantlets were used as control. *P. minus* roots were harvested from JA-treated plantlets at day-1, day-3 and day-6 for GC-MS analysis. Each experiment was performed in triplicate where each treatment contained 10 plantlets. The experimental designed was done based on factorial 5 x 3 (JA concentration x treatment period). The volatile compounds and other secondary metabolites induced by JA stress were extracted from 2g of roots by Solid Phase Micro Extraction (SPME). The extracted compounds were then purified and separated by gas chromatography in Shimadzu AQ5050P with HP-5MS non-polar column (30m x 0.25mm x 0.25µM) and detected by quadrupole mass spectrometry. The parameters for GC-MS analysis were as follow: injection temperature 220ºC; detector temperature 280ºC; column temperature 50ºC – 3 min, 20ºC/min - 100ºC, hold 3 min, 30ºC/min - 250ºC, hold 3 min; flow rate 1.3ml/min; injection volume 1µl; injection method – split ration and mass spectrometry was operated in scan mode. Finally the compounds detected by MS were compared against the GC-MS Nist. 147 library according to similarity index (SI) and retention time (RT). Only compounds with SI unit more than 80 and present consistently in two or more replicate were considered for further analysis. The results from qualitative analysis showed β-caryophyllene present abundantly and consistently in the sample. Hence, it was selected as a marker compounds which is known as single point external standard for quantitative analysis. The data obtained were analysed with SAS (Statistical Analysis Systems) statistic program version 9.0 at significant level p < 0.05. General Linear Modelling (GLM) and Duncan analysis were performed according to experimental design and the appropriate data obtained.

### **2.3.1 Phytochemical analysis of** *P. minus* **roots**

The chemical compounds from some species in the Polygonum family have been studied decades ago, for example *P. minus* (Karim 1987) and *P. odoratum* (Dung et al. 1995; Hunter et al. 1997). From our experiment, 30 compounds were successfully detected by GC-MS from

Alteration of Abiotic Stress Responsive Genes

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 57

period that could alter and increase the production of volatile compounds significantly, which is more than 2-fold compared to control will be chosen for subsequent subtractive screening. This step was to ensure that the differentially expressed genes are the genes coded for enzymes involved in volatile compounds induced by JA. We divided the compounds induced by JA into three categories. First group represents the compounds that increase in kesum roots treated with JA (nonane, heptane, β-caryophyllene, trans-αbergamotene, β-farnesene, α-caryophyllene and pentanoic acid). Second group represents compounds that decrease or not detected in kesum roots after JA treatment (pbenzoquinone, phenol, α-panasinsen, octane, undecane and 1,2-benzenedicarboxylic acid) whereas the third group shows compounds that have slight increment after JA elicitation (octadecanal). The chromatogram profile for JA-induced compounds was shown in Figure 2.

Retention Time Compounds Total Peak Area (%) 8.027 Nonane 1.65 13.599 Heptane 1.11 13.752 Octadecanal 3.08 13.922 β-caryophyllene 17.57 14.007 Trans-α-bergamotene 2.13 14.128 β-farnesene 2.84 14.193 α-caryophyllene 9.50 14.256 p-benzoquinone 1.85 14.532 Phenol 2.73 14.656 α-panasinsen 1.82 15.063 Pentanoic acid 1.47 15.556 Octane 1.42 15.633 Heptane 0.44 16.072 Undecane 0.52 16.517 1,2-benzenedicarboxylic acid 0.52 16.596 Nonane 0.44

Among the elevated compounds, β-caryophyllene was found to be the most dominant sesquiterpene compound in kesum roots and it presents consistently in every experiment replicate. It has also been found in many other plant species such as *Elsholtzia argyi* flower (Peng & Yang 2004), *Salvia officinalis* flower (Dewick 2001), carrot seed oil (Ozcan & Chalchat 2007) and *Artemisia absinthium* essential oil (Judpentiene & Mockute 2004). This compound is the major ingredient for plant aroma (Peng & Yang 2004) and it is always used in the perfumery and aromatherapy industry (Dewick 2001). Therefore it was chosen as a marker compounds for further quantitative analysis where JA concentration and treatment period could be determined. For this purpose, pure β-caryophyllene was purchased from Sigma Ltd. as an external standard and diluted to 100ppm to obtain the standard peak area. The number of β-caryophyllene analytes from the JA-treated root samples was calculated by comparing their respective peak area with the standard peak area. All data collected were performed with ANOVA analysis. The increment of β-caryophyllene analytes was considered significant at the significant level of p < 0.05. Figure 3 below shows the effect of

Table 2. Volatile compounds detected in kesum root extracts.

the *P. minus* roots extract based on the comparison of similarity index (SI) and retention time (RT) with the database in NIST 147 library. The chromatogram profile for non-treated plant was shown in Figure 1.

Fig. 1. Chromatogram profile for volatile compounds extracted from non-treated kesum roots extract using HP-5MS non-polar column (30m length x 0.25mm diameter x 0.25µM thickness). Injection temperature: 220ºC; Detection temperature: 280 ºC; Column temperature: 50 ºC, 3 min; 20ºC/min - 100ºC, 3 min; 30ºC/min - 250ºC, 3 min. Flow rate: 1.3ml/min. Injection volume: 1µl. Injection method: split ratio. Type of detector: quadrupole. Mass spectrometry: scan mode. TIC: Total ion chromatogram.

Of 30 compounds detected, only 16 compounds were found to have SI value more than 80. These compounds were shown in Table 2 according to their own RT. The dominant compounds found in kesum roots extract are β-caryophyllene (17.57%), acetic acid (11.07%), α-caryophyllene (9.50%), octadecanal (3.08%), β-farnesene (2.84%), phenol (2.73%) and trans-α-bergamotene (2.13%). The volatile extracts consisted of 32.85% sesquiterpenes (βcaryophyllene, trans-α-bergamotene, β-farnesene, α-caryophyllene and α-panasinsene) and 5.58% alkanes (nonane, heptanes, octane and undecane). Only one aliphatic aldehyde was detected in kesum root extracts, which is known as octadecanal (3.07%).

### **2.3.2 The effect of JA elicitation on the production of volatile compounds in** *P. minus*  **roots**

We were looking into two factors that will affect JA elicitation on kesum, which is the JA concentration and treatment period. Four concentrations of JA (50µM, 100µM, 150µM and 200µM) and three treatment period (1, 3 and 6 days) were tested to ensure that the concentration of JA is not too high or the treatment period is not too long to cause plant death. This is because JA was proven to be the negative regulator for plant growth and development by creating a stress environmental condition to plants (Gadzovska et al. 2007). The optimum JA concentration and treatment period need to be carefully determined to ensure that the volatile compounds could be altered to the maximum level of production compared to control sample. Only the combination of JA concentration and treatment

the *P. minus* roots extract based on the comparison of similarity index (SI) and retention time (RT) with the database in NIST 147 library. The chromatogram profile for non-treated plant

Fig. 1. Chromatogram profile for volatile compounds extracted from non-treated kesum roots extract using HP-5MS non-polar column (30m length x 0.25mm diameter x 0.25µM

temperature: 50 ºC, 3 min; 20ºC/min - 100ºC, 3 min; 30ºC/min - 250ºC, 3 min. Flow rate:

Of 30 compounds detected, only 16 compounds were found to have SI value more than 80. These compounds were shown in Table 2 according to their own RT. The dominant compounds found in kesum roots extract are β-caryophyllene (17.57%), acetic acid (11.07%), α-caryophyllene (9.50%), octadecanal (3.08%), β-farnesene (2.84%), phenol (2.73%) and trans-α-bergamotene (2.13%). The volatile extracts consisted of 32.85% sesquiterpenes (βcaryophyllene, trans-α-bergamotene, β-farnesene, α-caryophyllene and α-panasinsene) and 5.58% alkanes (nonane, heptanes, octane and undecane). Only one aliphatic aldehyde was

**2.3.2 The effect of JA elicitation on the production of volatile compounds in** *P. minus* 

We were looking into two factors that will affect JA elicitation on kesum, which is the JA concentration and treatment period. Four concentrations of JA (50µM, 100µM, 150µM and 200µM) and three treatment period (1, 3 and 6 days) were tested to ensure that the concentration of JA is not too high or the treatment period is not too long to cause plant death. This is because JA was proven to be the negative regulator for plant growth and development by creating a stress environmental condition to plants (Gadzovska et al. 2007). The optimum JA concentration and treatment period need to be carefully determined to ensure that the volatile compounds could be altered to the maximum level of production compared to control sample. Only the combination of JA concentration and treatment

thickness). Injection temperature: 220ºC; Detection temperature: 280 ºC; Column

1.3ml/min. Injection volume: 1µl. Injection method: split ratio. Type of detector: quadrupole. Mass spectrometry: scan mode. TIC: Total ion chromatogram.

detected in kesum root extracts, which is known as octadecanal (3.07%).

was shown in Figure 1.

**roots**

period that could alter and increase the production of volatile compounds significantly, which is more than 2-fold compared to control will be chosen for subsequent subtractive screening. This step was to ensure that the differentially expressed genes are the genes coded for enzymes involved in volatile compounds induced by JA. We divided the compounds induced by JA into three categories. First group represents the compounds that increase in kesum roots treated with JA (nonane, heptane, β-caryophyllene, trans-αbergamotene, β-farnesene, α-caryophyllene and pentanoic acid). Second group represents compounds that decrease or not detected in kesum roots after JA treatment (pbenzoquinone, phenol, α-panasinsen, octane, undecane and 1,2-benzenedicarboxylic acid) whereas the third group shows compounds that have slight increment after JA elicitation (octadecanal). The chromatogram profile for JA-induced compounds was shown in Figure 2.


Table 2. Volatile compounds detected in kesum root extracts.

Among the elevated compounds, β-caryophyllene was found to be the most dominant sesquiterpene compound in kesum roots and it presents consistently in every experiment replicate. It has also been found in many other plant species such as *Elsholtzia argyi* flower (Peng & Yang 2004), *Salvia officinalis* flower (Dewick 2001), carrot seed oil (Ozcan & Chalchat 2007) and *Artemisia absinthium* essential oil (Judpentiene & Mockute 2004). This compound is the major ingredient for plant aroma (Peng & Yang 2004) and it is always used in the perfumery and aromatherapy industry (Dewick 2001). Therefore it was chosen as a marker compounds for further quantitative analysis where JA concentration and treatment period could be determined. For this purpose, pure β-caryophyllene was purchased from Sigma Ltd. as an external standard and diluted to 100ppm to obtain the standard peak area. The number of β-caryophyllene analytes from the JA-treated root samples was calculated by comparing their respective peak area with the standard peak area. All data collected were performed with ANOVA analysis. The increment of β-caryophyllene analytes was considered significant at the significant level of p < 0.05. Figure 3 below shows the effect of

Alteration of Abiotic Stress Responsive Genes

identify and clone the transcripts regulated by JA stress.

**3. Differentially expressed genes induced by JA** 

Subtractive screening is an efficient approach to clone the genes which are being expressed in one population but not being expressed or slightly expressed in another population. In this study, cDNA derived from kesum roots treated with 150µM JA for 3 days was served as tester whereas the cDNA derived from non-treated kesum roots was served as driver for subtracted cDNA library construction. Only forward subtraction was done as we were interested in identifying genes up-regulated by JA. A total of 1,344 white colonies were randomly picked from the subtracted cDNA library and screened by PCR using M13 forward and reverse universal primers to confirm the presence of cDNA inserts. PCR amplification revealed that 960 colonies were single stranded clones with the insert sizes ranging from 250bp to 1,200bp. These clones were subsequently hybridized against

**3.1 Identification of JA-responsive genes** 

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 59

Day-1 observation indicate that the production of β-caryophyllene was occur at a much lower level and inconsistent because the standard deviation between replicates differ greatly (coefficient of variance = 27.86). The overall production of this sesquiterpene compound at all JA concentration was lower than control. The ANOVA analysis showed that no significant increase (p > 0.5) was observed in the production of β-caryophyllene in kesum roots treated with all four JA concentrations compared to control. This might be because of the kesum plantlets might need time to adapt to the stressful environment. The development of kesum root cells might be retarded after treated with JA which would suppress the primary metabolism and subsequently activated the secondary metabolism in root cells (Chen & Chen 2000; Wang et al. 2001). The level of production in day-3 was much higher and could be found in all three replicates for each JA concentration tested. The production of β-caryophyllene increased after treated with JA from 50µM to 150µM but decrease again at 200µM. Significant increase (p < 0.05) was seen when *P. minus* was treated with 150µM JA, which is 203.45 + 114.79µg/g compared to 25.59 + 8.96µg/g in control roots (coefficience of variance = 73.28). The production of this sesquiterpene compound increase about 1.2-fold, 2.1-fold and 8 fold in the roots treated with 50µM, 100µM and 150µM JA respectively compared to control sample when kesum plantlets established defense mechanism against abiotic stress created by JA elicitation. However, exposure of kesum to higher concentration of JA inhibited the production of β-caryophyllene where a decrease of 1.4-fold of this metabolite was observed in 200µM JA treatment. Necrosis was observed when the leaves and roots of kesum turned brownish as JA is an inhibitor to roots growth (Wasternack 2007). In day-6, β-caryophyllene present consistently in two or more replicates in every treatment. However, ANOVA analysis showed that the level of production decrease significantly (p > 0.05) compared to day-3 in all four treatments (coefficience of variance = 34.21). This situation might be caused by over exposure of kesum plantlets to JA stress. Our observation is similar to a study done by Sanchez-Sampedro et al. (2005) where they found that the production of silymarin in *Silybum marianum* increased to the maximum level after 3 days of MeJA treatment but decrease significantly after 7 days. Therefore, we concluded that kesum roots treated with 150µM JA for 3 days could produce βcaryophyllene at maximum level and we assume that the biosynthesis of other metabolites such as alkanes, aldehydes, alcohols and acids could also be enhance. Hence the RNA extracted from this treatment was subtracted against the non-treated kesum roots RNA to

JA elicitation on the production of β-caryophyllene analytes in both JA-treated and nontreated kesum roots.

Fig. 2. Chromatogram profile for volatile compounds extracted from 150µM JA-treated kesum roots extract using HP-5MS non-polar column (30m length x 0.25mm diameter x 0.25µM thickness). Injection temperature: 220ºC; Detection temperature: 280 ºC; Column temperature: 50 ºC, 3 min; 20ºC/min - 100ºC, 3 min; 30ºC/min - 250ºC, 3 min. Flow rate: 1.3ml/min. Injection volume: 1µl. Injection method: split ratio. Type of detector: quadrupole. Mass spectrometry: scan mode. TIC: Total ion chromatogram.

Fig. 3. The effect of JA treatment on β-caryophyllene in kesum roots. The values with different alphabet (a-c) are different significantly (p < 0.05). Data represent mean for triplicate with standard deviation.

JA elicitation on the production of β-caryophyllene analytes in both JA-treated and non-

Fig. 2. Chromatogram profile for volatile compounds extracted from 150µM JA-treated kesum roots extract using HP-5MS non-polar column (30m length x 0.25mm diameter x 0.25µM thickness). Injection temperature: 220ºC; Detection temperature: 280 ºC; Column temperature: 50 ºC, 3 min; 20ºC/min - 100ºC, 3 min; 30ºC/min - 250ºC, 3 min. Flow rate:

1.3ml/min. Injection volume: 1µl. Injection method: split ratio. Type of detector: quadrupole. Mass spectrometry: scan mode. TIC: Total ion chromatogram.

Fig. 3. The effect of JA treatment on β-caryophyllene in kesum roots. The values with different alphabet (a-c) are different significantly (p < 0.05). Data represent mean for

triplicate with standard deviation.

treated kesum roots.

Day-1 observation indicate that the production of β-caryophyllene was occur at a much lower level and inconsistent because the standard deviation between replicates differ greatly (coefficient of variance = 27.86). The overall production of this sesquiterpene compound at all JA concentration was lower than control. The ANOVA analysis showed that no significant increase (p > 0.5) was observed in the production of β-caryophyllene in kesum roots treated with all four JA concentrations compared to control. This might be because of the kesum plantlets might need time to adapt to the stressful environment. The development of kesum root cells might be retarded after treated with JA which would suppress the primary metabolism and subsequently activated the secondary metabolism in root cells (Chen & Chen 2000; Wang et al. 2001). The level of production in day-3 was much higher and could be found in all three replicates for each JA concentration tested. The production of β-caryophyllene increased after treated with JA from 50µM to 150µM but decrease again at 200µM. Significant increase (p < 0.05) was seen when *P. minus* was treated with 150µM JA, which is 203.45 + 114.79µg/g compared to 25.59 + 8.96µg/g in control roots (coefficience of variance = 73.28). The production of this sesquiterpene compound increase about 1.2-fold, 2.1-fold and 8 fold in the roots treated with 50µM, 100µM and 150µM JA respectively compared to control sample when kesum plantlets established defense mechanism against abiotic stress created by JA elicitation. However, exposure of kesum to higher concentration of JA inhibited the production of β-caryophyllene where a decrease of 1.4-fold of this metabolite was observed in 200µM JA treatment. Necrosis was observed when the leaves and roots of kesum turned brownish as JA is an inhibitor to roots growth (Wasternack 2007). In day-6, β-caryophyllene present consistently in two or more replicates in every treatment. However, ANOVA analysis showed that the level of production decrease significantly (p > 0.05) compared to day-3 in all four treatments (coefficience of variance = 34.21). This situation might be caused by over exposure of kesum plantlets to JA stress. Our observation is similar to a study done by Sanchez-Sampedro et al. (2005) where they found that the production of silymarin in *Silybum marianum* increased to the maximum level after 3 days of MeJA treatment but decrease significantly after 7 days. Therefore, we concluded that kesum roots treated with 150µM JA for 3 days could produce βcaryophyllene at maximum level and we assume that the biosynthesis of other metabolites such as alkanes, aldehydes, alcohols and acids could also be enhance. Hence the RNA extracted from this treatment was subtracted against the non-treated kesum roots RNA to identify and clone the transcripts regulated by JA stress.

### **3. Differentially expressed genes induced by JA**

### **3.1 Identification of JA-responsive genes**

Subtractive screening is an efficient approach to clone the genes which are being expressed in one population but not being expressed or slightly expressed in another population. In this study, cDNA derived from kesum roots treated with 150µM JA for 3 days was served as tester whereas the cDNA derived from non-treated kesum roots was served as driver for subtracted cDNA library construction. Only forward subtraction was done as we were interested in identifying genes up-regulated by JA. A total of 1,344 white colonies were randomly picked from the subtracted cDNA library and screened by PCR using M13 forward and reverse universal primers to confirm the presence of cDNA inserts. PCR amplification revealed that 960 colonies were single stranded clones with the insert sizes ranging from 250bp to 1,200bp. These clones were subsequently hybridized against

Alteration of Abiotic Stress Responsive Genes

either under natural conditions or JA stress.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 61

190 strongly hybridized clones were picked from Reverse Northern results and sent for sequencing. Of these, 174 clones were readable sequence in which 130 clones were unigenes that showed significant similarity to cDNA sequences in the NCBI database (E-value < 10-5), 18 clones did not show any similarity to any known sequences and 26 clones had no significant results (Table 3). All of the 130 unigenes were deposited into NCBI and could be found in dbEST with accession numbers starting from GR505448 to GR505519. These clones were then classified into 11 categories according to their putative molecular functions (Figure 5). The largest set of genes was assigned to the stress-related genes (25%). This was followed by the following groups: other metabolism (20%), unknown genes (9%), transcription factors (8%), amino acid metabolism (6%), signal transduction and kinase (5%), carbohydrate metabolism (2%), transporter (2%), energy (2%), and regulation of gene expression (2%). Clones that showed similarity to the cDNA sequences from other plant species, but did not have any specific function, were assigned to the 'other' category (19%). The functional categories showed that these cDNAs might be involved in different biological processes. Clones that showed no similarity to any sequences in the GenBank were classified as hypothetical proteins and their putative structure and functions were predicted using bioinformatics software, which will be discussed later in this chapter. These clones may represent unique genes that were transcribed in response to the JA treatment that are involved in the metabolic pathway elicited by JA. We focused further analysis on clones that represent genes involved in biosynthesis of aromatic compounds in kesum,

The largest group (25%) was assigned to stress-related genes. Exogenous JA application has been known as a stress treatment to plants and served as a stimulus to activate the expression of genes involved in the synthesis of plant secondary metabolites. As expected, a large number of clones encoded for stress-related genes were identified upon JA elicitation in this study. The complexity of kesum roots response towards JA elicitation suggested that many genes involved in plant defence mechanism against stress. A few clones were found to have homology with abiotic stress related genes as a defence response of kesum root cells towards JA treatment, including glutathione S-transferase from *Glycine max* (GR505459) and putative glutathione S-transferase T1 from *Lycopersicon esculentum* (GR505461), heat shock protein (GR505458), anionic peroxidise H from *Zea mays* (GR505463) and peroxidise 1 from *Scutellaria baicalensis* (GR505464), ELI3-1 (GR505453) and auxin induced protein (GR505454). Generally plant response towards pathogen or herbivore attack would activate a series of mechanism, including synthesizing anthocyanin and oligolignol, pathogenesis proteins (PR), generation of reactive oxygen species and formation of plant cell walls (Pauwels et al. 2009). The increase of gluthathione S-transferase (GST) was associated with hormone homeostasis or anthocyanin isolation in vacuole because GST played a role as auxin, cytokinin and anthocyanin transporter. When kesum roots were exposed to JA, excessive anthocyanin will be synthesized. The equilibrium of hormone in kesum root cells could be achieved by transporting the excessive anthocyanin into vacuole to be removed. This step was catalyzed by glutathione S-transferase enzyme (Moons 2003). On the other hand, peroxidase transcripts could be linked to generation of reactive oxygen species such as H2O2 and other metabolites like phenylpropanoids (Thimmaraju et al. 2006). Therefore we predicted that the peroxidase induced by JA was responsible for volatile compounds production such as sesquiterpenes in kesum roots. ELI3-1 gene is a type of elicitor activated gene and it responds to a wide range of elicitors (Ellard-Ivey & Douglas 1996). In this study, ELI3-1 was induced by JA, as well as heat shock protein which functions as a defence

unsubtracted tester cDNA and unsubtracted driver cDNA by Reverse Northern hybridization to reduce false positives. Our results showed that of these 960 clones, 195 clones hybridized strongly, 213 clones hybridized moderately, while 552 clones hybridized weakly with the unsubtracted tester cDNA whereas almost all of the clones showed weak or no signal when compared with the unsubtracted driver probe (Figure 4).

Fig. 4. Reverse Northern analysis showing differential screening for putative cDNA clones altered by JA. (a) PCR products hybridized with DIG-labelled tester cDNA. (b) PCR products hybridized with DIG-labelled driver cDNA. Clones showing significant differential expression were pointed with red arrows. Negative control was pointed with blue arrow.

a) JA-treated sample b) Control sample

unsubtracted tester cDNA and unsubtracted driver cDNA by Reverse Northern hybridization to reduce false positives. Our results showed that of these 960 clones, 195 clones hybridized strongly, 213 clones hybridized moderately, while 552 clones hybridized weakly with the unsubtracted tester cDNA whereas almost all of the clones showed weak or

a) JA-treated sample b) Control sample a) JA-treated sample b) Control sample

a) JA-treated sample b) Control sample Fig. 4. Reverse Northern analysis showing differential screening for putative cDNA clones altered by JA. (a) PCR products hybridized with DIG-labelled tester cDNA. (b) PCR products hybridized with DIG-labelled driver cDNA. Clones showing significant differential expression

were pointed with red arrows. Negative control was pointed with blue arrow.

no signal when compared with the unsubtracted driver probe (Figure 4).

190 strongly hybridized clones were picked from Reverse Northern results and sent for sequencing. Of these, 174 clones were readable sequence in which 130 clones were unigenes that showed significant similarity to cDNA sequences in the NCBI database (E-value < 10-5), 18 clones did not show any similarity to any known sequences and 26 clones had no significant results (Table 3). All of the 130 unigenes were deposited into NCBI and could be found in dbEST with accession numbers starting from GR505448 to GR505519. These clones were then classified into 11 categories according to their putative molecular functions (Figure 5). The largest set of genes was assigned to the stress-related genes (25%). This was followed by the following groups: other metabolism (20%), unknown genes (9%), transcription factors (8%), amino acid metabolism (6%), signal transduction and kinase (5%), carbohydrate metabolism (2%), transporter (2%), energy (2%), and regulation of gene expression (2%). Clones that showed similarity to the cDNA sequences from other plant species, but did not have any specific function, were assigned to the 'other' category (19%). The functional categories showed that these cDNAs might be involved in different biological processes. Clones that showed no similarity to any sequences in the GenBank were classified as hypothetical proteins and their putative structure and functions were predicted using bioinformatics software, which will be discussed later in this chapter. These clones may represent unique genes that were transcribed in response to the JA treatment that are involved in the metabolic pathway elicited by JA. We focused further analysis on clones that represent genes involved in biosynthesis of aromatic compounds in kesum, either under natural conditions or JA stress.

The largest group (25%) was assigned to stress-related genes. Exogenous JA application has been known as a stress treatment to plants and served as a stimulus to activate the expression of genes involved in the synthesis of plant secondary metabolites. As expected, a large number of clones encoded for stress-related genes were identified upon JA elicitation in this study. The complexity of kesum roots response towards JA elicitation suggested that many genes involved in plant defence mechanism against stress. A few clones were found to have homology with abiotic stress related genes as a defence response of kesum root cells towards JA treatment, including glutathione S-transferase from *Glycine max* (GR505459) and putative glutathione S-transferase T1 from *Lycopersicon esculentum* (GR505461), heat shock protein (GR505458), anionic peroxidise H from *Zea mays* (GR505463) and peroxidise 1 from *Scutellaria baicalensis* (GR505464), ELI3-1 (GR505453) and auxin induced protein (GR505454). Generally plant response towards pathogen or herbivore attack would activate a series of mechanism, including synthesizing anthocyanin and oligolignol, pathogenesis proteins (PR), generation of reactive oxygen species and formation of plant cell walls (Pauwels et al. 2009). The increase of gluthathione S-transferase (GST) was associated with hormone homeostasis or anthocyanin isolation in vacuole because GST played a role as auxin, cytokinin and anthocyanin transporter. When kesum roots were exposed to JA, excessive anthocyanin will be synthesized. The equilibrium of hormone in kesum root cells could be achieved by transporting the excessive anthocyanin into vacuole to be removed. This step was catalyzed by glutathione S-transferase enzyme (Moons 2003). On the other hand, peroxidase transcripts could be linked to generation of reactive oxygen species such as H2O2 and other metabolites like phenylpropanoids (Thimmaraju et al. 2006). Therefore we predicted that the peroxidase induced by JA was responsible for volatile compounds production such as sesquiterpenes in kesum roots. ELI3-1 gene is a type of elicitor activated gene and it responds to a wide range of elicitors (Ellard-Ivey & Douglas 1996). In this study, ELI3-1 was induced by JA, as well as heat shock protein which functions as a defence

Alteration of Abiotic Stress Responsive Genes

**GR505467** 457 S-adenosyl-L-methionine

**GR505468** 446 S-adenosyl-L-methionine

**GR505469** 443 S-adenosyl-L-methionine

**GR505471** 741 S-adenosyl-L-homocystein

**GR505474** 328 Dihydrolipoamide

**GR505475** 460 Nodulin-35 (N-35) gene

**GR505477** 520 Glycosyltransferase family

**GR505478** 524 Cytochrome oxidase subunit 1

**GR505479** 391 Cytochrome oxidase subunit 1 (COI)

**GR505484** 748 Glucan-endo-1,3-beta-

**GR505485** 430 Type-AAA ATPase family

**GR505486** 393 Glyseraldehyde-3-phosphate

synthetase

synthetase

synthetase

hydrolase

dehydrogenase 1

**GR505476** 742 Nodulin family protein (NLP *Gossypium* 

protein 47

(COI) gene

**GR505480** 557 Cytochrome c oxidase *Gossypium* 

glucosidase

protein

dehydrogenase

**GR505487** 399 Root-specific metal transporter *Lycopersicon* 

**GR505489** 377 Ribosomal protein S12 *Fagopyrum* 

Table 3. Putative JA-induced cDNA sequences in *P. minus* roots.

**GR505490** 675 18S rRNA gene *Polygonum* sp.

**Acid amino metabolism** 

**Other metabolism**

**Energy**

**Transporter** 

**Regulation of gene expression** 

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 63

**GR505473** 313 Glyoxal oxidase-related mRNA *Arabidopsis thaliana* 2 5e-06

encoding a subunit of uricase II

**GR505482** 401 NADH dehydrogenase *Beta vulgaris* 7 0.0 **GR505483** 344 Urate oxidase *Vitis vinifera* 1 1e-20

**GR505472** 952 Lipoxigenase ( lox gene) *Capsicum annuum* 1 7e-47

**GR505488** 291 Auxin efflux carrier protein *Zea mays* 2 1e-11

*Beta vulgaris* 3 1e-135

*Actinidia chinensis* 3 2e-29

*Arabidopsis thaliana* 1 3e-20

*Glycine max* 1 3e-09

*Arabidopsis thaliana* 1 2e-05

*Persicaria maculosa* 4 0.0

*Plumbago* sp. 3 6e-163

*Nicotina tabacum* 1 5e-29

*Arabidopsis thaliana* 1 1e-41

*Zea mays* 2 3e-11

1 5e-12

1 8e-162

2 0.0

1 6e-18

1 0.0

1 4e-49

3 0.0

*Elaeagnus umbrellata* 

*hirsutum*

*barbadense*

*esculentum*

*esculentum*

Soltis

*Mesembrayanthemu m crystallinum* 


**GR505449** 265 F-box containing protein *Populus tremula* 1 3e-12

**GR505470** 423 GAMYB-binding protein (gbp5) *Hordeum vulgare* 1 2e-25 **GR505481** 298 ERF-like transcription factor *Coffea canephora* 1 2e-09

**GR505514** 285 Protein kinase *Malus domestica* 2 1e-55

**GR505519** 712 Calmodulin-binding protein *Arabidopsis thaliana* 2 7e-116

**GR505452** 539 MeJA-elicited hairy roots culture *Panax ginseng* 4 3e-22

**GR505454** 589 Auxin-induced protein *Nicotina tabacum* 7 6e-13

**GR505457** 406 Cell cultures in osmotic stress *Bouteloua gracilis* 3 0.0 **GR505458** 669 Gene for heat-shock protein *Glycine max* 2 3e-17

**GR505463** 747 Anionic peroxidase H *Zea mays* 1 1e-09

**GR505465** 684 Alcohol dehydrogenase *Prunus armeniaca* 1 3e-68 **GR505448** 603 Alcohol dehydrogenase 1 (adh1) *Zea mays* 1 1e-75 **GR505466** 436 β-fructofuranosidase *Arabidopsis thaliana* 1 2e-14

**GR505460** 595 Kelch repeat-containing F-box

**GR505492** 583 BURP domain containing

**GR505503** 583 BURP domain containing

**GR505518** 563 Multicopy supressor IRA1

**GR505451** 666 MeJA-elicited root cell

**GR505453** 503 ELI3-1 (ELICITOR ACTIVATED

**GR505455** 269 EST from mild drought-stressed leaves

**GR505456** 267 cDNA clone from senescing

**GR505459** 656 Glutathione S-transferase

**GR505461** 570 Putative glutathione S-transferase T1

**GR505462** 716 cDNA clones from water stress

GENE 3)

leaves

(GST14)

seedlings

**GR505464** 546 Peroxidase 1 *Scutellaria* 

family protein

protein

protein

(MSI1)

suspension culture

(bp) Similarity Organism

Number of clones

*Arabidopsis thaliana* 1 1e-22

*Solanum tuberosum* 2 9e-05

*Phaseolus vulgaris* 4 8e-06

*Arabidopsis thaliana* 1 3e-48

*Medicago trunculata* 1 2e-18

*Arabidopsis thaliana* 1 2e-29

*Populus tremula* 1 5e-16

*Populus tremula* 1 3e-05

*Glycine max* 7 2e-21

*Zea mays* 1 2e-07

3 3e-10

1 3e-48

*Lycopersicon esculentum* 

*baicalensis* 

E-Value

Gene Bank Accession Size

**Transcription factor**

**Signal transduction** 

**& kinase** 

**Stress-related** 

**Carbohydrate metabolism** 


Table 3. Putative JA-induced cDNA sequences in *P. minus* roots.

Alteration of Abiotic Stress Responsive Genes

herbivory biotic stress.

kesum roots.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 65

oxidase transcripts. JA elicitation was also believed to induce the expression of urate oxidase and activates the production of H2O2 that resulted in hypersensitive cell death. The functions of glyoxal oxidase and dihydrolipoamide dehydrogenase in kesum roots under JA stress were yet to be discovered. Another unigene that encoded for lipoxygenase, the first enzyme in the oxylipin pathway for JA biosynthesis (Devitt et al. 2006) was also found in this study. It has been identified as the enzyme that involved in the production pathways of volatile compounds as the indirect plant defensive response to herbivory (Kessler and Baldwin 2001). Thus, it is believed that the lipoxygenase expression was associated with other volatile compounds detected by GC-MS, such as the alkanes, aldehydes and alcohols. This result suggests that there is a crosstalk between abiotic stress triggered by JA and other

Another group of cDNA sequences were associated with transcription factor (8%). For example, F-box containing TIR1 protein (GR505449), Kelch-repeat containing F-box family protein (GR505460), GAMYB-binding protein (gbp5) (GR505470), ERF-like transcription factor (GR505481) and BURP domain containing protein from *Solanum tuberosum*  (GR505492) and *Phaseolus vulgaris* (GR505503). The F-box containing TIR1 protein (Parry & Estelle 2006) and Kelch-repeat containing F-box protein have been proven to be activated by JA in plant cells. Naturally F-box containing TIR 1 protein is a receptor to auxin. In plants, auxin is activated by auxin responsive factor (ARF) but inhibited by Aux/IAA protein. It was predicted that the expression of F-box containing TIR1 transcripts could activate the degradation of Aux/IAA protein so that auxin could be synthesized to equilibrate the hormones content in kesum roots. The Kelch-repeat containing F-box family protein was thought to be interacted with other proteins which involved in protein degradation process through ubiquitin-dependent pathway. Protein degradation is an important process in regulating cell cycle, transcription and signal transduction as a defence mechanism in kesum (Sun et al. 2007). The GAMYB-binding protein, BURP domain and ERF-like transcription factor induced by JA in this study were believed to be elements that regulate JA signalling in

Genes that were categorised into amino acid metabolism (6%) include cDNA clones coded for enzymes involved in phenylpropoanoids biosynthesis pathway, namely S-adenosyl-Lmethionine sinthase from *Beta vulgaris* (GR505467), *Actinidia chinensis* (GR505468) and *Elaeagnus umbrellata* (GR505469), and S-adenosyl-L-homocystein hydrolase (GR505471) (Dewick 2001). The results of this study suggested that S-adenosyl methionine synthase and S-adenosyl homocystein hydrolase induced by JA could activate the production of aromatic compounds in kesum roots using aromatic amino acids as precursor. Carbohydrate metabolism (2%) or carbon-containing compounds covered alcohol dehydrogenase gene from *Prunus armeniaca* (GR505568), alcohol dehydrogenase 1 from *Zea mays* (GR505568) and β-fructofuranosidase (GR505466). The up-regulation of transcription of alcohol dehydrogenase may be associated with the biosynthesis of phenylpropenes, another important group of volatiles (Devitt et al. 2006). This result suggests that the genes involved in the biosynthesis of secondary metabolites can be induced by JA elicitation in kesum roots. Genes categorized under signal transduction and kinase (5%) include kinase protein (GR505514), Multicopy suppressor IRA1 (MSI1) (GR505518) and calmodulin-binding protein (CaMBP) (GR505519). Kinase protein could modify other proteins or enzymes through phosphorylation of serine, threonine or tyrosine residues (Zheng et al. 2004). The kinase proteins found in this study might function in phosphotylation protein as a response

Fig. 5. Classification of clones based on their putative molecular functions.

response towards environmental stress. Besides, JA is known to be a phytohormone that regulates many plant physiological processes and it could interact with other hormone such as salicylic acid, absisic acid, auxin and giberellin in controlling plant growth and development (Creelman & Mulpuri 2002; Pauwels et al. 2009). Apart from that, some clones also showed homology to cDNA sequences in hairy roots of *Medicago trunculata* (GR505451) and *Panax ginseng* (GR505452) treated with MeJA. Other clones that showed homology to cDNA sequences of plants grown under drought stress (GR505455), osmotic stress (GR505457), water stress (GR505462) and leaves senescence (GR505456) were also identified. This result implies that many different stress factors will lead to the same gene expression (Sandermann et al. 1998). This result also suggests that JA, or its derivative MeJA, are signal molecules that regulate kesum defense responses in response to stressful environments, resulting in the activation of defense-related genes in plants.

The next group of transcripts which showed putative functions in plant growth and development were categorised under other metabolism (20%). Any changes in the primary metabolism will lead to plant defence response to stress (Ingram 7 Bartels 1996). Transcripts that were induced by JA in kesum roots include glyoxal oxidase (GR505473), dihydrolipoamide dehydrogenase (GR505474), Nodulin-35 gene (GR505475), nodulin family protein (NLP) (GR505476), cytochrome oxidase subunit I from *Persicaria maculosa*  (GR505478) and *Plumbago sp.* (GR505479), cytochrome c oxidase from *Gossypium barbadense*  (GR505480), urate oxidase (GR505483), glucan-endo-1,3-beta-glucoxidase (GR505484), glycosyltransferase family protein 47 (GR505477) and NADH dehydrogenase (GR505482). It was predicted that the Nodulin-35 and nodulin family protein were being expressed to ensure the normal development of root nodules as JA might inhibit roots elongation (Gadzovska et al. 2007). Glucan-endo-1,3-beta-glucosidase and glycosyltransferase family protein 47 were also being expressed to form kesum root cell walls. Besides, it was predicted that the generation of reactive oxygen species or secondary metabolites had affected the respiration process in kesum root cells and thus leading to the increase of cytochrome

Fig. 5. Classification of clones based on their putative molecular functions.

resulting in the activation of defense-related genes in plants.

response towards environmental stress. Besides, JA is known to be a phytohormone that regulates many plant physiological processes and it could interact with other hormone such as salicylic acid, absisic acid, auxin and giberellin in controlling plant growth and development (Creelman & Mulpuri 2002; Pauwels et al. 2009). Apart from that, some clones also showed homology to cDNA sequences in hairy roots of *Medicago trunculata* (GR505451) and *Panax ginseng* (GR505452) treated with MeJA. Other clones that showed homology to cDNA sequences of plants grown under drought stress (GR505455), osmotic stress (GR505457), water stress (GR505462) and leaves senescence (GR505456) were also identified. This result implies that many different stress factors will lead to the same gene expression (Sandermann et al. 1998). This result also suggests that JA, or its derivative MeJA, are signal molecules that regulate kesum defense responses in response to stressful environments,

The next group of transcripts which showed putative functions in plant growth and development were categorised under other metabolism (20%). Any changes in the primary metabolism will lead to plant defence response to stress (Ingram 7 Bartels 1996). Transcripts that were induced by JA in kesum roots include glyoxal oxidase (GR505473), dihydrolipoamide dehydrogenase (GR505474), Nodulin-35 gene (GR505475), nodulin family protein (NLP) (GR505476), cytochrome oxidase subunit I from *Persicaria maculosa*  (GR505478) and *Plumbago sp.* (GR505479), cytochrome c oxidase from *Gossypium barbadense*  (GR505480), urate oxidase (GR505483), glucan-endo-1,3-beta-glucoxidase (GR505484), glycosyltransferase family protein 47 (GR505477) and NADH dehydrogenase (GR505482). It was predicted that the Nodulin-35 and nodulin family protein were being expressed to ensure the normal development of root nodules as JA might inhibit roots elongation (Gadzovska et al. 2007). Glucan-endo-1,3-beta-glucosidase and glycosyltransferase family protein 47 were also being expressed to form kesum root cell walls. Besides, it was predicted that the generation of reactive oxygen species or secondary metabolites had affected the respiration process in kesum root cells and thus leading to the increase of cytochrome oxidase transcripts. JA elicitation was also believed to induce the expression of urate oxidase and activates the production of H2O2 that resulted in hypersensitive cell death. The functions of glyoxal oxidase and dihydrolipoamide dehydrogenase in kesum roots under JA stress were yet to be discovered. Another unigene that encoded for lipoxygenase, the first enzyme in the oxylipin pathway for JA biosynthesis (Devitt et al. 2006) was also found in this study. It has been identified as the enzyme that involved in the production pathways of volatile compounds as the indirect plant defensive response to herbivory (Kessler and Baldwin 2001). Thus, it is believed that the lipoxygenase expression was associated with other volatile compounds detected by GC-MS, such as the alkanes, aldehydes and alcohols. This result suggests that there is a crosstalk between abiotic stress triggered by JA and other herbivory biotic stress.

Another group of cDNA sequences were associated with transcription factor (8%). For example, F-box containing TIR1 protein (GR505449), Kelch-repeat containing F-box family protein (GR505460), GAMYB-binding protein (gbp5) (GR505470), ERF-like transcription factor (GR505481) and BURP domain containing protein from *Solanum tuberosum*  (GR505492) and *Phaseolus vulgaris* (GR505503). The F-box containing TIR1 protein (Parry & Estelle 2006) and Kelch-repeat containing F-box protein have been proven to be activated by JA in plant cells. Naturally F-box containing TIR 1 protein is a receptor to auxin. In plants, auxin is activated by auxin responsive factor (ARF) but inhibited by Aux/IAA protein. It was predicted that the expression of F-box containing TIR1 transcripts could activate the degradation of Aux/IAA protein so that auxin could be synthesized to equilibrate the hormones content in kesum roots. The Kelch-repeat containing F-box family protein was thought to be interacted with other proteins which involved in protein degradation process through ubiquitin-dependent pathway. Protein degradation is an important process in regulating cell cycle, transcription and signal transduction as a defence mechanism in kesum (Sun et al. 2007). The GAMYB-binding protein, BURP domain and ERF-like transcription factor induced by JA in this study were believed to be elements that regulate JA signalling in kesum roots.

Genes that were categorised into amino acid metabolism (6%) include cDNA clones coded for enzymes involved in phenylpropoanoids biosynthesis pathway, namely S-adenosyl-Lmethionine sinthase from *Beta vulgaris* (GR505467), *Actinidia chinensis* (GR505468) and *Elaeagnus umbrellata* (GR505469), and S-adenosyl-L-homocystein hydrolase (GR505471) (Dewick 2001). The results of this study suggested that S-adenosyl methionine synthase and S-adenosyl homocystein hydrolase induced by JA could activate the production of aromatic compounds in kesum roots using aromatic amino acids as precursor. Carbohydrate metabolism (2%) or carbon-containing compounds covered alcohol dehydrogenase gene from *Prunus armeniaca* (GR505568), alcohol dehydrogenase 1 from *Zea mays* (GR505568) and β-fructofuranosidase (GR505466). The up-regulation of transcription of alcohol dehydrogenase may be associated with the biosynthesis of phenylpropenes, another important group of volatiles (Devitt et al. 2006). This result suggests that the genes involved in the biosynthesis of secondary metabolites can be induced by JA elicitation in kesum roots. Genes categorized under signal transduction and kinase (5%) include kinase protein (GR505514), Multicopy suppressor IRA1 (MSI1) (GR505518) and calmodulin-binding protein (CaMBP) (GR505519). Kinase protein could modify other proteins or enzymes through phosphorylation of serine, threonine or tyrosine residues (Zheng et al. 2004). The kinase proteins found in this study might function in phosphotylation protein as a response

Alteration of Abiotic Stress Responsive Genes

**3.2.1 Computational analysis of unknown genes** 

**3.2.2 Protein characterization by physicochemical properties** 

2010).

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 67

The comparison of DNA or protein sequences from various organisms using computational methods is a powerful tool in protein study. By finding similarities between sequences, functional inference of newly sequenced genes can be achieved, new members of gene families can be predicted and evolutionary relationship can be explored. Computational analysis can quickly analyze and assign hypothetical proteins and able to generally predict their tentative biochemical functions (Lubec et al. 2005, Galperin 2001, Hoskeri 2010). Fundamentally, the prediction of functional inference is achievable by standard homologybased gene annotation complemented by genomic-context approaches (von Mering et al. 2003, Mellor et al. 2002, Marcotte et al. 1999) and, in some cases, requires structural intervention (Kolker et al. 2004). The combination of these approaches is intuitive and usually applies to various circumstances. Even though predictions can sometimes reliably infer the function of hypothetical proteins (Aravind and Koonin 1999), predictions do not provide necessary information regarding the exact biochemical function of a protein. Thus, predictions must still be validated through wet-lab experiments. However, computational analysis provides a faster and cheaper alternative to wet-lab experiments. Here, we cover the computational predictions of a set of hypothetical proteins obtained from the substracted cDNA library of a *P. minus* root that was treated with jasmonic acid (Gor et al.

The unknown protein dataset discovered from a subtracted cDNA library of *P. minus* roots elicited with jasmonic acid were first translated to protein sequences for detailed bioinformatic analysis. The sequences were then examined for the existence of signal peptides using a signal peptide prediction tool. Knowledge of the existence of a signal peptide in a protein sequence is essential to defining and characterizing the protein. If there is a detectable signal peptide in a sequence, the signal peptide region must be cut off before the sequence can be used for further bioinformatics analysis. The sequences were compared to the databases of non-redundant proteins to detect any homologous sequences. The sequences with no significant outputs from the similarity search were further analyzed using preliminary structure-prediction analysis to identify a possible fold category. This information provided useful insights into the functional inference of these sequences. The analysis was performed using an in-house analysis portal called the Hypothetical Protein Analysis System (HPAS), which provided a systematic functional annotation procedure. The HPAS consisted of various tools for signal peptide prediction (SignalP 3.0) (Bendtsen et al. 2004), analysis of physicochemical properties (ProtParam (Gasteiger et al. 2003) and ProtScale (Yu et al. 2010)), topology analysis (Psortb (Bagos et al. 2008), SOSUI (Hirokawa et al. 1998), HMMTOP (Tusnady and Simon 2001), SignalP (Bendtsen et al. 2004), LipoP (Rahman et al. 2008)) and similarity search and annotation (NPSA@BLASTP (Altschul et al. 1990), NPSA@PSI-BLAST (Altschul et al. 1997), MPSrch (Agarwal et al. 1998), SSEARCH(Mazumder et al. 2008) and InterProScan (Zdobnov et al. 2001)). The HPAS covered all of the possible aspects of a protein sequence and, through a series of analytical tools, used all of the protein's characteristics to determine the protein's predicted functions.

ProtParam was used to compute the physicochemical properties of these hypothetical proteins. Here, a few selected physicochemical properties were highlighted; molecular

towards JA elicitation. Another plant response against stress is the increased of free calcium in cytosol as Ca2+ ion plays an important role in signal transduction that activated plant defence genes (Reddy et al. 2008). Ca2+ ion could activate calmodulin protein (CaM). The interaction between Ca2+/CaM and target molecules could lead to plant response towards environmental stress by activation of calmodulin-binding protein (CaMBPs) (Zielinski 1998). Besides, MSI1 has also been proven to function in signal transduction mechanism (Zheng et al. 2004). Therefore, the increase expression of these transcripts could possibly be linked to JA-induced signal transduction pathway.

AAA-type ATPase family protein mRNA (GR505485) and glyceraldehydes-3-phosphate dehydrogenase (GR505486) were categorized under energy group (2%). The increment of these transcripts was mainly due to energy consumption in regulation of plant metabolisms under JA stress. Root-specific metal transporter (GR505487) and auxin efflux carrier protein (GR505488) were classified into transporter group (2%). The discovery of root-specific metal transporter in JA-treated kesum roots may suggest that *P. minus* could be a suitable plant for phytoremediation of metal contamination in soil and further investigation need to be done by focusing to this aspect. The expression of auxin efflux carrier protein could be linked to auxin induced protein and F-box protein and it was predicted that the interaction of these three components could stabilize JA content in kesum roots by auxin synthesis. Next, S12 ribosomal protein (GR505489) and rRNA 18S gene (GR505490) were classified into regulation of gene expression group (2%). A few cDNA clones which represent ribosomal proteins such as 60S, 40S and 30S were known to be gene sequences respond towards stress. These ribosomal proteins play important roles in *de novo* protein synthesis (Machida et al. 2008).

Clones which have no significant similarity with any sequences in the databases were categorized as unknown genes (9%) and into others group (19%) that include cDNA clones that have no similarity with any nucleotide, mRNA or EST sequences in NCBI database (GR505498, GR505499, GR505500, GR505501, GR505502, GR505504, GR505505, GR505506, GR505507, GR505508, GR505509, GR505510, GR505511, GR505512, GR505513, GR505515, GR505516, GR505517), These sequences could be considered as novel genes induced by JA in kesum roots and further characterization must be done to identify their function in plant stress response, JA signalling and secondary metabolites production. The relationship between these clones and JA elicitation is yet to be identified and bioinformatics analysis has been carried out to investigate possible classification of these unknown sequences.

### **3.2 Discovery of unknown and novel cDNA sequences discovered during JA elicitation**

A substantial fraction of the genes in the EST dataset encode for unknown proteins (also termed as hypothetical proteins) and about half the proteins in most genomes are candidates for hypothetical proteins (Minion et al. 2004). Hypothetical proteins are proteins that are predicted from nucleic acid sequences but have no corresponding experimental protein (Lubec et al. 2005). They are characterized by low identity to known annotated proteins. Thus, their functions remain unknown, and they pose a challenge to functional genomics and biology in general (Galperin 2001). Hypothetical proteins are utmost importance to complete genomic and proteomic information. Detailed knowledge on hypothetical proteins offers presentation of new structures and functions, contributing to the rising of new domains and motifs, revelation on a series of additional pathways hence completing fragmentary knowledge on the mosaic of proteins intrinsically.

towards JA elicitation. Another plant response against stress is the increased of free calcium in cytosol as Ca2+ ion plays an important role in signal transduction that activated plant defence genes (Reddy et al. 2008). Ca2+ ion could activate calmodulin protein (CaM). The interaction between Ca2+/CaM and target molecules could lead to plant response towards environmental stress by activation of calmodulin-binding protein (CaMBPs) (Zielinski 1998). Besides, MSI1 has also been proven to function in signal transduction mechanism (Zheng et al. 2004). Therefore, the increase expression of these transcripts could possibly be linked to

AAA-type ATPase family protein mRNA (GR505485) and glyceraldehydes-3-phosphate dehydrogenase (GR505486) were categorized under energy group (2%). The increment of these transcripts was mainly due to energy consumption in regulation of plant metabolisms under JA stress. Root-specific metal transporter (GR505487) and auxin efflux carrier protein (GR505488) were classified into transporter group (2%). The discovery of root-specific metal transporter in JA-treated kesum roots may suggest that *P. minus* could be a suitable plant for phytoremediation of metal contamination in soil and further investigation need to be done by focusing to this aspect. The expression of auxin efflux carrier protein could be linked to auxin induced protein and F-box protein and it was predicted that the interaction of these three components could stabilize JA content in kesum roots by auxin synthesis. Next, S12 ribosomal protein (GR505489) and rRNA 18S gene (GR505490) were classified into regulation of gene expression group (2%). A few cDNA clones which represent ribosomal proteins such as 60S, 40S and 30S were known to be gene sequences respond towards stress. These ribosomal proteins play important roles in *de novo* protein synthesis (Machida et al.

Clones which have no significant similarity with any sequences in the databases were categorized as unknown genes (9%) and into others group (19%) that include cDNA clones that have no similarity with any nucleotide, mRNA or EST sequences in NCBI database (GR505498, GR505499, GR505500, GR505501, GR505502, GR505504, GR505505, GR505506, GR505507, GR505508, GR505509, GR505510, GR505511, GR505512, GR505513, GR505515, GR505516, GR505517), These sequences could be considered as novel genes induced by JA in kesum roots and further characterization must be done to identify their function in plant stress response, JA signalling and secondary metabolites production. The relationship between these clones and JA elicitation is yet to be identified and bioinformatics analysis has

been carried out to investigate possible classification of these unknown sequences.

**3.2 Discovery of unknown and novel cDNA sequences discovered during JA** 

fragmentary knowledge on the mosaic of proteins intrinsically.

A substantial fraction of the genes in the EST dataset encode for unknown proteins (also termed as hypothetical proteins) and about half the proteins in most genomes are candidates for hypothetical proteins (Minion et al. 2004). Hypothetical proteins are proteins that are predicted from nucleic acid sequences but have no corresponding experimental protein (Lubec et al. 2005). They are characterized by low identity to known annotated proteins. Thus, their functions remain unknown, and they pose a challenge to functional genomics and biology in general (Galperin 2001). Hypothetical proteins are utmost importance to complete genomic and proteomic information. Detailed knowledge on hypothetical proteins offers presentation of new structures and functions, contributing to the rising of new domains and motifs, revelation on a series of additional pathways hence completing

JA-induced signal transduction pathway.

2008).

**elicitation** 

The comparison of DNA or protein sequences from various organisms using computational methods is a powerful tool in protein study. By finding similarities between sequences, functional inference of newly sequenced genes can be achieved, new members of gene families can be predicted and evolutionary relationship can be explored. Computational analysis can quickly analyze and assign hypothetical proteins and able to generally predict their tentative biochemical functions (Lubec et al. 2005, Galperin 2001, Hoskeri 2010). Fundamentally, the prediction of functional inference is achievable by standard homologybased gene annotation complemented by genomic-context approaches (von Mering et al. 2003, Mellor et al. 2002, Marcotte et al. 1999) and, in some cases, requires structural intervention (Kolker et al. 2004). The combination of these approaches is intuitive and usually applies to various circumstances. Even though predictions can sometimes reliably infer the function of hypothetical proteins (Aravind and Koonin 1999), predictions do not provide necessary information regarding the exact biochemical function of a protein. Thus, predictions must still be validated through wet-lab experiments. However, computational analysis provides a faster and cheaper alternative to wet-lab experiments. Here, we cover the computational predictions of a set of hypothetical proteins obtained from the substracted cDNA library of a *P. minus* root that was treated with jasmonic acid (Gor et al. 2010).

### **3.2.1 Computational analysis of unknown genes**

The unknown protein dataset discovered from a subtracted cDNA library of *P. minus* roots elicited with jasmonic acid were first translated to protein sequences for detailed bioinformatic analysis. The sequences were then examined for the existence of signal peptides using a signal peptide prediction tool. Knowledge of the existence of a signal peptide in a protein sequence is essential to defining and characterizing the protein. If there is a detectable signal peptide in a sequence, the signal peptide region must be cut off before the sequence can be used for further bioinformatics analysis. The sequences were compared to the databases of non-redundant proteins to detect any homologous sequences. The sequences with no significant outputs from the similarity search were further analyzed using preliminary structure-prediction analysis to identify a possible fold category. This information provided useful insights into the functional inference of these sequences. The analysis was performed using an in-house analysis portal called the Hypothetical Protein Analysis System (HPAS), which provided a systematic functional annotation procedure. The HPAS consisted of various tools for signal peptide prediction (SignalP 3.0) (Bendtsen et al. 2004), analysis of physicochemical properties (ProtParam (Gasteiger et al. 2003) and ProtScale (Yu et al. 2010)), topology analysis (Psortb (Bagos et al. 2008), SOSUI (Hirokawa et al. 1998), HMMTOP (Tusnady and Simon 2001), SignalP (Bendtsen et al. 2004), LipoP (Rahman et al. 2008)) and similarity search and annotation (NPSA@BLASTP (Altschul et al. 1990), NPSA@PSI-BLAST (Altschul et al. 1997), MPSrch (Agarwal et al. 1998), SSEARCH(Mazumder et al. 2008) and InterProScan (Zdobnov et al. 2001)). The HPAS covered all of the possible aspects of a protein sequence and, through a series of analytical tools, used all of the protein's characteristics to determine the protein's predicted functions.

### **3.2.2 Protein characterization by physicochemical properties**

ProtParam was used to compute the physicochemical properties of these hypothetical proteins. Here, a few selected physicochemical properties were highlighted; molecular

Alteration of Abiotic Stress Responsive Genes

hypothetical proteins of P. minus Huds.

output of the BLAST search.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 69

BNM2 is induced at the beginning of microspore embryogenesis, whereas the corresponding protein remains confined to the seed, where it is localized in the protein storage vacuoles (Zheng et al. 1992, Boutilier et al. 1994. Teerawanichpan et al. 2009, Treacy et 1997). USP from *Vicia faba* L., known as an abundant non-storage seed protein, is expressed during the early stage of zygotic embryogenesis (Bassuner et al. 1998) and at the very beginning of in vitro embryogenesis (21). However, PG1 is a non-catalytic -subunit of the polygalacturonase isozyme from the ripening tomato and plays an important role in regulating pectin metabolism (Zheng et al. 1992, Watson et al. 1994). In contrast, RD22 is a drought-induced protein in Arabidopsis thaliana and is often used as a reference for drought-stress treatment in different plants (Yamaguchi-Shinozaki et al. 1993). To date, this is the first report of the prediction of the BURP-domain-containing protein from the

Psi-BLAST was then used to identify remote homologs, especially for the sequences with no significant hits from the BLAST search output. A detailed sequence analysis of these sequences allowed us to provide a tentative characterization of GR505450. A distant homolog was identified as an elongation factor 1-gamma from *Danio rerio* (zebrafish) with an e-value of 2e-30. Unlike the BLAST search, Psi-BLAST identified the homolog for the hypothetical proteins GR505494, GR505505, GR505506, GR505507, GR505508, GR505509, GR505512, GR505515 and GR505517 as BURP-domain-containing protein 17 (B9G9L9). The homolog of GR505510 was BURP-domain-containing protein 5 (Q0JEP3), and the homolog of GR505511 was BURP-domain-containing protein 3 (Q942D4). Notably, for GR505511, the BLAST search identified a similarity to dehydration-responsive protein RD22 (Q08298). In this case, the BLAST output was favorable because the percentage of sequence identity between GR505511 and Q08298 is 39%. In general, Psi-BLAST was able to successfully identify distant homologs from the same protein family group and, hence, corroborate the

Furthermore, there were a few more sequences (GR505491, GR505493, GR505495, GR505496, GR505497, GR505498, GR505499, GR505500, GR505502, GR50513 and GR505516) that had no significant hits from either the BLAST or Psi-BLAST runs. These sequences were then analyzed with two different similarity software packages (MPSrch and SSearch). The significance of the MPSrch results depends on the score value (i.e., a higher score implies more significant results). However, for the SSearch results, both the e-value and the SW score play important roles in choosing a significant hit. Both of these programs identified a putative uncharacterized protein from *Oryza sativa subsp. indica* (A2XQP1\_ORYSI) and *Sorghum bicolor* (C5XJ38) for GR505494 and from *Oryza sativa subsp. indica* (A2XQP1\_ORYSI) and *Glycine max* (C6TCE4) for GR505510. For GR505491, MPSrch identified a putative uncharacterized protein (D0ND35\_PHYIN) from *Phytophthora infestans T30-4* as its homolog, while SSearch found no matches. GR505493 had no significant matches from either the BLAST or Psi-BLAST runs. Notably, MPSrch and SSearch detected Nacetylglucosaminyltransferase (Q70MW8) from *Bradyrhizobium sp. ISLU256* as similar to GR505493. N-acetylglucosaminyltransferase (EC.2.4.1.-) is a resident Golgi-enzyme that is essential for the processing of high mannose to hybrid and complex glycans (Strasser et al. 2005). For GR505500, the first two programs failed to detect any similarity matches, but MPSearch identified a match with a predicted protein (B7G4S6\_PHATR) from *Phaeodactylum tricornutum CCAP 1055/1*, and SSearch identified galactoside-O-acetyltransferase (Q465V0) from *Methanosarcina barkeri*. This enzyme is usually found in bacteria, and it is interesting to experimentally investigate the existence of this protein in a plant. There were no significant

weight, pI, instability index, aliphatic index and GRAVY (grand average of hydropathy). A GRAVY index greater than zero indicates a hydrophobic protein (Kyte and Doolittle 1982). Notably, only one sequence in this dataset (GR505502) had a GRAVY value (0.528) greater than zero. The other proteins were predicted to be hydrophilic. The aliphatic value refers to the relative volume occupied by aliphatic side chains (Ala, Val, Ile and Leu) and is considered to be a positive factor for increased thermal stability of globular proteins (Ikai 1980). Both GR505495 and GR505502 had the highest aliphatic indices (115.98 and 111.2, respectively). The stability index provides an estimate of the stability of a protein *in vitro*. An instability index higher than 40 indicates an unstable protein. Our results showed that five sequences were predicted to be unstable (GR505491, GR505497, GR505498, GR505499 and GR505502). Different protein localizations usually imply different biological functions. The prediction of subcellular localization is relevant to inferring possible functions, annotating genomes, designing proteomics experiments and characterizing pharmacological targets (Lubec et al. 2005). The prediction of the protein type from its primary sequence or the determination of whether an uncharacterized protein is a membrane protein is important in both bioinformatics and proteomics. For this purpose, a few programs were used (Psortb (Bagos et al. 2008), SOSUI (Hirokawa et al. 1998), HMMTOP (Tusnady and Simon 2001), SignalP (Bendtsen et al. 2004), LipoP (Rahman et al. 2008)) to predict the subcellular localizations of the hypothetical proteins. Two sequences were predicted to be membrane proteins (GR505495 and GR505450, with two and one transmembrane helices, respectively). A polypeptide can be a membrane protein if it contains at least one transmembrane helix. HMMTOP predicted transmembrane regions for both sequences, at residues 106-130 and 137- 159 for GR505495 and residues 39-62 for GR505450. Table 4 shows the physicochemical analysis of the *P. minus* Huds hypothetical proteins achieved using various tools from HPAS and public databases. The consensus results were significant and were selected for further analysis (namely, the molecular responses of *P. minus* Huds roots to jasmonic acid induction).

### **3.2.3 Similarity search**

Four programs are consecutively used for a similarity search analysis. Table 5 provides all results from the analysis. In the first round, BLAST was used to find sequences that were similar to the hypothetical proteins. If BLAST did not find any significant hits for the hypothetical sequences, then Psi-BLAST was used. MPSrch and SSearch were then used for the sequences that had no significant matches from the previous program. BLAST was able to reveal similarities to BURP-domain-containing protein 3 for GR505494, GR505505, GR505506, GR505507, GR505508, GR505509, GR505510, GR505512, GR505515 and GR505517. The sequence motif of the BURP-domain-containing protein family has been described previously (Hattori et al. 1998), and many plant species (but not other organisms) that contain this domain have been identified. The BURP-domain-containing protein consists of several modules, such as an N-terminal hydrophobic transit peptide, a short conserved segment, an optional segment consisting of repeating units that are unique to each protein and the BURP domain at the C-terminus. The BURP-domain-containing protein consists of four typical members, BNM2, USP, RD22 and PG1. Thus far, this domain has been found only in plants, suggesting that its function may be plant-specific. The BURP-domaincontaining protein family has been found in various plant species, but their specific functions are still being explored. Based on their existence in various plants at various stages and in various locations, many BURP family members are involved in maintaining normal plant metabolism and development. For example, in the oilseed rape (*Brassica napes* L.),

weight, pI, instability index, aliphatic index and GRAVY (grand average of hydropathy). A GRAVY index greater than zero indicates a hydrophobic protein (Kyte and Doolittle 1982). Notably, only one sequence in this dataset (GR505502) had a GRAVY value (0.528) greater than zero. The other proteins were predicted to be hydrophilic. The aliphatic value refers to the relative volume occupied by aliphatic side chains (Ala, Val, Ile and Leu) and is considered to be a positive factor for increased thermal stability of globular proteins (Ikai 1980). Both GR505495 and GR505502 had the highest aliphatic indices (115.98 and 111.2, respectively). The stability index provides an estimate of the stability of a protein *in vitro*. An instability index higher than 40 indicates an unstable protein. Our results showed that five sequences were predicted to be unstable (GR505491, GR505497, GR505498, GR505499 and GR505502). Different protein localizations usually imply different biological functions. The prediction of subcellular localization is relevant to inferring possible functions, annotating genomes, designing proteomics experiments and characterizing pharmacological targets (Lubec et al. 2005). The prediction of the protein type from its primary sequence or the determination of whether an uncharacterized protein is a membrane protein is important in both bioinformatics and proteomics. For this purpose, a few programs were used (Psortb (Bagos et al. 2008), SOSUI (Hirokawa et al. 1998), HMMTOP (Tusnady and Simon 2001), SignalP (Bendtsen et al. 2004), LipoP (Rahman et al. 2008)) to predict the subcellular localizations of the hypothetical proteins. Two sequences were predicted to be membrane proteins (GR505495 and GR505450, with two and one transmembrane helices, respectively). A polypeptide can be a membrane protein if it contains at least one transmembrane helix. HMMTOP predicted transmembrane regions for both sequences, at residues 106-130 and 137- 159 for GR505495 and residues 39-62 for GR505450. Table 4 shows the physicochemical analysis of the *P. minus* Huds hypothetical proteins achieved using various tools from HPAS and public databases. The consensus results were significant and were selected for further analysis (namely, the molecular responses of *P. minus* Huds roots to jasmonic acid induction).

Four programs are consecutively used for a similarity search analysis. Table 5 provides all results from the analysis. In the first round, BLAST was used to find sequences that were similar to the hypothetical proteins. If BLAST did not find any significant hits for the hypothetical sequences, then Psi-BLAST was used. MPSrch and SSearch were then used for the sequences that had no significant matches from the previous program. BLAST was able to reveal similarities to BURP-domain-containing protein 3 for GR505494, GR505505, GR505506, GR505507, GR505508, GR505509, GR505510, GR505512, GR505515 and GR505517. The sequence motif of the BURP-domain-containing protein family has been described previously (Hattori et al. 1998), and many plant species (but not other organisms) that contain this domain have been identified. The BURP-domain-containing protein consists of several modules, such as an N-terminal hydrophobic transit peptide, a short conserved segment, an optional segment consisting of repeating units that are unique to each protein and the BURP domain at the C-terminus. The BURP-domain-containing protein consists of four typical members, BNM2, USP, RD22 and PG1. Thus far, this domain has been found only in plants, suggesting that its function may be plant-specific. The BURP-domaincontaining protein family has been found in various plant species, but their specific functions are still being explored. Based on their existence in various plants at various stages and in various locations, many BURP family members are involved in maintaining normal plant metabolism and development. For example, in the oilseed rape (*Brassica napes* L.),

**3.2.3 Similarity search** 

BNM2 is induced at the beginning of microspore embryogenesis, whereas the corresponding protein remains confined to the seed, where it is localized in the protein storage vacuoles (Zheng et al. 1992, Boutilier et al. 1994. Teerawanichpan et al. 2009, Treacy et 1997). USP from *Vicia faba* L., known as an abundant non-storage seed protein, is expressed during the early stage of zygotic embryogenesis (Bassuner et al. 1998) and at the very beginning of in vitro embryogenesis (21). However, PG1 is a non-catalytic -subunit of the polygalacturonase isozyme from the ripening tomato and plays an important role in regulating pectin metabolism (Zheng et al. 1992, Watson et al. 1994). In contrast, RD22 is a drought-induced protein in Arabidopsis thaliana and is often used as a reference for drought-stress treatment in different plants (Yamaguchi-Shinozaki et al. 1993). To date, this is the first report of the prediction of the BURP-domain-containing protein from the hypothetical proteins of P. minus Huds.

Psi-BLAST was then used to identify remote homologs, especially for the sequences with no significant hits from the BLAST search output. A detailed sequence analysis of these sequences allowed us to provide a tentative characterization of GR505450. A distant homolog was identified as an elongation factor 1-gamma from *Danio rerio* (zebrafish) with an e-value of 2e-30. Unlike the BLAST search, Psi-BLAST identified the homolog for the hypothetical proteins GR505494, GR505505, GR505506, GR505507, GR505508, GR505509, GR505512, GR505515 and GR505517 as BURP-domain-containing protein 17 (B9G9L9). The homolog of GR505510 was BURP-domain-containing protein 5 (Q0JEP3), and the homolog of GR505511 was BURP-domain-containing protein 3 (Q942D4). Notably, for GR505511, the BLAST search identified a similarity to dehydration-responsive protein RD22 (Q08298). In this case, the BLAST output was favorable because the percentage of sequence identity between GR505511 and Q08298 is 39%. In general, Psi-BLAST was able to successfully identify distant homologs from the same protein family group and, hence, corroborate the output of the BLAST search.

Furthermore, there were a few more sequences (GR505491, GR505493, GR505495, GR505496, GR505497, GR505498, GR505499, GR505500, GR505502, GR50513 and GR505516) that had no significant hits from either the BLAST or Psi-BLAST runs. These sequences were then analyzed with two different similarity software packages (MPSrch and SSearch). The significance of the MPSrch results depends on the score value (i.e., a higher score implies more significant results). However, for the SSearch results, both the e-value and the SW score play important roles in choosing a significant hit. Both of these programs identified a putative uncharacterized protein from *Oryza sativa subsp. indica* (A2XQP1\_ORYSI) and *Sorghum bicolor* (C5XJ38) for GR505494 and from *Oryza sativa subsp. indica* (A2XQP1\_ORYSI) and *Glycine max* (C6TCE4) for GR505510. For GR505491, MPSrch identified a putative uncharacterized protein (D0ND35\_PHYIN) from *Phytophthora infestans T30-4* as its homolog, while SSearch found no matches. GR505493 had no significant matches from either the BLAST or Psi-BLAST runs. Notably, MPSrch and SSearch detected Nacetylglucosaminyltransferase (Q70MW8) from *Bradyrhizobium sp. ISLU256* as similar to GR505493. N-acetylglucosaminyltransferase (EC.2.4.1.-) is a resident Golgi-enzyme that is essential for the processing of high mannose to hybrid and complex glycans (Strasser et al. 2005). For GR505500, the first two programs failed to detect any similarity matches, but MPSearch identified a match with a predicted protein (B7G4S6\_PHATR) from *Phaeodactylum tricornutum CCAP 1055/1*, and SSearch identified galactoside-O-acetyltransferase (Q465V0) from *Methanosarcina barkeri*. This enzyme is usually found in bacteria, and it is interesting to experimentally investigate the existence of this protein in a plant. There were no significant

Alteration of Abiotic Stress Responsive Genes

**Peptide ID** 

GR505450 No significant match

GR505491 No significant match

GR505493 No significant match

GR505495 No significant match

GR505496 No significant match

GR505497 No significant match

BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

GR505494

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 71

Elongation factor 1-gamma;

Organism = *Danio rerio* (Zebrafish)

No sequence selected in the PSI-BLAST model

No sequence found by PSI-BLAST

BURP-domaincontaining protein

Organism = *Oryza sativa subsp. japonica*

No sequence selected in the PSI-BLAST model

No sequence selected in the PSI-BLAST model

No sequence found by PSI-BLAST

17;

**BLAST Psi-BLAST MPSrch SSearch Description Description Description Description** 

Putative

protein;

Putative

protein; Organism =

*T30-4*

nsferase; Organism = *Bradyrhizobium sp.* 

*ISLU256*

Putative

protein;

(Rice)

Putative

protein;

*HTCC2148*

Putative

protein; Score = 93; Identity = 11.4; Identifier = A8URE8\_9AQUI;

uncharacterized

Organism = *Oryza sativa subsp. indica* 

Predicted protein; Organism =

*Nematostella vectensis* (Starlet sea anemone)

uncharacterized

uncharacterized

Organism = *marine gamma proteobacterium* 

N-

uncharacterized

uncharacterized

*Phytophthora infestans* 

acetylglucosaminyltra

Organism = *Glycine max* (Soybean)

Probable glutathione Stransferase; Organism = *Nicotiana tabacum* (Common tobacco)

No significant

acetylglucosaminyl transferase; Organism = *Bradyrhizobium sp.* 

match

*ISLU256*

Putative

protein Sb03g033760; Organism = *Sorghum bicolor* (Sorghum)

Putative

protein;

*gamma* 

Putative

protein; E-value = 0.45; SW Score = 127; Bit Score = 38.3; Identifier =

uncharacterized

Predicted protein; Organism = *Nematostella vectensis* (Starlet sea anemone)

uncharacterized

*proteobacterium HTCC2148*

uncharacterized

Organism = *marine* 

N-


Table 4. Physicochemical properties of hypothetical proteins from the root of *P. minus*.

**ProtParam** 

**Positive residues**

**Instability index** 

**Aliphatic index** 

(Stable) 67.6 -0.269

(Stable) 76.19 -0.148

(Stable) 115.98 0.346

(Stable) 83.41 -0.176

(Unstable) 66.56 -0.541

(Unstable) 43.02 -0.723

(Unstable) 50.46 -1.064

(Stable) 70.18 -0.367

(Unstable) 111.2 0.528

(Stable) 71.61 -0.29

(Stable) 74.01 -0.205

(Stable) 67.99 -0.348

(Stable) 69.55 -0.202

(Stable) 67.99 -0.348

(Stable) 78.17 -0.06

(Stable) 78.35 -0.045

(Stable) 81.4 -0.16

(Unstable) 56.24 -0.5

**GRAV Y** 

**Negative residues**

GR505494 201 22009.5 6.06 23 19 35.5 (Stable) 69.25 -0.277

GR505506 167 18241 6.89 16 16 31.5 (Stable) 73.47 -0.12 GR505507 166 18520.6 6.04 19 16 34.5 (Stable) 75.66 -0.245

GR505511 135 14905.9 6.03 18 15 28.6 (Stable) 61.26 -0.485

GR505515 167 18256 7.58 16 17 33.9 (Stable) 71.14 -0.166

Table 4. Physicochemical properties of hypothetical proteins from the root of *P. minus*.

**Peptide ID** 

**Amino acid count**

**Molecular weight** 

**pI value**

GR505450 121 14082.1 4.73 21 14 33.85

GR505491 85 9656.2 6.39 7 6 69.9

GR505493 63 6679.5 9.52 4 7 23.04

GR505495 164 18007.6 6.98 13 13 23.11

GR505496 91 10096.5 5.45 13 12 36.17

GR505497 151 16836.1 9.46 9 15 41.19

GR505498 43 4594 6.91 4 4 43.57

GR505499 87 9811.1 9 10 13 43.43

GR505500 164 18010.1 5.18 18 12 25.48

GR505502 50 5511.6 4.46 6 4 46.33

GR505505 174 19184.4 7.11 20 20 26.11

GR505508 167 18383.3 5.83 20 16 32.49

GR505509 159 17609.2 6.18 19 17 35.51

GR505510 157 16928.4 6.83 16 16 27.45

GR505512 159 17609.2 6.18 19 17 35.51

GR505513 126 13267.9 8.96 8 11 23.75

GR505516 127 13339 8.96 8 11 24.97

GR505517 171 18937.5 7.9 18 19 26.83


Alteration of Abiotic Stress Responsive Genes

Organism = *Oryza sativa subsp. japonica*

BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

Dehydrationresponsive protein RD22; Organism = *Arabidopsis thaliana*

BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

BURP-domaincontaining protein

GR505513 No significant match

GR505508

GR505509

GR505510

GR505511

GR505512

GR505515

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 73

Organism = *Oryza sativa subsp. indica*

uncharacterized

Organism = *Oryza sativa subsp. indica*

*sibiricum*

Putative

protein Sb03g033760; Organism = *Sorghum bicolor* (Sorghum)

Putative

protein; Organism = *Glycine max* (Soybean)

uncharacterized

BURP-domaincontaining protein; Organism = *Brassica napus* (Rape)

RD22-like protein; Organism = *Polygonum sibiricum*

Putative coat protein; Organism = *Elderberry latent* 

RD22-like protein; Organism = *Polygonum* 

*virus*

uncharacterized

RD22-like protein; Organism = *Polygonum sibiricum*

(Rice)

Putative

protein;

(Rice)

Putative

protein;

(Rice)

Putative

protein;

(Rice)

Putative

protein;

(Rice)

Putative

protein;

(Rice)

*latent virus*

Putative

protein;

uncharacterized

uncharacterized

Organism = *Oryza sativa subsp. indica*

uncharacterized

Organism = *Oryza sativa subsp. indica*

uncharacterized

Organism = *Oryza sativa subsp. indica*

uncharacterized

Organism = *Oryza sativa subsp. indica*

Putative coat protein; Organism = *Elderberry* 

Organism = *Oryza sativa subsp. japonica*

BURP-domaincontaining protein

BURP-domaincontaining protein

BURP-domaincontaining protein

BURP-domaincontaining protein

BURP-domaincontaining protein

No sequence selected in the PSI-BLAST model

BURP-domaincontaining protein

Organism = *Oryza sativa subsp. japonica*

Organism = *Oryza sativa subsp. japonica*

Organism = *Oryza sativa subsp. japonica*

Organism = *Oryza sativa subsp. japonica*

Organism = *Oryza sativa subsp. japonica*

17;

17;

5;

3;

17;

17;


No sequence found by PSI-BLAST

No sequence selected in the PSI-BLAST model

No sequence selected in the PSI-BLAST model

No sequence found by PSI-BLAST

BURP-domaincontaining protein

BURP-domaincontaining; protein

BURP-domaincontaining protein

Organism = *Oryza sativa subsp. japonica*

Organism = *Oryza sativa subsp. japonica*

17;

17

17;

GR505498 No significant match

GR505499 No significant match

GR505500 No significant match

GR505502 No significant match

> BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

> BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

> BURP-domaincontaining protein 3;

GR505505

GR505506

GR505507

Organism =

*5-R1-1*

Putative

protein;

(Rice)

Putative

protein; Organism =

*1055/1*

Putative

protein;

(Rice)

Putative

protein;

(Rice)

Putative

protein;

uncharacterized

Organism = *Oryza sativa subsp. indica*

uncharacterized

*Sphingomonas wittichii* (strain RW1 / DSM 6014 / JCM 10273)

Predicted protein; Organism = *Phaeodactylum tricornutum CCAP* 

Predicted protein; Organism = *Paracoccidioides brasiliensis (strain Pb03)*

uncharacterized

Organism = *Oryza sativa subsp. indica*

uncharacterized

Organism = *Oryza sativa subsp. indica*

uncharacterized

*Hydrogenivirga sp. 128-*

A5Z3S2; Organism = *Eubacterium ventriosum ATCC* 

*27560*

Putative

protein; Organism = *Ruminococcus gnavus ATCC* 

*29149*

Putative

protein; Organism = *Sphingomonas wittichii* (strain RW1 / DSM 6014 / JCM 10273)

uncharacterized

uncharacterized

Galactoside-Oacetyltransferase; Organism = *Methanosarcina barkeri* (strain Fusaro / DSM 804)

No significant

uncharacterized

RD22-like protein; Organism = *Polygonum sibiricum*

RD22-like protein; Organism = *Polygonum* 

match

Putative

protein Sb03g033760; Organism = *Sorghum bicolor* (Sorghum)


Alteration of Abiotic Stress Responsive Genes

Treated Untreated

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 75

(peroxidase, POD) and the clone involved in protein degradation pathway was GR505460, which had a cDNA sequence similar to the kelch-repeat containing F-box family protein (Fbox). Ubiquitin 11, an endogenous gene expressed constitutively in plant was selected as internal control for normalization of gene expression. In general, the expression patterns were consistent with the results of the Reverse Northern analysis. The expression patterns can be divided into three types: (1) strong upregulation in JA-treated roots and slight up-regulation in normal roots, i.e., GR505453 and GR505459; (2) strong up-regulation in JA-treated roots but very little or no expression in normal roots, i.e., GR505465, GR505467, GR505471, GR505464, and GR505460; and (3) slight upregulation in JA-treated roots compared to non-treated roots, i.e., GR505472. The expression level of Ubiquitin 11 did not differ between samples (Fig. 6).

Fig. 6. Semi-quantitative RT-PCR analysis of expression patterns of genes responsive to jasmonic acid metabolism of JA-treated P. minus roots subtracted library. Expression pattern for each selected clone was examined in both JA-treated and non-treated roots' samples. Ubiquitin 11 was used as a control to demonstrate equal cDNA used as templates.

dehydrogenase); GR505472 (LOX lipoxygenase); GR505467 (SAMS S-adenosyl-L-methionine synthetase) and GR505471 (SHH S-adenosyl-L-homocysteine hydrolase). Clones related to abiotic stress are GR505453 (ELI3-1 ELI3-1 gene), GR505459 (GST glutathione S-transferase) and GR505464 (POD peroxidase). GR505460 is a clone similar to F-box protein (kelch repeat

Clones involved in aromatic compounds biosynthesis are GR505465 (ADH alcohol

containing F-box family protein)


Table 5. Results of the similarity search using four similarity search programs (BLAST, Psi-BLAST, MPSrch and SSearch)

matches for GR505502, except for one match from MPSrch, but the score was low. Thus, thissequence is a good candidate for a structure-prediction approach for making a functional inference. Neither GR505513 nor GR505516 had matches from the BLAST or Psi-BLAST runs. Using MPSrch and SSearch, both sequences were predicted to be similar to a putative coat protein (Q911J7) from the elderberry latent virus. Even though the scores from both programs were reasonably low, at least the output can provide some insight into the functions of these hypothetical proteins and a basis for experimentation. A number of hypothetical proteins obtained from the roots of *P. minus* Huds were computationally identified as similar to at least one fully characterized domain. However, the functional interpretation of these proteins is limited. Notably, despite our best efforts, we were unable to provide functional annotations for GR505498 or GR505502. In addition, functional changes over evolutionary time (Devos and Valencia 2000, Todd et al. 2001) and database errors (Brenner 1999) confound the reliable computational predictions of the precise functions of these newly discovered genes. Further experimental evidence is required to successfully deduce their molecular roles.

### **3.3 Effect of JA elicitation on gene expression**

RT-PCR analysis was performed to compare the transcripts expression between control root sample and JA-treated root samples. To verify whether the gene expression corresponding to the cDNA sequences generated by SSH were differentially expressed in *P. minus* under JA stress, four clones involved in the biosynthesis of aromatic compounds, three clones related to abiotic stress, and one clone representing a transcription factor were examined. Those clones were selected based on the nearest E-value to zero and molecular functions identified by BLAST. The clones that showed similarity to genes associated with aromatic compound biosynthesis were GR505472 (lipoxygenase, LOX), GR505465 (alcohol dehydrogenase, ADH), GR505467 (S-adenosyl-L-methionine synthetase, SAMS) and GR505471 (S-adenosyl-Lhomocysteine hydrolase, SHH). The clones that were similar to abiotic stress response genes were GR505453 (ELI3-1), GR505459 (glutathione S-transferase, GST) and GR505464

Organism = *Oryza sativa subsp. indica*

Putative coat protein; Organism = *Elderberry*  *sibiricum* 

Putative coat protein; Organism = *Elderberry latent* 

*virus*

Putative

protein Sb03g033760; Organism = *Sorghum bicolor* (Sorghum)

uncharacterized

(Rice)

*latent virus*

Putative

protein;

(Rice)

Table 5. Results of the similarity search using four similarity search programs (BLAST, Psi-

matches for GR505502, except for one match from MPSrch, but the score was low. Thus, thissequence is a good candidate for a structure-prediction approach for making a functional inference. Neither GR505513 nor GR505516 had matches from the BLAST or Psi-BLAST runs. Using MPSrch and SSearch, both sequences were predicted to be similar to a putative coat protein (Q911J7) from the elderberry latent virus. Even though the scores from both programs were reasonably low, at least the output can provide some insight into the functions of these hypothetical proteins and a basis for experimentation. A number of hypothetical proteins obtained from the roots of *P. minus* Huds were computationally identified as similar to at least one fully characterized domain. However, the functional interpretation of these proteins is limited. Notably, despite our best efforts, we were unable to provide functional annotations for GR505498 or GR505502. In addition, functional changes over evolutionary time (Devos and Valencia 2000, Todd et al. 2001) and database errors (Brenner 1999) confound the reliable computational predictions of the precise functions of these newly discovered genes. Further experimental evidence is required to

RT-PCR analysis was performed to compare the transcripts expression between control root sample and JA-treated root samples. To verify whether the gene expression corresponding to the cDNA sequences generated by SSH were differentially expressed in *P. minus* under JA stress, four clones involved in the biosynthesis of aromatic compounds, three clones related to abiotic stress, and one clone representing a transcription factor were examined. Those clones were selected based on the nearest E-value to zero and molecular functions identified by BLAST. The clones that showed similarity to genes associated with aromatic compound biosynthesis were GR505472 (lipoxygenase, LOX), GR505465 (alcohol dehydrogenase, ADH), GR505467 (S-adenosyl-L-methionine synthetase, SAMS) and GR505471 (S-adenosyl-Lhomocysteine hydrolase, SHH). The clones that were similar to abiotic stress response genes were GR505453 (ELI3-1), GR505459 (glutathione S-transferase, GST) and GR505464

uncharacterized

Organism = *Oryza sativa subsp. indica*

Organism = *Oryza sativa subsp. japonica*

No sequence selected in the PSI-BLAST model

BURP-domaincontaining protein

Organism = *Oryza sativa subsp. japonica*

17;

Organism = *Oryza sativa subsp. japonica*

BURP-domaincontaining protein 3; Organism = *Oryza sativa subsp. japonica*

BLAST, MPSrch and SSearch)

successfully deduce their molecular roles.

**3.3 Effect of JA elicitation on gene expression** 

GR505516 No significant match

GR505517

(peroxidase, POD) and the clone involved in protein degradation pathway was GR505460, which had a cDNA sequence similar to the kelch-repeat containing F-box family protein (Fbox). Ubiquitin 11, an endogenous gene expressed constitutively in plant was selected as internal control for normalization of gene expression. In general, the expression patterns were consistent with the results of the Reverse Northern analysis. The expression patterns can be divided into three types: (1) strong upregulation in JA-treated roots and slight up-regulation in normal roots, i.e., GR505453 and GR505459; (2) strong up-regulation in JA-treated roots but very little or no expression in normal roots, i.e., GR505465, GR505467, GR505471, GR505464, and GR505460; and (3) slight upregulation in JA-treated roots compared to non-treated roots, i.e., GR505472. The expression level of Ubiquitin 11 did not differ between samples (Fig. 6).

Fig. 6. Semi-quantitative RT-PCR analysis of expression patterns of genes responsive to jasmonic acid metabolism of JA-treated P. minus roots subtracted library. Expression pattern for each selected clone was examined in both JA-treated and non-treated roots' samples. Ubiquitin 11 was used as a control to demonstrate equal cDNA used as templates. Clones involved in aromatic compounds biosynthesis are GR505465 (ADH alcohol dehydrogenase); GR505472 (LOX lipoxygenase); GR505467 (SAMS S-adenosyl-L-methionine synthetase) and GR505471 (SHH S-adenosyl-L-homocysteine hydrolase). Clones related to abiotic stress are GR505453 (ELI3-1 ELI3-1 gene), GR505459 (GST glutathione S-transferase) and GR505464 (POD peroxidase). GR505460 is a clone similar to F-box protein (kelch repeat containing F-box family protein)

Alteration of Abiotic Stress Responsive Genes

signaling and must be further investigated.

compounds in kesum.

al. 2000).

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 77

these fatty acids may have served as substrates for the production of volatile aromatic

Fig. 8. Alcohol and aldehyde production by oxylipin pathway in tomato. (Source: Baldwin et

Apart from that, seven clones showed similarity to S-adenosyl-L-methionine synthetase (87% identity similar to *Beta vulgaris*) and one clone was similar to S-adenosyl-Lhomocysteine hydrolase (87% identity similar to *Mesembrayanthemum crystallinum*). These clones were involved in the synthesis of aromatic shikimic acid through the shikimate pathway. Both of these clones demonstrated the type 2 expression pattern, where their expression in kesum roots was only up-regulated upon JA elicitation and no expression was observed under control conditions. Shikimic acid is the precursor for phenylpropanoid biosynthesis, another important group of secondary metabolites (Dewick 2001). Both Sadenosyl-L-methionine synthetase (SAMS, EC 2.5.1.6) and S-adenosyl-L-homocysteine hydrolase (SHH, EC 3.3.1.1) are enzymes that play a role in the synthesis of S-adenosyl-Lmethionine (SAM), the main donor of the methyl group for many specific methyl transferase reactions, such as the transmethylation of alkaloids (Kutchan 1995). In plants, SAM is also associated with the production of phenylpropanoids (Kawalleck et al. 1992). Again, our data corroborate previous reports that identified flavonoids in leaves of the Polygonum family (including P. minus), which are synthesized by the phenylpropanoid metabolic pathway (Urones et al. 1990), P. stagninum (Datta et al. 2002) and P. hydropiper (Peng et al. 2003). The data suggest that the genes that are normally expressed in leaves could be triggered by JA in roots. On the other hand, SAM is also the intermediate molecule in ethylene and polyamine biosynthesis (Ravanel et al. 1998). Nevertheless, the expression of these two genes was also shown to be up-regulated in the petunia flower and they were linked to the production of benzenoid, a compound that contributes to the flower's scent (Schuurink et al. 2006). Hence, it was believed that the expression of the SAMS and SHH genes found in this study was related to the production of phenylpropanoids, alkaloids, ethylene or polyamine after JA elicitation. The activated-methyl cycle was predicted to be closely related to JA

### **3.4 Correlation study between phytochemistry profiling and transcriptomic dataset 3.4.1 Genes involved in aromatic compounds production**

A total of 11 cDNA sequences found in this study showed significant similarity with enzymes associated with biosynthesis of volatile compounds from plants. These sequences are found to be involved in acyl lipid catabolism pathway (2 ADH clones and 1 LOX clone) and shikimate pathway (7 SAMS clones and 1 SHH clone). GR505471 clone showed 81% identity similar to lipoxygenase gene isolated from *Capsicum annuum*. Lipoxygenase (LOX, EC 1.13.11.12) catalyzes deoxygenation of linoleic and linolenic acid to hydroperoxide in oxylipin pathway (Devitt 2006). Oxylipin is a common name for oxidized compounds derived from fatty acid though enzymatic reaction. Examples of oxidized compounds are hydroperoxide fatty acid, hydroxyl fatty acid, epoxy fatty acid, keto fatty acid, volatile aldehyde and cyclic compounds. This oxylipin pathway will affect plant aroma and taste (Yilmaz 2000). Lipoxygenase has been isolated from cucumber infected by spider mite and the expression of this gene was linked with the production of volatile compound named (Z)- 3-hexynyl acetate (Mercke et al. 2004). It has also been isolated from papaya (Devitt et al. 2006) and apple (Dixon & Hewett 2000). Thus, we the expression of lipoxygenase was believed to be associated with the production of aldehyde in kesum, such as octadecanal which contribute to the aromatic flavour of kesum. Beside, GR505464 clone showed 72% identity similar to alcohol dehydrogenase from *Prunus armeniaca*. Alcohol dehydrogenase (ADH, EC 1.1.1.1) identified in this study might be involved in phenylpropanoids formation, another group of volatile aromatic compounds (Devitt et al. 2006). All the alkanes identified by GC-MS in kesum root extract could be oxidized into alcohols, aldehydes and acids homolog to the alkanes by using NAD and NADH as cofactor (Figure 7) (Dixon & Hewett 2000). ADH has also been identified in papaya (Devitt et al. 2006), apple (Dixon & Hewett 2000), corn (Walker et al. 1987) and grapevine (Torregrosa et al. 2008). It was believed that the expression of both LOX and ADH were involved in the oxidation of alkanes into alcohols, aldehydes and acids in kesum roots.

$$\text{RCH}\_2\text{CH}\_3 \xrightarrow[\text{O}\_2]{\text{NADH}} \text{RCH}\_2\text{CH}\_2\text{OH} \xrightarrow[\text{ADH}]{\text{NAD}} \text{RCH}\_2\text{CH} \xrightarrow{\text{NAD}} \text{RCH}\_2\text{COOH}$$

Fig. 7. Oxidation pathway of alkanes.

The identification of lipoxygenase and alcohol dehydrogenase in our study have further confirmed the results obtained by Karim (1987), who reported that approximately 76% of the essential oil in kesum leaves were comprised of aliphatic aldehydes, such as decanal and dodecanal (Karim 1987). Our results also strengthened the GC–MS data previously reported for *P. odoratum* leaves (Du˜ng et al. 1995; Hunter et al. 1997). Naturally these two genes may occur in kesum roots but are not routinely expressed. However, our study showed that their expression in roots could be up-regulated by exposure to JA. According to a model proposed by Yilmaz (2001) in a tomato-ripening study, lipoxygenase will catalyze the deoxygenation of linoleic and linolenic acid into hydroperoxides and subsequently to aldehydes and alcohols. Alcohol dehydrogenase will then catalyze the oxidation of aldehydes to the respective alcohols or vice versa (Figure 8) (Yilmaz 2001). In this study, JA elicitation may have caused the release of free fatty acids from the root cell membranes and

A total of 11 cDNA sequences found in this study showed significant similarity with enzymes associated with biosynthesis of volatile compounds from plants. These sequences are found to be involved in acyl lipid catabolism pathway (2 ADH clones and 1 LOX clone) and shikimate pathway (7 SAMS clones and 1 SHH clone). GR505471 clone showed 81% identity similar to lipoxygenase gene isolated from *Capsicum annuum*. Lipoxygenase (LOX, EC 1.13.11.12) catalyzes deoxygenation of linoleic and linolenic acid to hydroperoxide in oxylipin pathway (Devitt 2006). Oxylipin is a common name for oxidized compounds derived from fatty acid though enzymatic reaction. Examples of oxidized compounds are hydroperoxide fatty acid, hydroxyl fatty acid, epoxy fatty acid, keto fatty acid, volatile aldehyde and cyclic compounds. This oxylipin pathway will affect plant aroma and taste (Yilmaz 2000). Lipoxygenase has been isolated from cucumber infected by spider mite and the expression of this gene was linked with the production of volatile compound named (Z)- 3-hexynyl acetate (Mercke et al. 2004). It has also been isolated from papaya (Devitt et al. 2006) and apple (Dixon & Hewett 2000). Thus, we the expression of lipoxygenase was believed to be associated with the production of aldehyde in kesum, such as octadecanal which contribute to the aromatic flavour of kesum. Beside, GR505464 clone showed 72% identity similar to alcohol dehydrogenase from *Prunus armeniaca*. Alcohol dehydrogenase (ADH, EC 1.1.1.1) identified in this study might be involved in phenylpropanoids formation, another group of volatile aromatic compounds (Devitt et al. 2006). All the alkanes identified by GC-MS in kesum root extract could be oxidized into alcohols, aldehydes and acids homolog to the alkanes by using NAD and NADH as cofactor (Figure 7) (Dixon & Hewett 2000). ADH has also been identified in papaya (Devitt et al. 2006), apple (Dixon & Hewett 2000), corn (Walker et al. 1987) and grapevine (Torregrosa et al. 2008). It was believed that the expression of both LOX and ADH were involved in the oxidation of

The identification of lipoxygenase and alcohol dehydrogenase in our study have further confirmed the results obtained by Karim (1987), who reported that approximately 76% of the essential oil in kesum leaves were comprised of aliphatic aldehydes, such as decanal and dodecanal (Karim 1987). Our results also strengthened the GC–MS data previously reported for *P. odoratum* leaves (Du˜ng et al. 1995; Hunter et al. 1997). Naturally these two genes may occur in kesum roots but are not routinely expressed. However, our study showed that their expression in roots could be up-regulated by exposure to JA. According to a model proposed by Yilmaz (2001) in a tomato-ripening study, lipoxygenase will catalyze the deoxygenation of linoleic and linolenic acid into hydroperoxides and subsequently to aldehydes and alcohols. Alcohol dehydrogenase will then catalyze the oxidation of aldehydes to the respective alcohols or vice versa (Figure 8) (Yilmaz 2001). In this study, JA elicitation may have caused the release of free fatty acids from the root cell membranes and

**3.4 Correlation study between phytochemistry profiling and transcriptomic dataset** 

**3.4.1 Genes involved in aromatic compounds production** 

alkanes into alcohols, aldehydes and acids in kesum roots.

Fig. 7. Oxidation pathway of alkanes.

these fatty acids may have served as substrates for the production of volatile aromatic compounds in kesum.

Fig. 8. Alcohol and aldehyde production by oxylipin pathway in tomato. (Source: Baldwin et al. 2000).

Apart from that, seven clones showed similarity to S-adenosyl-L-methionine synthetase (87% identity similar to *Beta vulgaris*) and one clone was similar to S-adenosyl-Lhomocysteine hydrolase (87% identity similar to *Mesembrayanthemum crystallinum*). These clones were involved in the synthesis of aromatic shikimic acid through the shikimate pathway. Both of these clones demonstrated the type 2 expression pattern, where their expression in kesum roots was only up-regulated upon JA elicitation and no expression was observed under control conditions. Shikimic acid is the precursor for phenylpropanoid biosynthesis, another important group of secondary metabolites (Dewick 2001). Both Sadenosyl-L-methionine synthetase (SAMS, EC 2.5.1.6) and S-adenosyl-L-homocysteine hydrolase (SHH, EC 3.3.1.1) are enzymes that play a role in the synthesis of S-adenosyl-Lmethionine (SAM), the main donor of the methyl group for many specific methyl transferase reactions, such as the transmethylation of alkaloids (Kutchan 1995). In plants, SAM is also associated with the production of phenylpropanoids (Kawalleck et al. 1992). Again, our data corroborate previous reports that identified flavonoids in leaves of the Polygonum family (including P. minus), which are synthesized by the phenylpropanoid metabolic pathway (Urones et al. 1990), P. stagninum (Datta et al. 2002) and P. hydropiper (Peng et al. 2003). The data suggest that the genes that are normally expressed in leaves could be triggered by JA in roots. On the other hand, SAM is also the intermediate molecule in ethylene and polyamine biosynthesis (Ravanel et al. 1998). Nevertheless, the expression of these two genes was also shown to be up-regulated in the petunia flower and they were linked to the production of benzenoid, a compound that contributes to the flower's scent (Schuurink et al. 2006). Hence, it was believed that the expression of the SAMS and SHH genes found in this study was related to the production of phenylpropanoids, alkaloids, ethylene or polyamine after JA elicitation. The activated-methyl cycle was predicted to be closely related to JA signaling and must be further investigated.

Alteration of Abiotic Stress Responsive Genes

defence response against abiotic stress.

**5. Acknowledgement** 

Gor Mian Chee.

**6. References** 

**4. Conclusion** 

secondary metabolites production in response to JA.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 79

the cell cycle, transcription, and signal transduction, as a mechanism for the root cells to adapt to JA elicitation stress (Sun et al. 2007). The functions of these proteins have been demonstrated in Arabidopsis and they may serve as transcription factors in genes expressed in response to JA treatment. While these proteins all play the same role in regulating JA, they also regulate species-specific secondary metabolite pathways (Pauwels et al. 2009). These proteins must be characterized and examined for the mechanism that drives

Our results showed that there is a close relationship between abiotic stress and the expression of genes involved in the biosynthesis of secondary metabolites. The subtractive cDNA library data set presented here provides the first collection of a set of JA responsive genes that may be involved in the secondary metabolite production in *P. minus* roots and also those participating in plant-defense mechanisms. These genes include dehydrogenase, lipoxygenase, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase, glutathione S-transferase, peroxidase, ELI3-1, and a transcription factor, F-box family protein. Identification of genes associated with flavour volatiles and the production of other aromatic compounds will provide a better understanding of the secondary metabolite biosynthetic pathways and their regulation in *P. minus*. Furthermore, the observed stressrelated genes induced by JA elicitation indicate that plants respond to abiotic stresses in parallel with the biosynthesis of certain secondary metabolites. Characterization of these genes will be studied in details using *E. coli* expression system. Besides, their functions could be explored with GC or HPLC to confirm the synthesis of the corresponding compounds whereas their ability to cope with abiotic stress could be performed by culturing *P. minus* plantlets in various stress conditions. In this study, we concluded that the JAresponsive genes might be the genes associated with volatile compounds production as a

We would like to thanks to the the Ministry of Higher Education of Malaysia (MOHE) for funding the project under the Fundamental Research Grant Scheme (FRGS) awarded to Ismanizan Ismail. Our gratitude also goes to the Ministry of Science, Technology and Innovation of Malaysia for National Science Foundation Fellowship (NSF) awarded to Miss

Agarwal, P. and States, D.J. 1998. Comparative accuracy of methods for protein sequence

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. 1990. Basic local alignment

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J.

1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search

similarity search. *Bioinformatics.* 14(1): 40-47.

programs. *Nucleic Acids Res.* 25(17): 3389-3402.

search tool. *J Mol Biol.* 215(3): 403-410.

### **3.4.2 Genes related to abiotic stress**

Clones encoding the genes related to abiotic stress,such as glutathione S-transferase (GST), ELI3-1 and peroxidase (POD) were evaluated to investigate the correlation between JA stress and other stress factors. Both GST and ELI3-1 demonstrated a type 1 expression pattern. Ten clones were similar to GST in this subtracted library. GST (EC 2.5.1.18) is a cytosolic enzyme found in all eukaryotes. This gene is always detected in stressed plants, such as heavy metal and salt-treated rice seedlings (Moons 2003), water-stressed maize seedlings (Zheng et al. 2004), drought-stressed horse gram (Chandra Obul Reddy et al. 2008) and fungus-elicited rice seedlings (Xiong et al. 2001). Therefore, and not surprisingly, the expression of GST was observed in normal kesum roots because the in vitro culture itself was a stress condition for kesum plantlets. It was strongly up-regulated in JAtreated roots as a defense mechanism, suggesting an overlap in the plant responses of plants to various stress factors. Also, GST catalyzes the conjugation between synthetic electrophilic compounds and the glutathione tripeptide (c-glutamyl-cysteinyl-glycine, GSH). The polar Sglutathionylated product will be actively transported into the vacuole by an ATP-binding cassette. Thus, GST is part of the detoxification mechanism in plants. In fact, it is the main ingredient for a variety of commercial herbicides (Andrews et al. 2005). Therefore, it is crucial to investigate kesum as a potential plant for phytoremediation purposes. ELI3-1 is another gene related to the abiotic stress caused by JA elicitation that will lead to phytoalexin and pathogenesis-related protein accumulation, phenolic compound production, and cell wall reconstruction. Seventeen of the ELI genes were identified in parsley cells cultured and treated with the Phytophthora megasperma fungus (Trezzini et al. 1993). In addition, MeJA elicitation has successfully activated ELI3, TyrDC, HRGP, and BMT genes in parsley (Ellard-Ivey and Douglas 1996). This observation proved that the expression of ELI3-1 could also be induced by JA elicitation. Peroxidase (POD, EC 1.11.1.7) plays an important role in the oxidation process, such as in peroxidative ooxidative oxidation and catalytic hydrosilylation (Umaya and Kobayashi 2003; Veitch 2004). It is an enzyme that catalyzes the oxidation of phenylpropanoids (Thimmaraju et al. 2006). It also functions in the plant-defense system, and it could be triggered by an elicitor (Go´mez-Va´squez et al. 2004; Perera and Jones 2004). The clones that were similar to POD in this study showed a type 2 expression pattern in the RT-PCR analysis. Its expression has been shown to induce the production of terpenoids in cucumbers infected by spider mites via oxidative degradation (Mercke et al. 2004). Our observation corroborates the results reported in kesum (Karim 1987) and *P. odoratum* (Du˜ng et al. 1995; Hunter et al. 1997), where various sesquiterpene hydrocarbons and their oxygenated compounds were identified. Therefore, it was predicted that stress stimuli, such as JA, could regulate the induction of important classes of plant secondary metabolites in kesum. In addition, its oxidative reaction showed that POD can be used as a component in the reagent for clinical diagnosis and various laboratory experiments (Thimmaraju et al. 2006) and thus increases the value of kesum.

### **3.4.3 Transcription factor activated by JA**

Interestingly, the kelch-repeat containing F-box family protein and the TIR1 protein that is contained in the F-box found in this subtractive cDNA library are induced by JA (Craig and Tyers 1999; Parry and Estelle 2006). The kelchrepeat containing F-box family protein is involved in the protein–protein interaction in the ubiquitin protein degradation process via the ubiquitin-mediated pathway. The protein degradation process is important to regulate the cell cycle, transcription, and signal transduction, as a mechanism for the root cells to adapt to JA elicitation stress (Sun et al. 2007). The functions of these proteins have been demonstrated in Arabidopsis and they may serve as transcription factors in genes expressed in response to JA treatment. While these proteins all play the same role in regulating JA, they also regulate species-specific secondary metabolite pathways (Pauwels et al. 2009). These proteins must be characterized and examined for the mechanism that drives secondary metabolites production in response to JA.

### **4. Conclusion**

78 Plants and Environment

Clones encoding the genes related to abiotic stress,such as glutathione S-transferase (GST), ELI3-1 and peroxidase (POD) were evaluated to investigate the correlation between JA stress and other stress factors. Both GST and ELI3-1 demonstrated a type 1 expression pattern. Ten clones were similar to GST in this subtracted library. GST (EC 2.5.1.18) is a cytosolic enzyme found in all eukaryotes. This gene is always detected in stressed plants, such as heavy metal and salt-treated rice seedlings (Moons 2003), water-stressed maize seedlings (Zheng et al. 2004), drought-stressed horse gram (Chandra Obul Reddy et al. 2008) and fungus-elicited rice seedlings (Xiong et al. 2001). Therefore, and not surprisingly, the expression of GST was observed in normal kesum roots because the in vitro culture itself was a stress condition for kesum plantlets. It was strongly up-regulated in JAtreated roots as a defense mechanism, suggesting an overlap in the plant responses of plants to various stress factors. Also, GST catalyzes the conjugation between synthetic electrophilic compounds and the glutathione tripeptide (c-glutamyl-cysteinyl-glycine, GSH). The polar Sglutathionylated product will be actively transported into the vacuole by an ATP-binding cassette. Thus, GST is part of the detoxification mechanism in plants. In fact, it is the main ingredient for a variety of commercial herbicides (Andrews et al. 2005). Therefore, it is crucial to investigate kesum as a potential plant for phytoremediation purposes. ELI3-1 is another gene related to the abiotic stress caused by JA elicitation that will lead to phytoalexin and pathogenesis-related protein accumulation, phenolic compound production, and cell wall reconstruction. Seventeen of the ELI genes were identified in parsley cells cultured and treated with the Phytophthora megasperma fungus (Trezzini et al. 1993). In addition, MeJA elicitation has successfully activated ELI3, TyrDC, HRGP, and BMT genes in parsley (Ellard-Ivey and Douglas 1996). This observation proved that the expression of ELI3-1 could also be induced by JA elicitation. Peroxidase (POD, EC 1.11.1.7) plays an important role in the oxidation process, such as in peroxidative ooxidative oxidation and catalytic hydrosilylation (Umaya and Kobayashi 2003; Veitch 2004). It is an enzyme that catalyzes the oxidation of phenylpropanoids (Thimmaraju et al. 2006). It also functions in the plant-defense system, and it could be triggered by an elicitor (Go´mez-Va´squez et al. 2004; Perera and Jones 2004). The clones that were similar to POD in this study showed a type 2 expression pattern in the RT-PCR analysis. Its expression has been shown to induce the production of terpenoids in cucumbers infected by spider mites via oxidative degradation (Mercke et al. 2004). Our observation corroborates the results reported in kesum (Karim 1987) and *P. odoratum* (Du˜ng et al. 1995; Hunter et al. 1997), where various sesquiterpene hydrocarbons and their oxygenated compounds were identified. Therefore, it was predicted that stress stimuli, such as JA, could regulate the induction of important classes of plant secondary metabolites in kesum. In addition, its oxidative reaction showed that POD can be used as a component in the reagent for clinical diagnosis and various laboratory experiments (Thimmaraju et al. 2006) and thus increases

Interestingly, the kelch-repeat containing F-box family protein and the TIR1 protein that is contained in the F-box found in this subtractive cDNA library are induced by JA (Craig and Tyers 1999; Parry and Estelle 2006). The kelchrepeat containing F-box family protein is involved in the protein–protein interaction in the ubiquitin protein degradation process via the ubiquitin-mediated pathway. The protein degradation process is important to regulate

**3.4.2 Genes related to abiotic stress** 

the value of kesum.

**3.4.3 Transcription factor activated by JA** 

Our results showed that there is a close relationship between abiotic stress and the expression of genes involved in the biosynthesis of secondary metabolites. The subtractive cDNA library data set presented here provides the first collection of a set of JA responsive genes that may be involved in the secondary metabolite production in *P. minus* roots and also those participating in plant-defense mechanisms. These genes include dehydrogenase, lipoxygenase, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase, glutathione S-transferase, peroxidase, ELI3-1, and a transcription factor, F-box family protein. Identification of genes associated with flavour volatiles and the production of other aromatic compounds will provide a better understanding of the secondary metabolite biosynthetic pathways and their regulation in *P. minus*. Furthermore, the observed stressrelated genes induced by JA elicitation indicate that plants respond to abiotic stresses in parallel with the biosynthesis of certain secondary metabolites. Characterization of these genes will be studied in details using *E. coli* expression system. Besides, their functions could be explored with GC or HPLC to confirm the synthesis of the corresponding compounds whereas their ability to cope with abiotic stress could be performed by culturing *P. minus* plantlets in various stress conditions. In this study, we concluded that the JAresponsive genes might be the genes associated with volatile compounds production as a defence response against abiotic stress.

### **5. Acknowledgement**

We would like to thanks to the the Ministry of Higher Education of Malaysia (MOHE) for funding the project under the Fundamental Research Grant Scheme (FRGS) awarded to Ismanizan Ismail. Our gratitude also goes to the Ministry of Science, Technology and Innovation of Malaysia for National Science Foundation Fellowship (NSF) awarded to Miss Gor Mian Chee.

### **6. References**


Alteration of Abiotic Stress Responsive Genes

Plant Sci 2002 , 162:59-77

477.

107.

3397-3405.

*Biology* 72: 299-328.

Society of Plant Biologists.

tags. *Plant Science* 170: 356-363.

England: John Wiley & Sons, LTD.

*Academy of Sciences USA* 93(12): 6025-6030.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 81

Chen, H. & Chen, F. 2000. Effects of yeast elicitor on the growth and secondary metabolism

Chesnokov Y, Meister A, and Manteuffel R (2002) A chimeric green fluorescent protein gene

Chong, T. M., Abdullah, M. A., Fadzillah, N. M., Lai, O. M. & Lajis, N. H. 2005. Jasmonic

Chong, T. M., Abdullah, M. A., Lai, O. M., Fadzillah, M. N. & Lajis, N. H. 2005. Effective

Craig, K. L. & Tyers, M. 1999. The F-box: a new motif for ubiquitin dependent proteolysis in

Creelman, R. A., Tierney, M. L. & Mullet, J. E. 1992. Jasmonic acid/ methyl jasmonate

Datta, B. K., Datta, S. K., Rashid, M. A. & Sarker, S. D. 2002. Flavonoids from *Polygnoum stagninum* (Polygonaceae). *Biochemical Systematics and Ecology* 30: 693-696. Degenhardt, J. & Gershenzon, J. 2000. Demonstration and characterization of (*E*)-nerolidol

Devitt, L. C., Sambridge, T., Holton, T. A., Mitchelson, K. & Dietzgen, R. G. 2006. Discovery

Devos, D. and Valencia, A. 2000. Practical limits of function prediction. *Proteins.* 41(1): 98-

Dewick, P. M. 2001. *Medicinal Natural Products. A Biosynthetic Approach*. Ed. ke-2. hlm. 184.

Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B.,

Dixon, J. & Hewett, E. 2000. Factors affecting apple aroma/flavour volatile concentration: a review. *New Zealand Journal of Crop and Horticultural Science* 28: 155-173. Dornenburg, H. & Knorr, D. 1996. Production of the phenolic flavour compounds with cultured cells and tissue of *Vanilla planifolia* species. *Food Biotechnology* 10: 75-92. Drawert, F., Berger, R. G. & Godelmann, R. 1984. Regioselective biotransformation of valencene in cell suspension cultures of *Citrus* spp. *Plant Cell Reports* 3: 37-40.

Dixon, R. A. 2001. Natural products and plant disease resistance. *Nature* 411: 843-847.

4,8-dimethyl-1,3,7-nonatriene biosynthesis. *Planta* 210: 815-822.

suspension culture. *Process Biochemistry* 35: 837 – 840.

of a high-tanshinone-producing line of the Ti transformed *Salvia miltiorrhiza* cells in

as an embryonic marker in transgenic cell culture of *Nicotiana plumbaginifolia* Viv.

acid elicitation of anthraquinones with some associated enzymic and non-enzymic antioxidant responses in *Morinda elliptica*. *Enzyme and Microbial Technology* 36: 469-

elicitation factors in *Morinda elliptica* cell suspension culture. *Process Biochemistry* 40:

cell cycle regulation and signal transduction. *Progress in Biophysics and Molecular* 

accumulate in wounded soybean hypocotyls and modulate wound-induced gene expression. *Proceedings of the National Acaddemy of Sciences USA* 89: 4938-4941. Creelman, R. A. & Mulpuri, R. 2002. The oxylipin pathway in arabidopsis. Dlm: Somerville,

C. & Meyerozitz, E. M. (pnyt.). *The Arabidopsis Book*, hlm. 1-24. America: American

synthase from maize: a herbivore-inducible terpene synthase participating in (*3E*)-

of genes associated with fruit ripening in *Carica papaya* using expressed sequence

Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D. & Siebert, P. D. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. *Proceedings of the National* 


Andrews, C. J., Cummins, I., Skipsey, M., Grundy, N. M., Jepson, I., Townson, J. & Edwards,

Apostol, L., Heinstein, P. F. & Low, P. S. 1989. Rapid stimulation of an oxidative burst during elicitation of cultures plant cells. *Plant Physiology* 190: 109-116. Aravind L and Koonin EV (1999) Gleaning non-trivial structural, functional and

Armero, J. & Tena, M. 2001. Possible role of plasma membrane H+-ATPase in the elicitation

Bagos, P.G., Tsirigos, K.D., Liakopoulos, T.D. and Hamodrakas, S.J. 2008. Prediction of

Balandrin, M.J. & Klocke, J.A. 1988. Medicinal, aromatic and industrial materials from

Baldwin, E. A., Scott, J. W., Shewmaker, C. K. & Schuch, W. 2000. Flavor trivia and tomato

Barz, W., Daniel, S., Hinderer, W., Jaques, U., Kessmann, H., Koster, J. & Tiemann, K. 1988.

Bassüner R, Bäumlein H, Huth A, Jung R, Wobus U, Rapoport TA, Saalbach G, Müntz K

Bendtsen, J.D., Nielsen, H., von Heijne, G. and Brunak, S. 2004. Improved prediction of

Boscaiu, M., Lull, C., Lidon, A., Bautista, I., Donat, P., Mayoral, O. & Vicente, O. 2008. Plant responses to abiotic stress in their natural habitats. *Horticulture*. 65(1): 53-58. Bourgaud, F., Gravot, A., Milesi, S. & Gontier, E. 2001. Review - Production of plant secondary metabolites: a historical perspective. *Plant Science* 161: 839-851. Boutilier, K.A., Gines, M.J., DeMoor, J.M., Huang, B., Baszczynski, C.L., Iyer, V.N. and Miki,

*and Aromatic Plant*. Jil. 4. hlm. 1-36. Berlin: Springer-Verlag.

compounds. *Horticultural Science* 35(6): 1013-1022.

product. Plant Mol Biol 1998 , 11:321-334.

signal peptides: SignalP 3.0. *J Mol Biol.* 340(4): 783-795.

Brenner S E (1999) Errors in genome annotation. Trends Genet, 15:132-133.

*Physiology* 82: 205-219.

Biol., 287, 1023–1040.

Springer-Verlag.

180.

seedling. *Plant Science* 161: 791-798.

Model. *J Proteome Res.* 7(12): 5082-5093.

R. 2005. Purification and characterisation of a family of glutathione transferases with roles in herbiside detoxification in soybean (*Glycine max* L.); selective enhancement by herbicides and herbicide safeners. *Pesticide Biochemistry and* 

evolutionary information about proteins by iterative database searches. J. Mol.

of phytoalexin and related isoflavone root secretion in chickpea (*Cicer arietinum* L.)

lipoprotein signal peptides in Gram-positive bacteria with a Hidden Markov

plants. Dlm: Bajai, Y. P. S. (pnyt.). *Biotechnology in Agriculture and Forestry. Medicinal* 

aroma: biochemistry and possible mechanism for control of important aroma

Elicitation and metabolism of phytoalexins in plant cell cultures. Dlm: Pais, M., Mavituna, F. & Novais, J. (pnyt.). *Plant Cell Biotechnology*, hlm. 211-230. Berlin:

(1998) Abundant embryonic mRNA in field bean(*Vicia faba L*.) codes for a new class of seed proteins: cDNA cloning and characterization of the primary translation

B.L. 1994. Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis. *Plant Mol Biol.* 26(6): 1711-1723. Bouwmeester, H. J., Verstappen, F. W., Posthumus, M. A. & Dicke, M. 1999. Spider mite-

induced (3S)-(E)-nerolidol synthase activity in cucumber and lima bean. The first dedicated step in acyclic C11-homoterpene biosynthesis. *Plant Physiology* 121: 173-

Chae, Y. A., Yu, H. S., Song, J. S, Chun, H. K. & Park, S. U. 2000. Indigo production in hairy root cultures of *Polygonum tinctorium* Lour. *Biotechnology Letters* 22: 1527-1530.


Alteration of Abiotic Stress Responsive Genes

Systems Biology. 3(2):50-51.

604.

1898.

12: 27-30.

*Microbiology and Biotechnology* 76: 753-760.

*Naltional Academy of Sciences USA* 89: 4713-4717.

emissions in nature. *Science* 291: 2141-2144.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 83

Hoskeri, JH, Krishna, V & Amruthavalli C (2010) Functional annotation of conserved

Huang, X. W., Li, Y. X., Niu, Q. H. & Zhang, K. Q. 2007. Suppression subtractive

Hunter, M. V., Brophy, J. J., Ralph, B. J. & Elenvenu, F. E. 1997. Composition of *Polygonum* 

Ikai, A. 1980. Thermostability and aliphatic index of globular proteins. *J Biochem.* 88(6): 1895-

Ingram, J . & Bartels, D. 1996. The molecular basis of dehydration tolerance in plants. *Annual* 

Judpentienë, A. & Mockutë, D. 2004. Chemical composition of essential oils of *Artemisia absinthium* L. (wormwood) growing wild in Vilnius. *CHEMIJA*. 15(4): 64-68. Karim, B. Y. 1987. Kesom oil- A natural source of aliphatic aldehydes. *Perfumer and Flavorist* 

Kawalleck, P., Plesch, G., Hahlbrock, K. & Somssich, I. E. 1992. Induction by fungal elicitor

Kessler, A. & Baldwin, I. T. 2001. Defensive function of herbivore-induced plant volatile

Kolker, E., Makarova, K.S., Shabalina, S., Picone, A.F., Purvine, S., Holzman, T., Cherny, T.,

Korkina, L.G. 2007. Phenylpropanoids as naturally occurring antioxidants: From plant defense to human health. Cellular and Molecular Biology. 53(1): 15-25. Kubigsteltig, I., Laudert, D. & Weiler, E. W. 1999. Structure and regulation of the *Arabidopsis* 

Kutchan, T. M. 1995. Alkaloid Biosynthesis – The basis for metabolic engineering of

Kyte, J. and Doolittle, R.F. 1982. A simple method for displaying the hydropathic character

Longo, M. A. & Sanroman, M. A. 2006. Production of food aroma compounds: microbial and enzymatic methodologies. *Food Technology and Biotechnology* 44: 335-353. Low, P. S. & Merida, J. R. 1996. The oxidative burst in plant defense: function and signal

Lubec, G., Afjehi-Sadat, L., Yang, J.W. and John, J.P. 2005. Searching for hypothetical

Machida, T., Murase, H., Kato, E., Honjoh, K. I., Matsumoto, K., Miyamoto, T. & Iio, M. 2008.

suppression subtractive hybridization. *Plant Science* 175: 238-246.

proteins: theory and practice based upon original data and literature. *Prog* 

Isolation of cDNAs for hardening-induced genes from *Chlorella vulgaris* by

Haemophilus influenzae. *Nucleic Acids Res.* 32(8): 2353-2361.

*thaliana* allene oxide synthase gene. *Planta* 208: 463-471.

medicinal plants. *The Plant Cell* 7(7): 1059-1070.

transduction. *Physiologia Plantarum* 96: 533-542.

of a protein. *J Mol Biol.* 157(1): 105-132.

*Neurobiol.* 77(1-2): 90-127.

of *S-*adenosil-L-methionine synthetase and *S*-adenosyl-L-homocysteine hydrolase mRNAs in cultured cells and leaves of *Petroselinum crispum*. *Proceedings of the* 

Armbruster, D., Munson, R.S., Jr., Kolesov, G., Frishman, D. and Galperin, M.Y. 2004. Identification and functional analysis of 'hypothetical' genes expressed in

*Review of Plant Physiology and Plant Molecular Biology* 47: 377-403.

hypothetical proteins in *Rickettsia massiliae* MTU5. Journal of Computer Science &

hybridization (SSH) and its modifications in microbiological research. *Applied* 

*odoratum* Lour. From Southern Australia. *Journal of Essential Oil Research* 9(5): 603-


Dung, N. X., Hac, L. V. & Leclercq, P. A. 1995. Volatile constituents of the aerial parts of Vietnamese *Polygonum odoratum* L. *Journal of Essential Oil Research* 7: 339-340. Ellard-Ivey, M. & Douglas, C. J. 1996. Role of jasmonate in the elicitor- and wound-inducible

Feussner, I & Wasternack, C. 2002. The lipoxygenase pathway. *Annual Review of Plant Biology* 

Fowler, M. W. & Scragg, A. H. 1988. Natural products from higher plants and plant cell

Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, K. &

Gadzovska, S., Maury, S., Delaunay, A., Spasenoski, M., Joseph, C. & Hagege, D. 2007.

Galperin, M.Y. 2001. Conserved 'hypothetical' proteins: new hints and new puzzles. *Comp* 

Galperin, M.Y. and Koonin, E.V. 2004. 'Conserved hypothetical' proteins: prioritization of

Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D. and Bairoch, A. 2003.

Gelli, A., Higgins, V. J. & Blumwald, E. 1997. Activation of plant membrane Ca2+ permeable channels by race-specific fungal elicitors. *Plant Physiology* 113: 269-279. Gleba, D., Borisjuk, N.V., Borisjuk, L.G., Kneer, R., Poulev, A., Skarzhinskaya, M.,

Gor MC, Ismail I, Wan-Mustapha WA, Zainal Z, Mohd-Noor N, Othman R, Mohamed-

Gundlach, H., Muller, M. J., Kutchan, T. M. & Zenk, M. H. 1992. Jasmonic acid is a signal

Hattori J, Boutilier KA, van Lookeren Campagne MM, Miki BL (1998) A conserved BURP

Hirokawa, T., Boon-Chieng, S. and Mitaku, S. 1998. SOSUI: classification and secondary structure prediction system for membrane proteins. *Bioinformatics.* 14(4): 378-379.

targets for experimental study. *Nucleic Acids Res.* 32(18): 5452-5463.

*biotechnology*. Jil. 18. hlm. 165-177. Berlin: Springer-Verlag.

*Opinion in Plant Biology*. 9: 436-442.

*Nucleic Acids Res.* 31(13): 3784-3788.

Plant 2010, DOI 10.1007/s11738-010-0546-2.

*of Sciences USA* 89: 2389-2393.

Mol Gen Genet 1998;259:424-428

*Organ Culture* 89: 1-13.

94(1): 87-97.

*Funct Genomics.* 2(1): 14-18.

183-192.

6: 372-378.

expression of defense genes in parsley and transgenic tobacco. *Plant Physiology* 112:

culture. Dlm: Pais, M.S.S., Mavituna, F. & Novais, J.M. (pnyt.). *Plant cell* 

Shinozaki, K. 2006. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signalling networks. *Current* 

Jasmonic acid elicitation of *Hypericum perforatum* L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones. *Plant Cell Tissue and* 

ExPASy: The proteomics server for in-depth protein knowledge and analysis.

Dushenkov, S., Logendra, S., Gleba & Y.Y., Raskin, I. 1999. Use of plant roots for phytoremediation and molecular farming. Proc Natl Acad Sci USA 96:5973–5977 Gόmez-Vásquez, R., Day, R., Bushmann, H., Randles, S., Beeching, J. R. & Cooper, R. M.

2004. Phenylpropanoids, phenylalanine ammonia lyase and peroxidases in elicitorchallenged cassava (*Manihot esculenta*) suspension cells and leaves. *Annals of Botany* 

Hussein ZA (2010) Identification of cDNAs for jasmonic acid-responsive genes in *Polygonum minus* roots by suppression subtractive Hybridization. Acta Physiol

transducer in elicitor-induced plant cell cultures. *Proceedings of the National Academy* 

domain defines a novel group of plant proteins with unusual primary structures.


Alteration of Abiotic Stress Responsive Genes

Oxford: Clarendon.

24(11):2377-2382.

*Sciences USA* 95: 7805-7812.

*Biotechnology* 8: 154-159.

*Biotechnology* 4: 407-414.

*Today* 13: 161-171.

plants. *Biotechnologia Aplicada*. 22: 1-10.

*Zhejiang University Science B* 6(2): 91-95.

*Experimentalis et Applicata* 111(3): 165-171.

*of Medicinal Chemistry* 45: 2542-2547.

*medicinal plants*. Jil. 29. hlm. 19-45. New York: Plenum.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 85

Peng, H. Y. & Yang, X. E. 2004. Volatile constituents in flowers of *Elsholtzia argyi* and their

Perera, M. R. & Jones, M. G. K. 2004. Expression of the peroxidase gene promoter (*Shpx6b*)

Pezzuto, J. M. 1995. Natural product cancer chemoprotective agents. Dlm: Arnason, J.T.,

Phillipson, J. D. 1990. Plants as source of valuable products. Dlm: Charlwood, B. V. &

Poulev, A., O'Neal, J. M., Logendra, S., Pouleva, R. B., Timeva, V., Garvey, A. S., Gleba, D.,

Rahman O, Cummings SP, Harrington DJ and Sutcliffe IC (2008) Methods for the

Ramachandra Rao, S. & Ravishankar, G. A. 2002. Plant cell cultures: Chemical factories of

Ravanel, S., Gakiere, B., Job, D. & Douce, R. 1998. The specific features of methionine

Reddy, C. O. P.,Sairanganayakulu, G., Thippeswamy, M., Sudhakar Reddy, P., Reddy, M. K.

Rodriguez, M., Canales, E. & Borrás-Hidalgo, O. 2005. Molecular aspect of abiotic stress in

Romeis, T. 2001. Protein kinases in the plant defense response. *Current Opinion in Plant* 

Saklani, A. & Kutty, S. K. 2008. Plant-derived compounds in clinical trials. *Drug Discovery* 

Sanchez-Sampedro, M. A., Fernandez-Tarrago, J. & Corchete, P. 2005. Yeast extract and

Sandermann, H., Ernst, D., Heller, W. & Langebartels, C. 1998. Ozone: An abiotic elicitor of

Schuurink, R. C., Haring, M. A. & Clark, D. G. 2006. Regulation of volatile benzenoid

biosynthesis in petunia flowers. *Trends in Plant Science* 11(1): 20-25.

*marianum* (L.) Gaertn. *Journal of Biotechnology* 119: 60-69.

plant defence reactions. *Trends in Plant Sci*ence 3: 47-50.

methyl jasmonate-induced silymarin production in cell cultures of *Silybum* 

secondary metabolites. *Biotechnology Advances* 20: 101-153.

variation: a possible utilization of plant resources after phytoremediation. *Journal of* 

from *Stylosanthes humulis* in transgenic plants during insect attack. *Entomologia* 

Mata, R. & Romeo, J. T. (pnyt.). *Recent advances in phytochemistry. Phytochemistry of* 

Rhodes, M. J. C. (pnyt.). *Secondary products from plant tissue culture*, hlm. 1-21.

Jenkins, I. S., Halpern, B. T., Kneer, R., Cragg, G. M. & Raskin, I. 2003. Elicitation, a new window into plant chemodiversity and phytochemical drug discovery. *Journal* 

bioinformatic identification of bacterial lipoproteins encoded in the genomes of Gram-positive bacteria. World Journal of Microbiology and Biotechnology

biosynthesis and metabolism in plants. *Proceedings of the National Academy of* 

& Chinta Sudhakar. 2008. Identification of stress-induced genes from the drought tolerant semi-arid legume crop horsegram (*Macrotyloma uniflorum* (Lam.) Verdc.) through analysis of subtracted expressed sequence tags. *Plant Science* 175: 372-384. Roberts, S. C. & Shuler, M. L. 1997. Large scale plant cell culture. *Current opinion in* 


Marcotte, E.M., Pellegrini, M., Thompson, M.J., Yeates, T.O. and Eisenberg, D. 1999. A

Matsuda, H., Shimoda, H., Morikawa, T & Yoshikawa, M. 2001. Phytoestrogens from the

Mazumder, R., Vasudevan, S. and Nikolskaya, A.N. 2008. Protein functional annotation by

Mellor JC, Yanai I, Clodfelter KH, Mintseris J, DeLisi C, 2002. Predictome: a database of putative functional links between proteins. Nucleic Acids Res. 30, 306–309. Menke, F. L. H., Parchmann, S., Mueller, M. J., Kijne, J. W. & Memelink, J. 1999. Involvement

Mercke, P., Kappers, I. F., Verstappen, F. W. A., Vorst, O., Dicke, M. & Bouwmeester, H. J.

Miersh, O. & Wasternack, C. 2000. Octadecanoid and jasmonate signalling in tomato

Moons, A. 2003. *Osgstu3* and *Osgstu4*, encoding tau class glutathione S-transferase are heavy

Nakao, M., Ono, K. & Takio, S. 1999. The effect of calcium on flavonol production in cell suspension culture of *Polygonum hydropiper*. *Plant Cell Reports* 18: 107-111. Namdeo, A. G. 2007. Plant cell elicitation for production of secondary metabolites: A review.

Oksman-Caldenteyl, K. M. & Inze, D. 2004. Plant cell factories in the post-genomic era: new

Őzcan, M. M. & Chalchat, J. C. 2007. Chemical composition of carrot seeds (*Daucus carota* L.)

Palazon, J. et al. 2003. Inhibition of paclitaxel and baccatin III accumulation by mevinolin

Paré, P. W. & Tumlinson, J. H. 1997. De novo biosynthesis of volatiles induced by insect

Parry, G. & Estelle, M. 2006. Auxin receptors: a new role for F-box proteins. *Current Opinion* 

Pauwels, L., Inze, D. & Goossens, A. 2009. Jasmonate-inducible gene: what does it mean?

Peng, Z. F., Strack, D., Baumert, A., Subramaniam, R., Goh, N. K., Chia, T. F., Tan, S. N. &

Chia, L. S. 2003. Antioxidant flavonoids from leaves of *Polyognum hydropiper* L.

herbivory in cotton plants. *Plant Physiology* 114: 1161-1167.

jasmonate biosynthesis. *Biological Chemistry* 381: 715-722.

402(6757): 83-86.

*Letters* 11: 1839-1842.

homology. *Methods Mol Biol.* 484(465-490.

*roseus*. *Plant Physiology* 119: 1289-1296.

roots. *FEBS Letters* 553: 427-432.

*Pharmacognosy Reviews* 1 (1): 69-79.

*Aceites* 58 (4): 359-365.

*in Cell Biology* 18: 152-156.

*Phytochemistry* 62: 219-228.

*Trends in Plant Science* 14(2): 87-91.

101: 157-163.

440.

combined algorithm for genome-wide prediction of protein function. *Nature.*

roots of *Polygonum cuspidatum* (Polygonaceae): Structure-requirement of hydroxyanthraquinones for estrogenic activity. *Bioorganic and Medicinal Chemistry* 

of the octadecanoid pathway and protein phosphorylation in fungal elicitorinduced expression of terpenoid indole alkaloid biosynthetic genes in *Catharanthus* 

2004. Combined transcript and metabolite analysis reveals genes involved in spider mite induced volatile formation in cucumber plants. *Plant Physiology* 135: 2012-2024.

(*Lycopersicon esculentum* Mill.) leaves: endogenous jasmonates do not induce

metal- and hypoxic stress-induced and differentially salt stress-responsive in rice

ways to produce designer secondary metabolites. *Trends in Plant Science* 9(9): 433-

cultivated in Turkey: characterization of the seed oil and essential oil. *Grasas y* 

and fosmidomycin in suspension cultures of *Taxus baccata*. *Journal of Biotechnology* 


Alteration of Abiotic Stress Responsive Genes

*Phytochemistry* 60: 289-293.

*and Biotechnology* 55: 404 – 410.

Oxford: Blackwell Publishing Ltd.

ripening. *Plant Cell.* 6(11): 1623-1634.

*Molecular Plant-Microbe Interaction* 14(5): 685-692.

31(1): 258-261.

17-25.

*Physiology* 38(7): 776-782.

*Journal of Biology* 25: 351-360.

in *Polygonum minus* Roots by Jasmonic Acid Elicitation 87

Von Mering, C., Huynen, M., Jaeggi, D., Schmidt, S., Bork, P. and Snel, B. 2003. STRING: a

Walker, J. C., Howard, E. A., Dennis, E. S. & Peacock, W. J. 1987. DNA sequences required

Walker, T. S., Bais, H. P. & Vivanco, J. M. 2002. Jasmonic acid-induced hypericin production

Wan Hassan, W. E. 2007. Healing Herbs of Malaysia. Cetak ulang. Kuala Lumpur: Federal

Wang, C., Wu, J. & Mei, X. 2001. Enhancement of Taxol production and excretion in *Taxus* 

Wasternack, C. 2006. Oxilipins: biosynthesis, signal transduction and action. Dlm: Hedden,

Wasternack, C. 2007. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress responses, growth and development. *Annals of Botany* 100: 681-697. Watson, C.F., Zheng, L. and DellaPenna, D. 1994. Reduction of tomato polygalacturonase

Xiong, L. Z., Lee, M. W., Qi, M. & Yang, Y. N. 2001. Identification of defense-related rice

Yamaguchi-Shinozaki, K. and Shinozaki, K. 1993. The plant hormone abscisic acid mediates

Yazaki, K., Takade, K. & Tabata, M. 1997. Effects of methyl jasmonate on shikonin and

Yilmaz, E. 2000. Oxylipin pathway in the biosynthesis of fresh tomato volatiles. *Turkish* 

Yu, N.Y., Wagner, J.R., Laird, M.R., Melli, G., Rey, S., Lo, R., Dao, P., Sahinalp, S.C., Ester,

Yu, R. M., Ma, N., Yan, C. Y. & Zhao, Y. 2006. Effects of exogenous phytohormones on hairy

Zdobnov, E.M. and Apweiler, R. 2001. InterProScan--an integration platform for the signature-recognition methods in InterPro. *Bioinformatics.* 17(9): 847-848. Zhao, J. et al. 2005. Elicitor signal transduction leading to production of plant secondary

capabilities for all prokaryotes. *Bioinformatics.* 26(13): 1608-1615.

hairy root cultures. *Chinese Journal of Biotechnology* 22(4): 619-623.

metabolites. *Biotechnology Advances* 23: 283-333.

*the National Academy of Science USA* 84: 6624-6628.

Land Development Authority (FELDA).

database of predicted functional associations between proteins. *Nucleic Acids Res.*

for anaerobic expression of the maize alcohol dehydrogenase 1 gene. *Proceedings of* 

in cell suspencion cultures of *Hypericum perforatum* L. (St. John's wort).

*chinensis* cell culture by fungal elicitation and medium renewal. *Applied Microbiology* 

P. & Thomas, S. (pnyt.). *Plant hormone signaling*. *Annual Plant Reviews*. hlm. 185-228.

beta subunit expression affects pectin solubilization and degradation during fruit

genes by suppression subtractive hybridization and differential screening.

the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. *Mol Gen Genet.* 238(1-2):

dihydroechinofuran production in *Lithospermum* cell cultures. *Plant and Cell* 

M., Foster, L.J. and Brinkman, F.S. 2010. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive

root growth of *Polygonum multiflorum* and biosynthesis of anthraquinones in its


Sharon, M. et al. 1998. Effects of methyl jasmonate and elicitor on the activation of

Song, W. C., Funk, C. D. & Brash, K. A. R. 1993. Molecular cloning of an allene oxide

Strasser, R., Stadlmann, J., Svoboda, B., Altmann, F., Glossl, J. and Mach, L. 2005. Molecular

Sun, Y. J., Zhou, X. F. & Ma, H. 2007. Genome-wide analysis of kelch repeat-containing F-

Teerawanichpan Prapapan, Xia Qun, Caldwell SarahJ, Datla Raju, Selvaraj Gopalan (2009)

Thimmaraju, R., Bhagyalakshmi, N., Venkatachalam, L., Sreedhar, R. V. & Ravishankar, G.

Todd, A.E., Orengo, C.A. and Thornton, J.M. 2001. Evolution of function in protein superfamilies, from a structural perspective. *J Mol Biol.* 307(4): 1113-1143. Torregrosa, L., Pradal, M., Souquet, J. M., Rambert, M., Gunata, Z. & Tesniere, C. 2008.

Townsley, P. M. 1972. Chocolate from plant cells. *Journal of the Institute of Canadian Science* 

Treacy, B.K., Hattori, J., Prud'homme, I., Barbour, E., Boutilier, K., Baszczynski, C.L., Huang,

Trezzini, G. F., Horrichs, A. & Somssich, I. E. 1993. Isolation of putative defense-related

Tusnady, G.E. and Simon, I. 2001. The HMMTOP transmembrane topology prediction

Urones, J. G., Marcos, I. S., Perez, B. G. & Barcala, P. B. 1990. Flavonoids from *Polygonum* 

Vanisree, M., Lee, C. Y., Lo, S. F., Nalawade, S. M., Lin, C. Y. & Tsay H. S. 2004. Studies on

Vanisree, M. & Tsay, H. S. 2004. Plant cell cultures – An alternatie and efficient source for

Vazquez-Flota, A. & De Luca, V. 1998. Jasmonate modulates development and lightregulated alkaloid biosynthesis in *Catharanthus roseus*. *Phytochemistry* 49: 395-402.

plant tissue cultures. *Botanical Bulletin of Academia Sinica* 45: 1-22.

tobacco cell cultures. *Plant Science* 123: 13-19.

2009 , 71:331-343.

*Plant Science* 174: 149-155.

*Mol Biol.* 34(4): 603-611.

*Molecular Biology* 21: 385-389.

server. *Bioinformatics.* 17(9): 849-850.

*minus. Phytochemistry* 29(11): 3687-3689.

*of Applied Science and Engineering* 2(1): 29-48.

*and Technology Aliment* 7: 76-78.

Sticher, O. 1998. Getting to the root of ginseng. *Chemical Technology* 28: 26-32.

box family. *Journal of Integrative Plant Biology* 49(6): 940-952.

*Beta vulgaris* L. *Electronic Journal of Biotechnology* 9(5): 512-520.

plants lacking complex N-glycans. *Biochem J.* 387(Pt 2): 385-391.

phenylalanine ammonia lyse and the accumulation of scopoletin and scopolin in

synthase. A cytochrome P-450 specialized for metabolism of fatty acid hydroperoxides. *Proceedings of the National Academy of Sciences USA* 98: 8519-8523.

basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana

Protein storage vacuoles of *Brassica napus* zyotic embryos accumulate a BURP domain protein and perturbation of its production distorts the PSV. Plant Mol Biol

A. 2006. Elicitation of peroxidase activity in genetically transformed root cultures of

Manipulation of *VvAdh* to investigate its function in grape berry development.

B., Johnson, D.A. and Miki, B.L. 1997. Bnm1, a Brassica pollen-specific gene. *Plant* 

genes from *Arabisopsis thaliana* and expression in fungal elicitor-treated cells. *Plant* 

the production of some important secondary metabolites from medicinal plants by

the production of biologically important secondary metabolites. *International Journal* 


**4** 

*Brazil* 

**Transcriptomics of Sugarcane** 

**Osmoprotectants Under Drought** 

WL Burnquist2, AM Benko-Iseppon1 and EA Kido1

*2Center of Sugarcane Technology (CTC)*

RLO Silva1, JRC Ferreira Neto1, V Pandolfi1, SM Chabregas2,

Sugarcane (*Saccharum* spp.) is an alogamous plant from the Poaceae family and the Andropogoneae tribe (Daniels & Roach, 1987). This crop covers more than 23 million hectares worldwide, representing about 0.5 % of the total global area used for agriculture, with a production of 1.6 billion metric tons of crushable stems (FAOSTAT, 2009). Brazil is the world's largest producer, contributing with two-thirds of total sugar production - about 31 million tons per year - of which 19.5 million tons are exported (UNICA, 2009). Sugarcane, its derivatives and by products have received great attention, due to their multiple uses, with emphasis on the ethanol production, representing an important renewable biofuel source. It has been estimated that sugarcane ethanol fuel may replace up to 10.0 % of the world's refined petroleum products consumption in the next 15 to 20 years (Goldemberg, 2007). Despite its importance and similarity to other important agronomic crops, the sugarcane production has been adversely influenced by many environmental factors such as

Abiotic stresses are among the main causes of losses in the productivity of the major crops worldwide (Bray *et al*., 2000), a scenario where drought figures as the most significant stress, causing negative impacts on crop adaptation and productivity. Besides, this condition can exacerbate the effect of other stresses (biotic or abiotic) to which the plants may be submitted. Although breeding activities have provided significant progress for the understanding of the physiological and molecular responses of plants to water deficit, there is still a large gap between yields in optimal and stressful conditions (Cattivelli *et al*., 2008). Essays regarding plant responses to drought stress have been published applying technologies of functional genomics (Wang *et al.,* 2011). These evaluations provided important insights into molecular and biochemical mechanisms in the study of drought tolerance in various crops and model species. Plants are able to ''perceive'' the external stimuli by multiple sensors, recognizing adverse situations and invoking signal transduction cascades and consequently secondary messengers, activating stress responsive genes (Grennan, 2006), resulting in both molecular and physiological responses. Among the mechanisms developed by plants to face the adverse conditions generated under drought, the accumulation of osmoprotectants compounds are often recognized as a mitigation mechanism of the negative consequences of water deficit (Choluj *et al*., 2008).

**1. Introduction** 

harsh climate and soil conditions.

*1Federal University of Pernambuco (UFPE), Department of Genetics* 


### **Transcriptomics of Sugarcane Osmoprotectants Under Drought**

RLO Silva1, JRC Ferreira Neto1, V Pandolfi1, SM Chabregas2, WL Burnquist2, AM Benko-Iseppon1 and EA Kido1 *1Federal University of Pernambuco (UFPE), Department of Genetics 2Center of Sugarcane Technology (CTC) Brazil* 

### **1. Introduction**

88 Plants and Environment

Zheng L, Heupel RC, DellaPenna D (1992) The beta subunit of tomato fruit

Zheng, J., Zhao, J. F., Tao, Y. Z., Wang, J. H., Liu, Y. J., Fu, J. J., Jin, Y., Gao, P., Zhang, J. P.,

Zielinski, R. E. 1998. Calmodulin and calmodulin-binding proteins in plants. *Annual Review* 

unique structural features. Plant Cell 1992 , 4:1147-1156.

*of Plant Physiology and Plant Molecular Biology* 49: 697-725.

*Biology* 55: 807-823.

polygalacturonase isoenzyme 1: isolation, characterization, and identification of

Bai, Y. F. & Wang, G. Y. 2004. Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray. *Plant Molecular* 

> Sugarcane (*Saccharum* spp.) is an alogamous plant from the Poaceae family and the Andropogoneae tribe (Daniels & Roach, 1987). This crop covers more than 23 million hectares worldwide, representing about 0.5 % of the total global area used for agriculture, with a production of 1.6 billion metric tons of crushable stems (FAOSTAT, 2009). Brazil is the world's largest producer, contributing with two-thirds of total sugar production - about 31 million tons per year - of which 19.5 million tons are exported (UNICA, 2009). Sugarcane, its derivatives and by products have received great attention, due to their multiple uses, with emphasis on the ethanol production, representing an important renewable biofuel source. It has been estimated that sugarcane ethanol fuel may replace up to 10.0 % of the world's refined petroleum products consumption in the next 15 to 20 years (Goldemberg, 2007). Despite its importance and similarity to other important agronomic crops, the sugarcane production has been adversely influenced by many environmental factors such as harsh climate and soil conditions.

> Abiotic stresses are among the main causes of losses in the productivity of the major crops worldwide (Bray *et al*., 2000), a scenario where drought figures as the most significant stress, causing negative impacts on crop adaptation and productivity. Besides, this condition can exacerbate the effect of other stresses (biotic or abiotic) to which the plants may be submitted. Although breeding activities have provided significant progress for the understanding of the physiological and molecular responses of plants to water deficit, there is still a large gap between yields in optimal and stressful conditions (Cattivelli *et al*., 2008). Essays regarding plant responses to drought stress have been published applying technologies of functional genomics (Wang *et al.,* 2011). These evaluations provided important insights into molecular and biochemical mechanisms in the study of drought tolerance in various crops and model species. Plants are able to ''perceive'' the external stimuli by multiple sensors, recognizing adverse situations and invoking signal transduction cascades and consequently secondary messengers, activating stress responsive genes (Grennan, 2006), resulting in both molecular and physiological responses. Among the mechanisms developed by plants to face the adverse conditions generated under drought, the accumulation of osmoprotectants compounds are often recognized as a mitigation mechanism of the negative consequences of water deficit (Choluj *et al*., 2008).

Transcriptomics of Sugarcane Osmoprotectants Under Drought 91

profit from the high resolution power of SuperSAGE® coupled to the Illumina® sequencing in a SuperTag Digital Gene Expression (STDGE) profile (GenXPro GmbH, Frankfurt, Germany) trying to characterize the transcriptome of drought-stressed sugarcane roots after 24 hours of submission to this stress, aiming to elect a best group of tags to be validated by RTqPCR. For this purpose, a high-throughput transcriptome project as a joint Brazilian initiative from UFPE (Federal University of Pernambuco) and CTC (Sugarcane Technology Center) was carried out. The project generated a large amount of gene candidates from different important categories considering the response against this kind of stress. In the present chapter an overview regarding the identification, categorization and differential expression of osmoprotectants will be evaluated in sugarcane, compared with the up to date knowledge concerning this crop and related species. Considering its role in world's economy and biotechnological potential, the identification and expression profile of responsive osmoprotectant coding genes in sugarcane may be helpful to unravel the basic mechanisms of stress tolerance, bringing valuable evidences for sugarcane improvement.

Biological stress may be defined as an adverse environmental condition that inhibits the normal operation of a biological system such as plants (Jones & Jones, 1989). The life in the terrestrial condition represents a challenge to plants, and often have to occupy environments that are not the most appropriated for their development, being also subjected to frequent environmental changes in their native conditions. A large number of abiotic factors associated with plant-water relations – such as drought, salinity, chilling, frost and flooding – negatively affect the overall growth of terrestrial plants, leading to stunted form, metabolic changes, reduced yields, germination problems and even plant death under extreme conditions (Smith and Bhavel, 2007). Among the mentioned factors, drought stands out, bringing the most serious threat in view of the existing freshwater shortage in many regions of the world, bringing serious limitations to the agriculture

Notwithstanding the availability of more than 150 definitions for drought in the literature (Boken, 2005), it is often defined in terms of available humidity as compared with a normal value, with the severity correlating in function to the time and magnitude of the exposition to a deficient humidity (Smith & Pethley, 2009). Among the diverse types reported, some can be highlighted as meteorological, hydrological, socioeconomic and agricultural drought (Boken, 2005; Smith & Pethley, 2009). According to Boken (2005), the meteorological drought occurs when seasonal or annual precipitation falls below its long-term average; the hydrological drought when the meteorological drought is prolonged and causes local shortage of surface and groundwater; the socioeconomic drought is a manifestation of continued drought of severe intensity that shatters the economy and sociopolitical situation in a region, while the agricultural drought sets, due to soil moisture stress, a significant

The agricultural drought is the most important nowadays and will be here focused. Agriculture is by far the largest consumer of water, representing for 80.0 % of the freshwater consumption worldwide (Jury & Vaux, 2005). Economic losses associated to water availability reached about one billion dollars, in 2009, only in the United States (Anderson *et al*., 2009). Actually, one-third of the world population lives in areas with water shortages. This is specially serious considering other adverse factors such as the high levels of

**2. Drought: Understanding the problem** 

decline in crop yields (production per unit area).

(Jury & Vaux, 2005).

Osmoprotectants are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume (Yancey, 2001), and may play other roles regarding tolerance, as proteins stabilizing and antioxidant action (Rathinasabapathi, 2000). They include sugars, mainly fructose and sucrose, sugar alcohols (like myo-inositol), complex sugars (like trehalose and fructans) and charged metabolites (like glycinebetaine, proline and ectoine) (Yancey, 2005).

Previous information regarding sugarcane osmoprotectants under stress was reported. For example Suriyan & Chalermpol (2009) analyzed diverse parameters in sugarcane submitted to iso-osmotic salt and water-deficit stresses. Among the physiological alterations, an increase in the proline content in stressed-leaves was positively correlated to the reported stresses, indicating a key role of proline in osmoregulation and antioxidant defense mechanisms. Also Rasheed *et al*. (2010) investigated the possible roles of proline and glycine betaine (GB) in mitigating the effect of chilling stress in the sprouting nodal buds of sugarcane. To accomplish this evaluation, they performed a pre-treatment with proline bud chips and GB, obtaining a substantial reduction in the H2O2 production and an increase in the synthesis efficiency of soluble sugars, protecting developing tissues from the effects of chilling stress. Recent molecular research works, regarding drought and salinity performance in sugarcane, were carried out using techniques based on molecular hybridization such as SSH [Subtractive Hybridization Suppressive] (Patade *et al*., 2010) and micro/macroarrays (Rodrigues *et al*., 2009). In a general view, the main limitations of these methods regard their low sensibility and specificity (Shimkets, 2004). Moreover, in relation to micro/macroarrays, in spite of their high performance and broad use, the inability to analyze and discover new genes have been reported (Wang *et al*., 2009). Techniques based on sequencing [i.e. SAGE (Velculescu *et al*., 1995) and derivatives] take advantage of the available frequencies of fragments (tags) representing transcripts expressed in the sample, by the assumption that a short and defined tag contains the information needed to identify the corresponding cDNA (Velculescu *et al*., 1995). Thus, besides constituting an open architecture analysis (i.e., allowing the discovery of new genes), the abundance of tags found for a given gene provides an estimative of their transcription in the sample. In this context, the SuperSAGE technique (Matsumura *et al*., 2003) stands out for its efficiency in generating transcription profiles, especially with the actual association to the high performance sequencing platforms [Pyrosequencer (454 Roche®), Solexa (Illumina®) and SOLiD (Applied Biosystems®)].

The SuperSAGE method is characterized by the generation of 26 bp tags by using the type III restriction enzyme *Eco*P15I (Matsumura *et al*., 2003). This technique presents itself as one of the most modern tools of functional genomics and provides some advantages such as improvements in tag-to-gene annotation, simultaneous analysis of two interacting eukaryotic organisms, full-length cDNAs amplification using tags as primers, potential use of tags via RNA interference (RNAi) in gene function studies, identification of antisense and rare transcripts, and identification of transcripts with alternative splicing (Matsumura *et al*., 2006). It also provides a global and quantitative transcriptomic analysis based on the study of the entire transcriptome produced in a given time under a given stimulus. This technique has been successfully applied in plant species such as rice (Matsumura *et al*., 2003), banana (Coemans *et al*., 2005), chili pepper (Hamada *et al*., 2008), chickpea (Molina *et al.*, 2008; 2011), tobacco (Gilardoni *et al*., 2010) and tropical crops (cowpea, soybean, sugarcane; Kido *et al*., 2010). Some of them using the association of SuperSAGE with a high-throughput sequencing platform (HT-SuperSAGE; Matsumura *et al*., 2010). In the present work we

Osmoprotectants are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume (Yancey, 2001), and may play other roles regarding tolerance, as proteins stabilizing and antioxidant action (Rathinasabapathi, 2000). They include sugars, mainly fructose and sucrose, sugar alcohols (like myo-inositol), complex sugars (like trehalose and fructans) and charged metabolites (like glycinebetaine, proline

Previous information regarding sugarcane osmoprotectants under stress was reported. For example Suriyan & Chalermpol (2009) analyzed diverse parameters in sugarcane submitted to iso-osmotic salt and water-deficit stresses. Among the physiological alterations, an increase in the proline content in stressed-leaves was positively correlated to the reported stresses, indicating a key role of proline in osmoregulation and antioxidant defense mechanisms. Also Rasheed *et al*. (2010) investigated the possible roles of proline and glycine betaine (GB) in mitigating the effect of chilling stress in the sprouting nodal buds of sugarcane. To accomplish this evaluation, they performed a pre-treatment with proline bud chips and GB, obtaining a substantial reduction in the H2O2 production and an increase in the synthesis efficiency of soluble sugars, protecting developing tissues from the effects of chilling stress. Recent molecular research works, regarding drought and salinity performance in sugarcane, were carried out using techniques based on molecular hybridization such as SSH [Subtractive Hybridization Suppressive] (Patade *et al*., 2010) and micro/macroarrays (Rodrigues *et al*., 2009). In a general view, the main limitations of these methods regard their low sensibility and specificity (Shimkets, 2004). Moreover, in relation to micro/macroarrays, in spite of their high performance and broad use, the inability to analyze and discover new genes have been reported (Wang *et al*., 2009). Techniques based on sequencing [i.e. SAGE (Velculescu *et al*., 1995) and derivatives] take advantage of the available frequencies of fragments (tags) representing transcripts expressed in the sample, by the assumption that a short and defined tag contains the information needed to identify the corresponding cDNA (Velculescu *et al*., 1995). Thus, besides constituting an open architecture analysis (i.e., allowing the discovery of new genes), the abundance of tags found for a given gene provides an estimative of their transcription in the sample. In this context, the SuperSAGE technique (Matsumura *et al*., 2003) stands out for its efficiency in generating transcription profiles, especially with the actual association to the high performance sequencing platforms [Pyrosequencer (454 Roche®), Solexa (Illumina®) and

The SuperSAGE method is characterized by the generation of 26 bp tags by using the type III restriction enzyme *Eco*P15I (Matsumura *et al*., 2003). This technique presents itself as one of the most modern tools of functional genomics and provides some advantages such as improvements in tag-to-gene annotation, simultaneous analysis of two interacting eukaryotic organisms, full-length cDNAs amplification using tags as primers, potential use of tags via RNA interference (RNAi) in gene function studies, identification of antisense and rare transcripts, and identification of transcripts with alternative splicing (Matsumura *et al*., 2006). It also provides a global and quantitative transcriptomic analysis based on the study of the entire transcriptome produced in a given time under a given stimulus. This technique has been successfully applied in plant species such as rice (Matsumura *et al*., 2003), banana (Coemans *et al*., 2005), chili pepper (Hamada *et al*., 2008), chickpea (Molina *et al.*, 2008; 2011), tobacco (Gilardoni *et al*., 2010) and tropical crops (cowpea, soybean, sugarcane; Kido *et al*., 2010). Some of them using the association of SuperSAGE with a high-throughput sequencing platform (HT-SuperSAGE; Matsumura *et al*., 2010). In the present work we

and ectoine) (Yancey, 2005).

SOLiD (Applied Biosystems®)].

profit from the high resolution power of SuperSAGE® coupled to the Illumina® sequencing in a SuperTag Digital Gene Expression (STDGE) profile (GenXPro GmbH, Frankfurt, Germany) trying to characterize the transcriptome of drought-stressed sugarcane roots after 24 hours of submission to this stress, aiming to elect a best group of tags to be validated by RTqPCR. For this purpose, a high-throughput transcriptome project as a joint Brazilian initiative from UFPE (Federal University of Pernambuco) and CTC (Sugarcane Technology Center) was carried out. The project generated a large amount of gene candidates from different important categories considering the response against this kind of stress. In the present chapter an overview regarding the identification, categorization and differential expression of osmoprotectants will be evaluated in sugarcane, compared with the up to date knowledge concerning this crop and related species. Considering its role in world's economy and biotechnological potential, the identification and expression profile of responsive osmoprotectant coding genes in sugarcane may be helpful to unravel the basic mechanisms of stress tolerance, bringing valuable evidences for sugarcane improvement.

### **2. Drought: Understanding the problem**

Biological stress may be defined as an adverse environmental condition that inhibits the normal operation of a biological system such as plants (Jones & Jones, 1989). The life in the terrestrial condition represents a challenge to plants, and often have to occupy environments that are not the most appropriated for their development, being also subjected to frequent environmental changes in their native conditions. A large number of abiotic factors associated with plant-water relations – such as drought, salinity, chilling, frost and flooding – negatively affect the overall growth of terrestrial plants, leading to stunted form, metabolic changes, reduced yields, germination problems and even plant death under extreme conditions (Smith and Bhavel, 2007). Among the mentioned factors, drought stands out, bringing the most serious threat in view of the existing freshwater shortage in many regions of the world, bringing serious limitations to the agriculture (Jury & Vaux, 2005).

Notwithstanding the availability of more than 150 definitions for drought in the literature (Boken, 2005), it is often defined in terms of available humidity as compared with a normal value, with the severity correlating in function to the time and magnitude of the exposition to a deficient humidity (Smith & Pethley, 2009). Among the diverse types reported, some can be highlighted as meteorological, hydrological, socioeconomic and agricultural drought (Boken, 2005; Smith & Pethley, 2009). According to Boken (2005), the meteorological drought occurs when seasonal or annual precipitation falls below its long-term average; the hydrological drought when the meteorological drought is prolonged and causes local shortage of surface and groundwater; the socioeconomic drought is a manifestation of continued drought of severe intensity that shatters the economy and sociopolitical situation in a region, while the agricultural drought sets, due to soil moisture stress, a significant decline in crop yields (production per unit area).

The agricultural drought is the most important nowadays and will be here focused. Agriculture is by far the largest consumer of water, representing for 80.0 % of the freshwater consumption worldwide (Jury & Vaux, 2005). Economic losses associated to water availability reached about one billion dollars, in 2009, only in the United States (Anderson *et al*., 2009). Actually, one-third of the world population lives in areas with water shortages. This is specially serious considering other adverse factors such as the high levels of

Transcriptomics of Sugarcane Osmoprotectants Under Drought 93

Additionally, microarray platforms have been also used to evaluate sugarcane expression profile. The first ones were used to investigate gene expression differences between immature and maturing stems of sugarcane (Casu *et al*., 2003; 2004). Using glass microarrays the authors assayed up to 4,715 non-redundant random ESTs derived from immature and maturing stems, and also from roots. The most recent report using this method made use of a custom cDNA microarray (3,598) to profile the effect of elevated CO2 on sugarcane leaves (De Souza *et al*., 2008). Regarding open architecture transcriptomics technologies – which analyze the entire population of transcripts produced in a given time under a given stimulus – a single literature report is available for sugarcane, in an approach developed by Calsa & Figueira (2007). The authors used standard 14 bp SAGE to characterize the sugarcane mature leaf transcriptome, generating 9,482 valid tags, with 5,227 unique sequences, from which 3,659 (70.0 %) matched at least one sugarcane assembled sequence with putative

Despite the relative data abundance for sugarcane transcriptome covering different conditions, there is still a restricted number of publications regarding the analysis of molecular behavior under drought, whilst most available evidences come from technologies based on hybridization or cDNA (EST) sequencing. Rocha *et al*. (2007) in a cDNA microarrays approach to profile expression of 1,545 genes involved in signaling processes in plants submitted to drought, phosphate starvation, herbivory and N2-fixing endophytic bacteria, identified 485 differentially expressed candidates after exposition of the plants to water shortage. In another approach Gupta *et al*. (2010) using cDNA libraries associated to the RTqPCR validation of drought related genes, revealed differences greater than 2-fold regarding the expression of given genes during dehydration stress. The most recent report was carried out by Iskandar *et al*. (2011) that investigated whether the degree of expression of eight stress-related genes - *P5CS, OAT, AS, PST5, TF1, LEA, POX* and *dehydrin* – was correlated with the sucrose content in the sugarcane culm, and whether such genes were also responsive to water deficit stress. Almost all selected genes were upregulated, with exception of *POX* that was downregulated after 15 days of water deficit stress. However, subsequent analysis revealed a different transcriptional profile to that, showing a correlation with the sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit, but did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis

indicated that proline was negatively correlated with sucrose concentration.

on the metabolic pathways involved in acclimation process to water deficit.

adaptive responses include a significant accumulation of compatible solutes.

**4. Drought and osmoprotectants in plants** 

Since drought is a complex feature discovery of genes associated to tolerance processes is urgent, being one of the most important and difficult challenges. A larger number of studies is made necessary, aiming to understand this issue in sugarcane, enriching the knowledge

Water deficit, caused by "lack of water" or by other environmental stresses like extreme temperatures or salinity (Bartels & Souer, 2004), has been great problems for agriculture, affecting virtually every aspect of plant physiology and metabolism. As a consequence of these stresses, a range of adaptive responses including morphological (Jaleel *et al*., 2009), physiological (Harb *et al*., 2010) and biochemical changes (Ahmadi *et al*., 2010) enabled plants to tolerate and survive at such adverse conditions. Similar cellular and molecular

function.

atmospheric CO2, climate change scenarios and predictions of future global warming, all of them increasing drought incidence, frequency and severity. Significant further problems are predicted for food production due to the limited availability of suitable freshwater for agriculture.

According to Hazell & Wood (2008), the climate change will affect different localities in different ways, with potential benefits to some important food growing areas as the Canadian Prairies, but making agriculture more difficult in some other regions as many drought prone areas in Africa and Americas. Such predictions also reinforce that agricultural systems have considerable capacity to adapt to climate change, but this poses many challenges that are not yet fully understood, with urgent research efforts necessary to identify the best ways to adapt.

### **3. Sugarcane and drought: Current outlook and use of science in search of solutions**

Sugarcane is one of the world's major food crops, providing about 75.0 % of the sugar harvested for human consumption [Food and Agriculture Organization (FAO) statistics]. The crop is closely associated with sustainability as it is presented as a renewable energy source including ethanol and electricity (Tew & Cobill, 2008). Despite this status, sugarcane suffers severe losses due to the availability of soil water. In Brazil, the largest global sugarcane producer (Henry, 2010), the state of São Paulo accounts for about 60.0 % of the country production, suffering losses of around 10.0 % due to periods of drought, as reported for harvests in 2010 (UNICA, 2011). Thus, it is necessary to generate new tolerant varieties to this stress. Currently, breeding programs for sugarcane are being developed in different countries by public and private institutions, and also by cooperative systems formed by producers. Most projects involve the performance of controlled crossings which are costly and bring long term results, reaching sometimes more than 15 years (Cesnik & Miocque, 2004).

An alternative way to promote the breeding is associated with the analysis and identification of specific genes involved in given metabolic processes. The traditional method for identifying a gene responsible for a particular trait includes the initial demonstration that the trait is inheritable, followed by isolation of a candidate gene that is postulated to be responsible for that trait. This "single gene" approach is fundamentally flawed for many traits since it is assumed that every trait is governed by a single gene. According to Casu *et al*. (2010) genomics and all of the related "omics" techniques, e.g. transcriptomics, break this formula and rely on the in-depth assembly of large amounts of data followed by data-mining to determine connections between a particular trait and any number of associated genes.

Transcriptomics data are available for sugarcane, and have been generated mainly by EST (Expressed Sequence Tag) sequencing, or using methodologies based on probe hybridization arrays, using known genes from other crops. Among the available collections of ESTs for sugarcane, the sequences generated by the SUCEST project should be highlighted. This project regards a large consortium of Brazilian researchers who sequenced approximately 238,000 redundant ESTs from 26 diverse cDNA libraries (Vettore *et al*., 2001), representing the largest effort to generate information using new technologies for this species. This endeavor brought a broader panel when compared to previous available ESTs produced by other consortia in countries like Australia (Casu *et al*., 2003, 2004; Bower *et al*., 2005) and the US (Ma *et al*., 2004).

atmospheric CO2, climate change scenarios and predictions of future global warming, all of them increasing drought incidence, frequency and severity. Significant further problems are predicted for food production due to the limited availability of suitable freshwater for

According to Hazell & Wood (2008), the climate change will affect different localities in different ways, with potential benefits to some important food growing areas as the Canadian Prairies, but making agriculture more difficult in some other regions as many drought prone areas in Africa and Americas. Such predictions also reinforce that agricultural systems have considerable capacity to adapt to climate change, but this poses many challenges that are not yet fully understood, with urgent research efforts necessary to

**3. Sugarcane and drought: Current outlook and use of science in search of** 

results, reaching sometimes more than 15 years (Cesnik & Miocque, 2004).

Sugarcane is one of the world's major food crops, providing about 75.0 % of the sugar harvested for human consumption [Food and Agriculture Organization (FAO) statistics]. The crop is closely associated with sustainability as it is presented as a renewable energy source including ethanol and electricity (Tew & Cobill, 2008). Despite this status, sugarcane suffers severe losses due to the availability of soil water. In Brazil, the largest global sugarcane producer (Henry, 2010), the state of São Paulo accounts for about 60.0 % of the country production, suffering losses of around 10.0 % due to periods of drought, as reported for harvests in 2010 (UNICA, 2011). Thus, it is necessary to generate new tolerant varieties to this stress. Currently, breeding programs for sugarcane are being developed in different countries by public and private institutions, and also by cooperative systems formed by producers. Most projects involve the performance of controlled crossings which are costly and bring long term

An alternative way to promote the breeding is associated with the analysis and identification of specific genes involved in given metabolic processes. The traditional method for identifying a gene responsible for a particular trait includes the initial demonstration that the trait is inheritable, followed by isolation of a candidate gene that is postulated to be responsible for that trait. This "single gene" approach is fundamentally flawed for many traits since it is assumed that every trait is governed by a single gene. According to Casu *et al*. (2010) genomics and all of the related "omics" techniques, e.g. transcriptomics, break this formula and rely on the in-depth assembly of large amounts of data followed by data-mining to determine connections between a particular trait and any

Transcriptomics data are available for sugarcane, and have been generated mainly by EST (Expressed Sequence Tag) sequencing, or using methodologies based on probe hybridization arrays, using known genes from other crops. Among the available collections of ESTs for sugarcane, the sequences generated by the SUCEST project should be highlighted. This project regards a large consortium of Brazilian researchers who sequenced approximately 238,000 redundant ESTs from 26 diverse cDNA libraries (Vettore *et al*., 2001), representing the largest effort to generate information using new technologies for this species. This endeavor brought a broader panel when compared to previous available ESTs produced by other consortia in countries like Australia (Casu *et al*., 2003, 2004; Bower *et al*.,

agriculture.

**solutions** 

identify the best ways to adapt.

number of associated genes.

2005) and the US (Ma *et al*., 2004).

Additionally, microarray platforms have been also used to evaluate sugarcane expression profile. The first ones were used to investigate gene expression differences between immature and maturing stems of sugarcane (Casu *et al*., 2003; 2004). Using glass microarrays the authors assayed up to 4,715 non-redundant random ESTs derived from immature and maturing stems, and also from roots. The most recent report using this method made use of a custom cDNA microarray (3,598) to profile the effect of elevated CO2 on sugarcane leaves (De Souza *et al*., 2008). Regarding open architecture transcriptomics technologies – which analyze the entire population of transcripts produced in a given time under a given stimulus – a single literature report is available for sugarcane, in an approach developed by Calsa & Figueira (2007). The authors used standard 14 bp SAGE to characterize the sugarcane mature leaf transcriptome, generating 9,482 valid tags, with 5,227 unique sequences, from which 3,659 (70.0 %) matched at least one sugarcane assembled sequence with putative function.

Despite the relative data abundance for sugarcane transcriptome covering different conditions, there is still a restricted number of publications regarding the analysis of molecular behavior under drought, whilst most available evidences come from technologies based on hybridization or cDNA (EST) sequencing. Rocha *et al*. (2007) in a cDNA microarrays approach to profile expression of 1,545 genes involved in signaling processes in plants submitted to drought, phosphate starvation, herbivory and N2-fixing endophytic bacteria, identified 485 differentially expressed candidates after exposition of the plants to water shortage. In another approach Gupta *et al*. (2010) using cDNA libraries associated to the RTqPCR validation of drought related genes, revealed differences greater than 2-fold regarding the expression of given genes during dehydration stress. The most recent report was carried out by Iskandar *et al*. (2011) that investigated whether the degree of expression of eight stress-related genes - *P5CS, OAT, AS, PST5, TF1, LEA, POX* and *dehydrin* – was correlated with the sucrose content in the sugarcane culm, and whether such genes were also responsive to water deficit stress. Almost all selected genes were upregulated, with exception of *POX* that was downregulated after 15 days of water deficit stress. However, subsequent analysis revealed a different transcriptional profile to that, showing a correlation with the sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit, but did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis indicated that proline was negatively correlated with sucrose concentration.

Since drought is a complex feature discovery of genes associated to tolerance processes is urgent, being one of the most important and difficult challenges. A larger number of studies is made necessary, aiming to understand this issue in sugarcane, enriching the knowledge on the metabolic pathways involved in acclimation process to water deficit.

### **4. Drought and osmoprotectants in plants**

Water deficit, caused by "lack of water" or by other environmental stresses like extreme temperatures or salinity (Bartels & Souer, 2004), has been great problems for agriculture, affecting virtually every aspect of plant physiology and metabolism. As a consequence of these stresses, a range of adaptive responses including morphological (Jaleel *et al*., 2009), physiological (Harb *et al*., 2010) and biochemical changes (Ahmadi *et al*., 2010) enabled plants to tolerate and survive at such adverse conditions. Similar cellular and molecular adaptive responses include a significant accumulation of compatible solutes.

Transcriptomics of Sugarcane Osmoprotectants Under Drought 95

1995). Based on the correlation between GB accumulation and stress tolerance, progress in exogenous GB application (Jokinen *et al*., 1999), cloning and expression of GB encoding enzymes has been achieved (Sakamoto & Murata, 2002; Quan *et al*., 2004). Examples of these genes include *CMO* (Tabuchi *et al*., 2005), *BADH* (Wood *et al.*, 1996); *CDH* and *BADH* (Landfald & Strøm, 1986), which were cloned from different organisms and introduced into transgenic plants. For example, in transgenic cotton (transformed with *bet*A gene) GB expression induced the protection of the cell membrane integrity from drought stress damage, being also active in osmotic adjustment (Lv *et al*., 2007). Huang *et al*. (2000) transformed three different species (*Arabidopsis thaliana, Brassica napus* and *Nicotiana tabacum*) with the *COX* gene from *Arthrobacter pascens*, an elucidative approach considering that these plants are non accumulators of this osmoprotector. The highest levels of betaine in independent transgenic plants were de 10- to 20-fold lower than the levels found in natural betaine producers. Further, it was observed that the supplementation of choline is necessary to allow the accumulation of physiologically relevant amounts of betaine. Despite of that, the authors reported the acquisition of a moderate stress tolerance (drought and salinity) in some but not all betaine-producing transgenic lines, based on the relative shoot growth; while the responses to salinity, drought, and freezing stresses were variable among the three transformed species. The results lead to the supposition that higher efficiencies would be achieved in species that naturally produce this osmoprotector (Huang *et al*., 2000). Similar results were achieved by Shirasawa *et al.* (2006) after transformation of rice plants (*Oryza sativa*) – also a non accumulator of GB - with the choline monooxygenase gene from spinach. Enhanced tolerance to salt stress and temperature stress in the seedling stage was observed, however the CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity. Considering sugarcane, Patade *et al*. (2008) observed the accumulation of free proline and glycine betaine in embryogenic sugarcane calli *(Saccharum officinarum* L.; cv. CoC-671) after NaCl stress. The gradual increase in glycine betaine was positively correlated with the concentrations of NaCl (up to 213.9 mM). Indeed, such osmoprotector was also higher as compared to proline content, in all stress conditions tested (NaCl treatments: 42.8 to 256.7 mM). However, in the higher NaCl concentration, proline

Proline (Pro) is a proteinogenic amino acid essential for primary metabolism. It is considered one of the most important osmolytes, being accumulated in a large number of species in response to stress damage (for a review, see Hare & Cress, 1997). Under abiotic stress condition, proline accumulation is involved in the maintenance of turgor, promoting continued growth in case of low water potential in the soil (Mullet & Whitsitt, 1996). The accumulation of this important osmolyte, upon osmotic stress, is well documented in a large number of different organisms, including, protozoa (Poulin *et al.*, 1987), eubacteria (Csonka, 1989) and marine invertebrates (Burton, 1991). In addition to its role in the osmotic adjustment mechanism, other important functions have been attributed to proline (Bartels & Sunkar, 2005) such as protection of plasma membrane integrity, enhancing of different enzymes activity (Sharma & Dubey, 2005; Mishra & Dubey, 2006), regulation of nitrogen and carbon reservoir

In higher plants, proline biosynthesis may proceed from two different ways: either via glutamate, by successive reductions catalyzed by pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), respectively (Hu *et al*., 1992; Savouré *et al*.,

(Kishor *et al*., 2005) and as scavenger of free radicals (Smirnoff & Cumbes, 1989).

was not observed.

**4.2 Proline** 

Compatible solutes regard a variety of low-molecular-weight organic compounds, electrically neutral molecules, soluble in water and nontoxic at high cellular concentrations (Yancey, 2001). Such osmolytes include a variety of simple sugars (e.g. fructose and glucose), sugar alcohols (glycerol and methylated inositols) and complex sugars (trehalose, raffinose and fructans), while other include quaternary amino acid derivatives (proline, glycine betaine, b-alanine betaine, proline betaine), tertiary amines (ectoine; 1,4,5,6 tetrahydro-2-methyl-4-carboxy-lpyrimidine) and sulfonium compounds (choline o-sulfate, dimethyl sulfonium propironate) (Rhodes & Hanson, 1993; Vinocur & Altman, 2005). Compatible solute accumulation in response to osmotic stress is a ubiquitous process in organisms as diverse as bacteria, plants and animals (Bohnert & Jensen, 1996). These osmoprotectants compounds are typically confined mainly to the cytosol, chloroplasts, and other cytoplasmic compartments (Rontein *et al*., 2002), protecting plants in different ways, including: stress defense by osmotic adjustment (helping the cells to maintain their hydrated state and turgor maintenance), stabilization of proteins and enzymes, induction of stress proteins and acceleration of reactive oxygen species scavenging systems (Bohnert & Jensen 1996; Ashraf & Foolad, 2007). In plants that naturally accumulate osmoprotectants, the level of these compounds are highest under stress extension (Rhodes & Hanson, 1993). So, changes in plant drought-induced gene expressions have been revealed, and many genes have been isolated from numerous species, playing important roles in both initial stress response and in establishing plant stress tolerance (Shinozaki & Yamaguchi-Shinozaki, 2007). Proline, glycine betaine, sugars and sugar alcohols are examples of compatible solutes encoded by some of these stress-inducible genes that function in cellular osmotic adjustment, promoting drought tolerance, guaranteeing plasma membrane integrity, without disrupting the protein function (Bartels & Sunkar, 2005). So, the osmotic adjustment by accumulation of these compounds has been proposed as an important mechanism to overcome the negative consequences of water deficit in crop production (Rathinasabapathi, 2000; Choluj *et al*., 2008). Based on accumulation of these compounds to be associated to high levels of tolerance in plants, and considering that their beneficial effects are generally not species-specific (Rontein *et al*., 2002), considerable progress has been achieved in investigations using transgenic plants overexpressing selected osmoprotectants conferring abiotic stress tolerance (Ashraf & Foolad, 2007; Chen & Murata, 2008). Some of them are reviewed below.

### **4.1 Glycine betaine**

Glycine betaine (GB) is a quaternary ammonium compound (QAC) synthesized by a great variety of organisms, including plants, animals and microorganisms (Rhodes & Hanson, 1993). In most organisms GB is synthesized either by the oxidation (or dehydrogenation) of choline or by the N-methylation of glycine. However, the pathway from choline to glycine betaine has been the main GB-accumulation pathway in plant species (Weretilnyk *et al*., 1989). In this pathway choline is converted to betaine aldehyde by choline monooxygenase (CMO) (Rathinasabapathi *et al*., 1997), which is then converted to GB by betaine aldehyde dehydrogenase (BADH) (Vojtechova *et al*., 1997). Similarly to proline (and other osmoprotectants in plants) GB is one of the most extensively studied compatible solutes, being upregulated after drought (Ma *et al*., 2007), salinity (Kern & Dyer, 2004), low temperature (Zhang *et al*., 2010) and oxidative stresses (Liu *et al*., 2011). *In vitro* assays indicate that GB acts as an osmoprotector, stabilizing both the quaternary structure of proteins and the highly ordered membrane structure under adverse conditions (Gorham, 1995). Based on the correlation between GB accumulation and stress tolerance, progress in exogenous GB application (Jokinen *et al*., 1999), cloning and expression of GB encoding enzymes has been achieved (Sakamoto & Murata, 2002; Quan *et al*., 2004). Examples of these genes include *CMO* (Tabuchi *et al*., 2005), *BADH* (Wood *et al.*, 1996); *CDH* and *BADH* (Landfald & Strøm, 1986), which were cloned from different organisms and introduced into transgenic plants. For example, in transgenic cotton (transformed with *bet*A gene) GB expression induced the protection of the cell membrane integrity from drought stress damage, being also active in osmotic adjustment (Lv *et al*., 2007). Huang *et al*. (2000) transformed three different species (*Arabidopsis thaliana, Brassica napus* and *Nicotiana tabacum*) with the *COX* gene from *Arthrobacter pascens*, an elucidative approach considering that these plants are non accumulators of this osmoprotector. The highest levels of betaine in independent transgenic plants were de 10- to 20-fold lower than the levels found in natural betaine producers. Further, it was observed that the supplementation of choline is necessary to allow the accumulation of physiologically relevant amounts of betaine. Despite of that, the authors reported the acquisition of a moderate stress tolerance (drought and salinity) in some but not all betaine-producing transgenic lines, based on the relative shoot growth; while the responses to salinity, drought, and freezing stresses were variable among the three transformed species. The results lead to the supposition that higher efficiencies would be achieved in species that naturally produce this osmoprotector (Huang *et al*., 2000). Similar results were achieved by Shirasawa *et al.* (2006) after transformation of rice plants (*Oryza sativa*) – also a non accumulator of GB - with the choline monooxygenase gene from spinach. Enhanced tolerance to salt stress and temperature stress in the seedling stage was observed, however the CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity. Considering sugarcane, Patade *et al*. (2008) observed the accumulation of free proline and glycine betaine in embryogenic sugarcane calli *(Saccharum officinarum* L.; cv. CoC-671) after NaCl stress. The gradual increase in glycine betaine was positively correlated with the concentrations of NaCl (up to 213.9 mM). Indeed, such osmoprotector was also higher as compared to proline content, in all stress conditions tested (NaCl treatments: 42.8 to 256.7 mM). However, in the higher NaCl concentration, proline was not observed.

### **4.2 Proline**

94 Plants and Environment

Compatible solutes regard a variety of low-molecular-weight organic compounds, electrically neutral molecules, soluble in water and nontoxic at high cellular concentrations (Yancey, 2001). Such osmolytes include a variety of simple sugars (e.g. fructose and glucose), sugar alcohols (glycerol and methylated inositols) and complex sugars (trehalose, raffinose and fructans), while other include quaternary amino acid derivatives (proline, glycine betaine, b-alanine betaine, proline betaine), tertiary amines (ectoine; 1,4,5,6 tetrahydro-2-methyl-4-carboxy-lpyrimidine) and sulfonium compounds (choline o-sulfate, dimethyl sulfonium propironate) (Rhodes & Hanson, 1993; Vinocur & Altman, 2005). Compatible solute accumulation in response to osmotic stress is a ubiquitous process in organisms as diverse as bacteria, plants and animals (Bohnert & Jensen, 1996). These osmoprotectants compounds are typically confined mainly to the cytosol, chloroplasts, and other cytoplasmic compartments (Rontein *et al*., 2002), protecting plants in different ways, including: stress defense by osmotic adjustment (helping the cells to maintain their hydrated state and turgor maintenance), stabilization of proteins and enzymes, induction of stress proteins and acceleration of reactive oxygen species scavenging systems (Bohnert & Jensen 1996; Ashraf & Foolad, 2007). In plants that naturally accumulate osmoprotectants, the level of these compounds are highest under stress extension (Rhodes & Hanson, 1993). So, changes in plant drought-induced gene expressions have been revealed, and many genes have been isolated from numerous species, playing important roles in both initial stress response and in establishing plant stress tolerance (Shinozaki & Yamaguchi-Shinozaki, 2007). Proline, glycine betaine, sugars and sugar alcohols are examples of compatible solutes encoded by some of these stress-inducible genes that function in cellular osmotic adjustment, promoting drought tolerance, guaranteeing plasma membrane integrity, without disrupting the protein function (Bartels & Sunkar, 2005). So, the osmotic adjustment by accumulation of these compounds has been proposed as an important mechanism to overcome the negative consequences of water deficit in crop production (Rathinasabapathi, 2000; Choluj *et al*., 2008). Based on accumulation of these compounds to be associated to high levels of tolerance in plants, and considering that their beneficial effects are generally not species-specific (Rontein *et al*., 2002), considerable progress has been achieved in investigations using transgenic plants overexpressing selected osmoprotectants conferring abiotic stress tolerance (Ashraf & Foolad, 2007; Chen & Murata, 2008). Some of them are

Glycine betaine (GB) is a quaternary ammonium compound (QAC) synthesized by a great variety of organisms, including plants, animals and microorganisms (Rhodes & Hanson, 1993). In most organisms GB is synthesized either by the oxidation (or dehydrogenation) of choline or by the N-methylation of glycine. However, the pathway from choline to glycine betaine has been the main GB-accumulation pathway in plant species (Weretilnyk *et al*., 1989). In this pathway choline is converted to betaine aldehyde by choline monooxygenase (CMO) (Rathinasabapathi *et al*., 1997), which is then converted to GB by betaine aldehyde dehydrogenase (BADH) (Vojtechova *et al*., 1997). Similarly to proline (and other osmoprotectants in plants) GB is one of the most extensively studied compatible solutes, being upregulated after drought (Ma *et al*., 2007), salinity (Kern & Dyer, 2004), low temperature (Zhang *et al*., 2010) and oxidative stresses (Liu *et al*., 2011). *In vitro* assays indicate that GB acts as an osmoprotector, stabilizing both the quaternary structure of proteins and the highly ordered membrane structure under adverse conditions (Gorham,

reviewed below.

**4.1 Glycine betaine** 

Proline (Pro) is a proteinogenic amino acid essential for primary metabolism. It is considered one of the most important osmolytes, being accumulated in a large number of species in response to stress damage (for a review, see Hare & Cress, 1997). Under abiotic stress condition, proline accumulation is involved in the maintenance of turgor, promoting continued growth in case of low water potential in the soil (Mullet & Whitsitt, 1996). The accumulation of this important osmolyte, upon osmotic stress, is well documented in a large number of different organisms, including, protozoa (Poulin *et al.*, 1987), eubacteria (Csonka, 1989) and marine invertebrates (Burton, 1991). In addition to its role in the osmotic adjustment mechanism, other important functions have been attributed to proline (Bartels & Sunkar, 2005) such as protection of plasma membrane integrity, enhancing of different enzymes activity (Sharma & Dubey, 2005; Mishra & Dubey, 2006), regulation of nitrogen and carbon reservoir (Kishor *et al*., 2005) and as scavenger of free radicals (Smirnoff & Cumbes, 1989).

In higher plants, proline biosynthesis may proceed from two different ways: either via glutamate, by successive reductions catalyzed by pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), respectively (Hu *et al*., 1992; Savouré *et al*.,

Transcriptomics of Sugarcane Osmoprotectants Under Drought 97

(Kaplan *et al*., 2004; Peters *et al*., 2007). In the case of the halophyte *Mesembryanthemum crystallinum* (common ice plant) – that possesses a remarkable tolerance against drought, high salinity, and cold stress – inositol is methylated to D-ononitol and subsequently epimerized to D-pinitol. This plant accumulates a large amount of these inositol derivatives

Throughout the biological kingdom, myo-inositol is synthesized by a two-step pathway that is unofficially known as the "Loewus pathway". The first step is the conversion of Dglucose-6-P to D-myo-inositol (1)-Monophosphate, 1D-MI-1-P, which is catalyzed by an Lmyo-inositol 1-phosphate synthase (MIPS) (Majumder *et al*., 1997), followed by its specific dephosphorylation to free myo-inositol by the Mg++ dependent L-Myo-inositol 1-phosphate phosphatase (IMP) (Parthasarathy *et al.*, 1994). Due to the potential of myo-inositol, some transgenic plants expressing this substance have been generated, mainly using MIPS

Majee *et al*. (2004) reported on the isolation of the *PINO1* gene (also known as *PcINO1*, encoding an l-myo-inositol 1-phosphate synthase) from the wild halophytic rice relative *Porteresia coarctata*. This gene was expressed in tobacco plants, conferring them the capacity of growth in 200–300 mM NaCl with retention of ∼ 40–80 % of the photosynthetic competence with concomitant increased inositol production when compared with unstressed control. Additionally, *PINO1* transgenics showed *in vitro* salt-tolerance,

Das-Chatterjee *et al*. (2006) carried out a functional introgression of *PcINO1* and *OsINO1* genes (this last regarding the corresponding homologue from the cultivated rice that encodes for a salt-sensitive MIPS protein) in distantly related organisms, as prokaryotes (*Escherichia coli*) to eukaryotes (yeast: *Schizosaccharomyces pombe*; plants: *Oryza sativa* and *Brassica juncea*) analyzing the tolerance of these transgenic lines under salinity stress. The results confirmed the role of the *PcINO1* gene, conferring salt tolerance to various levels of complexity, from prokaryotes to different eukaryotes, including higher plants, leading to an unabated production of inositol and survival under NaCl stress. Patra *et al*. (2010), in turn, held introgression and functional expression studies in tobacco plants using *PcINO1* (a) and *McIMTI* (b) [inositol methyl transferase, IMTI, from the common ice plant *M. crystallinum*] genes. After submission of the obtained transgenic lines to saline and oxidative stresses it was observed that all plants presented higher performances in terms of growth potential and photosynthetic activity and were less prone to oxidative and salt stresses when compared to the controls. Physiological experiments demonstrated the superiority of the *PcINO1–McIMT1* double transgenic plants to withstand the salt stress accompanied by the accumulation of both myo-inositol and methylated inositols in the system over the

Trehalose is a non-reducing α,α-1,1-linked glucose disaccharide that functions as an energy source and a storage form of more reactive glucose in lower organisms (Galinski, 1993). At least three different pathways for the biological synthesis of trehalose have been reported (Elbein *et al*., 2003). In plants, the synthesis of this sugar occurs normally by the formation of the trehalose-6-phosphate (T6P) from the UDP-glucose and glucose-6-phosphate, a reaction catalyzed by the trehalose 6-phosphate synthase (TPS). Afterwards the T6P is dephosphorylated by the trehalose-6-phosphate phosphatase (TPP) resulting in the

during the stress (Adams *et al*., 1992; Vernon *et al*., 1993).

confirming *in planta* functional expression of this gene.

transgenic plants with either of the single gene(s).

formation of free trehalose (Wingler, 2002).

**4.4 Trehalose** 

enzyme or inositol derived enzymes.

1995) or ornithine pathway, by ornithine d-aminotransferase (OAT) (Mestichelli *et al*., 1979). Here the first pathway will be discussed, since it is considered the main pathway during osmotic stress in plants (Bartels & Sunkar, 2005; Parida *et al.*, 2008) especially considering the drought response. Under water deficit, proline is synthesized from the glutamate by two intermediates. In the first step, the glutamate is reduced to glutamic acid-5 semialdehyde (GSA) by P5CS. The GSA produced is converted into pyrroline-5-carboxylate (P5C) (Hu *et al*., 1992; Savouré *et al*., 1995) which is then reduced by P5CR to proline (Zhang *et al*., 1995). Proline induction in response to abiotic stresses has been related for many angiosperms (Mohammadkhani & Heidari, 2008; Székely *et al*., 2008), revealing a positive relationship between proline accumulation and stress tolerance in this group. Kishor *et al*. (1995) reported the overexpression of a *Vigna aconitifolia P5CS1* gene in tobacco plants, leading to increased levels of proline (10- to 18-fold when compared to the control plants), enhancing root biomass, growth rhythm and tolerance under drought-stress. The importance of proline metabolism in the process of drought tolerance was evidenced by Ronde *et al*. (2000) in soybean plants (*Glycine max*). The authors reported the suppression of proline synthesis in transgenic soybean plants containing the *P5CS* gene in the antisense direction. Transformed plants presented increased sensitivity to water deficit, as compared with the wild type. In cotton under drought-stress, Parida *et al.* (2008) verified an induction of proline levels by the upregulation of P5CS and downregulation of proline dehydrogenase (PDH), indicating a possible involvement of proline production in the development of drought tolerance. Osmotic adjustment through proline accumulation was reported as a primary response of drought stressed sugarcane (*S. officinarum*) plantlets (Errabii *et al*., 2006).

On the other hand, reports suggested that the increase in proline concentration is related to protective symptoms under severe water stress rather than an osmoregulatory function. In transgenic wheat plants, the higher accumulation of proline (when compared to the wild type) conferred drought stress tolerance by increasing the antioxidant metabolism rather than increasing osmotic adjustment (Vendruscolo *et al*., 2007). In sugarcane transformed with the *V. aconitifolia P5CS* gene, it was observed that after nine days without irrigation proline content in transgenic plants was on the average 2.5-fold higher than in the controls. However, no osmotic adjustment was observed in plants overproducing proline during the water-deficit period, suggesting a role of proline as component of the antioxidative response system rather than as a promoter of osmotic adjustment (Molinari *et al*., 2007). Indeed, the hypothesis of the protective role played by proline under severe drought stress was also supported by Gomes *et al*. (2010), who evaluated the water stress effect on osmotic potential, proline accumulation and cell membrane stability in leaflets of the coconut palm (*Cocos nucifera* L.).

### **4.3 Myo-inositol**

Inositol is a cyclohexanehexol, a cyclic carbohydrate with six hydroxyl groups, one on each carbon ring. Among the nine types of existing steroisomers, myo-inositol is the most abundant in the nature, being also important for the biosynthesis of a wide variety of compounds including inositol phosphates, glycosylphosphatidylinositols, phosphatidylinositides, inositol esters, and ethers in plants (Murthy, 2006). Besides the own myo-inositol, other related or derived molecules are also important osmoprotectors. Myoinositol serves as a substrate for the formation of galactinol, the galactosyl-donor that plays a key role in the formation of raffinose family oligosaccharides (RFOs, raffinose, stachyose, verbascose) from sucrose. RFOs accumulate in plants under different stress conditions

1995) or ornithine pathway, by ornithine d-aminotransferase (OAT) (Mestichelli *et al*., 1979). Here the first pathway will be discussed, since it is considered the main pathway during osmotic stress in plants (Bartels & Sunkar, 2005; Parida *et al.*, 2008) especially considering the drought response. Under water deficit, proline is synthesized from the glutamate by two intermediates. In the first step, the glutamate is reduced to glutamic acid-5 semialdehyde (GSA) by P5CS. The GSA produced is converted into pyrroline-5-carboxylate (P5C) (Hu *et al*., 1992; Savouré *et al*., 1995) which is then reduced by P5CR to proline (Zhang *et al*., 1995). Proline induction in response to abiotic stresses has been related for many angiosperms (Mohammadkhani & Heidari, 2008; Székely *et al*., 2008), revealing a positive relationship between proline accumulation and stress tolerance in this group. Kishor *et al*. (1995) reported the overexpression of a *Vigna aconitifolia P5CS1* gene in tobacco plants, leading to increased levels of proline (10- to 18-fold when compared to the control plants), enhancing root biomass, growth rhythm and tolerance under drought-stress. The importance of proline metabolism in the process of drought tolerance was evidenced by Ronde *et al*. (2000) in soybean plants (*Glycine max*). The authors reported the suppression of proline synthesis in transgenic soybean plants containing the *P5CS* gene in the antisense direction. Transformed plants presented increased sensitivity to water deficit, as compared with the wild type. In cotton under drought-stress, Parida *et al.* (2008) verified an induction of proline levels by the upregulation of P5CS and downregulation of proline dehydrogenase (PDH), indicating a possible involvement of proline production in the development of drought tolerance. Osmotic adjustment through proline accumulation was reported as a primary response of drought stressed sugarcane (*S.* 

On the other hand, reports suggested that the increase in proline concentration is related to protective symptoms under severe water stress rather than an osmoregulatory function. In transgenic wheat plants, the higher accumulation of proline (when compared to the wild type) conferred drought stress tolerance by increasing the antioxidant metabolism rather than increasing osmotic adjustment (Vendruscolo *et al*., 2007). In sugarcane transformed with the *V. aconitifolia P5CS* gene, it was observed that after nine days without irrigation proline content in transgenic plants was on the average 2.5-fold higher than in the controls. However, no osmotic adjustment was observed in plants overproducing proline during the water-deficit period, suggesting a role of proline as component of the antioxidative response system rather than as a promoter of osmotic adjustment (Molinari *et al*., 2007). Indeed, the hypothesis of the protective role played by proline under severe drought stress was also supported by Gomes *et al*. (2010), who evaluated the water stress effect on osmotic potential, proline accumulation and cell membrane stability in leaflets of the coconut palm (*Cocos* 

Inositol is a cyclohexanehexol, a cyclic carbohydrate with six hydroxyl groups, one on each carbon ring. Among the nine types of existing steroisomers, myo-inositol is the most abundant in the nature, being also important for the biosynthesis of a wide variety of compounds including inositol phosphates, glycosylphosphatidylinositols, phosphatidylinositides, inositol esters, and ethers in plants (Murthy, 2006). Besides the own myo-inositol, other related or derived molecules are also important osmoprotectors. Myoinositol serves as a substrate for the formation of galactinol, the galactosyl-donor that plays a key role in the formation of raffinose family oligosaccharides (RFOs, raffinose, stachyose, verbascose) from sucrose. RFOs accumulate in plants under different stress conditions

*officinarum*) plantlets (Errabii *et al*., 2006).

*nucifera* L.).

**4.3 Myo-inositol** 

(Kaplan *et al*., 2004; Peters *et al*., 2007). In the case of the halophyte *Mesembryanthemum crystallinum* (common ice plant) – that possesses a remarkable tolerance against drought, high salinity, and cold stress – inositol is methylated to D-ononitol and subsequently epimerized to D-pinitol. This plant accumulates a large amount of these inositol derivatives during the stress (Adams *et al*., 1992; Vernon *et al*., 1993).

Throughout the biological kingdom, myo-inositol is synthesized by a two-step pathway that is unofficially known as the "Loewus pathway". The first step is the conversion of Dglucose-6-P to D-myo-inositol (1)-Monophosphate, 1D-MI-1-P, which is catalyzed by an Lmyo-inositol 1-phosphate synthase (MIPS) (Majumder *et al*., 1997), followed by its specific dephosphorylation to free myo-inositol by the Mg++ dependent L-Myo-inositol 1-phosphate phosphatase (IMP) (Parthasarathy *et al.*, 1994). Due to the potential of myo-inositol, some transgenic plants expressing this substance have been generated, mainly using MIPS enzyme or inositol derived enzymes.

Majee *et al*. (2004) reported on the isolation of the *PINO1* gene (also known as *PcINO1*, encoding an l-myo-inositol 1-phosphate synthase) from the wild halophytic rice relative *Porteresia coarctata*. This gene was expressed in tobacco plants, conferring them the capacity of growth in 200–300 mM NaCl with retention of ∼ 40–80 % of the photosynthetic competence with concomitant increased inositol production when compared with unstressed control. Additionally, *PINO1* transgenics showed *in vitro* salt-tolerance, confirming *in planta* functional expression of this gene.

Das-Chatterjee *et al*. (2006) carried out a functional introgression of *PcINO1* and *OsINO1* genes (this last regarding the corresponding homologue from the cultivated rice that encodes for a salt-sensitive MIPS protein) in distantly related organisms, as prokaryotes (*Escherichia coli*) to eukaryotes (yeast: *Schizosaccharomyces pombe*; plants: *Oryza sativa* and *Brassica juncea*) analyzing the tolerance of these transgenic lines under salinity stress. The results confirmed the role of the *PcINO1* gene, conferring salt tolerance to various levels of complexity, from prokaryotes to different eukaryotes, including higher plants, leading to an unabated production of inositol and survival under NaCl stress. Patra *et al*. (2010), in turn, held introgression and functional expression studies in tobacco plants using *PcINO1* (a) and *McIMTI* (b) [inositol methyl transferase, IMTI, from the common ice plant *M. crystallinum*] genes. After submission of the obtained transgenic lines to saline and oxidative stresses it was observed that all plants presented higher performances in terms of growth potential and photosynthetic activity and were less prone to oxidative and salt stresses when compared to the controls. Physiological experiments demonstrated the superiority of the *PcINO1–McIMT1* double transgenic plants to withstand the salt stress accompanied by the accumulation of both myo-inositol and methylated inositols in the system over the transgenic plants with either of the single gene(s).

### **4.4 Trehalose**

Trehalose is a non-reducing α,α-1,1-linked glucose disaccharide that functions as an energy source and a storage form of more reactive glucose in lower organisms (Galinski, 1993). At least three different pathways for the biological synthesis of trehalose have been reported (Elbein *et al*., 2003). In plants, the synthesis of this sugar occurs normally by the formation of the trehalose-6-phosphate (T6P) from the UDP-glucose and glucose-6-phosphate, a reaction catalyzed by the trehalose 6-phosphate synthase (TPS). Afterwards the T6P is dephosphorylated by the trehalose-6-phosphate phosphatase (TPP) resulting in the formation of free trehalose (Wingler, 2002).

Transcriptomics of Sugarcane Osmoprotectants Under Drought 99

(up to 8.805–12.863 mg/g fresh weight), whereas trehalose was undetectable in nontransgenic plants. Trehalose accumulation in these plants resulted in increased drought tolerance, as shown by the drought physiological indexes, such as the rate of bound water/free water, plasma membrane permeability, malondialdehyde content, chlorophyll a

Besides a review of the up to date evaluations, the present preview analyzes the sugarcane transcriptome under drought, using a combination of high-throughput transcriptome profiling by SuperSAGE with the Solexa® sequencing technology, allowing the *in silico* identification of potential tags related to osmoprotectants in response to this stress. In the scope of this report four libraries have been generated by the Bulked-Extremes SuperTag Digital Gene Expression (BE-STDGE) method (GenXPro GmbH, Frankufurt, Germany), using bulked root tissues from four drought-tolerant materials as compared with four bulked drought-sensible genotypes, aiming to generate a panel of differentially expressed stress-responsive genes. Both groups were submitted to the same experimental conditions at the glasshouses of CTC (a Brazilian Sugarcane Technology Center, in Piracicaba, state of São Paulo, Brazil), including 24 hours of water deficit stress as compared with non stressed controls. The SuperSAGE libraries produced 8,787,315 tags (26 bp) that, after exclusion of singlets, allowed the identification of 205,975 unitags. Most relevant BlastN matches (42 ≤ Score ≤ 52; intact CATG sequence and plus/plus alignments) comprised 567,420 tags, regarding 75,404 unitags with 164,860 different ESTs, most of them matching to sequences of the genus *Saccharum*. The coverage of the transcriptome by the tags, considering the number of tags per genotype in relation to the number of expected transcripts per cell (500,000; Kamalay & Goldberg, 1980), was 6.5 times for the tolerant and 5.8 for the sensible bulk, i.e., the number of expected transcripts in a single copy per cell should be represented by around six tags in each library. Coverage of this magnitude permits a comprehensive

and b contents, and activity of SOD and POD of the excised leaves.

**5. SuperSAGE: Looking for osmoprotectants in sugarcane** 

evaluation of a given transcriptome, including rare expressed transcripts.

Regarding BADH (EC 1.2.1.8) seven unitags were identified. Most of them (six) were upregulated (UR) in the first comparison (I) that refers to the bulk of tolerant accessions under stress *versus (vs)* non stressed tolerant control (Table 1). From these UR unitags, only one (SD186519) was also UR in the comparison II (sensible bulks under stress *vs* the sensible control; Table 1). This unitag was aligned (BlastN) with accession gb A275267.1 and TC139975 (SOGI, *Sacharum officinarum* Gene Index, release 3). The first EST refers to a cDNA showing a perfect match while the second regarded a transcript similar to a BADH (UniRef100\_Q6BD86) with a single mismatch. Two other UR unitags in the tolerant bulk (SD161066 and SD158219) showed contrasting expression (DR) as compared with the susceptible bulk (comparison II; Table 1). The SD158219 unitag aligned with the same TC139975 of SOGI database, in the 3'UTR region regarding the BADH cDNA of maize (gb BT067636.1). Alignments of tags in the 3'UTR region are expected when using this methodology that is based on cDNAs originated from the vicinity of the poli-A tail of

**5.1 Betaine aldehyde dehydrogenase (BADH)** 

RNAs.

Although trehalose is widely distributed in the nature (including prokaryotes and eukaryotes) this sugar has been isolated from a few plant species, being identified in ripening fruits of species from the Apiacea family, in the leaves of *Selaginella lepidophylla* and its relatives (Goddijn & van Dun, 1999) as well as in *Arabidopsis thaliana* (Wingler *et al*., 2002). According to Elbein *et al*. (2003) in yeast and plants trehalose may serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from denaturation caused by a variety of stress conditions, including desiccation. Kaushik & Bhat (2003) demonstrated that this sugar is an exceptional stabilizer of proteins, while Fait *et al*. (2006) considered its role in the maintenance of the conformation of both storage and housekeeping proteins during dehydration in seeds of *A. thaliana*.

Considering the evidences in favor of a positive role of this protein under abiotic stress, trehalose has been widely evaluated in expression assays. Garg *et al*. (2002) showed that trehalose overproduction has considerable potential for improving abiotic stress tolerance in rice transgenic plants, which accumulated increased amounts of it and showed high levels of tolerance to salt, drought, and low-temperature stresses, as compared with the nontransformed controls. Resurrection plants (*S. lepidophylla*) have the ability to withstand almost complete water loss in their vegetative tissues, being able to remain alive in the dried state for several years and regaining full functionality upon re-hydration (Scott, 2000). Such capacity is associated with an accumulation of trehalose in plant leaves (Iturriaga *et al*., 2000).

Almeida *et al*. (2005) transformed tobacco plants with the *AtTPS1* gene from *Arabidopsis*. The transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Additionally, the transgenic plants were submitted to drought, desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 °C and 35 °C). The transformed plants revealed a reduced increase of drought tolerance and dehydration but exhibited a considerable tolerance to osmotic and temperature stresses, indicating that the heterologous expression of *TPS1* gene from *Arabidopsis* can be successfully used to increase abiotic stress in plants.

Zhang *et al*. (2005) transformed tobacco plants with the trehalose synthase gene from *Grifola frondosa*, submitting transformed plants to drought and salinity stresses (MS medium containing 1 % NaCl). Compared with non-transgenic plants, the transgenic ones were able to accumulate high levels of trehalose, which were increased up to 2.126–2.556 mg/g fresh weight, although levels were undetectable in non-transgenic plants. This trehalose accumulation resulted in increased tolerance to drought and salinity improving physiological performance, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, the activity of superoxide dismutase (SOD) and peroxidase (POD) in excised leaves.

Some evaluations of trehalose activity have been also carried out in sugarcane. Wang *et al*. (2005) transferred the trehalose synthase gene from *G. frondosa* to sugarcane, analyzing the tolerance of the transgenics to osmotic stress [PEG8000 17.4 % (w/v)]. While the nontransformed plants began turning yellow at the third day, with wilting and drying extending from old leaves to young leaves in seven days, all transgenic plants kept green and began turning yellow only at the seventh day, indicating their improvement regarding osmotic stress tolerance. Zhang *et al*. (2006) carried out a similar approach using the same gene in sugarcane, generating transgenic plants that accumulated high levels of trehalose

Although trehalose is widely distributed in the nature (including prokaryotes and eukaryotes) this sugar has been isolated from a few plant species, being identified in ripening fruits of species from the Apiacea family, in the leaves of *Selaginella lepidophylla* and its relatives (Goddijn & van Dun, 1999) as well as in *Arabidopsis thaliana* (Wingler *et al*., 2002). According to Elbein *et al*. (2003) in yeast and plants trehalose may serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from denaturation caused by a variety of stress conditions, including desiccation. Kaushik & Bhat (2003) demonstrated that this sugar is an exceptional stabilizer of proteins, while Fait *et al*. (2006) considered its role in the maintenance of the conformation of both storage and

Considering the evidences in favor of a positive role of this protein under abiotic stress, trehalose has been widely evaluated in expression assays. Garg *et al*. (2002) showed that trehalose overproduction has considerable potential for improving abiotic stress tolerance in rice transgenic plants, which accumulated increased amounts of it and showed high levels of tolerance to salt, drought, and low-temperature stresses, as compared with the nontransformed controls. Resurrection plants (*S. lepidophylla*) have the ability to withstand almost complete water loss in their vegetative tissues, being able to remain alive in the dried state for several years and regaining full functionality upon re-hydration (Scott, 2000). Such capacity is

Almeida *et al*. (2005) transformed tobacco plants with the *AtTPS1* gene from *Arabidopsis*. The transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Additionally, the transgenic plants were submitted to drought, desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 °C and 35 °C). The transformed plants revealed a reduced increase of drought tolerance and dehydration but exhibited a considerable tolerance to osmotic and temperature stresses, indicating that the heterologous expression of *TPS1* gene

Zhang *et al*. (2005) transformed tobacco plants with the trehalose synthase gene from *Grifola frondosa*, submitting transformed plants to drought and salinity stresses (MS medium containing 1 % NaCl). Compared with non-transgenic plants, the transgenic ones were able to accumulate high levels of trehalose, which were increased up to 2.126–2.556 mg/g fresh weight, although levels were undetectable in non-transgenic plants. This trehalose accumulation resulted in increased tolerance to drought and salinity improving physiological performance, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, the activity of superoxide dismutase (SOD) and

Some evaluations of trehalose activity have been also carried out in sugarcane. Wang *et al*. (2005) transferred the trehalose synthase gene from *G. frondosa* to sugarcane, analyzing the tolerance of the transgenics to osmotic stress [PEG8000 17.4 % (w/v)]. While the nontransformed plants began turning yellow at the third day, with wilting and drying extending from old leaves to young leaves in seven days, all transgenic plants kept green and began turning yellow only at the seventh day, indicating their improvement regarding osmotic stress tolerance. Zhang *et al*. (2006) carried out a similar approach using the same gene in sugarcane, generating transgenic plants that accumulated high levels of trehalose

associated with an accumulation of trehalose in plant leaves (Iturriaga *et al*., 2000).

from *Arabidopsis* can be successfully used to increase abiotic stress in plants.

peroxidase (POD) in excised leaves.

housekeeping proteins during dehydration in seeds of *A. thaliana*.

(up to 8.805–12.863 mg/g fresh weight), whereas trehalose was undetectable in nontransgenic plants. Trehalose accumulation in these plants resulted in increased drought tolerance, as shown by the drought physiological indexes, such as the rate of bound water/free water, plasma membrane permeability, malondialdehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves.

### **5. SuperSAGE: Looking for osmoprotectants in sugarcane**

Besides a review of the up to date evaluations, the present preview analyzes the sugarcane transcriptome under drought, using a combination of high-throughput transcriptome profiling by SuperSAGE with the Solexa® sequencing technology, allowing the *in silico* identification of potential tags related to osmoprotectants in response to this stress. In the scope of this report four libraries have been generated by the Bulked-Extremes SuperTag Digital Gene Expression (BE-STDGE) method (GenXPro GmbH, Frankufurt, Germany), using bulked root tissues from four drought-tolerant materials as compared with four bulked drought-sensible genotypes, aiming to generate a panel of differentially expressed stress-responsive genes. Both groups were submitted to the same experimental conditions at the glasshouses of CTC (a Brazilian Sugarcane Technology Center, in Piracicaba, state of São Paulo, Brazil), including 24 hours of water deficit stress as compared with non stressed controls. The SuperSAGE libraries produced 8,787,315 tags (26 bp) that, after exclusion of singlets, allowed the identification of 205,975 unitags. Most relevant BlastN matches (42 ≤ Score ≤ 52; intact CATG sequence and plus/plus alignments) comprised 567,420 tags, regarding 75,404 unitags with 164,860 different ESTs, most of them matching to sequences of the genus *Saccharum*. The coverage of the transcriptome by the tags, considering the number of tags per genotype in relation to the number of expected transcripts per cell (500,000; Kamalay & Goldberg, 1980), was 6.5 times for the tolerant and 5.8 for the sensible bulk, i.e., the number of expected transcripts in a single copy per cell should be represented by around six tags in each library. Coverage of this magnitude permits a comprehensive evaluation of a given transcriptome, including rare expressed transcripts.

### **5.1 Betaine aldehyde dehydrogenase (BADH)**

Regarding BADH (EC 1.2.1.8) seven unitags were identified. Most of them (six) were upregulated (UR) in the first comparison (I) that refers to the bulk of tolerant accessions under stress *versus (vs)* non stressed tolerant control (Table 1). From these UR unitags, only one (SD186519) was also UR in the comparison II (sensible bulks under stress *vs* the sensible control; Table 1). This unitag was aligned (BlastN) with accession gb A275267.1 and TC139975 (SOGI, *Sacharum officinarum* Gene Index, release 3). The first EST refers to a cDNA showing a perfect match while the second regarded a transcript similar to a BADH (UniRef100\_Q6BD86) with a single mismatch. Two other UR unitags in the tolerant bulk (SD161066 and SD158219) showed contrasting expression (DR) as compared with the susceptible bulk (comparison II; Table 1). The SD158219 unitag aligned with the same TC139975 of SOGI database, in the 3'UTR region regarding the BADH cDNA of maize (gb BT067636.1). Alignments of tags in the 3'UTR region are expected when using this methodology that is based on cDNAs originated from the vicinity of the poli-A tail of RNAs.

Transcriptomics of Sugarcane Osmoprotectants Under Drought 101

Alt Tra: Alternative transcript version; Chr: chromosome; put: putative; exp.: expressed; Align: Alignment Length; Mis: Mismatch; Orient: Orientation. BADH (Betaine aldehyde dehydrogenase); P5CS (Delta(1)-pyrroline-5-carboxylate synthetase); P5CR (Delta(1)-

pyrroline-5-carboxylate reductase); MIPS (Myo-inositol 1-phosphate synthase); TPS (Trehalose-6-phosphate synthase); TPP (Trehalose-

phosphatase protein).

Table 2. BlastN results of SuperSAGE osmoprotectants-related tags from sugarcane roots

under hydric deficif against cDNAs of *Sorghum bicolor* (Phytozome database).


Alt Tra: Alternative transcript version; Chr: chromosome; put: putative; exp.: expressed; Align: Alignment Length; Mis: Mismatch; Orient: Orientation. BADH (Betaine aldehyde dehydrogenase); P5CS (Delta(1)-pyrroline-5-carboxylate synthetase); P5CR (Delta(1)-pyrroline-5-carboxylate reductase); MIPS (Myo-inositol 1-phosphate synthase); TPS (Trehalose-6-phosphate synthase); TPP (Trehalosephosphatase protein)

Table 1. Comparison of sugarcane SuperSAGE libraries showing tags annotated as osmoprotectant-relative, the respective fold change, and regulation of the tags (*p* ≤ 0.05).

On the other hand, the SD161066 unitag aligned with two mismatches in the TC24905 of the PAVIGI (*Panicum virgatum* Gene Index, release 1). This TC presented an annotation against a partially homologue (85 %) *Zea mays* sequence of betaine aldehyde dehydrogenase.

All the unitags were aligned against the *Sorghum bicolor* genome available at the Phytozome site (http://www.phytozome.net/) and the respective cDNAs. From seven BADH unitags three mapped on the genome (SD161066 at chromosome 7; SD160278 and SD7041 both at chromosome 6) (see the loci at the Table 2). As mentioned by Ming *et al*. (1998), the levels and patterns of chromosome structural rearrangement in *Saccharum* and *Sorghum* based in their close relationship, high degree of colinearity, and cross-hybridization of DNA probes, all impel use of the small genome of *Sorghum* to guide molecular mapping and positional cloning in *Saccharum*.

For each identified locus a single transcript was identified in *Sorghum* (Table 2). Further, as shown in Table 2, the unitag SD7041 presented a perfect BlastN alignment (score 52) of +/- (plus/minus) type against the transcript Sb06g019200.1, the same cDNA that aligned to another unitag in the +/+ sense (in this last case presenting some mismatches). A detailed analysis of the +/- alignment revealed its positioning in a complementary 3'UTR region in


Table 2. BlastN results of SuperSAGE osmoprotectants-related tags from sugarcane roots under hydric deficif against cDNAs of *Sorghum bicolor* (Phytozome database).

100 Plants and Environment

**Comparison I II III IV Libraries DTS/DTC DSS/DSC DTS/DSS DTC/DSC Tag Annotation FC Reg FC Reg FC Reg FC Reg**  SD186519 BADH 2.2 UR 1.6 UR 1.80 UR 1.33 ns SD161066 BADH 1.3 UR -1.4 DR 2.81 UR 1.58 UR SD158219 BADH 6.4 UR -5.3 DR 6.36 UR -5.32 DR SD160278 BADH 2.0 UR -1.4 ns 3.44 UR 1.27 ns SD167799 BADH 2.1 UR -1.7 ns 1.57 ns -2.20 DR SD167796 BADH 3.4 UR 1.1 ns 2.04 ns -1.51 ns SD7041 BADH 1.2 ns -1.3 ns 2.69 UR 1.64 ns SD68048 P5CS 3.3 UR 4.7 UR 2.0 ns 2.8 UR SD130985 P5CS 5.8 UR -1.1 ns 1.2 ns -5.2 ns SD154736 P5CR 1.3 ns -1.1 ns 1.3 ns -1.1 ns SD175871 P5CR 1.2 ns -1.7 ns 1.3 ns -1.6 ns SD50849 MIPS 1.3 UR -2.9 DR 2.22 UR -1.71 DR SD50847 MIPS 1.1 ns -2.3 DR -1.03 ns -2.50 DR SD134872 MIPS 1.1 ns -3.2 DR # ns -3.41 ns SD61158 TPS 2.4 UR 1.8 ns 3.62 UR 2.80 UR SD146286 TPS 2.5 ns 2.8 UR 1.28 ns 1.41 ns SD267553 TPS -1.3 ns -2.7 ns -1.76 ns -3.73 DR SD6994 TPP 6.3 UR -1.8 DR 7.37 UR -1.50 ns SD25600 TPP 2.4 UR # ns 2.38 UR # ns SD190162 TPP 7.1 UR 1.5 ns 8.50 UR 1.76 ns Alt Tra: Alternative transcript version; Chr: chromosome; put: putative; exp.: expressed; Align: Alignment Length; Mis: Mismatch; Orient: Orientation. BADH (Betaine aldehyde dehydrogenase); P5CS (Delta(1)-pyrroline-5-carboxylate synthetase); P5CR (Delta(1)-pyrroline-5-carboxylate reductase); MIPS (Myo-inositol 1-phosphate synthase); TPS (Trehalose-6-phosphate synthase); TPP (Trehalose-

Table 1. Comparison of sugarcane SuperSAGE libraries showing tags annotated as osmoprotectant-relative, the respective fold change, and regulation of the tags (*p* ≤ 0.05).

partially homologue (85 %) *Zea mays* sequence of betaine aldehyde dehydrogenase.

On the other hand, the SD161066 unitag aligned with two mismatches in the TC24905 of the PAVIGI (*Panicum virgatum* Gene Index, release 1). This TC presented an annotation against a

All the unitags were aligned against the *Sorghum bicolor* genome available at the Phytozome site (http://www.phytozome.net/) and the respective cDNAs. From seven BADH unitags three mapped on the genome (SD161066 at chromosome 7; SD160278 and SD7041 both at chromosome 6) (see the loci at the Table 2). As mentioned by Ming *et al*. (1998), the levels and patterns of chromosome structural rearrangement in *Saccharum* and *Sorghum* based in their close relationship, high degree of colinearity, and cross-hybridization of DNA probes, all impel use of the small genome of *Sorghum* to guide molecular mapping and positional

For each identified locus a single transcript was identified in *Sorghum* (Table 2). Further, as shown in Table 2, the unitag SD7041 presented a perfect BlastN alignment (score 52) of +/- (plus/minus) type against the transcript Sb06g019200.1, the same cDNA that aligned to another unitag in the +/+ sense (in this last case presenting some mismatches). A detailed analysis of the +/- alignment revealed its positioning in a complementary 3'UTR region in

phosphatase protein)

cloning in *Saccharum*.

pyrroline-5-carboxylate reductase); MIPS (Myo-inositol 1-phosphate synthase); TPS (Trehalose-6-phosphate synthase); TPP (Trehalose-

phosphatase protein).

Transcriptomics of Sugarcane Osmoprotectants Under Drought 103

Concerning MIPS (EC 5.5.1.4), from three annotated unitags only one (SD50849) was overexpressed (UR) in the comparison I, while all three unitags were repressed (DR) in the comparison II (Table 1). All three unitags mapped in the chromosome 1 of *Sorghum* in the *locus* Sb01g044290, with three predicted alternative transcripts. Two of the tags presented perfect alignments (score 52) with the referred *locus*, one of them (SD134872) aligned in the CDS of the three predicted transcripts, while the other (SD50847) aligned in the region covering the transition from CDS to the first four bases of the 3'UTR. The remaining unitag (SD50849) presented a mismatch with the *Sorghum* genome and also with the respective transcript (Table 2). Interestingly, this unitag represents a possible single base polymorphism (A/G substitution) compared to unitag SD50847. Regarding this polymorphism, sequencing errors are not probable, especially considering the tag frequency. Both unitags were the most frequent in the SuperSAGE libraries, varying from 37 to 59 tpm, while the unitag SD134872 presented less than 3 tpm. This potential SNP and its relation to the differential expression in the tolerant (comparison I) is worth additional

Three unitags have been annotated for TPS (EC 2.4.1.15) one of them UR in the comparison I (SD61158) the second UR in comparison II (SD146286) and the third (SD267553) not varying significantly among the different compared conditions (Table 1). All three unitags mapped against the *Sorghum* genome in the chromosomes 4 and 9 (Table 2). Considering the matching region of chromosome 4, a single *Sorghum* transcript was associated (Sb04g035560.1) similar to a putative uncharacterized protein. Compared to this transcript, the SD146286 unitag presented a substitution (G/A) in a CDS region, while the unitag SD61158 presented two G/A substitutions. From the three unitags, SD61158 was the most expressed, varying from 16 to 39 tpm, while the other two tags were less frequent (< 4 tpm). The alignment against chromosome 9 revealed two mismatches as compared with the

For trehalose-phosphatase (EC 3.1.3.12) three unitags were UR in the comparison I (Table 1), while one of them (SD6994) was also DR in the comparison II. The mentioned unitag and the SD190162 unitag presented the highest FCs (6.3 and 7.1) for comparison I (Table 1) with the related ESTs sharing 91 % identity in 230 aligned bases. From all three unitags, SD6994 was the most expressed (13 to 83 tpm), followed by SD190162 (3 to 20 tpm) and by SD25600 (< 3 tpm). All three unitags mapped in the sorghum genome, in the chromosome 7, aligning with the transcript Sb07g020270.1 (Table 2) annotated as a putative trehalose-6-phosphate synthase. The most expressed unitag was SD6994 aligned perfectly with a *Sorghum* transcript at the 3'UTR portion of the sequence. Besides, the unitag SD25600 is similar to the SD6994, with a single A/C substitution. The third unitag (SD190162) also aligned in the 3'UTR of the same *sorghum* transcript in a more distant position in comparison to the 3' end than SD6994 presenting a single mismatch to the *Sorghum* transcript (T in *Sorghum* and C in the tag). If the alignment was perfect, one could argument that it was the consequence of an incomplete digestion by the *Nla*III enzyme. However, in the present work a double digestion was

**5.4 Myo-inositol 1-phosphate synthase (MIPS)** 

efforts for its validation in the future.

unitag SD267553.

**5.5 Trehalose-6-phosphate synthase (TPS)** 

**5.6 Trehalose-phosphatase protein (TPP)** 

carried out, avoiding this error source.

the reverse strand as compared with the genome and the transcript, suggesting a putative NAT (natural antisense transcript). NATs are naturally occurring RNA transcripts that are complementary to other endogenous RNA transcripts. They may regard Cis-natural antisense transcripts (cis-NATs) when transcribed at the same genomic loci of other genes, but from the opposite direction, while trans-NATs are transcribed from different genomic *loci* (Lavorgna *et al.*, 2004). Such antisense transcripts do not compose an uniform group, but present some features in common, with emphasis on the complementarity to the sense genic sequences that may (or not) codify proteins (Faghihi & Wahlestedt, 2009). NATs have been reported in different types of expression assays, including SAGE (Quéré *et al.*, 2004), LongSAGE (Obermeier *et al*., 2009) and SuperSAGE (Molina *et al*., 2008).

An annotation against the Phytozome regarding the BADH unitags identified them as aldehyde dehydrogenase, a protein family that includes BADH. Considering their absolute frequency observed (from six to 95 tags per million – tpm - in the tolerant bulk), the sensitivity of the SuperSAGE methodology in detecting rare transcripts could be verified in posterior assays.

For most UR unitags (four out of six) in the tolerant bulk (Table 1), the amount of BADH tags after the drought tolerant stresses (DTS) was significantly higher (comparison III) than that observed for the drought sensible stressed (DSS) bulk. In the absence of stress (comparison IV), both bulks, that are genetically diverse, presented variable expression, depending on the unitag. Still, a single unitag (SD7041) presented no significant expression differences after stress in both comparisons (I and III).

### **5.2 Delta(1)-pyrroline-5-carboxylate synthetase (P5CS)**

With respect to P5CS (EC 2.7.2.11) two unitags were induced in the comparison I (stressed tolerant *vs* tolerant control), while in the comparison II (sensible bulk) a unitag (SD68048) appeared induced (Table 1). For this unitag the observed difference among both bulks under stress was not significant at the studied level (*p* ≤ 0.05; comparison III), revealing similar amounts in both bulks 24 hours after drought stress. The same unitag (SD68048) was also UR in the comparison IV (both controls without stress), being significantly most represented than in the tolerant bulk (Table 1).

In turn, the SD130985 unitag was UR in the comparison I (tolerant bulk stressed *vs* control), presenting a higher FC than that estimated for the unitag SD68048 (5.8 *vs* 3.3). Both unitags aligned to the 3'UTR region of the associated ESTs with a single mismatch (data not shown). Considering the alignment against the *Sorghum* genome, both tags mapped, with SD68048 in the chromosome 3 (associated with a transcript) while SD130985 mapped in the chromosome 9 in a region corresponding to two alternative transcripts with different sizes regarding their UTR portions, with no consequences to the CDS and the final protein. The absolute frequencies of these unitags in the sugarcane transcriptome via SuperSAGE varied from three to 19 tpm (tags per million) considering their presence in the tolerant bulk.

### **5.3 Delta(1)-pyrroline-5-carboxylate reductase (P5CR)**

For P5CR (EC 1.5.1.2) two unitags have been observed, but these presented no significant variation in the analyzed SuperSAGE comparisons, being therefore not commented here. After mapping both unitags against the *Sorghum* genome, only the SD154736 tag mapped in the chromosome 3 (Table 2) in a CDS region of an identified transcript.

### **5.4 Myo-inositol 1-phosphate synthase (MIPS)**

102 Plants and Environment

the reverse strand as compared with the genome and the transcript, suggesting a putative NAT (natural antisense transcript). NATs are naturally occurring RNA transcripts that are complementary to other endogenous RNA transcripts. They may regard Cis-natural antisense transcripts (cis-NATs) when transcribed at the same genomic loci of other genes, but from the opposite direction, while trans-NATs are transcribed from different genomic *loci* (Lavorgna *et al.*, 2004). Such antisense transcripts do not compose an uniform group, but present some features in common, with emphasis on the complementarity to the sense genic sequences that may (or not) codify proteins (Faghihi & Wahlestedt, 2009). NATs have been reported in different types of expression assays, including SAGE (Quéré *et al.*, 2004),

An annotation against the Phytozome regarding the BADH unitags identified them as aldehyde dehydrogenase, a protein family that includes BADH. Considering their absolute frequency observed (from six to 95 tags per million – tpm - in the tolerant bulk), the sensitivity of the SuperSAGE methodology in detecting rare transcripts could be verified in

For most UR unitags (four out of six) in the tolerant bulk (Table 1), the amount of BADH tags after the drought tolerant stresses (DTS) was significantly higher (comparison III) than that observed for the drought sensible stressed (DSS) bulk. In the absence of stress (comparison IV), both bulks, that are genetically diverse, presented variable expression, depending on the unitag. Still, a single unitag (SD7041) presented no significant expression

With respect to P5CS (EC 2.7.2.11) two unitags were induced in the comparison I (stressed tolerant *vs* tolerant control), while in the comparison II (sensible bulk) a unitag (SD68048) appeared induced (Table 1). For this unitag the observed difference among both bulks under stress was not significant at the studied level (*p* ≤ 0.05; comparison III), revealing similar amounts in both bulks 24 hours after drought stress. The same unitag (SD68048) was also UR in the comparison IV (both controls without stress), being significantly most represented

In turn, the SD130985 unitag was UR in the comparison I (tolerant bulk stressed *vs* control), presenting a higher FC than that estimated for the unitag SD68048 (5.8 *vs* 3.3). Both unitags aligned to the 3'UTR region of the associated ESTs with a single mismatch (data not shown). Considering the alignment against the *Sorghum* genome, both tags mapped, with SD68048 in the chromosome 3 (associated with a transcript) while SD130985 mapped in the chromosome 9 in a region corresponding to two alternative transcripts with different sizes regarding their UTR portions, with no consequences to the CDS and the final protein. The absolute frequencies of these unitags in the sugarcane transcriptome via SuperSAGE varied from three to 19 tpm (tags per million) considering their presence in the tolerant bulk.

For P5CR (EC 1.5.1.2) two unitags have been observed, but these presented no significant variation in the analyzed SuperSAGE comparisons, being therefore not commented here. After mapping both unitags against the *Sorghum* genome, only the SD154736 tag mapped in

LongSAGE (Obermeier *et al*., 2009) and SuperSAGE (Molina *et al*., 2008).

differences after stress in both comparisons (I and III).

than in the tolerant bulk (Table 1).

**5.2 Delta(1)-pyrroline-5-carboxylate synthetase (P5CS)** 

**5.3 Delta(1)-pyrroline-5-carboxylate reductase (P5CR)** 

the chromosome 3 (Table 2) in a CDS region of an identified transcript.

posterior assays.

Concerning MIPS (EC 5.5.1.4), from three annotated unitags only one (SD50849) was overexpressed (UR) in the comparison I, while all three unitags were repressed (DR) in the comparison II (Table 1). All three unitags mapped in the chromosome 1 of *Sorghum* in the *locus* Sb01g044290, with three predicted alternative transcripts. Two of the tags presented perfect alignments (score 52) with the referred *locus*, one of them (SD134872) aligned in the CDS of the three predicted transcripts, while the other (SD50847) aligned in the region covering the transition from CDS to the first four bases of the 3'UTR. The remaining unitag (SD50849) presented a mismatch with the *Sorghum* genome and also with the respective transcript (Table 2). Interestingly, this unitag represents a possible single base polymorphism (A/G substitution) compared to unitag SD50847. Regarding this polymorphism, sequencing errors are not probable, especially considering the tag frequency. Both unitags were the most frequent in the SuperSAGE libraries, varying from 37 to 59 tpm, while the unitag SD134872 presented less than 3 tpm. This potential SNP and its relation to the differential expression in the tolerant (comparison I) is worth additional efforts for its validation in the future.

### **5.5 Trehalose-6-phosphate synthase (TPS)**

Three unitags have been annotated for TPS (EC 2.4.1.15) one of them UR in the comparison I (SD61158) the second UR in comparison II (SD146286) and the third (SD267553) not varying significantly among the different compared conditions (Table 1). All three unitags mapped against the *Sorghum* genome in the chromosomes 4 and 9 (Table 2). Considering the matching region of chromosome 4, a single *Sorghum* transcript was associated (Sb04g035560.1) similar to a putative uncharacterized protein. Compared to this transcript, the SD146286 unitag presented a substitution (G/A) in a CDS region, while the unitag SD61158 presented two G/A substitutions. From the three unitags, SD61158 was the most expressed, varying from 16 to 39 tpm, while the other two tags were less frequent (< 4 tpm). The alignment against chromosome 9 revealed two mismatches as compared with the unitag SD267553.

### **5.6 Trehalose-phosphatase protein (TPP)**

For trehalose-phosphatase (EC 3.1.3.12) three unitags were UR in the comparison I (Table 1), while one of them (SD6994) was also DR in the comparison II. The mentioned unitag and the SD190162 unitag presented the highest FCs (6.3 and 7.1) for comparison I (Table 1) with the related ESTs sharing 91 % identity in 230 aligned bases. From all three unitags, SD6994 was the most expressed (13 to 83 tpm), followed by SD190162 (3 to 20 tpm) and by SD25600 (< 3 tpm). All three unitags mapped in the sorghum genome, in the chromosome 7, aligning with the transcript Sb07g020270.1 (Table 2) annotated as a putative trehalose-6-phosphate synthase. The most expressed unitag was SD6994 aligned perfectly with a *Sorghum* transcript at the 3'UTR portion of the sequence. Besides, the unitag SD25600 is similar to the SD6994, with a single A/C substitution. The third unitag (SD190162) also aligned in the 3'UTR of the same *sorghum* transcript in a more distant position in comparison to the 3' end than SD6994 presenting a single mismatch to the *Sorghum* transcript (T in *Sorghum* and C in the tag). If the alignment was perfect, one could argument that it was the consequence of an incomplete digestion by the *Nla*III enzyme. However, in the present work a double digestion was carried out, avoiding this error source.

Transcriptomics of Sugarcane Osmoprotectants Under Drought 105

Anderson, D.P.; Welch, J.M. & Robinson, J. (2009) Drought Impact on Agriculture

http://agecoext.tamu.edu/fileadmin/user\_upload/Documents/Resources/Public

Ashraf, M. & Foolad, M.R. (2007) Roles of glycine betaine and proline in improving plant

Bartels, D. & Souer, E. (2004) Molecular responses of higher plants to dehydration. In: *Plant* 

Bartels, D. & Sunkar, R. (2005) Drought and salt tolerance in plants. *Critical Reviews in Plant* 

Bohnert, H.J. & Jensen, R.G. (1996) Strategies for engineering water-stress tolerance in

Boken, V.K. (2005) Agricultural Drought and Its Monitoring and Prediction: Some Concepts.

Bower, N.I.; Casu, R.E.; Maclean, D.J.; Reverter, A.; Chapman, S.C. & Manners, J.M. (2005)

Bray, E.A.; Bailey-Serres, J. & Weretilnyk, E. (2000) Responses to abiotic stresses. In:

Burton, R.S. (1991) Regulation of proline synthesis in osmotic response: effects of protein

Calsa Jr , T. & Figueira, A. (2007) Serial analysis of gene expression in sugarcane (*Saccharum*

Casu, R.E.; Dimmock, C.M.; Chapman, S.C.; Grof, C.P.L.; McIntyre, C.L.; Bonnett, G.D. &

Casu, R.E.; Grof, C.P.L.; Rae, A.L.; McIntyre, C.L.; Dimmock, C.M. & Manners, J.M. (2003)

Casu, R.E.; Hotta, C.T. & Souza, G.M. (2010) Functional Genomics: Transcriptomics of

abiotic stress resistance. *Environmental and Experimental Botany*, Vol. *59*, (March

*Responses to Abiotic Stress*, Hirt, H. & Shinozaki, K. (Eds), Springer-Verlag, Berlin

plants. *Trends in Biotechnology*. Vol. 14, (March 1996), No. 3, pp. 89–97, ISSN 0167-

In: *Monitoring and Predicting Agricultural Drought: A Global Study*, Boken, V.K.; Cracknell, A.P. & Heathcote, R.L. (Eds.), 3-10, Oxford University Press, ISBN 978-0-

Transcriptional response of sugarcane roots to methyl jasmonate. *Plant Science*, Vol.

*Biochemistry and Molecular Biology of Plants*, Gruissem, W.; Buchannan, B. & Jones, R. (Eds.), pp. 1158-1249, American Society of Plant Physiologists, ISBN 978-094-308-

synthesis inhibitors. *Journal of Experimental Zoology*, Vol. 259, (August 1991), No. 2,

spp.). *Plant Molecular Biology*, Vol. 63, (April 2007), No. 6, pp. 745–762, ISSN 1573-

Manners, J.M.M. (2004) Identication of differentially expressed transcripts from maturing stem of sugarcane by *in silico* analysis of stem expressed sequence tags and gene expression proling. *Plant Molecular Biology*, Vol. 54, (March 2004), No. 4,

Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis. *Plant Molecular Biology*, Vol. 52, (May 2003), No. 2, pp. 371–386,

Sugarcane — Current Status and Future Prospects. In: *Genetics, Genomics And Breeding of Sugarcane*, Henry, R.J. & Kole, C. (Eds.), 167-190, CRC Press, ISBN

Approaches \$1 Billion Early in 2009. 05.05.2011, Available from:

ations/DroughtImpact.pdf.

19-516234-9, New York, USA.

839-6, Rockville, USA.

pp. 272–277, ISSN 1932-5231.

pp. 503–517, ISSN 1573-5028.

9781578086849, New York, USA.

ISSN 1573-5028.

Heidelberg.

7799.

5028.

2007), No. 2, pp. 206-216, ISSN 0098-8472.

*Sciences*, Vol. 24, No. 1, pp. 23-58, ISSN 1549-7836.

168, (March 2005), No. 3, pp. 761–772, ISSN 0168-9452.

### **6. Concluding remarks**

The present review highlights how scarce information about sugarcane osmoprotectants are at physiological, genomic and transcriptomic levels. Many crops lack the ability to efficiently synthesize some types of osmoprotectants that are naturally accumulated by stress-tolerant plants. Our SuperSAGE data revealed that all procured osmoprotectants categories are present and expressed in sugarcane. However, most of them are discretely expressed in roots after 24 hours of drought stress and also considering the same tissue in non-stressed controls. These discrete expression and their fold changes, detected by SuperSAGE, would probably remain undetected using other transcriptomics approaches, justifying the scarce previous informations about this protein group in sugarcane. Some identified candidates may have an osmoprotectant role in the initial response against drought in this crop and deserve additional evaluations. Hence, as shown by different research approaches in plants lacking osmoprotectants, their transgenic expression represented dramatic differences in the tolerance and survival to abiotic stresses including drought, salinity and freezing, what may be the case of sugarcane. The present chapter brings the first overview of the sugarcane transcriptome under drought with a combination of the high-throughput transcriptome profiling SuperSAGE technology coupled with a nextgeneration sequencing platform. This approach allowed the identification of some potential target osmoprotectants candidates in the drought stress response. Validation procedures as well as transient expression assays are planned for future works, aiming to collaborate with breeding and biotechnological approaches for benefit of the sugarcane culture, especially facing the scenario of future climate change.

### **7. Acknowledgments**

The authors thank Prof. Dr. Gunter Kahl (Frankfurt University, Germany), Dr. Bjorn Rotter, Ruth Jungmann, Nico Kretzdorn and Dr. Peter Winter (GenXPro GmbH, Germany) for scientific and technical advices. This work has been funded by Brazilian intitutions: Financier for Studies and Projects (FINEP), Foundation for Science and Technology of Pernambuco State (FACEPE), National Council for Scientific and Technological Development (CNPq) and Sugarcane Technology Center (CTC).

### **8. References**


The present review highlights how scarce information about sugarcane osmoprotectants are at physiological, genomic and transcriptomic levels. Many crops lack the ability to efficiently synthesize some types of osmoprotectants that are naturally accumulated by stress-tolerant plants. Our SuperSAGE data revealed that all procured osmoprotectants categories are present and expressed in sugarcane. However, most of them are discretely expressed in roots after 24 hours of drought stress and also considering the same tissue in non-stressed controls. These discrete expression and their fold changes, detected by SuperSAGE, would probably remain undetected using other transcriptomics approaches, justifying the scarce previous informations about this protein group in sugarcane. Some identified candidates may have an osmoprotectant role in the initial response against drought in this crop and deserve additional evaluations. Hence, as shown by different research approaches in plants lacking osmoprotectants, their transgenic expression represented dramatic differences in the tolerance and survival to abiotic stresses including drought, salinity and freezing, what may be the case of sugarcane. The present chapter brings the first overview of the sugarcane transcriptome under drought with a combination of the high-throughput transcriptome profiling SuperSAGE technology coupled with a nextgeneration sequencing platform. This approach allowed the identification of some potential target osmoprotectants candidates in the drought stress response. Validation procedures as well as transient expression assays are planned for future works, aiming to collaborate with breeding and biotechnological approaches for benefit of the sugarcane culture, especially

The authors thank Prof. Dr. Gunter Kahl (Frankfurt University, Germany), Dr. Bjorn Rotter, Ruth Jungmann, Nico Kretzdorn and Dr. Peter Winter (GenXPro GmbH, Germany) for scientific and technical advices. This work has been funded by Brazilian intitutions: Financier for Studies and Projects (FINEP), Foundation for Science and Technology of Pernambuco State (FACEPE), National Council for Scientific and Technological

Adams, P.; Thomas, J.C.; Vernon, D.M., Bohnert, H.J. & Jensen, R.G. (1992) Distinct cellular

Ahmadi A.; Emam Y. & Pessarakli M. (2010) Biochemical Changes In Maize Seedlings

Almeida, A.M.; Villalobos, E.; Araújo, S.S.; Leyman, B.; Van Dijck, P.; Alfaro-Cardoso, L.;

and organismic responses to salt stress. *Plant and Cell Physiology*, Vol. 33, (December

Exposed To Drought Stress Conditions At Different Nitrogen Levels. *Journal of* 

Fevereiro, P.S.; Torné, J.M. & Santos, D.M. (2005) Transformation of tobacco with an *Arabidopsis thaliana* gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses. *Euphytica*, Vol. 146, (November 2005) No. 1-2, pp. 165-176,

**6. Concluding remarks** 

facing the scenario of future climate change.

Development (CNPq) and Sugarcane Technology Center (CTC).

1992), No. 8, pp. 1215-1223, ISSN 0032-0781.

*Plant Nutrition*, Vol. 33, No. 4, pp. 541-556, ISSN 1532-4087.

**7. Acknowledgments** 

**8. References** 

ISSN 1573-5060.


Transcriptomics of Sugarcane Osmoprotectants Under Drought 107

Galinski, E.A. (1993) Compatible solutes of halophilic eubacteria: molecular principles,

Garg, A.K.; Ju-Kon, K., Owens, T.G.; Ranwala, A.P., Choi, Y.D.; Kochian, L.V. & Wu, R.J.

Gilardoni, P.A.; Schuck, S.; Jungling, R.; Rotter, B.; Baldwin, I.T. & Bonaventure, G. (2010)

Goddijn, O.J. & van Dun, K. (1999) Trehalose metabolism in plants. *Trends in Plant Science* ,

Goldemberg, J. (2007) Ethanol for a sustainable energy future. *Science*, Vol. 315, (February

Gomes, F.P.; Oliva, M.A.; Mielke, M.S.; Almeida, A-A.F. & Aquino, L.A. (2010) Osmotic

Gorham, J. (1995) Betaines in higher plants - biosynthesis and role in stress metabolism. In*:* 

Grennan, A.K. (2006) Abiotic Stress in Rice. An "Omic" Approach. *Plant Physiology*, Vol. 140,

Gupta, V.; Raghuvanshi, S.; Gupta,A.; Saini, N.; Gaur, A.; Khan, S.; Gupta, R.S.; Singh, J.;

Harb, A.; Krishnam, A.; Ambavaram, M.M.R. & Pereira, A. (2010) Molecular and

Hare, P.D. & Cress, W.A. (1997). Metabolic implications of stress-induced proline

Hazell, P. & Wood, S. (2008) Drivers of change in global agriculture. Philosophical

49, (July 1993), No. 6-7, pp. 487-496, ISSN 1420-682X.

*Plant Biology*, Vol. 10, (April 2010), pp.1-16, ISSN 1471-2229.

Vol. 4, (August 1999) , No. 8, pp. 315–319, ISSN 1360-1385.

2007), pp. 808–810, ISSN 1095-9203.

2010), No. 3, pp. 379-384, ISSN 0304-4238.

(April 2006), pp. 1139-1141, ISSN 1532-2548.

No. 3, pp. 1254-1271, ISSN 1532-2548.

6490.

England.

ISSN 1610-739X.

79-102, ISSN 1573-5087.

515, ISSN 1471-2970.

water-solute interaction, stress protection. *Cellular and Molecular Life Sciences*, Vol.

(2002). Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. *Proceedings of National Academy of Sciences of the United States of America,* Vol. 99, (December 2002), No. 25, pp. 15898-15903, ISSN 1091-

SuperSAGE analysis of the *Nicotiana attenuata* transcriptome after fatty acid-amino acid elicitation (FAC): identication of early mediators of insect responses. *BMC* 

adjustment, proline accumulation and cell membrane stability in leaves of *Cocos nucifera* submitted to drought stress. *Scientia Horticulturae*, Vol. 126, (September

*Amino Acids and their Derivates in Higher Plants*, Wallsgrove R.M. (Ed.), pp. 173–203, Society of Experimental Biology Seminar Series, ISBN 9780511721809, Cambridge,

Duttamajumder, S.K.; Srivastava, S.; Suman, A.; Khurana, J.P.; Kapur, R. & Tyagi, A.K. (2010) The water-deficit stress- and red-rot-related genes in sugarcane. *Functional and Integrative Genomics*, Vol. 10, (May 2010), No. 2, ISSN 1438-7948. Hamada, H.; Matsumura, H.; Tomita, R.; Terauchi, R.; Suzuki, K. & Kobayashi, K. (2008)

SuperSAGE revealed different classes of early resistance response genes in capsicum chinense plants harboring L-3-resistance gene infected with pepper mild mottle virus. *Journal of General Plant Pathology*, Vol. 74, (August 2008), pp. 313–321.

Physiological Analysis of Drought Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth. *Plant Physiology*, Vol. 154, (August 2010),

accumulation in plants. *Plant Growth Regulation*, Vol. 21, (February 1997), No. 2, pp.

Transactions of the Royal Society B, Vol. 363, (February 2008), No. 1491, pp. 495–


Cattivelli, L.; Rizza, F.; Badeck, F-W.; Mazzucotelli, E.; Mastrangelo, A.M.; Francia, E.;

Cesnik, R & Miocque J. (2004) *Melhoramento da cana-de-açúcar: marco sucro-alcooleiro no Brasil*. Embrapa Informações Tecnológicas, ISBN 85-7383-282-7, Brasília, Brasil. Chen, T.H.H. & Murata, N. (2008). Glycinebetaine: an effective protectant against abiotic

Chołuj, D.; Karwowska, R.; Ciszewska, A. & Jasińska, M. (2008) Influence of long-term

Coemans, B.; Matsumura, H.; Terauchi, R.; Remy, S.; Swennen, R. & Sagi, L. (2005)

Csonka, L.N. (1989) Physiological and genetic responses of bacteria to osmotic stress.

Daniels, J. & Roach B.T. (1987) Taxonomy and evolution. In: *Sugarcane Improvement Through Breeding*, Heinz D.J., (Ed.), pp. 7-84, Elsevier, ISBN 0444427694, New York, USA. Das-Chatterjee, A.D.; Goswami, L.; Maitra, Susmita.; Dastidar, K.G.; Ray, S & Majumder,

De Souza A.P.; Gaspar, M.; Silva, E.A.; Ulian, E.C.; Waclawovsky, A.J.; Nishiyama Jr , M.Y.;

Errabii, T.; Gandonou, C.B.; Essalmani, H.; Abrini J.; Idaomar M. & Skali-Senhaji N. (2006)

Faghihi, M.A. & Wahlestedt, C. (2009) Regulatory roles of natural antisense transcripts.

Fait, A.; Angelovici, R.; Less, H.; Ohad, I.; Urbanczyk-Wochniak, E.; Fernie, A.R. & Galili, G.

FAOSTAT (2009) Food and Agriculture Organization of the United Nations. In: FAO Statistical Databases. 05.04.2010, Available from: http://www.faostat.fao.org.

Vol. 5, (August 2006), No. 16, pp. 1488-1493, ISSN 1684-5315.

Vol. 105, (January 2008), No. 1-2, pp. 1–14, ISSN 0378-4290.

111, (August 2005), pp. 1118-1126, ISSN 0040-5752.

ISSN 1360-1385.

147, ISSN 1098-5557.

3988, ISSN 1873-3468.

1460-2423.

ISSN 1471-0080.

839-854, ISSN 1532-2548.

5881.

Caterina, M.; Tondelli, A. & Stanca, A.M. (2008) Drought tolerance improvement in crop plants: An integrated view from breeding to genomics. *Field Crops Research,*

stress in plants. *Trends in Plant Science*, Vol 13, (September 2008), No. 9, pp. 449-505,

drought stress on osmolyte accumulation in sugar beet (*Beta vulgaris* L.) plants. *Acta Physiologiae Plantarum*, Vol. 30, (September 2008), No. 5, pp. 679-687, ISSN 0137-

SuperSAGE combined with PCR walking allows global gene expression profiling of banana (*Musa acuminata*), a non model organism. *Theoretical Applied Genetics*, Vol.

*Microbiology and Molecular Biology Reviews*, Vol. 53, (March 1989), No. 1, pp. 121–

A.L. (2006) Introgression of a novel salt-tolerant L-myo-inositol 1-phosphate synthase from *Porteresia coarctata* (Roxb.) Tateoka (PcINO1) confers salt tolerance to evolutionary diverse organisms. *Febs Letters*, Vol. 580, (July 2006), No. 16, pp. 3980-

Santos, R.V.; Teixeira, M.M.; Souza, G.M. & Buckeridge, M.S. (2008) Elevated CO2 increases photosynthesis, biomass and productivity, and modies gene expression in sugarcane. *Plant, Cell and Environment*, Vol. 31, pp. 1116–1127, ISSN 1365-3040. Elbein, A.D.; Pan, Y.T.; Pastuszak, I. & Carroll, D. (2003) New insights on trehalose: a

multifunctional molecule. *Glycobiology*, Vol. 13, (April 2003), No. 4, pp. 17R-27, ISSN

Growth, proline and ion accumulation in sugarcane callus cultures under droughtinduced osmotic stress and its subsequent relief. *African Journal of Biotechnology*,

*Nature Reviews Molecular Cell Biology*, Vol. 10, (September 2009), No. 9, pp. 637-643,

(2006) *Arabidopsis* seed development and germination is associated with temporally distinct metabolic switches. *Plant Physiology*, Vol. 142, (November 2006), No. 3, pp.


Transcriptomics of Sugarcane Osmoprotectants Under Drought 109

Kishor, P.B.K.; Hong, Z.; Miao, G.H.; Hu, C.A.A, & Verma, D.P.S. (1995) Overexpression of

Kishor, P.B.K.; Sangam, S.; Amrutha, R.N.; Laxmi, P.S.; Naidu, K.R.; Rao, K.R.S.S.; Rao, S.;

Landfald, B. & Strøm, A.R. (1986) Choline-glycine betaine pathway confers a high level of

Lavorgna, G.; Dahary, D.; Lehner, B.; Sorek, R.; Sanderson, C.M. & Casari, G. (2004) In

Liu J.; Wisniewski M.; Droby S.; Vero S.; Tian S. & Hershkovitz V. (2011) Glycine betaine

Lv, S.; Yang, A.; Zhang, K.; Wang, L. & Zhang, J. (2007) Increase of glycinebetaine synthesis

Ma, H.M.; Schulze, S.; Lee, S.; Yang, M.; Mirkov, E.; Irvine, J.; Moore, P. & Paterson, A.

Majee, M.; Maitra, S.; Dastidar, K.G.; Pattnaik, S.; Chatterjee, A.; Hait, N.C.; Das, K.P. &

Matsumura, H.; Reich, S.; Ito, A.; Saitoh, H.; Kamoun, S.; Winter, P.; Kah,l G.; Reuter, M.;

Matsumura, H.; Bin Nasir, K.H.; Yoshida, K.; Ito, A.; Kahl, G.; Krüger D.H. & Terauchi R.

Matsumura, H.; Yoshida, K.; Luo, S.; Kimura, E.; Fujibe, T.; Albertyn, Z.; Barrero, R.A.;

*Genetics*, Vol. 108, (March 2004), No. 5, pp. 851–863, ISSN 1432-2242. Ma, X.L.; Wang, Y.L.; Xie, S.L.; Wang, C. & Wang, W. (2007) Glycinebetaine application

*Physiology*, Vol. 54, (July 2007), No. 4, pp. 472-479, ISSN 1608-3407.

ISSN 1389-2037.

pp. 1387-1394, ISSN 1532-2548.

No. 3, pp. 424-438, ISSN 0011-3891.

No. 3, pp. 849-855, ISSN 1098-5530.

No. 3, pp. 233–248, ISSN 1572-9788.

*(*March 2011), No. 1, pp. 76-83, ISSN 1879-3460.

(December 2003), pp. 15718-15723, ISSN 1091-6490.

88-94, ISSN 0968-0004.

0006-3002.

1548-7105.

functional review. *Current Protein Peptide Science*, Vol. 11, (May 2010), pp. 220-230,

Δ'-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. *Plant Physiol*ogy, Vol. 108, (August 1995), No. 4,

Reddy, K.J; Theriappan. P. & Sreenivasulu, N. (2005) Regulation of proline biosynthesis, degradation,uptake and transport in higher plants: Its implications in plant growth and abiotic stress tolerance. *Current Science*, Vol. 88, (February 2005).

osmotic tolerance in *Escherichia coli*. *Journal of Bacteriology*, Vol. 165, (March 1986),

search of antisense. *Trends in Biochemical Sciences*, Vol. 29, (February 2004), No.2, pp.

improves oxidative stress tolerance and biocontrol efficacy of the antagonistic yeast *Cystofilobasidium infirmominiatum*. *International Journal of Food Microbiology*, Vol. 146,

improves drought tolerance in cotton. *Molecular Breeding*, Vol. 20, (October 2007),

(2004) An EST survey of the sugarcane transcriptome. *Theoretical and Applied* 

ameliorates negative effects of drought stress in tobacco. *Russian Journal of Plant* 

Majumder, A.L. (2004) A Novel Salt-tolerant L -myo-Inositol-1-phosphate Synthase from *Porteresia coarctata* (Roxb.) Tateoka, a Halophytic Wild Rice. *The Journal of Biological Chemistry*, Vol. 279, (July 2004), No. 27, pp. 28539–28552, ISSN 1083-351X. Majumder, A.L.; Johnson, M.D. & Henry, S.A. (1997) 1L-myoinositol-1-phosphate synthase.

*Biochimica et Biophysica Acta*, Vol. 1348, (September 1997), No. 1-2, pp. 245–256, ISSN

Krüger, D.H. & Terauchi, R. (2003) Gene expression analysis of plant host-pathogen interactions by SuperSAGE. *Proceedings of National Academy of Science*, Vol. 100,

(2006) SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays. *Nature Methods*, Vol. 3, (June 2006), No. 6, pp. 469-74, ISSN

Krüger D.H.; Kahl G.; Schroth, G.P. & Terauchi R. (2010) High-Throughput


Henry, R.J. (2010) Basic Information on the Sugarcane Plant, In: *Genetics, Genomics and* 

Hu, C.A.; Delauney, A.J. & Verma, D.P. (1992) A bifunctional enzyme (delta 1-pyrroline-5-

Huang, J.; Hirji, R.; Adam, L.; Rozwadowski, K.L.; Hammerlindl, J.K.; Keller, W.A. &

Iskandar, H.M.; Casu, R.E.; Fletcher, A.; Schmidt, S.; Xu, J.; Maclean, D.; Manners, J.M. &

Iturriaga, G..; Gaff, D.F. & Zentella, R. (2000) New desiccation tolerant plants, including a

Jaleel C.A.; Manivannan P.; Wahid A.; Farooq M.; Al-Juburi, H.J.; Somasundaram R. &

Jokinen, K.; Somersalo, S.; Mäkelä, P.; Urbano, P.; Rojo, C.; González, J.M.A.; Soler, J.; Usano,

Jones, H.G. & Jones, M.B. (1989) Introduction: some terminology and common

1–10, Cambridge University Press, ISBN 0521344239, Cambridge, England. Jury, W.A. & Vaux, H.Jr. (2005) The role of science in solving the world's emerging water

Kaplan, F.; Kopka, J.; Haskell, D.W.; Zhao, W.; Schiller, K.C.; Gatzke, N.; Sung, D.Y. & Guy,

*Physiology*, Vol. 136, (December 2004), No. 4, pp. 4159–4168, ISSN 1532-2548. Kaushik, J.K. & Bhat, R. (2003) Why Is Trehalose an Exceptional Protein Stabilizer? An

Kern, A.J. & Dyer, W.E. (2004) Glycine Betaine Biosynthesis Is Induced by Salt Stress but

Kido E.A.; Pandolfi V.; Houllou-Kido L.M.; Andrade P.P.; Marcelino F.C.; Nepomuceno

(November 2005), Vol. 102, No. 44, pp. 15715–15720, ISSN 1091-6490. Kamalay, J.C. & Goldberg, R.B. (1980) Regulation of structural gene expression in tobacco.

*Cell*, Vol. 19, (April 1980), No. 4, pp. 935–946, ISSN 0092-8674.

pp. 26458-26465, ISSN 1083-351X.

*Regulation*, Vol. 23, No. 9, pp. 9-19, ISSN 1435-8107.

*BMC Plant Biology*, Vol. 11, (January 2011), pp. 12, ISSN 1471-2229.

89, (October 1992), No. 19, pp. 9354–9358, ISSN 1091-6490.

122, (March 2000), No. 3, pp. 747-756, ISSN 1532-2548.

*Botany*, Vol. 48, No. 2, pp. 153–158, ISSN 0067-1924.

*Biology*, Vol. 11, No. 1, pp. 100–105, ISSN 1814-9596.

7572.

9781578086849, New York, USA.

*Breeding of Sugarcane*, Henry, R.J & Kole, C. (Eds.), 1-7, CRC Press, ISBN

carboxylate synthetase) catalyses the first two steps in proline biosynthesis in plants. *Proceedings of National Academy of Sciences of the United States of America*, Vol.

Selvarai, G. (2000) Genetic Engineering of Glycinebetaine Production toward Enhancing Stress Tolerance in Plants: Metabolic Limitations. *Plant Physiology*, Vol.

Bonnett, G.D. (2011) Identification of drought-response genes and a study of their expression during sucrose accumulation and water deficit in sugarcane culms.

grass in the central highlands of Mexico, accumulate trehalose. *Australian Journal of* 

Panneerselvam R. (2009) Drought Stress in Plants: A Review on Morphological Characteristics and Pigments Composition. *International Journal of Agriculture and* 

M.C.; Moure, J. & Moya M. (1999) Glycinebetaine from sugar beet enhances the yield of field-grown tomatoes. *Acta Horticulture*, Vol. 487, pp. 233–236, ISSN 0567-

mechanisms. In: *Plants Under Stress*, Jones, H.G.; Flowers, T.J. & Jones, M.B. (Eds.),

problems. *Proceedings of National Academy of Sciences of the United States of America*,

C.L. (2004) Exploring the temperature-stress metabolome of *Arabidopsis*. *Plant* 

Analysis of the Thermal Stability of Proteins in the Presence of the Compatible Osmolyte Trehalose. *The Journal of Biological Chemistry,* Vol. 278, (July 2003), No. 29,

Repressed by Auxinic Herbicides in *Kochia scoparia. Journal of Plant Growth* 

A.L.; Abdelnoor R.V.; Burnquist W.L. & Benko-Iseppon A.M. (2010) Plant antimicrobial peptides: an overview of SuperSAGE transcriptional profile and a functional review. *Current Protein Peptide Science*, Vol. 11, (May 2010), pp. 220-230, ISSN 1389-2037.


Transcriptomics of Sugarcane Osmoprotectants Under Drought 111

Parthasarathy, L.; Vadnal, R.E.; Parthasarathy, R. & Devi, C.S. (1994) Biochemical and

Patade, V.Y.; Rai, A.N. & Suprasanna, P. (2010) Expression analysis of sugarcane shaggy-like

Patade, V.Y.; Suprasanna, P. & Basat, V.A. (2008) Effects of salt stress in relation to osmotic

Peters, S.; Mundree, S.G.; Thomson, J.A.; Farrant, J.M. & Keller, F. (2007) Protection

Poulin, R.; Larochelle, J. & Hellebust, J.A. (1987) The regulation of amino acid metabolism

Quéré, R.M.L.; Manchon, L.; Lejeune, M.; Clément, O.; Pierrat, F.; Bonafoux, B.; Commes, T.;

Rasheed, R.; Wahid, A.; Ashraf, M. & Basra, S.M.A. (2010) Role of Proline and

Rathinasabapathi, B. (2000) Metabolic engineering for stress tolerance: installing

Rathinasabapathi, B.; Burnet, M.; Russell, B.L.; Gage, D.A; Liao, P.C.; Nye, G.J.; Golbeck, J.H.

Rocha, F.R.; Papini-Terzi, F.S.; Nishiyama Jr, M.Y.; Vêncio, R.Z.N.; Vicentini, R.; Duarte,

sugarcane. *BMC Genomics*, Vol. 8, (March 2007), pp. 71, ISSN 1471-2164.

*Zoology*, Vol. 243, (September 1987), No. 3, pp. 365–378, ISSN 1932-5231. Quan, R.; Shang, M.; Zhao, Y. & Zhang, J. (2004) Engineering of enhanced glycinebetaine

(November 2004), No. 1, pp. 477–486, ISSN 1467-7652.

(November 2004), No. 20, pp. e163, ISSN 1362-4962.

*Regulation*, Vol. 55, (July 2008) , No. 3, pp. 169-173, ISSN 1573-5087.

(September 2010), No. 1-4, pp. 143-152, ISSN 1615-6102.

*Sciences*, Vol. 54, No. 16, pp. 1127–1142, ISSN 0024-3205.

10.1007/s00709-010-0207-8, ISSN 1615-6102.

ISSN 0022-0957.

1814–9596.

6490.

pp. 709–716, ISSN 1095-8290.

increased level of *myo*-inositol and methylated inositol. *Protoplasma*, Vol. 245,

molecular properties of lithium-sensitive myo-inositol monophosphatase. *Life* 

kinase (SuSK) gene identified through cDNA subtractive hybridization in sugarcane (*Saccharum officinarum* L.). *Protoplasma*, (September 2010), DOI

adjustment on sugarcane (Saccharum officinarum L.) callus cultures. *Plant Growth* 

mechanisms in the resurrection plant *Xerophyta viscosa* (Baker): both sucrose and raffinose family oligosaccharides (RFOs) accumulate in leaves in response to water deficit. *Journal of Experimental Botany*, Vol. 58, (April 2007) No. 8, pp. 1947–1956,

during hyperosmotic stress in *Acanthamoetla castellanii. Journal of Experimental* 

synthesis improves drought tolerance in maize, *Plant Biotechnology Journal,* Vol. 2,

Piquemal, D. & Marti, J. (2004) Mining SAGE data allows large-scale, sensitive screening of antisense transcript expression. *Nucleic Acids Research*, Vol. 32,

Glycinebetaine in Improving Chilling Stress Tolerance in Sugarcane Buds at Sprouting. *International Journal of Agriculture & Biology*, Vol. 12, No. 1, pp. 1-8, ISSN

osmoprotectant synthesis pathways. *Annals of Botany*, Vol. 86, (October 2000), No. 4,

& Hanson, A.D. (1997). Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 94, (April 1997), No. 7, pp. 3454-3458, ISSN 1091-

R.D.C.; de Rosa Jr, V.E.; Vinagre, F.; Barsalobres, C.; Medeiros, A.H.; Rodrigues, F.A.; Ulian, E.C.; Zingaretti, S.M.; Galbiatti, J.A.; Almeida, R.S.; Figueira, A.V.O.; Hemerly, A.S.; Silva-Filho, M.C.; Menossi, M. & Souza, G.M. (2007) Signal transduction-related responses to phytohormones and environmental challenges in

SuperSAGE for Digital Gene Expression Analysis of Multiple Samples Using Next Generation Sequencing. *PloS One*, Vol. 5, (August 2010), No. 8, pp. e12010, ISSN 1932-6203.


Mestichelli, L.J.J.; Gupta, R.N. & Spenser, I.D. (1979) The Biosynthetic Route from Ornithine

Ming, R.; Liu, S-C.; Lin, Y-R..; da Silva, J.; Wilson, W.; Braga, D.; van Deynze, A.; Wenslaff,

Mishra, S. & Dubey, R.S. (2006) Inhibition of ribonuclease and protease activities in arsenic

*Physiology* Vol. 163, (September 2006), No. 9, pp. 927–936, ISSN 1618-1328. Mohammadkhani, N. & Heidari, R. (2008) Drought-induced Accumulation of Soluble

Molina, C.; Rotter, B.; Horres, R.; Sripada, M.U.; Besser, B.; Bellarmino, L.; Baum, M.;

Molina, C.; Zaman-Allah, M.; Khan, F.; Fatnassi, N.; Horres, R.; Rotter, B.; Steinhauer, D.;

Molinari, H.B.C.; Marur, C.J.; Daros, E.; Campos, M.K.F.; Carvalho, J.F.R.P.; Filho,

Mullet, J.E. & Whitsitt, M.S. (1996) Plant cellular responses to water decit. *Plant Growth Regulation*, Vol. 20, (November 1996), No. 2, pp. 119–124, ISSN 1573-5087. Murthy, P.P. (2006) Structure and Nomenclature of Inositol Phosphates, Phosphoinositides,

Obermeier, C.; Housseini, B.; Friedt, W. & Snowdon, R. (2009) Gene expression profiling via

Parida, A.K.; Dagaonkar, V.S.; Phalak, M.S. & Aurangabadkar, L.P. (2008) Differential

Patra, B.; Ray, S.; Richter, A. & Majumder, A.L. (2010) Enhanced salt tolerance of transgenic

*Genetics*, Vol. 150, (December 1998), pp. 1663-1682, ISSN 1943-2631.

1932-6203.

640~1X7, ISSN 1083-351X.

No. 3, pp. 448-453, ISSN 1818-4952.

0387275991, New York, USA.

ISSN 0137-5881.

(November 2008), pp 1-28, ISSN 1471-2164.

*Biology*, Vol. 11, (February 2011), pp. 31, ISSN 1471-2229.

Vol. 130, (June 2007), No. 2, pp. 218-229, ISSN 0031-9317.

*BMC Genomics*, Vol. 10, (July 2009), ISSN 1471-2164.

SuperSAGE for Digital Gene Expression Analysis of Multiple Samples Using Next Generation Sequencing. *PloS One*, Vol. 5, (August 2010), No. 8, pp. e12010, ISSN

to Proline. *The Journal of Biological Chemistry*, Vol. 254, (February 1979), No. 3, pp.

T.F.; Wu, K.K.; Moore, P.H.; Burnquist, W.; Sorrells, M.E. ; Irvine, J.E. & Paterson, A.H. (1998) Detailed Alignment of Saccharum and Sorghum Chromosomes: Comparative Organization of Closely Related Diploid and Polyploid Genomes.

exposed rice seedlings: role of proline as enzyme protectant. *Journal of Plant* 

Sugars and Proline in Two Maize Varieties. *World Applied Sciences Journal,* Vol. 3,

Matsumura, H.; Terauchi, R.; Kahl, G. & Winter, P. (2008) SuperSAGE: the drought stress-responsive transcriptome of chickpea roots. *BMC Genomics*, Vol. 9,

Amenc, L.; Drevon, J-J.; Winter, P. & Kahl, G. (2011) The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE. *BMC Plant* 

J.C.B.; Pereira, L.F. P.; Vieira, L.G.E. (2007) Evaluation of the stress-inducible production of proline in transgenic sugarcane (*Saccharum* spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. *Physiologia Plantarum*,

and Glycosylphosphatidylinositols, In: *Biology of Inositols and Phosphoinositides: Subcellular Biochemistry*, Majumder, L. & Biswas, B.B. (Eds.), 1-19, Springer, ISBN

LongSAGE in a non-model plant species: a case study in seeds of *Brassica napus*.

responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery. *Acta Physiologiae Plantarum*, Vol. 30, (September 2008), No. 5, pp. 619-627,

tobacco plants by co-expression of *PcINO1* and *McIMT1* is accompanied by

increased level of *myo*-inositol and methylated inositol. *Protoplasma*, Vol. 245, (September 2010), No. 1-4, pp. 143-152, ISSN 1615-6102.


Transcriptomics of Sugarcane Osmoprotectants Under Drought 113

Székely, G.; Abrahám, E.; Cséplo, Á.; Rigo, G.; Zsigmond, L.; Csiszár, J.; Ayaydin, F.;

Tabuchi, T.; Kawaguchi, Y.; Azuma, T.; Nanmori, T. & Yasuda, T. (2005) Similar Regulation

Tew, T.L. & Cobill, R.M. (2008) Genetic improvement of sugarcane (*Saccharum* spp) as an

UNICA (2011) União da indústria de Cana-de-açúcar. In: Dados e Cotações – Estatísticas. 05.05.2011, Available from http://www.unica.com.br/dadosCotacao/estatistica/. UNICA (2009) União da indústria de Cana-de-açúcar. In: Statistics of sugarcane sector. 18.12.2009, Available from http://www.unica.com.br/dadosCotacao/estatistica/. Velculescu, V.E.; Zhang, L.; Vogelstein, B. & Kinzler, K.W. (1995) Serial analysis of gene

Vendruscolo, E.C.G.; Schuster, I.; Pileggi, M.; Scapim, C.A. Molinari, H.B.C., Marur, C.J. &

Vernon D.; Ostrem J. & Bohnert H. (1993) Stress perception and response in a facultative

Vettore, A.L.; Silva, F.R.; Kemper, E.L. & Arruda, P. (2001) The libraries that made SUCEST. *Genetics and Molecular Biology*, Vol. 24, No. 1-4, pp. 1-7, ISSN 1415-4757. Vinocur, B. & Altman, A. (2005) Recent advances in engineering plant tolerance to abiotic

Vojtechova, M.; Hanson, A.D. & Munoz-Clares, R.A. (1997) Betaine-aldehyde

Wang, Z.; Gerstein, M. & Snyder, M. (2009) RNA-Seq: a revolutionary tool for

Wang, S.; Liang, D.; Shi, S.; Ma, F.; Shu, H & Wang, R. (2011) Isolation and Characterization

Vol. 29, No 1, (March 2011) pp. 125-134, ISSN 1572-9818.

(January 2009), No. 1, pp. 51-58, ISSN 1671-2927.

11–28, ISSN 1365-313X.

Springer, ISBN 0387708049, Germany.

10, pp. 1367-1376, ISSN 1618-1328.

(April 2005) No. 2, pp. 123-132, ISSN 1879-0356.

ISSN 1365-3040.

ISSN 1096-0384.

ISSN 0028-0836.

Iso-Osmotic Salt and Water-Deficit Stress. *Agricultural Sciences in China*, Vol. 8,

Strizhov, N.; Jásik, J.; Schmelzer, E.; Koncz, C. & Szabados, L. (2008) Duplicated *P5CS* genes of *Arabidopsis* play distinct roles in stress regulation and developmental control of proline biosynthesis. *The Plant Journal*, Vol. 53, (October 2008), No. 1, pp.

Patterns of Choline Monooxygenase, Phosphoethanolamine N-Methyltransferase and S-Adenosyl- L -Methionine Synthetase in Leaves of the *Halophyte Atriplex nummularia* L. *Plant and Cell Physiology*, Vol. 46, No. 3, pp. 505–513, ISSN 0032-0781.

energy crop In*: Genetic improvement of bioenergy crops*, Vermerris, W. (Ed), 273-294,

expression. *Science*, Vol. 270, No. 5235, (October 1995), pp. 484–487, ISSN 0036-8075.

Vieira, L.G. (2007) Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat. *Journal of Plant Physiology*, Vol. 164, (October 2007), No.

halophyte: the regulation of salinity-induced genes in *Mesembryanthemum crystallinum. Plant, Cell and Environment*, Vol. 16, (May 1993), No. 4, pp. 437-444,

stress: achievements and limitations. *Current Opinion in Biotechnology*, Vol. 16,

dehydrogenase from amaranth leaves efciently catalyzes the NAD-dependent oxidation of dimethylsulfoniopropionaldehyde to dimethyl-sulfoniopropionate. *Archives of Biochemistry and Biophysics*, Vol. 337, (January 1997), No. 1, pp. 81–88,

transcriptomics. *Nature Reviews Genetics*, Vol. 10, (January 2009), No 1, pp. 57-63,

of a Novel Drought Responsive Gene Encoding a Glycine-rich RNA-binding Protein in *Malus prunifolia* (Willd.) Borkh. *Plant Molecular Biology Reporter*,


Rodrigues, SA, Laia, M.L. & Zingaretti, S.M. (2009) Analysis of gene expression proles

Rhodes, D. & Hanson, A.D. (1993) Quaternary ammonium and tertiary sulphonium

Ronde, J.A.; Spreeth, M.H. & Cress, W.A. (2000) Effect of antisense L-D1-pyrroline-5-

Rontein, D.; Basset, G. & Hanson, A.D. (2002) Metabolic engineering of osmoprotectant

Sakamoto, A. & Murata, N. (2002) The role of glycine betaine in the protection of plants from

Savouré, A.; Jaoua, S.; Hua X.J.; Ardiles, W.; Van Montagu, M. & Verbruggen, N. (1995)

Scott, P. (2000) Resurrection plants and the secrets of eternal leaf. *Annals of Botany*, Vol. 85,

Sharma, P. & Dubey, R.S. (2005) Modulation of nitrate reductase activity in rice seedlings

Shimkets, R.A. (2004) Gene Expression Quantitation Technology Summary. In: *Gene* 

Shinozaki, K. & Yamaguchi-Shinozaki, K. (2007) Gene networks involved in drought stress

Shirasawa, K.; Takabe, T.; Takabe, T. & Kishitani, S. (2006) Accumulation of Glycinebetaine

Smirnoff, N. & Cumbes, Q.J. (1989) Hydroxyl radical scavenging activity of compatible solutes. *Phytochemistry*, Vol. 28, No. 4, pp. 1057-1060, ISSN 1873-3700. Smith, K. & Bhavel, M. (2007) Response of Plants to Salt and Water Stress and the Roles of

Suriyan, C. & Chalermpol, K. (2009) Proline Accumulation, Photosynthetic Abilities and

(February 2009), No. 2, pp. 286-302, ISSN 0168-9452.

*Biology*, Vol. 44, pp. 357–384, ISSN 1040-2519.

2002), No. 2, pp. 163-171, ISSN 1365-3040.

ISBN 1-59259-751-3, New Haven, USA.

2006), No. 3, pp. 565–571, ISSN 1095-8290.

Routledge, ISBN 0-203-88480-9, New York, USA.

pp. 221-227, ISSN 0022-0957.

(September 1995), No. 1, pp. 13–19, ISSN 1873-3468.

(February 2000), No. 2, pp. 159–166, ISSN 1095-8290.

1573-5087.

1328.

56, ISSN 1096-7184.

under water stress in tolerant and sensitive sugarcane plants. *Plant Science*, Vol. 176,

compounds in higher plants. *Annual Review of Plant Physiology and Plant Molecular* 

carboxylate reductase transgenic soybean plants subjected to osmotic and drought stress. *Plant Growth Regulation*, Vol. 32, (September 2000), No 1, pp. 13-26, ISSN

accumulation in plants. *Metabolic engineering*, Vol. 4, (January 2002), No. 1, pp. 49-

stress: clues from transgenic plants. *Plant, Cell and Environment*, Vol. 25, (February

Isolation, characterization, and chromosomal location of a gene encoding the delta 1-pyrroline-5-carboxylate synthetase in *Arabidopsis thaliana*. *FEBS Letters*, Vol. 372,

under aluminium toxicity and water stress: role of osmolytes as enzyme protectant. *Journal of Plant Physiology,* Vol. 162, (August 2005), No. 8, pp. 854–864, ISSN 1618-

*expression profile: methods and protocol*, Shimkets, R.A. (Ed.), pp. 1-12, Humana Press,

response and tolerance. *Journal of Experimental Botany*, Vol. 58, (January 2007), No. 2,

in Rice Plants that Overexpress Choline Monooxygenase from Spinach and Evaluation of their Tolerance to Abiotic Stress. *Annals of Botany,* Vol 98, (September

Aquaporins. In: *Plant Stress and Biotechnology*, Thangadurai, D.; Tang, W. & S-Q Song (Eds.), 90-104, Oxford Book Company, ISBN 8189473107, Jaipur, India. Smith, K. & Pethley, D. (2009) Hydrological Hazards: Droughts, In: *Environmental hazards:* 

*assessing risk and reducing disaster*, Smith, K. & Pethley, D. (Eds.), 262-284,

Growth Characters of Sugarcane (*Saccharum officinarum* L.) Plantlets in Response to

Iso-Osmotic Salt and Water-Deficit Stress. *Agricultural Sciences in China*, Vol. 8, (January 2009), No. 1, pp. 51-58, ISSN 1671-2927.


**5** 

*University of Murcia* 

*Spain* 

**Effect of UV Light on Secondary Metabolite Biosynthesis in Plant Cell Cultures Elicited** 

 **with Cyclodextrins and Methyl Jasmonate** 

All the chemical reactions that occur in the cells of a living organism are called metabolism. By these reactions, a large number of organic compounds, including sugars, amino acids, fatty acids, nucleotides and their polymers derived, that is, polysaccharides, proteins, lipids, RNA, DNA, etc ... are produced. These processes are essential and common to all organisms and are known as primary metabolism and related compounds are known as primary metabolites. In the case of plants, in addition to the primary metabolic pathways, other metabolic pathways are activated under certain situations and the compounds produced are called secondary metabolites. The role of secondary metabolites may seem irrelevant, but the truth is that the plant spends a great deal of energy in their synthesis and they have remained in the plant kingdom up to date. This is because that wide variety and high diversity of secondary metabolites have apparently evolved as a means for plants to interact with the environment and for the development of resistance against both abiotic and biotic stress. In fact, secondary metabolites are useful to protect plants against herbivores (insects and vertebrates), mammals, bacteria, fungi, viruses and even other competing plants. In addition, some plants use secondary metabolites to attract pollinators and seed dispersers, as signals for communication between plants and symbiotic microorganisms or for

*Daucus carota* L. (*Umbelliferae*) is a biennial herb, whose fruits (common name: wild carrot fruits) have been used in traditional Chinese medicine for the treatment of ancylostomiasis, dropsy, chronic kidney disease and bladder afflictions (Pant & Manandhar, 2007) due to a wide range of reported pharmacological effects, including antibacterial (Rossi et al., 2007), antifungal (Tavares et al., 2008), antihelminthic, hepatoprotective (Bishayee et al., 1995) and cytotoxic activities (Yang et al., 2008; Fu et al., 2009). Carrot roots contain a variety of carotenoids and anthocyanins that are responsible for the typical colour of the root. In addition, this vegetable also produces phenolic compounds such as scopoletin, *p*-hydroxy benzoic acid and the isocoumarin, 6-methoxymellein, all major components of the phytoalexin complex (Mercier et al., 2000). These compounds are induced in carrot by fungal infection, heavy metals or UV light (Marinelli et al., 1994), and therefore they are involved in plant defence responses. Recently, Sabater-Jara et al., (2008) have described the

protection against UV light and other physical stress (Wink 2003, 2008).

production of sterols in different cell cultures including *D. carota.* 

**1. Introduction** 

Lorena Almagro, Ana Belén Sabater-Jara, Sarai Belchí-Navarro, Francisco Fernández-Pérez, Roque Bru and María A. Pedreño


## **Effect of UV Light on Secondary Metabolite Biosynthesis in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate**

Lorena Almagro, Ana Belén Sabater-Jara, Sarai Belchí-Navarro, Francisco Fernández-Pérez, Roque Bru and María A. Pedreño *University of Murcia Spain* 

### **1. Introduction**

114 Plants and Environment

Wang, Z-Z.; Zhang, S-Z.; Ynag, B-P. & Li, Y-R. (2005) Trehalose Synthase Gene Transfer

Wingler, A. (2002) The function of trehalose biosynthesis in plants. *Phytochemistry*, Vol. 60,

Wood, A.J.; Saneoka, H.; Rhodes, D.; Joly, R.J. & Goldsbrough (1996) Betaine Aldehyde

Genes). *Plant Physiology*, Vol. 110, No. 4, pp. 1301-1308, ISSN 1532-2548. Yancey, P.H. (2001) Water stress, osmolytes and proteins. *Integrative and Comparative Genomics*, Vol. 41, (August 2001), No. 4, pp. 699–670, ISSN 1557-7023. Yancey, P.H. (2005) Organic osmolytes as compatible, metabolic and counteracting

*Biology*, Vol. 208, (August 2005), No. 15, pp. 2819-2830, ISSN 1477-9145. Zhang X-Y.; Liang C.; Wang G-P., Luo Y. & Wang W. (2010) The protection of wheat plasma

Zhang, C.S.; Lu, Q. & Verma, D. S. P. (1995) Removal of feedback inhibition of delta-1-

Zhang, S-Z.; Yang, B-P.; Feng, C-L.; Chen, R-K. & Luo, J-P (2006) Wen-Wei Cai 2 and Fei-Hu

*Plant Biology,* Vol. 48, (April 2006), No. 4, pp. 453−459, ISSN 1744-7909. Zhang, S-Z.; Yang, B-P, Feng, C-L. & Tang, H-L. (2005) Genetic Transformation of Tobacco

Vol. 54, (March 2010), No. 1, pp. 83-88, ISSN 1573-8264.

47, (May 2005), No. 5, pp. 579-587, ISSN 1744-7909.

(September 1995) , No. 35, pp. 20491-20496, ISSN 1083-351X.

(July 2002), No. 5, pp. 437–440, ISSN 1873-3700.

ISSN 1432-2048.

Mediated by *Agrobacterium tumefaciens* Enhances Resistance to Osmotic Stress in Sugarcane. *Sugar Tech,* Vol 7, (March 2005), No. 1, pp. 49-54, ISSN 0972-1525. Weretilnyk, E.A.; Bednarek, S.; McCue, K.F.; Rhodes, D. & Hanson, A.D. (1989) Comparative

biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons. *Planta*, Vol. 178, (June 1989), No. 3, pp. 342–352,

Dehydrogenase in *Sorghum* (Molecular Cloning and Expression of Two Related

cytoprotectants in high osmolarity and other stresses. *The Journal of Experimental* 

membrane under cold stress by glycine betaine overproduction. *Biologia Plantarum,* 

pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first 2 steps of praline biosynthesis in plants. *The Journal of Biological Chemistry*, Vol. 270,

Liu Expression of the *Grifola frondosa* Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane (*Saccharum officinarum* L.). *Journal of Integrative* 

with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco. *Journal of Integrative Plant Biology*, Vol. All the chemical reactions that occur in the cells of a living organism are called metabolism. By these reactions, a large number of organic compounds, including sugars, amino acids, fatty acids, nucleotides and their polymers derived, that is, polysaccharides, proteins, lipids, RNA, DNA, etc ... are produced. These processes are essential and common to all organisms and are known as primary metabolism and related compounds are known as primary metabolites. In the case of plants, in addition to the primary metabolic pathways, other metabolic pathways are activated under certain situations and the compounds produced are called secondary metabolites. The role of secondary metabolites may seem irrelevant, but the truth is that the plant spends a great deal of energy in their synthesis and they have remained in the plant kingdom up to date. This is because that wide variety and high diversity of secondary metabolites have apparently evolved as a means for plants to interact with the environment and for the development of resistance against both abiotic and biotic stress. In fact, secondary metabolites are useful to protect plants against herbivores (insects and vertebrates), mammals, bacteria, fungi, viruses and even other competing plants. In addition, some plants use secondary metabolites to attract pollinators and seed dispersers, as signals for communication between plants and symbiotic microorganisms or for protection against UV light and other physical stress (Wink 2003, 2008).

*Daucus carota* L. (*Umbelliferae*) is a biennial herb, whose fruits (common name: wild carrot fruits) have been used in traditional Chinese medicine for the treatment of ancylostomiasis, dropsy, chronic kidney disease and bladder afflictions (Pant & Manandhar, 2007) due to a wide range of reported pharmacological effects, including antibacterial (Rossi et al., 2007), antifungal (Tavares et al., 2008), antihelminthic, hepatoprotective (Bishayee et al., 1995) and cytotoxic activities (Yang et al., 2008; Fu et al., 2009). Carrot roots contain a variety of carotenoids and anthocyanins that are responsible for the typical colour of the root. In addition, this vegetable also produces phenolic compounds such as scopoletin, *p*-hydroxy benzoic acid and the isocoumarin, 6-methoxymellein, all major components of the phytoalexin complex (Mercier et al., 2000). These compounds are induced in carrot by fungal infection, heavy metals or UV light (Marinelli et al., 1994), and therefore they are involved in plant defence responses. Recently, Sabater-Jara et al., (2008) have described the production of sterols in different cell cultures including *D. carota.* 

Effect of UV Light on Secondary Metabolite Biosynthesis

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 117

the brain (Asada & Shuler, 1989). The dimeric terpenoid indole alkaloids, 3′,4′ anhydrovinblastine, vincristine, and vinblastine have powerful effects as anticancer drugs (Zhou et al., 2009). These dimeric terpenoid indole alkaloids are synthesized from vindoline and catharanthine (Fig. 2B). Catharanthine can be chemically coupled with vindoline to form the clinically important anticancer drug, vinblastine and so this could provide a novel and efficient way to produce vinblastine commercially. Vindoline is abundant in *C. roseus* plants, and catharanthine can be produced by *C. roseus* cell or hairy root cultures (Zhao et al., 2001), so a combination of both pathways could lead to a very high vinblastine production. Therefore, the production of catharanthine by various *C. roseus* cell types in culture and its mechanism of biosynthesis has been one of most extensively explored areas of plant cell or hairy root cultures in recent years. In this sense, the application of biotic or abiotic stimuli has been one of the most effective strategies for improving the productivity of

*Vitis vinifera* produces stilbenes, which are a small group of compounds characterized by a 1,2-diphenylethylene backbone, derived from the phenylpropanoid pathway. Most plant stilbenes have phytoalexin activity and are derivatives of the monomeric unit *trans*resveratrol (3,5,4´-trihydroxystilbene, Fig. 2C) although other structures are found in other plant families. In grape berries, stilbenes are synthesized under natural environmental conditions (Jeandet et al., 1991; Versari et al., 2001). The *cis*- and *trans*-isomers of resveratrol are mainly accumulated in the exocarp (skin) during all stages of development and are almost totally absent from pericarp (flesh). Monomers and oligomers of resveratrol are also constitutively present in the lignified organs of grapevine such as stems and roots (Jeandet et al., 2002). Therefore, both pre-existing high contents of stilbenes in plants and those synthesized after microbial attack are part of both constitutive and inducible defence responses. In addition to the well-known function of stilbenes as phytoalexins, these compounds may also be involved as chemical signals in allelopathy (Seigler et al., 2006), or in response to oxidative stress generated by UV irradiation (He et al., 2008; Privat et al., 2002; Teguo et al., 1998). The formation of stilbenes (namely viniferins in Vitis) is therefore considered to be a part of the general defense mechanisms since they also display strong antifungal and antimicrobial activities (Bru et al., 2006; Pezet et al*.,* 2004; Morales et al., 1998). In fact, *trans*-resveratrol, is found in both grapevine tissue and berries, and in cell cultures as

the result of both abiotic and biotic stress (Pezet et al*.,* 2003, 2004; Cantos et al., 2003).

Fig. 2. Structures of ajmalicine (A), catharanthine (B) and *trans*-resveratrol (C).

extensively studied natural products.

Since *trans*-resveratrol was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Siemann & Creasy, 1992), it is one of the most

terpenoid indole alkaloids from *C. roseus* cell cultures (Zhao et al., 2000).

Plant sterols, also called phytosterols, are isoprenoid-derived lipids that play essential roles in plant growth and development since they are integral components of the plant cell membranes and are responsible for its permeability and fluidity (Posé et al., 2009). In addition, phytosterols play an important role in cellular processes as precursors for brassinosteroids biosynthesis. They are also components of a wide variety of secondary metabolites such as the glycoalkaloids, cardenolides and saponins. Moreover, phytosterols have important pharmacological activities, including cholesterol-lowering, antitumor effects against lung, stomach, ovary and estrogen-dependent human breast cancer (Woyengo et al., 2009) and recently, they have reported to exert anti-atherosclerotic, anti-inflammatory and anti-oxidative activities in animals (Delgado-Zamarreño et al., 2009). The beneficial health effects of phytosterols have led to search potential strategies for enhancement these compounds from other natural sources. In this sense, the use of plant cell cultures has been developed as a promising alternative, especially when the production of bioactive compounds is difficult or unprofitable, or when it involves serious damage to the environment. In this way, campesterol, stigmasterol, β-sitosterol and fucosterol (Fig. 1), being major phytosterols found in plants, have been recently produced using plant cell systems (Sabater-Jara et al., 2010a,b; Bonfill et al., 2011; Lee et al., 2004 and Herchi et al., 2009).

Fig. 1. Structures of Campesterol(A), Stigmasterol (B), β-Sitosterol (C), and Fucosterol (D).

*Catharanthus roseus* (Madagascar periwinkle) is a perennial tropical plant belonging to the family *Apocynaceae* that produces more than 130 alkaloids (van der Heijden et al., 2004). The importance of this plant relies in its ability to synthesize a wide range of terpenoid indole alkaloids as part of its secondary metabolism. These compounds have vital roles as mediators of ecological interactions, and are very important for plant survival. They are involved in the defense against competitors, herbivores and pathogens, in attracting pollinators or symbionts and in the adaptation to both biotic and abiotic stress conditions. Although they are constitutive compounds, their levels may be enhanced by several factors. Some terpenoid indole alkaloids have a high added value because of their broad spectrum of pharmacological applications. Special attention has focused on the production of the antihypertensive monomeric terpenoid indole alkaloids serpentine and ajmalicine (Fig. 2A), which are used to combat heart arrhythmias and to improve the blood circulation in

Plant sterols, also called phytosterols, are isoprenoid-derived lipids that play essential roles in plant growth and development since they are integral components of the plant cell membranes and are responsible for its permeability and fluidity (Posé et al., 2009). In addition, phytosterols play an important role in cellular processes as precursors for brassinosteroids biosynthesis. They are also components of a wide variety of secondary metabolites such as the glycoalkaloids, cardenolides and saponins. Moreover, phytosterols have important pharmacological activities, including cholesterol-lowering, antitumor effects against lung, stomach, ovary and estrogen-dependent human breast cancer (Woyengo et al., 2009) and recently, they have reported to exert anti-atherosclerotic, anti-inflammatory and anti-oxidative activities in animals (Delgado-Zamarreño et al., 2009). The beneficial health effects of phytosterols have led to search potential strategies for enhancement these compounds from other natural sources. In this sense, the use of plant cell cultures has been developed as a promising alternative, especially when the production of bioactive compounds is difficult or unprofitable, or when it involves serious damage to the environment. In this way, campesterol, stigmasterol, β-sitosterol and fucosterol (Fig. 1), being major phytosterols found in plants, have been recently produced using plant cell systems (Sabater-Jara et al.,

Fig. 1. Structures of Campesterol(A), Stigmasterol (B), β-Sitosterol (C), and Fucosterol (D). *Catharanthus roseus* (Madagascar periwinkle) is a perennial tropical plant belonging to the family *Apocynaceae* that produces more than 130 alkaloids (van der Heijden et al., 2004). The importance of this plant relies in its ability to synthesize a wide range of terpenoid indole alkaloids as part of its secondary metabolism. These compounds have vital roles as mediators of ecological interactions, and are very important for plant survival. They are involved in the defense against competitors, herbivores and pathogens, in attracting pollinators or symbionts and in the adaptation to both biotic and abiotic stress conditions. Although they are constitutive compounds, their levels may be enhanced by several factors. Some terpenoid indole alkaloids have a high added value because of their broad spectrum of pharmacological applications. Special attention has focused on the production of the antihypertensive monomeric terpenoid indole alkaloids serpentine and ajmalicine (Fig. 2A), which are used to combat heart arrhythmias and to improve the blood circulation in

2010a,b; Bonfill et al., 2011; Lee et al., 2004 and Herchi et al., 2009).

the brain (Asada & Shuler, 1989). The dimeric terpenoid indole alkaloids, 3′,4′ anhydrovinblastine, vincristine, and vinblastine have powerful effects as anticancer drugs (Zhou et al., 2009). These dimeric terpenoid indole alkaloids are synthesized from vindoline and catharanthine (Fig. 2B). Catharanthine can be chemically coupled with vindoline to form the clinically important anticancer drug, vinblastine and so this could provide a novel and efficient way to produce vinblastine commercially. Vindoline is abundant in *C. roseus* plants, and catharanthine can be produced by *C. roseus* cell or hairy root cultures (Zhao et al., 2001), so a combination of both pathways could lead to a very high vinblastine production. Therefore, the production of catharanthine by various *C. roseus* cell types in culture and its mechanism of biosynthesis has been one of most extensively explored areas of plant cell or hairy root cultures in recent years. In this sense, the application of biotic or abiotic stimuli has been one of the most effective strategies for improving the productivity of terpenoid indole alkaloids from *C. roseus* cell cultures (Zhao et al., 2000).

*Vitis vinifera* produces stilbenes, which are a small group of compounds characterized by a 1,2-diphenylethylene backbone, derived from the phenylpropanoid pathway. Most plant stilbenes have phytoalexin activity and are derivatives of the monomeric unit *trans*resveratrol (3,5,4´-trihydroxystilbene, Fig. 2C) although other structures are found in other plant families. In grape berries, stilbenes are synthesized under natural environmental conditions (Jeandet et al., 1991; Versari et al., 2001). The *cis*- and *trans*-isomers of resveratrol are mainly accumulated in the exocarp (skin) during all stages of development and are almost totally absent from pericarp (flesh). Monomers and oligomers of resveratrol are also constitutively present in the lignified organs of grapevine such as stems and roots (Jeandet et al., 2002). Therefore, both pre-existing high contents of stilbenes in plants and those synthesized after microbial attack are part of both constitutive and inducible defence responses. In addition to the well-known function of stilbenes as phytoalexins, these compounds may also be involved as chemical signals in allelopathy (Seigler et al., 2006), or in response to oxidative stress generated by UV irradiation (He et al., 2008; Privat et al., 2002; Teguo et al., 1998). The formation of stilbenes (namely viniferins in Vitis) is therefore considered to be a part of the general defense mechanisms since they also display strong antifungal and antimicrobial activities (Bru et al., 2006; Pezet et al*.,* 2004; Morales et al., 1998). In fact, *trans*-resveratrol, is found in both grapevine tissue and berries, and in cell cultures as the result of both abiotic and biotic stress (Pezet et al*.,* 2003, 2004; Cantos et al., 2003).

Since *trans*-resveratrol was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Siemann & Creasy, 1992), it is one of the most extensively studied natural products.

Fig. 2. Structures of ajmalicine (A), catharanthine (B) and *trans*-resveratrol (C).

Effect of UV Light on Secondary Metabolite Biosynthesis

attenuation of UV-B radiation than flavonoids (Sheahan, 1996).

concentration was strongly enhanced by the UV treatment.

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 119

Accumulation of UV-absorbing compounds, mainly those of phenolic nature, is a typical defense mechanism of plants to increased UV radiation, and is the most common response produced by vascular plants (Searles et al., 2001). Derivatives of hydroxycinnamic acid are UV-absorbing compounds which have received less attention, probably because they have been considered as constitutive, rather than inducible, protective barrier, against UV-B radiation (Bornman et al., 1997). However, they absorb UV-B more effectively than flavonoids, the other UV-absorbing compounds, whose absorption peaks are shifted to the UV-A radiation. As a result, derivatives of hydroxycinnamic acid may provide greater

In plant cell cultures, UV light acts as an abiotic factor which stimulates the biosynthesis of secondary metabolites (Broeckling et al., 2005). Thus, it has been shown that UV-B light induces both the formation of dimeric terpenoid indole alkaloids and *tryptophan decarboxylase and strictosidine synthase* mRNA accumulation in *C. roseus* (Ouwerkek et al., 1999). Ramani & Jayabaskaran (2008) also observed the enhanced production of catharanthine and vindoline from *C. roseus* cell cultures, when cells were irradiated with UV-B for 5 min. In a similar way, Gläβgen et al., (1998) studied the effect of continuous irradiation with UV-containing white light (315-420 nm) on the anthocyanin content of *D. carota* cell cultures after 7 days of culture, and observed that the total anthocyanin

The effect of UV irradiation on stilbene content in grapevine cell cultures is little known and most of the research related with UV light has been directed at enhancing the stilbene content of grape berries (Adrian et al., 2000; Versari et al., 2001; Cantos et al., 2003), leaves (Langcake & Pryce 1977; Pezet et al., 2003) and callus tissue (Keller et al., 2000; Keskin & Kunter 2008, 2010). In addition, when Keller et al., (2000) studied stilbene accumulation in callus of grapevine irradiated with UV light, they found that only actively growing callus was capable of producing stilbenes (including *trans*-resveratrol), whereas old callus had lost this ability. This response was similar to that found in ripening grape berries, which

On the other hand, special attention has been paid to the use of chemical compounds such as β-cyclodextrins (CDs, Fig. 3) which are cyclic oligosaccharides consisting of seven α-Dglucopiranose residues linked by α (14) glucosidic bonds formed by the enzymatic modification of starch. These compounds chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack (Bru et al., 2006), thus they have been used to increase both the biosynthesis of *trans*-resveratrol and its secretion to the extracellular medium in *V. vinifera* cell cultures (Morales et al., 1998; Bru & Pedreño, 2003; Bru et al., 2006). Recently, Zamboni et al., (2009) reported that CDs trigger a signal transduction cascade which activates different families of transcription factors in grapevine cells, inducing a halt in cell division, reinforcement of the cell wall and the biosynthesis of *trans*-resveratrol and defence-related proteins. The method based on the use of CDs (Bru & Pedreño, 2003) differs from those that use other elicitors (Liswidowati et al., 1991) not only in the high levels of *trans*-resveratrol produced but also in the extraction process of this compound. Thus, in traditional elicitation methods, *trans*-resveratrol is extracted from elicited cells gives low yields, whereas in this new process, *trans*-resveratrol is secreted as it is produced by cells and recovered directly from the spent media with no biomass destruction. In addition, the high levels of *trans*-resveratrol accumulated in the culture medium were seen to have no toxic effect on the cell lines, allowing successful subcultures. This innovative elicitation process is mainly based on the CD characteristics.

gradually lose their potential for synthesizing stilbenes as they approach maturity.

Over the last 15 years, stilbenes, especially *trans*-resveratrol has received considerable interest, due to their biological activities and possible pharmacological applications. Hundreds of studies have reported the beneficial effects of *trans*-resveratrol on neurological (Okawara et al., 2007) and cardiovascular systems (Bradamante et al., 2004). One of the most striking biological activities of *trans*-resveratrol investigated during recent years has been its anticancer activity and it has been seen to prevent carcinogenesis in the stages of tumour initiation, promotion and progression (Pervaiz, 2003; Pezzuto, 2008). More results provide interesting insights into the effect of this compound on the lifespan of yeast, worms and flies, suggesting that *trans*-resveratrol could be regarded as a potential antiaging agent in treating age-related human diseases (De la Lastra & Villegas, 2005). In addition, effects described in mice subjected to a high-calorie diet (Baur et al., 2006) point to new approaches for treating not only age-related diseases but also obesity-related disorders (Kaeberlein & Rabinovitch, 2006). For these reasons, the wide ranging of pharmacological and clinical potential applications of *trans*-resveratrol and other stilbenes have been recently reviewed (Pezzuto, 2008; Shakibaei et al., 2009; Espín et al., 2007). That is why new strategies based on the use of *V. vinifera* cell cultures have been used to increase the level of *trans*-resveratrol production.

The close relationship between plant secondary metabolism and defence response is widely recognized. Plants not only respond to attack of pathogens, insects and herbivores or to other biotic and abiotic stresses but also to small molecules of different origin, called elicitors that trigger the same response in the plant as the pathogen or organism itself. When introduced in a living cell in small concentrations, elicitors are capable of redirecting the metabolism, leading to increased production of particular secondary metabolites. Then, elicitors are useful tools for improving the production of plant valuable secondary metabolites. In general, elicitors are classified on the basis of their origin and molecular structure. Biotic elicitors are derived from the pathogen or from the plant and can have a defined composition, when their molecular structures are known, or have a complex composition when they comprise several different molecular classes making impossible to define a unique chemical identity. On the other hand, abiotic elicitors have not a biological origin and are grouped in physical and chemicals factors (Vasconsuelo & Boland, 2007).

A wide array of external stimuli are capable of triggering changes in the plant cell, leading to a cascade of reactions that ultimately result in the formation and accumulation of secondary metabolites, which help plants to overcome the stress factors. Amongst these, ultraviolet (UV) light which is a minor part of the solar spectrum, represents an important ecological factor that influences the organisms and ecosystems, and it is related to the occurrence of some adaptive changes in organisms throughout the development of life on Earth.

UV radiation is divided into three regions: UV-C (wavelengths below 280 nm), UV-B (280- 315 nm) and UV-A (315-400 nm). UV-C is the most damaging, but it is almost completely absorbed by the stratosphere. By contrast, UV-B radiation is only partially absorbed by the stratospheric ozone layer, and UV-A is not absorbed at all. Therefore, a fraction of UV-B and all UV-A reaches the earth's surface, where they cause various biological effects. Moreover, the effectiveness of biological responses to UV radiation increases with decreasing wavelength, so these responses are normally dominated by the UV-B. However, in recent years UV-A action spectra have also been considered.

Over the last 15 years, stilbenes, especially *trans*-resveratrol has received considerable interest, due to their biological activities and possible pharmacological applications. Hundreds of studies have reported the beneficial effects of *trans*-resveratrol on neurological (Okawara et al., 2007) and cardiovascular systems (Bradamante et al., 2004). One of the most striking biological activities of *trans*-resveratrol investigated during recent years has been its anticancer activity and it has been seen to prevent carcinogenesis in the stages of tumour initiation, promotion and progression (Pervaiz, 2003; Pezzuto, 2008). More results provide interesting insights into the effect of this compound on the lifespan of yeast, worms and flies, suggesting that *trans*-resveratrol could be regarded as a potential antiaging agent in treating age-related human diseases (De la Lastra & Villegas, 2005). In addition, effects described in mice subjected to a high-calorie diet (Baur et al., 2006) point to new approaches for treating not only age-related diseases but also obesity-related disorders (Kaeberlein & Rabinovitch, 2006). For these reasons, the wide ranging of pharmacological and clinical potential applications of *trans*-resveratrol and other stilbenes have been recently reviewed (Pezzuto, 2008; Shakibaei et al., 2009; Espín et al., 2007). That is why new strategies based on the use of *V. vinifera* cell cultures have been used to increase the level of *trans*-resveratrol

The close relationship between plant secondary metabolism and defence response is widely recognized. Plants not only respond to attack of pathogens, insects and herbivores or to other biotic and abiotic stresses but also to small molecules of different origin, called elicitors that trigger the same response in the plant as the pathogen or organism itself. When introduced in a living cell in small concentrations, elicitors are capable of redirecting the metabolism, leading to increased production of particular secondary metabolites. Then, elicitors are useful tools for improving the production of plant valuable secondary metabolites. In general, elicitors are classified on the basis of their origin and molecular structure. Biotic elicitors are derived from the pathogen or from the plant and can have a defined composition, when their molecular structures are known, or have a complex composition when they comprise several different molecular classes making impossible to define a unique chemical identity. On the other hand, abiotic elicitors have not a biological origin and are grouped in physical and chemicals factors (Vasconsuelo &

A wide array of external stimuli are capable of triggering changes in the plant cell, leading to a cascade of reactions that ultimately result in the formation and accumulation of secondary metabolites, which help plants to overcome the stress factors. Amongst these, ultraviolet (UV) light which is a minor part of the solar spectrum, represents an important ecological factor that influences the organisms and ecosystems, and it is related to the occurrence of some adaptive changes in organisms throughout the development of life on

UV radiation is divided into three regions: UV-C (wavelengths below 280 nm), UV-B (280- 315 nm) and UV-A (315-400 nm). UV-C is the most damaging, but it is almost completely absorbed by the stratosphere. By contrast, UV-B radiation is only partially absorbed by the stratospheric ozone layer, and UV-A is not absorbed at all. Therefore, a fraction of UV-B and all UV-A reaches the earth's surface, where they cause various biological effects. Moreover, the effectiveness of biological responses to UV radiation increases with decreasing wavelength, so these responses are normally dominated by the UV-B. However, in recent

years UV-A action spectra have also been considered.

production.

Boland, 2007).

Earth.

Accumulation of UV-absorbing compounds, mainly those of phenolic nature, is a typical defense mechanism of plants to increased UV radiation, and is the most common response produced by vascular plants (Searles et al., 2001). Derivatives of hydroxycinnamic acid are UV-absorbing compounds which have received less attention, probably because they have been considered as constitutive, rather than inducible, protective barrier, against UV-B radiation (Bornman et al., 1997). However, they absorb UV-B more effectively than flavonoids, the other UV-absorbing compounds, whose absorption peaks are shifted to the UV-A radiation. As a result, derivatives of hydroxycinnamic acid may provide greater attenuation of UV-B radiation than flavonoids (Sheahan, 1996).

In plant cell cultures, UV light acts as an abiotic factor which stimulates the biosynthesis of secondary metabolites (Broeckling et al., 2005). Thus, it has been shown that UV-B light induces both the formation of dimeric terpenoid indole alkaloids and *tryptophan decarboxylase and strictosidine synthase* mRNA accumulation in *C. roseus* (Ouwerkek et al., 1999). Ramani & Jayabaskaran (2008) also observed the enhanced production of catharanthine and vindoline from *C. roseus* cell cultures, when cells were irradiated with UV-B for 5 min. In a similar way, Gläβgen et al., (1998) studied the effect of continuous irradiation with UV-containing white light (315-420 nm) on the anthocyanin content of *D. carota* cell cultures after 7 days of culture, and observed that the total anthocyanin concentration was strongly enhanced by the UV treatment.

The effect of UV irradiation on stilbene content in grapevine cell cultures is little known and most of the research related with UV light has been directed at enhancing the stilbene content of grape berries (Adrian et al., 2000; Versari et al., 2001; Cantos et al., 2003), leaves (Langcake & Pryce 1977; Pezet et al., 2003) and callus tissue (Keller et al., 2000; Keskin & Kunter 2008, 2010). In addition, when Keller et al., (2000) studied stilbene accumulation in callus of grapevine irradiated with UV light, they found that only actively growing callus was capable of producing stilbenes (including *trans*-resveratrol), whereas old callus had lost this ability. This response was similar to that found in ripening grape berries, which gradually lose their potential for synthesizing stilbenes as they approach maturity.

On the other hand, special attention has been paid to the use of chemical compounds such as β-cyclodextrins (CDs, Fig. 3) which are cyclic oligosaccharides consisting of seven α-Dglucopiranose residues linked by α (14) glucosidic bonds formed by the enzymatic modification of starch. These compounds chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack (Bru et al., 2006), thus they have been used to increase both the biosynthesis of *trans*-resveratrol and its secretion to the extracellular medium in *V. vinifera* cell cultures (Morales et al., 1998; Bru & Pedreño, 2003; Bru et al., 2006). Recently, Zamboni et al., (2009) reported that CDs trigger a signal transduction cascade which activates different families of transcription factors in grapevine cells, inducing a halt in cell division, reinforcement of the cell wall and the biosynthesis of *trans*-resveratrol and defence-related proteins. The method based on the use of CDs (Bru & Pedreño, 2003) differs from those that use other elicitors (Liswidowati et al., 1991) not only in the high levels of *trans*-resveratrol produced but also in the extraction process of this compound. Thus, in traditional elicitation methods, *trans*-resveratrol is extracted from elicited cells gives low yields, whereas in this new process, *trans*-resveratrol is secreted as it is produced by cells and recovered directly from the spent media with no biomass destruction. In addition, the high levels of *trans*-resveratrol accumulated in the culture medium were seen to have no toxic effect on the cell lines, allowing successful subcultures. This innovative elicitation process is mainly based on the CD characteristics.

Effect of UV Light on Secondary Metabolite Biosynthesis

elicited with CDs and MJ, alone or in combination.

2008).

**resveratrol** 

et al., 2009).

production of *trans*-resveratrol.

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 121

biosynthetic genes when used independently to treat grapevine cells. Such expression correlated with *trans*-resveratrol production in CD-treated cells but not in MJ-treated cells. In the combined treatment involving CDs and MJ, *trans*-resveratrol production which is secreted to the spent medium, reached a maximum value (360 mg/g DW) that was correlated with the maximum expression levels of stilbene biosynthetic genes, demonstrating the synergistic effect of the combination of MJ with CDs (Lijaveztky et al.,

Based on these evidences, the main objective of this chapter is to show the effect of UV light on the production of secondary metabolites in *C. roseus, V. vinifera* and *D. carota* cell cultures

The production of *trans*-resveratrol in cell cultures of *Vitis sp* has been analyzed by several groups (Kiselev 2011 and see therein). Analysis of *trans*-resveratrol production in untreated *Vitis* cell cultures (Teguo et al., 1996; Morales et al., 1998; Krisa et al., 1999; Tassoni et al., 2005) revealed a low level of *trans*-resveratrol accumulation, less than 0.01% DW or 2-3 mg/l. Therefore, various strategies such as the use of biotic and abiotic elicitors, addition of biosynthesis precursors and genetic transformation have been considered to improve the

Fig. 4 shows the level of *trans*-resveratrol (6.72 ± 1.03 mg/g FW) when Monastrell cell cultures were incubated with 50 mM CDs and 100 μM MJ, and how this value was higher than when cell cultures were treated only with CDs (3.15 ± 0.35 mg/g FW). However, very low amounts of *trans*-resveratrol were detected in the spent medium when grapevine cell cultures were elicited only with MJ, and no *trans*-resveratrol was detected in cell cultures treated with ethanol, the solvent in which MJ is delivered to the culture (data not shown). In a similar way, Krisa et al. (1999) described that the amount of total stilbenes secreted to the culture medium was negligible in both MJ and control cultures of three *V. vinifera* cultivars. These authors reported that piceid (glucosylated form of resveratrol) accumulation inside the grape cells of Cavernet-Sauvignon cultivar was notably induced when 25 µM MJ was added to the induction medium on day 6 (6.3 ± 0.2 mg piceid/g DW). Very low levels of piceid in cells (9.75 ± 1.17 µg/g DW) both in the presence of 25 µM MJ as when 25µM MJ and 50 mM CDs were jointly used as inducers (82.7 ± 10.9 μg/g DW, Lijavetzki et al., 2008). However, under the last conditions described above, the accumulation of *trans*-resveratrol in the extracellular medium was 212.6 ± 12.6 mg /g DW that is, 10.6 ± 0.6 mg/g FW (Pedreño

On the other hand, Kiselev et al. (2007) reported a high *trans*-resveratrol production in *V. amurensis* callus cultures transformed with the *rol*B gene of *Agrobacterium rhizogenes* (31.5 mg *trans*-resveratrol/g DW) which was 6.7 times lower than those obtained in the culture

UV-C light has been described as a physical inductor of stilbene biosynthesis in *Vitis sp*. As mentioned above, there are no reports on *trans*-resveratrol production in grapevine cell cultures elicited with UV-C light and most of the research related with UV-C light has been directed at enhancing the stilbene content of grape berries (Adrian et al., 2000; Versari et al., 2001; Cantos et al., 2003; González-Barrio et al., 2006), leaves (Douillet-Breuil et al., 1999; Pezet et al., 2003) and callus tissue (Keller et al., 2000; Keskin & Kunter 2008). Keller et al.,

medium under conditions described above using CDs and MJ jointly.

**2. Effect of UV light exposure, CDs and MJ on the production of** *trans***-**

They have a hydrophilic external surface and hydrophobic central cavity that can trap hydrophobic compounds, including *trans*-resveratrol. This hydrophobic cavity forms inclusion complexes, which in the case of CDs with *trans*-resveratrol, is of the 1:1 type, altering its physicochemical behaviour and making it a highly water-soluble compound (Morales et al., 1998).

This procedure has successful been applied to the production of phytosterols. Thus, Sabater-Jara et al., (2008) have developed a method based on the use of CDs to enhance phytosterol production by using *D. carota* cell cultures since these cyclic oligosaccharides are able to induce a cascade of cellular events that gives rise to the accumulation of phytosterols.

Methyl jasmonate (MJ, Fig. 3) is considered a key molecule in the signal transduction pathway involved in the induction of the biosynthesis of secondary metabolites which take part in plant defence reactions (Gundlach et al., 1992; Creelman & Mullet, 1997; Staswick et al., 1998; Chung et al., 2003). In this way, the application of MJ alone or in combination with CDs triggers the accumulation of secondary metabolites in *Solanaceae* cell cultures, e.g. capsidiol and solavetivone in *Capsicum annuum* (Ma, 2008; Sabater-Jara et al., 2010b). Lee-Parsons et al., (2004) showed that *C. roseus* cell cultures respond to MJ by increasing extracellular accumulation of ajmalicine whose production was dependent on MJ dose and elicitation time. Almagro et al., (2010) demonstrated that the maximum level of ajmalicine produced by cells and secreted to the media was reached when cell suspensions were incubated in the presence of MJ and CDs, production being around 2.2-fold higher than when cells were treated only with CDs.

Moreover, Tassoni et al., (2005) showed that MJ was highly effective in stimulating endogenous *trans*-resveratrol accumulation, as well as promoting its release into the extracellular medium of *V. vinifera cv* Barbera cell cultures. In this case, the endogenous *trans*-resveratrol accumulation was around 24 µg/g dry weight (DW) and the *trans*resveratrol secreted to the medium was over 8 µg/g DW. In a similar way, Belhadj et al., (2008) described the production of 0.6 mg *trans*-resveratrol/g DW in *V. vinifera* cv Gamay elicited with MJ in a culture medium with the sugar concentration increased. However, the most significant success in increasing *trans*-resveratrol content in grapevine cell cultures has been reached using CDs alone or in combination with MJ (Pedreño et al., 2009). Lijavetzki et al., (2008) analysed the effects of MJ, CDs and a combination of both on extracellular *trans*resveratrol production and the expression of stilbene biosynthetic genes in grapevine cell cultures. MJ and CDs significantly but transiently induced the expression of stilbene

Fig. 3. Structures of methyl jasmonate (A) and cyclodextrins (B).

They have a hydrophilic external surface and hydrophobic central cavity that can trap hydrophobic compounds, including *trans*-resveratrol. This hydrophobic cavity forms inclusion complexes, which in the case of CDs with *trans*-resveratrol, is of the 1:1 type, altering its physicochemical behaviour and making it a highly water-soluble compound

This procedure has successful been applied to the production of phytosterols. Thus, Sabater-Jara et al., (2008) have developed a method based on the use of CDs to enhance phytosterol production by using *D. carota* cell cultures since these cyclic oligosaccharides are able to induce a cascade of cellular events that gives rise to the accumulation of phytosterols. Methyl jasmonate (MJ, Fig. 3) is considered a key molecule in the signal transduction pathway involved in the induction of the biosynthesis of secondary metabolites which take part in plant defence reactions (Gundlach et al., 1992; Creelman & Mullet, 1997; Staswick et al., 1998; Chung et al., 2003). In this way, the application of MJ alone or in combination with CDs triggers the accumulation of secondary metabolites in *Solanaceae* cell cultures, e.g. capsidiol and solavetivone in *Capsicum annuum* (Ma, 2008; Sabater-Jara et al., 2010b). Lee-Parsons et al., (2004) showed that *C. roseus* cell cultures respond to MJ by increasing extracellular accumulation of ajmalicine whose production was dependent on MJ dose and elicitation time. Almagro et al., (2010) demonstrated that the maximum level of ajmalicine produced by cells and secreted to the media was reached when cell suspensions were incubated in the presence of MJ and CDs, production being around 2.2-fold higher than

Moreover, Tassoni et al., (2005) showed that MJ was highly effective in stimulating endogenous *trans*-resveratrol accumulation, as well as promoting its release into the extracellular medium of *V. vinifera cv* Barbera cell cultures. In this case, the endogenous *trans*-resveratrol accumulation was around 24 µg/g dry weight (DW) and the *trans*resveratrol secreted to the medium was over 8 µg/g DW. In a similar way, Belhadj et al., (2008) described the production of 0.6 mg *trans*-resveratrol/g DW in *V. vinifera* cv Gamay elicited with MJ in a culture medium with the sugar concentration increased. However, the most significant success in increasing *trans*-resveratrol content in grapevine cell cultures has been reached using CDs alone or in combination with MJ (Pedreño et al., 2009). Lijavetzki et al., (2008) analysed the effects of MJ, CDs and a combination of both on extracellular *trans*resveratrol production and the expression of stilbene biosynthetic genes in grapevine cell cultures. MJ and CDs significantly but transiently induced the expression of stilbene

(Morales et al., 1998).

when cells were treated only with CDs.

Fig. 3. Structures of methyl jasmonate (A) and cyclodextrins (B).

biosynthetic genes when used independently to treat grapevine cells. Such expression correlated with *trans*-resveratrol production in CD-treated cells but not in MJ-treated cells. In the combined treatment involving CDs and MJ, *trans*-resveratrol production which is secreted to the spent medium, reached a maximum value (360 mg/g DW) that was correlated with the maximum expression levels of stilbene biosynthetic genes, demonstrating the synergistic effect of the combination of MJ with CDs (Lijaveztky et al., 2008).

Based on these evidences, the main objective of this chapter is to show the effect of UV light on the production of secondary metabolites in *C. roseus, V. vinifera* and *D. carota* cell cultures elicited with CDs and MJ, alone or in combination.

### **2. Effect of UV light exposure, CDs and MJ on the production of** *trans***resveratrol**

The production of *trans*-resveratrol in cell cultures of *Vitis sp* has been analyzed by several groups (Kiselev 2011 and see therein). Analysis of *trans*-resveratrol production in untreated *Vitis* cell cultures (Teguo et al., 1996; Morales et al., 1998; Krisa et al., 1999; Tassoni et al., 2005) revealed a low level of *trans*-resveratrol accumulation, less than 0.01% DW or 2-3 mg/l. Therefore, various strategies such as the use of biotic and abiotic elicitors, addition of biosynthesis precursors and genetic transformation have been considered to improve the production of *trans*-resveratrol.

Fig. 4 shows the level of *trans*-resveratrol (6.72 ± 1.03 mg/g FW) when Monastrell cell cultures were incubated with 50 mM CDs and 100 μM MJ, and how this value was higher than when cell cultures were treated only with CDs (3.15 ± 0.35 mg/g FW). However, very low amounts of *trans*-resveratrol were detected in the spent medium when grapevine cell cultures were elicited only with MJ, and no *trans*-resveratrol was detected in cell cultures treated with ethanol, the solvent in which MJ is delivered to the culture (data not shown). In a similar way, Krisa et al. (1999) described that the amount of total stilbenes secreted to the culture medium was negligible in both MJ and control cultures of three *V. vinifera* cultivars. These authors reported that piceid (glucosylated form of resveratrol) accumulation inside the grape cells of Cavernet-Sauvignon cultivar was notably induced when 25 µM MJ was added to the induction medium on day 6 (6.3 ± 0.2 mg piceid/g DW). Very low levels of piceid in cells (9.75 ± 1.17 µg/g DW) both in the presence of 25 µM MJ as when 25µM MJ and 50 mM CDs were jointly used as inducers (82.7 ± 10.9 μg/g DW, Lijavetzki et al., 2008). However, under the last conditions described above, the accumulation of *trans*-resveratrol in the extracellular medium was 212.6 ± 12.6 mg /g DW that is, 10.6 ± 0.6 mg/g FW (Pedreño et al., 2009).

On the other hand, Kiselev et al. (2007) reported a high *trans*-resveratrol production in *V. amurensis* callus cultures transformed with the *rol*B gene of *Agrobacterium rhizogenes* (31.5 mg *trans*-resveratrol/g DW) which was 6.7 times lower than those obtained in the culture medium under conditions described above using CDs and MJ jointly.

UV-C light has been described as a physical inductor of stilbene biosynthesis in *Vitis sp*. As mentioned above, there are no reports on *trans*-resveratrol production in grapevine cell cultures elicited with UV-C light and most of the research related with UV-C light has been directed at enhancing the stilbene content of grape berries (Adrian et al., 2000; Versari et al., 2001; Cantos et al., 2003; González-Barrio et al., 2006), leaves (Douillet-Breuil et al., 1999; Pezet et al., 2003) and callus tissue (Keller et al., 2000; Keskin & Kunter 2008). Keller et al.,

Effect of UV Light on Secondary Metabolite Biosynthesis

accumulation although no cell browning was observed.

entering in their stationary phase.

treated cells (3.15 ± 0.35 mg/g FW).

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0

mg/g FW

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 123

*trans*-resveratrol levels found when Monastrell cell cultures were exposed to UV-C light (15 min) and elicited with CDs separately (0.16 ± 0.21 mg *trans*-resveratrol/g FW), and in combination with MJ (0.24 ± 0.29 mg *trans*-resveratrol/g FW), because these elicitation experiments were performed using 12-14 day old grapevine cell cultures which are just

Moreover, as shown in Fig. 4, Monastrell cell cultures treated with CDs and MJ, followed by short or long exposures to UV-C light, showed lower *trans*-resveratrol levels (5 min, 1.14 ± 0.36 and 30 min, 0.20 ± 0.29 mg *trans*-resveratrol/g FW) than UV-unexposed cells (6.72 ± 1.03 mg *trans*-resveratrol/g FW), so that UV-C light exposure was clearly detrimental to *trans*-resveratrol production. In fact, prolonged exposure to UV-C light (between 15 and 30 min, Fig.4) or even 120 min (data not shown) caused a drastic reduction in *trans*-resveratrol

However, when Monastrell cell cultures were jointly elicited with CDs and MJ and exposed to UV-A light (Fig. 5), the maximal level of *trans*-resveratrol was found at long exposures (30 min, 8.26 ± 0.48 mg/g FW, 90 min, 7.20 ± 1.14 mg/g FW) although no significant differences were found between CD+MJ-treated cells exposed to UV-A light at these long times and unexposed cells treated with the same chemicals elicitors (6.72 ± 1.03 mg/g FW). By the contrary, at short UV-A light exposures, a drop in the production of *trans*-resveratrol was observed and this decrease was more drastic when cells were elicited with CDs and MJ jointly (15 min, 3.18 ± 0.62 mg/g FW) than in CD-treated cells (2.20 ± 0.15 mg/g FW) in comparison with unexposed-cells (Fig. 5). In addition, when grapevine cell cultures were elicited with CDs and exposed to UV-A during 30 min, a slight increase in the production of *trans*-resveratrol (4.50 ± 0.30 mg/g FW) was detected in comparison with unexposed CD-

CD+UV-A CD+MJ+UV-A

0 5 15 30 60 90

Time (min)

Fig. 5. Effect of the exposure of Monastrell cell cultures to UV-A light (360 nm, 10μW/cm2)

in presence of CDs individually or in combination with MJ in cells elicited for 96 h. Elicitation experiments and analysis of *trans*-resveratrol in the culture medium were performed as described in the legend of the Fig.4. Values are given as the mean ± SD of

three replicates. Solid lines represent mg *trans*-resveratrol/g FW.

(2000) found that only actively growing calli of grapevine cv Cabernet-Sauvignon irradiated with UV-C light were capable of producing stilbenes, whereas old calli had lost this ability. Similar results were described by Keskin & Kunter (2008) working with Cabernet-Sauvignon callus cultures irradiated with UV-C light. They found that the effect of UV-C light on *trans*-resveratrol production was dependent on callus age since the highest *trans*resveratrol production was found in 12 day old calli (62.66 ± 0.40 μg *trans*-resveratrol/g FW) in comparison with those values obtained in 15 day old calli (18.12 ± 0.10 μg *trans*resveratrol/g FW) at the same irradiation time (15 min). In our experiments, Monastrell cell cultures treated with and without MJ and exposed to different UV-C light exposure times produced a negligible extracellular amount of *trans*-resveratrol and browning cell cultures (data not shown). The results obtained by Keskin & Kunter (2008) could explain the low

Fig. 4. Effect of UV-C light exposure on the production of *trans*-resveratrol in Monastrell cell cultures elicited in presence of CDs individually or in combination with MJ. Elicitation experiments were performed in triplicate using 14 day old *V. vinifera* cv Monastrell cell cultures. At zero time, 4 g of fresh weight (FW) of washed cells were transferred into 100 ml flasks and suspended in 20 ml of Gamborg B5 medium supplemented as described Bru et al., (2006), and in the presence of CDs alone or in combination with MJ. After that, cell cultures were maintained in a rotary shaker during 96 h at 25 °C in darkness. Control treatments without elicitors were always run in parallel (data not shown). In the other cases, elicitation was carried out under UV light at different exposure times in the presence of CDs alone or in combination with MJ. For this, flasks were opened in a laminar flow hood and exposed to UV-C light (254 nm, 10μW/cm2) at an irradiation distance of 15 cm. During this time and after irradiation, flasks were kept in continuous agitation for 96 h. After elicitation, cells were separated from the culture medium under a gentle vacuum and the spent medium of *V. vinifera* cell cultures was used for quantifying the *trans*-resveratrol. For this, aliquots of the spent medium of *V. vinifera* were diluted with water and methanol to a final concentration of 80% methanol (v/v). 20 µl of diluted and filtered samples were analysed in a HPLC-DAD as described Bru et al., (2006). *trans*-Resveratrol was identified (at 304 nm) and quantified by comparison with commercial *trans*-resveratrol of >99% purity. Values are given as the mean ± SD of three replicates.

(2000) found that only actively growing calli of grapevine cv Cabernet-Sauvignon irradiated with UV-C light were capable of producing stilbenes, whereas old calli had lost this ability. Similar results were described by Keskin & Kunter (2008) working with Cabernet-Sauvignon callus cultures irradiated with UV-C light. They found that the effect of UV-C light on *trans*-resveratrol production was dependent on callus age since the highest *trans*resveratrol production was found in 12 day old calli (62.66 ± 0.40 μg *trans*-resveratrol/g FW) in comparison with those values obtained in 15 day old calli (18.12 ± 0.10 μg *trans*resveratrol/g FW) at the same irradiation time (15 min). In our experiments, Monastrell cell cultures treated with and without MJ and exposed to different UV-C light exposure times produced a negligible extracellular amount of *trans*-resveratrol and browning cell cultures (data not shown). The results obtained by Keskin & Kunter (2008) could explain the low

CD+UV-C CD+MJ+UV-C

Fig. 4. Effect of UV-C light exposure on the production of *trans*-resveratrol in Monastrell cell cultures elicited in presence of CDs individually or in combination with MJ. Elicitation experiments were performed in triplicate using 14 day old *V. vinifera* cv Monastrell cell cultures. At zero time, 4 g of fresh weight (FW) of washed cells were transferred into 100 ml flasks and suspended in 20 ml of Gamborg B5 medium supplemented as described Bru et al., (2006), and in the presence of CDs alone or in combination with MJ. After that, cell cultures were maintained in a rotary shaker during 96 h at 25 °C in darkness. Control treatments without elicitors were always run in parallel (data not shown). In the other cases, elicitation was carried out under UV light at different exposure times in the presence of CDs alone or in combination with MJ. For this, flasks were opened in a laminar flow hood and exposed to UV-C light (254 nm, 10μW/cm2) at an irradiation distance of 15 cm. During this time and after irradiation, flasks were kept in continuous agitation for 96 h. After elicitation, cells were separated from the culture medium under a gentle vacuum and the spent medium of *V. vinifera* cell cultures was used for quantifying the *trans*-resveratrol. For this, aliquots of

0 5 15 30

Time (min)

the spent medium of *V. vinifera* were diluted with water and methanol to a final

given as the mean ± SD of three replicates.

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0

mg/g FW

concentration of 80% methanol (v/v). 20 µl of diluted and filtered samples were analysed in a HPLC-DAD as described Bru et al., (2006). *trans*-Resveratrol was identified (at 304 nm) and quantified by comparison with commercial *trans*-resveratrol of >99% purity. Values are *trans*-resveratrol levels found when Monastrell cell cultures were exposed to UV-C light (15 min) and elicited with CDs separately (0.16 ± 0.21 mg *trans*-resveratrol/g FW), and in combination with MJ (0.24 ± 0.29 mg *trans*-resveratrol/g FW), because these elicitation experiments were performed using 12-14 day old grapevine cell cultures which are just entering in their stationary phase.

Moreover, as shown in Fig. 4, Monastrell cell cultures treated with CDs and MJ, followed by short or long exposures to UV-C light, showed lower *trans*-resveratrol levels (5 min, 1.14 ± 0.36 and 30 min, 0.20 ± 0.29 mg *trans*-resveratrol/g FW) than UV-unexposed cells (6.72 ± 1.03 mg *trans*-resveratrol/g FW), so that UV-C light exposure was clearly detrimental to *trans*-resveratrol production. In fact, prolonged exposure to UV-C light (between 15 and 30 min, Fig.4) or even 120 min (data not shown) caused a drastic reduction in *trans*-resveratrol accumulation although no cell browning was observed.

However, when Monastrell cell cultures were jointly elicited with CDs and MJ and exposed to UV-A light (Fig. 5), the maximal level of *trans*-resveratrol was found at long exposures (30 min, 8.26 ± 0.48 mg/g FW, 90 min, 7.20 ± 1.14 mg/g FW) although no significant differences were found between CD+MJ-treated cells exposed to UV-A light at these long times and unexposed cells treated with the same chemicals elicitors (6.72 ± 1.03 mg/g FW). By the contrary, at short UV-A light exposures, a drop in the production of *trans*-resveratrol was observed and this decrease was more drastic when cells were elicited with CDs and MJ jointly (15 min, 3.18 ± 0.62 mg/g FW) than in CD-treated cells (2.20 ± 0.15 mg/g FW) in comparison with unexposed-cells (Fig. 5). In addition, when grapevine cell cultures were elicited with CDs and exposed to UV-A during 30 min, a slight increase in the production of *trans*-resveratrol (4.50 ± 0.30 mg/g FW) was detected in comparison with unexposed CDtreated cells (3.15 ± 0.35 mg/g FW).

Fig. 5. Effect of the exposure of Monastrell cell cultures to UV-A light (360 nm, 10μW/cm2) in presence of CDs individually or in combination with MJ in cells elicited for 96 h. Elicitation experiments and analysis of *trans*-resveratrol in the culture medium were performed as described in the legend of the Fig.4. Values are given as the mean ± SD of three replicates. Solid lines represent mg *trans*-resveratrol/g FW.

Effect of UV Light on Secondary Metabolite Biosynthesis

*roseus* cell cultures.

the production of catharanthine.

0,0 10,0 20,0 30,0 40,0 50,0 60,0 70,0 80,0 90,0

combination with MJ.

mg/g DW

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 125

extracts, Menke et al., 1999). If we consider that CDs act in a similar way to fungal elicitors because of they chemically resemble to the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack (Bru et al. 2006), results described by Peebles et al., (2009) could be agreed with the antagonistic effect observed in our experiments when only MJ and UV light (short and long exposures) were used to elicit *C.* 

As regards the production of catharanthine, its level increased when cells were treated with CDs separately or in combination with MJ and exposed to UV-C light both short and long exposure times (Fig. 6). However, the maximal level of catharanthine was observed when cells were exposed 30 min to UV-C irradiation and elicited both with CDs and CDs plus MJ, and no significant differences were found in the rest of experiments under UV light exposure (Fig. 6). There are not reports about how the exposition to UV-C affect to indole alkaloid production and only the effect of UV-B has been tested. In fact, Ramani & Jayabaskaran (2008) observed the enhanced production of catharanthine and vindoline from *C. roseus* cell cultures, in where increased their levels 3 and 12 fold, respectively when cells were irradiated with UV-B for 5 min. Although we do not test the effect of UV-B, these results are in accordance with our results since different UV-C exposition times increased

Catharanthine Ajmalicine

Fig. 6. Effect of different UV-C light exposure times on extracellular accumulation of ajmalicine and catharanthine in cell cultures of *C. roseus* elicited with CDs alone or in

Almagro et al., (2010). Values are given as the mean ± SD of three replicates.

*C. roseus* cv First Kiss Apricot cell cultures were obtained as described Almagro et al., (2010). Elicitation experiments were performed in triplicate using 10 day old *C. roseus* cell cultures. At zero time, cell cultures were elicited in the presence of CDs alone or in combination with MJ and they were maintained at 25°C in a rotary shaker at 110 rpm in darkness. UV light treatments were carried out as described in the legend of Fig. 4. Results were evaluated 96 h after treatments. Ajmalicine and catharanthine were extracted and analysed as described

All these results suggested that long UV-A light exposures (30-60 min) only increased slightly the levels of *trans*-resveratrol when cell cultures were elicited with CDs, and did not enhance *trans*-resveratrol levels when MJ was also present, so it seems that there is an antagonistic effect between MJ and UV-A light since short UV-A light irradiation decreased drastically the production of *trans*-resveratrol when cells were elicited in the presence of MJ. In addition, when grapevine cell cultures elicited with or without MJ in the absence of CDs, and exposed to UV-A light for 15 and 30 min, neither *trans*-resveratrol nor cell browning was detected (data not shown).

The results suggested that the effect of UV light on *trans*-resveratrol production was dependent not only on exposition time (short or long) and UV light type (C or A) but also on the presence of one or two chemical elicitors (CDs and/or MJ).

### **3. Effect of UV light exposure, CDs and MJ on the production of indole alkaloids**

Fig. 6. shows the effect of the exposure of *C. roseus* cell cultures to UV-C light in the presence of 50 mM CDs and 100 μM MJ, separately or in combination. As can be observed, the extracellular accumulation of ajmalicine is dependent on UV-C light time exposure since both short and long UV-C light exposures increased ajmalicine levels in all treatments. However, the maximal levels of ajmalicine were reached when *C. roseus* cell cultures were exposed at UV-C light during 30 min and these levels of production decreased as UV-C exposures increased. In fact, long UV-C light irradiation (60-90 min, Fig. 6) or even more (120 min, Almagro et al., 2010) caused a reduction in ajmalicine production in comparison with 30 min UV-C treatment (Fig. 6). However, 15 min UV-C irradiation was equivalent to long exposures (60-90 min) since no significant differences in ajmalicine production were found. In addition, short and long UV-C exposures had no stimulatory effect when *C. roseus* cell cultures were elicited only with MJ (data not shown). Similarly, control cells treated with short and long UV-C light showed similar low levels of ajmalicine to those unexposed control cells (data not shown). All these results suggested, besides the additive effect observed on ajmalicine accumulation provoked by the joint presence of CDs and MJ, that there was a synergism between these elicitors and UV-C light, and an antagonism between MJ and UV light (short and long exposures) when these elicitors are used to stimulate cells in the absence of CDs (data not shown). This fact may be due to the induction of some sort of stress that does not involve an increase in the production of ajmalicine, while UV-C light exposure may enhance the ajmalicine production but the elicitation in the presence of CDs is needed to provoke any enhancement. Zhao et al., (2001) also observed a synergistic effect on indole alkaloid accumulation in *C. roseus* cell cultures elicited with fungal preparations and chemicals. Among them, the combination of tetramethyl ammonium bromide and *Aspergillum niger* mycelial homogenate gave a maximal ajmalicine production of 0.84 ± 0.05 mg/g DW and an improved catharanthine accumulation of 0.57 ± 0.04 mg/g DW, values that are below those obtained in the best production conditions described above.

Peebles et al., (2009) demonstrated that the octadecanoid pathway, which is involved in the biosynthesis of jasmonates does not actively control the production of indole alkaloids under normal or UV-B stress conditions in *C. roseus* hairy roots. Their results also suggested that the role of the octadecanoid pathway in the abiotic or biotic stress response may differ, depending on the stress or culture type. In *C. roseus* cell cultures, the octadecanoid pathway was active when cells were exposed to biotic compounds (e.g. partially purified yeast

All these results suggested that long UV-A light exposures (30-60 min) only increased slightly the levels of *trans*-resveratrol when cell cultures were elicited with CDs, and did not enhance *trans*-resveratrol levels when MJ was also present, so it seems that there is an antagonistic effect between MJ and UV-A light since short UV-A light irradiation decreased drastically the production of *trans*-resveratrol when cells were elicited in the presence of MJ. In addition, when grapevine cell cultures elicited with or without MJ in the absence of CDs, and exposed to UV-A light for 15 and 30 min, neither *trans*-resveratrol nor cell browning

The results suggested that the effect of UV light on *trans*-resveratrol production was dependent not only on exposition time (short or long) and UV light type (C or A) but also on

Fig. 6. shows the effect of the exposure of *C. roseus* cell cultures to UV-C light in the presence of 50 mM CDs and 100 μM MJ, separately or in combination. As can be observed, the extracellular accumulation of ajmalicine is dependent on UV-C light time exposure since both short and long UV-C light exposures increased ajmalicine levels in all treatments. However, the maximal levels of ajmalicine were reached when *C. roseus* cell cultures were exposed at UV-C light during 30 min and these levels of production decreased as UV-C exposures increased. In fact, long UV-C light irradiation (60-90 min, Fig. 6) or even more (120 min, Almagro et al., 2010) caused a reduction in ajmalicine production in comparison with 30 min UV-C treatment (Fig. 6). However, 15 min UV-C irradiation was equivalent to long exposures (60-90 min) since no significant differences in ajmalicine production were found. In addition, short and long UV-C exposures had no stimulatory effect when *C. roseus* cell cultures were elicited only with MJ (data not shown). Similarly, control cells treated with short and long UV-C light showed similar low levels of ajmalicine to those unexposed control cells (data not shown). All these results suggested, besides the additive effect observed on ajmalicine accumulation provoked by the joint presence of CDs and MJ, that there was a synergism between these elicitors and UV-C light, and an antagonism between MJ and UV light (short and long exposures) when these elicitors are used to stimulate cells in the absence of CDs (data not shown). This fact may be due to the induction of some sort of stress that does not involve an increase in the production of ajmalicine, while UV-C light exposure may enhance the ajmalicine production but the elicitation in the presence of CDs is needed to provoke any enhancement. Zhao et al., (2001) also observed a synergistic effect on indole alkaloid accumulation in *C. roseus* cell cultures elicited with fungal preparations and chemicals. Among them, the combination of tetramethyl ammonium bromide and *Aspergillum niger* mycelial homogenate gave a maximal ajmalicine production of 0.84 ± 0.05 mg/g DW and an improved catharanthine accumulation of 0.57 ± 0.04 mg/g DW, values that are below those obtained in the best production conditions described above. Peebles et al., (2009) demonstrated that the octadecanoid pathway, which is involved in the biosynthesis of jasmonates does not actively control the production of indole alkaloids under normal or UV-B stress conditions in *C. roseus* hairy roots. Their results also suggested that the role of the octadecanoid pathway in the abiotic or biotic stress response may differ, depending on the stress or culture type. In *C. roseus* cell cultures, the octadecanoid pathway was active when cells were exposed to biotic compounds (e.g. partially purified yeast

**3. Effect of UV light exposure, CDs and MJ on the production of indole** 

the presence of one or two chemical elicitors (CDs and/or MJ).

was detected (data not shown).

**alkaloids** 

extracts, Menke et al., 1999). If we consider that CDs act in a similar way to fungal elicitors because of they chemically resemble to the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack (Bru et al. 2006), results described by Peebles et al., (2009) could be agreed with the antagonistic effect observed in our experiments when only MJ and UV light (short and long exposures) were used to elicit *C. roseus* cell cultures.

As regards the production of catharanthine, its level increased when cells were treated with CDs separately or in combination with MJ and exposed to UV-C light both short and long exposure times (Fig. 6). However, the maximal level of catharanthine was observed when cells were exposed 30 min to UV-C irradiation and elicited both with CDs and CDs plus MJ, and no significant differences were found in the rest of experiments under UV light exposure (Fig. 6). There are not reports about how the exposition to UV-C affect to indole alkaloid production and only the effect of UV-B has been tested. In fact, Ramani & Jayabaskaran (2008) observed the enhanced production of catharanthine and vindoline from *C. roseus* cell cultures, in where increased their levels 3 and 12 fold, respectively when cells were irradiated with UV-B for 5 min. Although we do not test the effect of UV-B, these results are in accordance with our results since different UV-C exposition times increased the production of catharanthine.

Fig. 6. Effect of different UV-C light exposure times on extracellular accumulation of ajmalicine and catharanthine in cell cultures of *C. roseus* elicited with CDs alone or in combination with MJ.

*C. roseus* cv First Kiss Apricot cell cultures were obtained as described Almagro et al., (2010). Elicitation experiments were performed in triplicate using 10 day old *C. roseus* cell cultures. At zero time, cell cultures were elicited in the presence of CDs alone or in combination with MJ and they were maintained at 25°C in a rotary shaker at 110 rpm in darkness. UV light treatments were carried out as described in the legend of Fig. 4. Results were evaluated 96 h after treatments. Ajmalicine and catharanthine were extracted and analysed as described Almagro et al., (2010). Values are given as the mean ± SD of three replicates.

Effect of UV Light on Secondary Metabolite Biosynthesis

Campesterol A

<sup>β</sup>-Sitosterol C

(Fig. 8).

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0

mg/g DW

mg/g DW

phytosterols/g DW.

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 127

identical to that of unexposed cells and its level decreased only when the exposition to UV-A was prolonged (Fig. 8). However, the addition of the third elicitor (UV-A) to cells elicited with CDs plus MJ provoked a biphasic response in the production of phytosterols reaching a maximal level when cell cultures were exposed 30 min to UV-A light. Also, the levels of fucosterol were lower than their derivatives, β-sitosterol and stigmasterol. It is also worth noting that the production of β-sitosterol was identical to that of campesterol

> 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0

mg/g DW

mg/g DW

Fucosterol B

Stigmasterol D

Fig. 7. Effect of different UV-C light exposure times on extracellular accumulation of phytosterols in cell cultures of *D. carota* elicited with CDs alone or in combination with MJ. *D. carota* calli were established in our laboratory in 2005 from root explants and they have been maintained at light at 25 ºC in 250 ml flasks containing 100 ml of Murashige & Skoog medium supplemented as described Sabater-Jara et al., (2008). *D. carota* cell cultures were initiated by inoculating friable callus pieces into 250 ml flasks containing 100 ml of the same

medium without agar and were maintained at 25ºC under a 16-h light/8-h dark

evaluated 96 h after treatments. Extraction, analysis and identification of different

Values are given as the mean ± SD of three replicates. Bars represent mg of different

photoperiod at 25°C in a rotary shaker at 110 rpm. Elicitation experiments were performed in triplicate using 10 day old *D. carota* as described in the legend to the Fig. 4. Results were

phytosterols in the culture medium were carried out as described Sabater-Jara et al., (2010b).

As regards the biosynthesis of major components of the phytoalexin complex described in *Daucus*, the isocoumarin, 6-methoxymellein was detected in those cell cultures jointly elicited with CDs plus MJ both UV-A irradiated and non-irradiated, and its level decreased as UV-A

In relation to the effect of UV-A, cell cultures treated with or without MJ and exposed to UV-A light were not able to produce indole alkaloids nor accumulate them in the culture medium (data not shown). However, when *C. roseus* cell cultures were elicited in the presence of CDs and exposed to UV-A light for 15 and 30 min, the production of ajmalicine increased in relation to non-exposed cells (data not shown) and this increase was similar to that observed in cell cultures elicited, in the same conditions, and irradiated with UV-C light (Fig. 6).

The combination of these three elicitors synergistically increased more the level of indole alkaloids than when each single or two elicitors are used. The reasons why this combination of three elicitors promoted best effects on indole alkaloid production are still not known. Because each chemical elicitor (CDs and/or MJ) or UV light stimulates indole alkaloid accumulation by different pathways, the mechanism for every treatment may be result of combining them since it depends on the interactions between physiological effects caused by the three elicitors.

### **4. Effect of UV light exposure, CDs and MJ on the production of phytosterols**

Fig. 7 shows the effect of different UV-C light exposure times on phytosterol production in *D. carota* cell cultures elicited in the presence of CDs alone or in combination with MJ. As can be observed in this figure, the production of these four phytosterols was greater when cell cultures were elicited in the presence of CDs alone than in those combined with MJ. In addition, control cells elicited with or without MJ and exposed to UV-C light for different exposure times (15, 30 and 60 min) did not produce any of these phytosterols (data not shown). When cell cultures were elicited only with CDs and exposed to UV-C irradiation, total production levels of phytosterols were kept identical to that of unexposed cells and these levels only decrease when the UV light exposure is prolonged (60 min). However, when the cells were elicited with CDs and MJ, a slight enhancement in the total content of phytosterols was observed as the UV-C light exposure times increased (Fig. 7). These results suggested that the production of phytosterols is sustained during UV exposure time through the joint presence of the three elicitors. In these conditions, production levels of fucosterol were lower than those of β-sitosterol and these, in turn, lower than those of stigmasterol. This low level of fucosterol can be explained by knowing its biosynthetic pathway. All of them are biosynthesized via the mevalonate-dependent isoprenoid pathway. After the synthesis of squalene catalyzed by squalene synthase and its epoxidation catalyzed by squalene epoxidase, 2,3-oxidosqualene is mainly cyclized to cycloartenol which requires cycloartenol synthase (Boutté & Grebe, 2009). Following conversion of 2,3 oxidosqualene to cycloartenol, the first cycle structure providing the basic sterol skeleton, the pathway is essentially linear until reaching 24-methylene lophenol. After formation of this compound, there is a bifurcation to either 24-methyl sterols, which include campesterol and its derivatives, the brassinosteroids, or 24-ethyl sterols, which include the structural sterols fucosterol, β-sitosterol and stigmasterol (Clouse, 2002; Posé et al., 2009).

The effect of different UV-A light exposure times on phytosterol production in *D. carota*  cell cultures elicited in the presence of CDs alone or in combination with MJ is shown in Fig. 8. Similarly to results described above, when *D. carota* cell cultures were elicited with CDs and exposed to different UV-A irradiation times, total phytosterol content remained

In relation to the effect of UV-A, cell cultures treated with or without MJ and exposed to UV-A light were not able to produce indole alkaloids nor accumulate them in the culture medium (data not shown). However, when *C. roseus* cell cultures were elicited in the presence of CDs and exposed to UV-A light for 15 and 30 min, the production of ajmalicine increased in relation to non-exposed cells (data not shown) and this increase was similar to that observed in cell cultures elicited, in the same conditions, and irradiated with UV-C light

The combination of these three elicitors synergistically increased more the level of indole alkaloids than when each single or two elicitors are used. The reasons why this combination of three elicitors promoted best effects on indole alkaloid production are still not known. Because each chemical elicitor (CDs and/or MJ) or UV light stimulates indole alkaloid accumulation by different pathways, the mechanism for every treatment may be result of combining them since it depends on the interactions between physiological effects caused

**4. Effect of UV light exposure, CDs and MJ on the production of phytosterols**  Fig. 7 shows the effect of different UV-C light exposure times on phytosterol production in *D. carota* cell cultures elicited in the presence of CDs alone or in combination with MJ. As can be observed in this figure, the production of these four phytosterols was greater when cell cultures were elicited in the presence of CDs alone than in those combined with MJ. In addition, control cells elicited with or without MJ and exposed to UV-C light for different exposure times (15, 30 and 60 min) did not produce any of these phytosterols (data not shown). When cell cultures were elicited only with CDs and exposed to UV-C irradiation, total production levels of phytosterols were kept identical to that of unexposed cells and these levels only decrease when the UV light exposure is prolonged (60 min). However, when the cells were elicited with CDs and MJ, a slight enhancement in the total content of phytosterols was observed as the UV-C light exposure times increased (Fig. 7). These results suggested that the production of phytosterols is sustained during UV exposure time through the joint presence of the three elicitors. In these conditions, production levels of fucosterol were lower than those of β-sitosterol and these, in turn, lower than those of stigmasterol. This low level of fucosterol can be explained by knowing its biosynthetic pathway. All of them are biosynthesized via the mevalonate-dependent isoprenoid pathway. After the synthesis of squalene catalyzed by squalene synthase and its epoxidation catalyzed by squalene epoxidase, 2,3-oxidosqualene is mainly cyclized to cycloartenol which requires cycloartenol synthase (Boutté & Grebe, 2009). Following conversion of 2,3 oxidosqualene to cycloartenol, the first cycle structure providing the basic sterol skeleton, the pathway is essentially linear until reaching 24-methylene lophenol. After formation of this compound, there is a bifurcation to either 24-methyl sterols, which include campesterol and its derivatives, the brassinosteroids, or 24-ethyl sterols, which include the structural

sterols fucosterol, β-sitosterol and stigmasterol (Clouse, 2002; Posé et al., 2009).

The effect of different UV-A light exposure times on phytosterol production in *D. carota*  cell cultures elicited in the presence of CDs alone or in combination with MJ is shown in Fig. 8. Similarly to results described above, when *D. carota* cell cultures were elicited with CDs and exposed to different UV-A irradiation times, total phytosterol content remained

(Fig. 6).

by the three elicitors.

identical to that of unexposed cells and its level decreased only when the exposition to UV-A was prolonged (Fig. 8). However, the addition of the third elicitor (UV-A) to cells elicited with CDs plus MJ provoked a biphasic response in the production of phytosterols reaching a maximal level when cell cultures were exposed 30 min to UV-A light. Also, the levels of fucosterol were lower than their derivatives, β-sitosterol and stigmasterol. It is also worth noting that the production of β-sitosterol was identical to that of campesterol (Fig. 8).

Fig. 7. Effect of different UV-C light exposure times on extracellular accumulation of phytosterols in cell cultures of *D. carota* elicited with CDs alone or in combination with MJ. *D. carota* calli were established in our laboratory in 2005 from root explants and they have been maintained at light at 25 ºC in 250 ml flasks containing 100 ml of Murashige & Skoog medium supplemented as described Sabater-Jara et al., (2008). *D. carota* cell cultures were initiated by inoculating friable callus pieces into 250 ml flasks containing 100 ml of the same medium without agar and were maintained at 25ºC under a 16-h light/8-h dark photoperiod at 25°C in a rotary shaker at 110 rpm. Elicitation experiments were performed in triplicate using 10 day old *D. carota* as described in the legend to the Fig. 4. Results were evaluated 96 h after treatments. Extraction, analysis and identification of different phytosterols in the culture medium were carried out as described Sabater-Jara et al., (2010b). Values are given as the mean ± SD of three replicates. Bars represent mg of different phytosterols/g DW.

As regards the biosynthesis of major components of the phytoalexin complex described in *Daucus*, the isocoumarin, 6-methoxymellein was detected in those cell cultures jointly elicited with CDs plus MJ both UV-A irradiated and non-irradiated, and its level decreased as UV-A

Effect of UV Light on Secondary Metabolite Biosynthesis

phenylpropanoid or flavonoid derivatives.

**5. Conclusion** 

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 129

Therefore, it is possible to think that, in the simultaneous treatment of carrot cells with UV-A light and elicitors, the chemical elicitor signal was dominant over the UV light signal, resulting in the UV induction or inhibition of different biosynthetic pathways which leads to

The application of biotic or abiotic stimuli has been one of the most effective strategies for improving the productivity of different useful secondary metabolites from plant cell cultures (Vasconsuelo & Boland, 2007; Zhao et al., 2005). The most frequently used elicitors are of fungal and yeast origin, jasmonic acid and MJ, chitosan, metal ions and UV light. More recently, special attention has been paid to the use of CDs as true elicitors, which act inducing defence responses, which include pathogenesis-related proteins and phytoalexin synthesis, especially the stilbenes like *trans*-resveratrol in *Vitis sp* (Morales et al., 1998; Bru et al., 2006; Zamboni et al., 2009; Martinez-Esteso et al. 2009), the accumulation of sesquiterpenes and phytosterols as well as pathogenesis-related proteins in *Capsicum sp* (Sabater-Jara et al., 2010b; Sabater-Jara et al., 2011), the increase of ajmalicine and catharanthine in *C. roseus* (Almagro et al., 2010) and the enhancement of the production of silymarin in *Silybum marianum* (L.) Gaernt cell cultures (Belchí-Navarro et al., 2011). The success in the production of secondary metabolites with the use of CDs lies in the properties of these compounds since they act not only as elicitors but also forming inclusion complexes (clathrates) with stilbenes (Morales et al., 1998), sesquiterpenes (Sabater-Jara et al., 2010b), indole alkaloids (Almagro et al, 2010) and silymarins (Belch**í**-Navarro et al., 2011), favouring the substantial accumulation of these metabolites and also preventing the toxic and/or

inhibitory effect of their extracellular accumulation on cells cultured in suspension.

Most of the publications concerning secondary metabolite production by means of plant cell cultures reported that the elicitation with MJ increased the accumulation of secondary metabolites (Zhao et al., 2005; Sánchez-Sampedro et al., 2005; Belchí-Navarro et al., 2011; Almagro et al., 2010; Lee-Parsons et al., 2004; Repka et al., 2004; Szabo et al., 1999; Tassoni et al., 2005; Yukimune et al., 1996; Mandujano-Chavez et al., 2000; Sabater-Jara et al., 2010a,b; Wang & Wu, 2005). In fact, the addition of a second elicitor, for instance MJ to cell cultures elicited with CDs increased significantly the production of *trans*-resveratrol, indole alkaloids and phytosterols in Monastrell grapevine, *C. roseus* and *D. carota* cell cultures, respectively. However, the effect of UV light on the production of these secondary metabolites is dependent not only on the exposition time (short or long) and UV light type (C or A), but also on the presence of one (CDs) or two chemical elicitors (CDs and MJ). Thus, the addition of a third elicitor (UV light) in Monastrell grapevine cell cultures, elicited with CDs separately or in combination with MJ, decreased (UV-C light, Fig. 4) or did not increase significantly the production of *trans*-resveratrol (UV-A light, Fig. 5). By the contrary, to achieve high ajmalicine production levels using a productive *C. roseus* cell line, the best operating conditions were elicitation using a combination of CDs and MJ and a cell exposition to UV light (A or C) during 30 min although this enhancement was also observed at all irradiation times tested (Fig. 6). Moreover, the production of phytosterols depended on exposition time (short or long) and UV light type (C or A) since the exposition of *D. carota* cell cultures to UV-C light did not increase phytosterol extracellular accumulation in CD- or CD plus MJ-treated cells (Fig. 7) whereas in cells elicited with CDs and MJ and exposed to UV-A during 30 min, an increase in the phytosterol production was observed (Fig. 8). The

exposure time increased (data not shown). Similarly, the accumulation of UV-absorbing compounds, mainly those of phenolic nature, that is, phenylpropanoid derivatives, were observed. In fact, eugenol and isoeugenol were identified both CD- and CD plus MJ-treated cells. The content of these phenols decreased when CD-treated cells were exposed to UV-A light. By the contrary, the levels of these compounds increased in cell cultures elicited with CDs plus MJ and exposed to a short UV-A irradiation (15 min), and they returned to decrease when UV-A exposure time is prolonged (60 min) (data not shown).

Gläβgen et al., (1998) reported that the biosynthesis and accumulation of anthocyanins in carrot cell cultures was strongly enhanced by continuous irradiation with UV-containing white light (315-420 nm) and that was preceded by the corresponding induction of the enzymes activities of the phenylpropanoid and flavonoid pathways. These authors also showed that the treatment with UV-A light and fungal elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV-A light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanins biosynthesis was suppressed. Their results indicated a coordinated regulation of the enzymes involved in anthocyanins biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the dominating role of the fungal elicitor signal over the UV stimulus.

Fig. 8. Effect of different UV-A light (360 nm, 10μW/cm2) exposure times on extracellular accumulation of phytosterols in cell cultures of *D. carota* elicited with CDs alone or in combination with MJ. Elicitation experiments, extraction, analysis and identification of different phytosterols in the culture medium were performed as described in the legend of the Fig. 7. Values are given as the mean ± SD of three replicates. Bars represent mg of different phytosterols/g DW.

Therefore, it is possible to think that, in the simultaneous treatment of carrot cells with UV-A light and elicitors, the chemical elicitor signal was dominant over the UV light signal, resulting in the UV induction or inhibition of different biosynthetic pathways which leads to phenylpropanoid or flavonoid derivatives.

### **5. Conclusion**

128 Plants and Environment

exposure time increased (data not shown). Similarly, the accumulation of UV-absorbing compounds, mainly those of phenolic nature, that is, phenylpropanoid derivatives, were observed. In fact, eugenol and isoeugenol were identified both CD- and CD plus MJ-treated cells. The content of these phenols decreased when CD-treated cells were exposed to UV-A light. By the contrary, the levels of these compounds increased in cell cultures elicited with CDs plus MJ and exposed to a short UV-A irradiation (15 min), and they returned to decrease

Gläβgen et al., (1998) reported that the biosynthesis and accumulation of anthocyanins in carrot cell cultures was strongly enhanced by continuous irradiation with UV-containing white light (315-420 nm) and that was preceded by the corresponding induction of the enzymes activities of the phenylpropanoid and flavonoid pathways. These authors also showed that the treatment with UV-A light and fungal elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV-A light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanins biosynthesis was suppressed. Their results indicated a coordinated regulation of the enzymes involved in anthocyanins biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the

Fig. 8. Effect of different UV-A light (360 nm, 10μW/cm2) exposure times on extracellular accumulation of phytosterols in cell cultures of *D. carota* elicited with CDs alone or in combination with MJ. Elicitation experiments, extraction, analysis and identification of different phytosterols in the culture medium were performed as described in the legend of the Fig. 7. Values are given as the mean ± SD of three replicates. Bars represent mg of

0,0 1,0 2,0 3,0 4,0 5,0 6,0

0,0 1,0 2,0 3,0 4,0 5,0 6,0

mg/g DW

mg/g DW

Fucosterol B

Stigmasterol D

different phytosterols/g DW.

0,0 1,0 2,0 3,0 4,0 5,0 6,0

0,0 1,0 2,0 3,0 4,0 5,0 6,0

mg/g DW

mg/g DW

when UV-A exposure time is prolonged (60 min) (data not shown).

dominating role of the fungal elicitor signal over the UV stimulus.

Campesterol A

<sup>β</sup>-Sitosterol C

The application of biotic or abiotic stimuli has been one of the most effective strategies for improving the productivity of different useful secondary metabolites from plant cell cultures (Vasconsuelo & Boland, 2007; Zhao et al., 2005). The most frequently used elicitors are of fungal and yeast origin, jasmonic acid and MJ, chitosan, metal ions and UV light.

More recently, special attention has been paid to the use of CDs as true elicitors, which act inducing defence responses, which include pathogenesis-related proteins and phytoalexin synthesis, especially the stilbenes like *trans*-resveratrol in *Vitis sp* (Morales et al., 1998; Bru et al., 2006; Zamboni et al., 2009; Martinez-Esteso et al. 2009), the accumulation of sesquiterpenes and phytosterols as well as pathogenesis-related proteins in *Capsicum sp* (Sabater-Jara et al., 2010b; Sabater-Jara et al., 2011), the increase of ajmalicine and catharanthine in *C. roseus* (Almagro et al., 2010) and the enhancement of the production of silymarin in *Silybum marianum* (L.) Gaernt cell cultures (Belchí-Navarro et al., 2011). The success in the production of secondary metabolites with the use of CDs lies in the properties of these compounds since they act not only as elicitors but also forming inclusion complexes (clathrates) with stilbenes (Morales et al., 1998), sesquiterpenes (Sabater-Jara et al., 2010b), indole alkaloids (Almagro et al, 2010) and silymarins (Belch**í**-Navarro et al., 2011), favouring the substantial accumulation of these metabolites and also preventing the toxic and/or inhibitory effect of their extracellular accumulation on cells cultured in suspension.

Most of the publications concerning secondary metabolite production by means of plant cell cultures reported that the elicitation with MJ increased the accumulation of secondary metabolites (Zhao et al., 2005; Sánchez-Sampedro et al., 2005; Belchí-Navarro et al., 2011; Almagro et al., 2010; Lee-Parsons et al., 2004; Repka et al., 2004; Szabo et al., 1999; Tassoni et al., 2005; Yukimune et al., 1996; Mandujano-Chavez et al., 2000; Sabater-Jara et al., 2010a,b; Wang & Wu, 2005). In fact, the addition of a second elicitor, for instance MJ to cell cultures elicited with CDs increased significantly the production of *trans*-resveratrol, indole alkaloids and phytosterols in Monastrell grapevine, *C. roseus* and *D. carota* cell cultures, respectively. However, the effect of UV light on the production of these secondary metabolites is dependent not only on the exposition time (short or long) and UV light type (C or A), but also on the presence of one (CDs) or two chemical elicitors (CDs and MJ). Thus, the addition of a third elicitor (UV light) in Monastrell grapevine cell cultures, elicited with CDs separately or in combination with MJ, decreased (UV-C light, Fig. 4) or did not increase significantly the production of *trans*-resveratrol (UV-A light, Fig. 5). By the contrary, to achieve high ajmalicine production levels using a productive *C. roseus* cell line, the best operating conditions were elicitation using a combination of CDs and MJ and a cell exposition to UV light (A or C) during 30 min although this enhancement was also observed at all irradiation times tested (Fig. 6). Moreover, the production of phytosterols depended on exposition time (short or long) and UV light type (C or A) since the exposition of *D. carota* cell cultures to UV-C light did not increase phytosterol extracellular accumulation in CD- or CD plus MJ-treated cells (Fig. 7) whereas in cells elicited with CDs and MJ and exposed to UV-A during 30 min, an increase in the phytosterol production was observed (Fig. 8). The

Effect of UV Light on Secondary Metabolite Biosynthesis

5266

ISSN 0897-5957

ISBN 978-052-1572-22-4, Cambridge, United Kingdon

PCT patent WO/2003/062406. US 2006/0205049 A1

(December 2005), pp. 65-71, ISSN 0021-8561

455-463, ISSN 0028-646X

1208-1214, ISSN 0021-8561

381, ISSN 1040-2519

ISSN 0021-8561

9422

2009), pp. 765-768, ISSN 0910-6340

2519

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 131

Boutté, Y. & Grebe, M. (2009). Cellular processes relying on sterol function in plants. Current

Bradamante, S.; Barenghi, L. & Villa, A. (2004). Cardiovascular protective effects of

Broeckling, CD.; Huhman, DV.; Farag, MA.; Smith, JT.; May, GD.; Mendes, P.; Dixon, RA. &

Bru, R.; Selles, S.; Casado-Vela, J.; Belchí-Navarro, S. & Pedreño, MA. (2006). Modified

Calderón, AA.; Zapata, JM.; Muñoz, R.; Pedreño, MA. & Ros-Barcelo, A. (1993). Resveratrol

Cantos, E.; Espín, JC.; Fernández, MJ.; Oliva, J. & Tomás-Barberán, FA. (2003). Postharvest

Chung, I.; Park, MR.; Chun, JC. & Yun, SJ. (2003). Resveratrol accumulation and resveratrol

Creelman, RA. & Mullet, JE. (1997). Biosynthesis and action of jasmonates in plants. *Annual* 

De la Lastra, C. & Villegas, I. (2005). Resveratrol as an anti-inflammatory and anti-aging

Delgado-Zamarreño, MM.; Bustamante-Rangel, M.; Martínez-Pelarda, D. & Carabías-

Douillet-Breuil, AC.; Jeandet, P.; Adrian, M. & Bessis, R. (1999). Changes in the phytoalexin

Espín, JC.; García-Conesa, MT. & Tomás-Barberán, FA. (2007). Nutraceuticals: Facts and

Vol.49, No.5, (May 2005), pp. 405–430, ISSN 1613-4125

*Botany,* Vol.53, No.410, (December 2004), pp. 323–326, ISSN 0022-0957 Bru, R. & Pedreño, MA. (2003). Method for the production of resveratrol in cell cultures.

*Environmental Change,* P.J. Lumsden, (Ed.), 157–168, Cambridge University Press,

Opinion in Plant Biology, Vol.12, No.6, pp. 705?713, (December 2009), ISSN 1369-

resveratrol. *Cardiovascular Drug Reviews*, Vol.22, No.3, (June 2006), pp. 169 –188,

Sumner, LW. (2005). Metabolic profiling of *Medicago trunculata* cell cultures reveals the effects of biotic and abiotic elicitors on metabolism*. Journal of Experimental* 

cyclodextrins are chemically defined glucan inducers of defense responses in grapevine cell cultures. *Journal of Agricultural and Food Chemistry,* Vol.54, No.1,

production as a part of the hypersensitive-like response of grapevine cells to an elicitor from *Trichoderma viride*. New Phytologist, Vol.124, No.3, (July 1993), pp.

UV-C irradiated grapes as potential source for producing stilbene-enriched red wines. *Journal of Agricultural and Food Chemistry*, Vol.51, No.5, (January 2003), pp.

synthase gene expression in response to abiotic stresses and hormones in peanut plants. *Plant Science*, Vol.164, No.1, (January 2003), pp. 103–109, ISSN 0168-9452 Clouse, S. (2002). Arabidopsis mutants reveal multiple roles for sterols in plant

development. *Plant Cell,* Vol.14, No.9, (September 2002), pp. 1995-2000, ISSN 1040-

*Review of Plant Physiology and Plant Molecular Biology*, Vol.48, (June 1997), pp. 355-

agent: mechanisms and clinical implications. *Molecular Nutrition & Food Research*,

Martínez, R. (2009). Analysis of β-sitosterol in seeds and nuts using pressurized liquid extraction and liquid chromatography. *Analytical Science,* Vol.25, No.6, (June

content of various *Vitis* spp. in response to ultraviolet C elicitation. *Journal of Agricultural and Food Chemistry*, Vol.47, No.10, (September 1999), pp. 4456-4461,

fiction. *Phytochemistry*, Vol.68, No.22, (September 2007), pp. 2986-3008, ISSN 0031-

mechanism by which the elicitor signal leads to suppression or activation of metabolite biosynthesis has yet to be investigated. All effects observed after application of the elicitors alone or in combination with UV light may be regulated at the level of both protein and mRNA of crucial enzymes whose activities increased or decreased. Further experiments are needed in order to elucidate the mechanism by which the joint action of three different elicitors in some cases, improves the production of secondary metabolites while in others, no effect is observed.

### **6. Acknowledgments**

Almagro L., Sabater-Jara AB., Fernández-Pérez F. held grants from the Ministerio de Ciencia e Innovación. We thank Pepita Alemán for help in maintaining Monastrell cell cultures. This work has been supported by Fundación Séneca (08799/PI/08) and Ministerio de Ciencia e Innovación (BIO2005-00332 and BIO2008-02941).

### **7. References**


mechanism by which the elicitor signal leads to suppression or activation of metabolite biosynthesis has yet to be investigated. All effects observed after application of the elicitors alone or in combination with UV light may be regulated at the level of both protein and mRNA of crucial enzymes whose activities increased or decreased. Further experiments are needed in order to elucidate the mechanism by which the joint action of three different elicitors in some cases, improves the production of secondary metabolites while in others,

Almagro L., Sabater-Jara AB., Fernández-Pérez F. held grants from the Ministerio de Ciencia e Innovación. We thank Pepita Alemán for help in maintaining Monastrell cell cultures. This work has been supported by Fundación Séneca (08799/PI/08) and Ministerio de Ciencia e

Adrian, M.; Jeandet, P.; Douillet-Breuil, AC.; Tesson, L. & Bessis, R. (2000). Stilbene content

Almagro, L.; López-Pérez, AJ. & Pedreño, MA. (2010). New method to enhance ajmalicine

Baur, JA.; Pearson, KJ.; Price, NL.; Jamieson, HA.; Lerin, C.; Kalra, A.; Prabhu, VV.; Allard,

Belchí-Navarro, S.; Pedreño, MA. & Corchete, P. (2011). Methyl jasmonate increases

Belhadj, A.; Telef, N. & Saigne, C. (2008). Effect of methyl jasmonate in combination with

Bishayee, A.; Sarkar, A. & Chatterjee, M. (1995). Hepatoprotective activity of carrot (*Daucus* 

Bornman, JF.; Reuber, S.; Cen, YP. & Weissenböck, G. (1997). Ultraviolet radiation as a stress

*Ethnopharmacology*, Vol.47, No.2 (July 1995), pp. 69-74, ISSN 0378-8741 Bonfill, M.; Mangas, S.; Moyano, E.; Cusido, RM. & Palazón, J. (2011). Production of

No.4, (April 2008), pp. 493-499, ISSN 0981-9428

of mature *Vitis vinifera* berries in response to UV-C elicitation. *Journal of Agricultural and Food Chemistry,* Vol.48, No.12, (November 2000), pp. 6103-6105, ISSN 0021-8561

production in *Catharanthus roseus* cell cultures based on the use of cyclodextrins. *Biotechnology Letters,* Vol.33, No.2, (October 2010), pp. 381-385, ISSN 0141-5492 Asada, M. & Shuler, ML. (1989). Stimulation of ajmalicine production and excretion from

*Catharanthus roseus*: effects of adsorption *in situ*, elicitors, and alginate immobilization. *Applied Microbiology and Biotechnology,* Vol.30, No.5, (May 1989),

JS.; Lopez-Lluch, G. & Lewis, K. (2006). Resveratrol improves health and survival of mice on a high-calorie diet. *Nature,* Vol.444, (November 2006), pp. 337-342, ISSN

silymarin production in *Silybum marianum* (L.) Gaernt cell cultures treated with βcyclodextrins. *Biotechnology Letters*, Vol.33, No.11, (January 2011), pp. 179-184, ISSN

carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures. *Plant Physiology and Biochemistry,* Vol.46,

*carota* L.) against carbon tetrachloride intoxication in mouse liver. *Journal of* 

centellosides and phytosterols in cell suspension cultures of *Centella asiatica. Plant Cell Tissue and Organ Culture*, Vol.104, No.1, (January 2011), pp. 61-67, ISSN 0167-

factor and the role of protective pigments. In: *Plants and UV-B: Responses to* 

no effect is observed.

**7. References** 

**6. Acknowledgments** 

Innovación (BIO2005-00332 and BIO2008-02941).

pp. 475–481, ISSN 0175-7598

0028-0836

0141-5492

6857

*Environmental Change,* P.J. Lumsden, (Ed.), 157–168, Cambridge University Press, ISBN 978-052-1572-22-4, Cambridge, United Kingdon


Effect of UV Light on Secondary Metabolite Biosynthesis

2004), pp. 976-984, ISSN 1471-9053

2004), pp. 1595-1599, ISSN 0141-5492

8, ISSN 1756-0500

ISSN 0141-5492

9861

0031-949X

(November 1999), pp. 1688-1690, ISSN 0163-3864

(February 1977), pp. 1193-1196, ISSN 0031-9422

No.2, (January 1991), pp. 307-314, ISSN 0032-0935

Vol.35, No.6, (April 1994), pp. 1457-1460, ISSN 0031-9422

No.2, (December 2009), pp. 331-341, ISSN 1874-3919

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 133

Langcake, P. & Pryce, RJ. (1977). The production of resveratrol and the viniferins by

Lee, MH.; Jeong, JH.; Seo, JW.; Shin, CG.; Kim, YS.; In, JG.; Yang, DC.; Yi, JS. & Choi, YE.

Lee-Parsons, CWT.; Ertürk, S. & Tengtrakool, J. (2004). Enhancement of ajmalicine

Lijavetzky, D.; Almagro, L.; Belchí-Navarro, S.; Martínez-Zapater, JM.; Bru, R. & Pedreño,

Liswidowati, FM.; Melchior, F.; Hohmann, F.; Schwer, B. & Kindl, H. (1991). Induction of

Ma, CJ. (2008). Cellulase elicitor induced accumulation of capsidiol in *Capsicum annuum* L.

Mandujano-Chavez, A.; Schoenbeck, MA.; Ralston, LF.; Lozoya-Gloria, E. & Chappell, J.

Marinelli, F.; Ronchi, VN. & Salvadori, P. (1994). Elicitor induction of enzyme activities and

Martinez-Esteso, MJ.; Sellés-Marchart, S.; Vera-Urbina, JC.; Pedreño, MA. & Bru, R. (2009).

Menke, FL.; Parchmann, S.; Mueller, MJ.; Kijne, JW. & Memelink, J. (1999). Involvement of

*Plant Physiology*, Vol.119, No.4 (April 1999), pp.1289-1296, ISSN 0032-0889 Mercier, J.; Roussel, D.; Charles, MT. & Arul, J. (2000). Systemic and local responses

Morales, M.; Bru, R.; García-Carmona, F.; Ros Barceló, A. & Pedreño, MA. (1998). Effect of

induction and 13C biolabelling. *Journal of Natural Products,* Vol.62, No.12,

grapevines in response to ultraviolet irradiations. *Phytochemistry,* Vol.16, No.8,

(2004). Enhanced triterpene and phytosterol biosynthesis in *Panax ginseng*  overexpressing squalene synthase gene. *Plant Cell Physiology*, Vol.45, No.8, (August

production in *Catharanthus roseus* cell cultures with methyl jasmonate is dependent on timing and dosage of elicitation. *Biotechnology Letters,* Vol.26, No.20, (October

MA. (2008). Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures. *BMC Research Notes*, Vol.1, No.132, (December 2008), pp. 1-

stilbene synthase by *Botrytis cinerea* in cultured grapevine cells. *Planta,* Vol.183,

suspension cultures. *Biotechnology Letters*, Vol.30, No.5, (May 2008), pp. 961–965,

(2000). Differential induction of sesquiterpene metabolism in tobacco cell suspension cultures by methyl jasmonate and fungal elicitor. *Archives of Biochemistry and Biophysics,* Vol.381, No.2, (September 200), pp. 285-294, ISSN 0003-

6-methoxymellein production in carrot cell suspension culture. *Phytochemistry*,

Changes of defense proteins in the extracellular proteome of grapevine (*Vitis vinifera* cv. Gamay) cell cultures in response to elicitors. *Journal of Proteomics,* Vol.73,

the octadecanoid pathway and protein phosphorylation in fungal elicitorinduced expression of terpenoid indole alkaloid biosynthetic genes in *Catharanthus roseus*.

associated with UV-induced and pathogen induced resistance to *Botrytis cinerea* in stored carrot. *Phytopathology,* Vol.90, No.9, (September 2000), pp. 981–986, ISSN

dimethyl-β-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before and after inoculation with shape *Xylophilus ampelinus*. *Plant Cell Tissue and Organ Culture,* Vol.53, No.3, (June 1998), pp. 179-187, ISSN 0167-6857


Fu, HW.; Zhang, L.; Yi, T. & Tian, JK. (2009). A new sesquiterpene from the fruits of *Daucus carota* L. *Molecules,* Vol.14, No.8, (August 2009), pp. 2862-2867, ISSN 1420-3049 Gläβgen, WE.; Rose, A.; Madlung, J.; Koch, W.; Gleitz, J. & Seitz, HU. (1998). Regulation of

González-Barrio, R.; Beltrán, D.; Cantos, E.; Gil, MI.; Espín, JC. & Tomás-Barberán, FA.

Gundlach, H.; Müller, MJ.; Kutchan, TM. & Zenk, MH. (1992). Jasmonic acid is a signal

Herchi, W.; Harrabi, S.; Sebei, K.; Rochut, S.; Boukhchina, S.; Pepe, C. & Kallel, H. (2009).

*and Biochemistry,* Vol.47, No.10, (October 2009), pp. 880-885, ISSN 0981-9428 Jeandet, P.; Bessis, R. & Gautheron, B. (1991). The production of resveratrol (3,5,4'-

Jeandet, P.; Douillet-Breuil, AC.; Bessis, R.; Debord, S.; Sbaghi, M. & Adrian, M. (2002).

*Food Chemistry*, Vol.50, No.10, (April 2002), pp. 2731–2741, ISSN 0021-8561 Kaeberlein, M. & Rabinovitch, PS. (2006). Medicine: grapes versus gluttony. *Nature,* Vol.16

Keller, M.; Steel, CC. & Creasy, GL. (2000). Stilbene accumulation in grapevine tissues:

Keskin, N. & Kunter, B. (2008). Production of *trans*-resveratrol in Cabernet Sauvignon (*Vitis* 

Keskin, N. & Kunter, B. (2010). Production of *trans*-resveratrol in callus tissue of öküzgözü

Kiselev, KV.; Dubrovina, AS.; Veselova, MV.; Bulgakov, VP.; Fedoreyev, SA. & Zhuravlev,

Krisa, S.; Larronde, F.; Budzinski, H.; Decendit, A.; Deffieux, G. & Mérillon, JM. (1999).

*Sciences,* Vol.20, No.3, (September 2010), pp. 197-200, ISSN 1018-7081 Kiselev, KV. (2011). Perspectives for production and application of resveratrol. *Applyed* 

2007), pp. 490–498, ISSN 0032-0935

No.14, (June 2008), pp. 5233–5241, ISSN 0022-3263

No.444, (November 2006), pp. 280-281, ISSN 0028-0836

2000), pp. 275-286, ISSN 0567-7572

pp. 193-196, ISSN 0042-7500

692, ISSN 0168-1656

ISSN 0021-8561

9254

7598

enzymes involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with ultraviolet light and fungal elicitors. *Planta,* Vol.204, No.4, (October

(2006). Comparison of Ozone and UV-C Treatments on the Postharvest Stilbenoid Monomers, Dimers and Trimers Induction in Var. 'Superior' White Table Grapes. *Journal of Agricultural and Food Chemistry*, Vol.54, No.12, (May 2006), pp. 4222-4228

transducer in elicitor-induced plant cell cultures. *Procedings of the National Academy of Science of the USA*, Vol.89, No.6, (March 1992), pp. 2389–2393, ISSN 0027-8424 He, S.; Wu, B.; Pan, Y. & Jiang, L. (2008). Stilbene oligomers from *Parthenocissus laetevirens*:

isolation, biomimetic synthesis, absolute configuration, and implication of antioxidative defense system in the plant. *Journal of Organic Chemistry,* Vol.73,

Phytosterols accumulation in the seeds of *Linum usitatissimum* L. *Plant Physiology* 

trihydroxystilbene) by grape berries in different developmental stages. *American Journal of Enology and Viticulture,* Vol.42, No.1, (May 1991), pp. 41–46, ISSN 0002-

Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism. *Journal of Agricultural and* 

Developmental and environmental effects. *Acta Horticulturae*, No.514, (August

*vinifera* L.) callus culture in response to ultraviolet-C irradiation. *Vitis,* Vol.47, No.4,

(*Vitis vinifera* L.) in response to ultraviolet-C irradiation*. Journal of Animal & Plant* 

*Microbiology and Biotechnology,* Vol.90, No.2, (March 2011), pp. 417-425, ISSN 0175-

YN. (2007). The rolB gene-induced overproduction of resveratrol in *Vitis amurensis* transformed cells. *Journal of Biotechnology*, Vol.128, No.3, (November 2006), pp. 681-

Stilbene production by *Vitis vinifera* cell suspension cultures: methyl jasmonate

induction and 13C biolabelling. *Journal of Natural Products,* Vol.62, No.12, (November 1999), pp. 1688-1690, ISSN 0163-3864


Effect of UV Light on Secondary Metabolite Biosynthesis

Valencia, Spain, July 4-9, 2010

2011), pp. 440-442, ISSN 1559-2316

1996), pp. 679-686, ISSN 0002-9122

(September 1998), pp. 747-754, ISSN 0960-7412

0168-1656

ISSN 1613-4125

ISSN 0002-9254

895-905, ISSN 0028-646X

2008), pp. 129-134, ISSN 0378-8741

in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate 135

*Food Chemistry*, Vol.5, No.18, (August 2007), pp. 7332–7336, ISSN 0021-8561 Sabater-Jara, AB.; Almagro, A.; Belchí-Navarro, S. & Pedreño, MA. (2010a). A new strategy

Sabater-Jara, AB.; Almagro, A.; Belchí-Navarro, S.; Ferrer, MA.; Ros-Barceló, A. & Pedreño,

Sabater-Jara, AB.; Almagro, L.; Bru, R. & Pedreño, MA. (2008). Use of cyclodextrins to

Sánchez-Sampedro, MA.; Fermández-Tárrago, J. & Corchete, P. (2005). Yeast extract and

Searles, PS.; Kropp, BR.; Flint, SD. & Caldwell, MM. (2001). Influence of solar UV-B

*Phytologist,* Vol.152, No.2, (November 2001), pp. 213-221, ISSN 0028-646X Seigler, DS. (2006). Basic pathways for the origin of allelopathic compounds, In: *Allelopathy:* 

Sheahan, JJ. (1996). Sinapate Esters Provide Greater UV-B Attenuation than Flavonoids in

Siemann, EH. & Creasy, LL. (1992). Concentration of the Phytoalexin Resveratrol in Wine.

Staswick, PE.; Yuen, GY. & Lehman, CC. (1998). Jasmonate signaling mutants of *Arabidopsis* 

Szabo, E.; Thelen, A. & Petersen, M. (1999). Fungal elicitor preparations and methyl

Tavares, AC.; Goncalves, MJ.; Cavaleiro, C.; Cruz, MT.; Lopes, MC.; Canhoto, J. & Salgueiro,

González, (Ed.), 11-58, Springer, ISBN 1-4020-4280-9-2, Netherlands. Shakibaei, M.; Harikumar, KB. & Aggarwal, BB. (2009). Resveratrol addiction: To die or not

*Daucus carota* L. essential oil against *Campylobacter jejuni*. *Journal of Agricultural and* 

to enhance the production of phytosterols in *Daucus carota* cell cultures, XVIII Congress of the Federation of European Societies of Plant Biology 2010, pp. 90,

MA. (2010b). Induction of sesquiterpenes, phytoesterols and extracellular pathogenesis-related proteins in elicited cell cultures of *Capsicum annuum. Journal of Plant Physiology*, Vol.167, No.15, (October 2010), pp. 1273-1281, ISSN 0176-1617 Sabater-Jara, AB.; Almagro, L.; Belchí-Navarro, S.; Barceló, AR. & Pedreño, MA. (2011).

Methyl jasmonate induces extracellular pathogenesis-related proteins in cell cultures of *Capsicum chinense*. *Plant Signaling and Behaviour*, Vol.6, No.3, (March

produce and extract phytoesterols from cell cultures. PCT patent WO/2010/049563

methyl jasmonate-induced silymarin in cell cultures of *Silybum marianum* (L.) Gaertn. *Journal of Biotechnology*, Vol.119, No.1, (September 2005), pp. 60-69, ISSN

radiation on peatland microbial communities of southern Argentinia. *New* 

*A Physiological Process with Ecological Implications*, M.J. Reigosa, N. Pedrol and L.

to die. *Molecular nutrition and food research*, Vol.53, No.1, (January 2009), pp. 115-128,

*Arabidopsis thaliana* (Brassicaceae). *American Journal of Botany,* Vol.83, No.6, (June

*American Journal of Enology and Viticulturae,* Vol.43, No.1, (July 1992), pp. 49-525,

are susceptible to the soil fungus *Pythium irregulare*. *Plant Journal*, Vol.15, No.6,

jasmonate enhance rosmarinic acid accumulation in suspension cultures of *Coleus blumei*. *Plant Cell Report*, Vol.18, No.6, (February 1999), pp. 485-489, ISSN 0721-7714 Tassoni, A.; Fornalé, S.; Franceschetti, M.; Musiani, F.; Micheal, AJ.; Perry, B. & Bagni, N.

(2005). Jasmonates and Na-orthovanadate promote resveratrol production in *Vitis vinifera* L. cv. Barbera cell cultures. *New Phytolgist,* Vol.166, No.3, (June 2005), pp.

LR. (2008). Essential oil of *Daucus carota* subsp. halophilus: Composition, antifungal activity and cytotoxicity. *Journal of Ethnopharmacology*, Vol.119, No.1, (September


Morel, G. (1970) Le problème de la transformation tumorale chez les végétaux. *Physiology* 

Okawara, M.; Katsuki, H.; Kurimoto, E.; Shibata, H.; Kume, T. & Akaike, A. (2006).

Ouwerkerk, PBF.; Trimborn, TO.; Hilliou, F. & Memelink, J. (1999). Nuclear factors GT-1 and

*& General Genetics,* Vol.261, No.4, (June 1999), pp. 610–622, ISSN 0026-8925 Pant, B. & Manandhar, S. (2007). *In vitro* propagation of carrot (*Daucus carota*). *Scientific* 

Pedreño, MA.; Belchí-Navarro, S.; Almagro, L. & Bru, R. (2009). Uso combinado de metil

Peebles, CAM.; Shanks, JV. & San, KY. (2009). The Role of the Octadecanoid Pathway in the

Pervaiz, S. (2003). Resveratrol: from grapevines to mammalian biology. *Journal of the* 

Pezet, R.; Gindro, K.; Viret, O. & Richter, H. (2004). Effects of resveratrol and pterostilbene

Pezet, R.; Perret, C.; Jean-Denis, JB.; Tabacchi, R.; Gindro, K. & Viret, O. (2003). δ-Viniferin, a

Pezzuto, JM. (2008). Resveratrol as an inhibitor of carcinogenesis. *Pharmaceutical Biology*,

Posé, D.; Castanedo, I.; Borsani, O.; Nieto, B.; Rosado, A.; Taconnat, L.; Ferrer, A.; Dolan, L.;

Privat, C.; Telo, JP.; Bernardes-Genisson, V.; Vieira, A.; Souchard, JP. & Nepveu, F. (2002).

Repka, V.; Fischerová, I. & Silhárova, K. (2004). Methyl jasmonate is a potent elicitor of

*Biologia Plantarum,* Vol.48, No.2, (May 2003), pp. 273-283, ISSN 0006-3134 Rossi, PG.; Bao, L.; Luciani, A.; Panighi, J.; Desjobert, JM.; Costa, J.; Casanova, J.; Bolla, JM. &

*Chemistry,* Vol.50, No.5, (January 2002), pp. 1213–1217, ISSN 0021-8561 Ramani, S. & Jayabaskaran, C. (2008). Enhanced catharathine and vindoline production in

*Signaling*, Vol.3, No.9, (April 2008), pp. 1-6, ISSN 1750-2187

Resveratrol protects dopaminergic neurons in midbrain slice culture from multiple insults. *Biochemical Pharmacology,* Vol.73, No.4, (February 2007), pp. 550–560, ISSN

3AF1 interact with multiple sequences within the promoter of the Tdc gene from Madagascar periwinkle: GT-1 is involved in UV light-induced expression. *Molecular* 

jasmonato y ciclodextrinas para la producción de resveratrol. PCT patent

Production of Terpenoid Indole Alkaloids in *Catharanthus roseus* Hairy Roots Under Normal and UV-B Stress Conditions. *Biotechnology and Bioengineering*, Vol.103,

*Federation of American Societies for Experimental Biology*, Vol.17, No.14, (November

on *Plasmopara viticola* zoospore mobility and disease development. *Vitis,* Vol.43,

resveratrol dehydrodimer: One of the major stilbenes synthesized by stressed grapevine leaves. *Journal of Agricultural and Food Chemistry,* Vol.51, No.18, (July

Valpuesta, V. & Botella, MA. (2009). Identification of the Arabidopsis dry2/sqe1-5 mutant reveals a central role for sterols in drought tolerance and regulation of reactive oxygen species*. Plant Journal*, Vol.59, No.1, (July 2009), pp. 63–76, ISSN

Antioxidant properties of trans-epsilon-viniferin as compared to stilbene derivatives in aqueous and nonaqueous media. *Journal of Agricultural and Food* 

suspension cultures of *Catharanthus roseus* by ultraviolet-B light. *Journal of Molecular* 

multiple defense responses in grapevine leaves and cell-suspension cultures.

Berti, L. (2007). (E)-methylisoeugenol and elemicin: antibacterial components of

*Végétale*, Vol.8, No.2, (January 1970), pp. 189-191, ISSN 0031-9368

*World*, Vol.5, No.5, (July 2007), pp. 51-53, ISSN 1996-8949

No.6, (April 2009), pp.1248-1254, ISSN 0006-3592

Vol.46, No.7, (July 2008), pp. 443-573, ISSN 1388-0209

2003), pp. 1975–1985, ISSN 0892-6638

No.3, pp. 145-148, ISSN 0042-7500

2003), pp. 5488-5492, ISSN 0021-8561

0006-2952.

WO/2009/106662

0960-7412

*Daucus carota* L. essential oil against *Campylobacter jejuni*. *Journal of Agricultural and Food Chemistry*, Vol.5, No.18, (August 2007), pp. 7332–7336, ISSN 0021-8561


**6** 

*Argentina* 

**Drought Tolerance and Stress Hormones:** 

Among environmental factors, water availability is probably the most limiting for crop quality and productivity, comprising economical output and human food supply (Roche et al., 2009). Water deficit is a multidimensional stress affecting plants at various levels of their organization (Yordanov et al., 2000). Thus, the effects of stress are often manifested at morpho-physiological, biochemical and molecular level, such as inhibition of growth (Bahrani et al., 2010), accumulation of compatible organic solutes (Sánchez-Díaz et al., 2008; DaCosta and Huang 2009), changes in phytohormones endogenous contents (Perales et al., 2005; Seki et al., 2007; Huang, 2008; Dobra et al., 2010), modifications in expression of stress responsive-genes (Xiong and Yang 2003; Yamaguchi-Shinozaki and Shinozaki, 2005; Huang et al., 2008), among others. Some of these responses are directly triggered by the changing water status of the tissues while others are brought about by plant hormones (Chaves et al., 2003). In this sense, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) are involved in a complex signal-transduction network that coordinates growth and development with plant responses to the environment (Agrawal et al., 2002; Jiang and

The aim of this chapter is to present the results of an actual progression of water stress tolerance, its associated hormones and the crosstalk between them in *Panicum virgatum*  (switchgrass), a member of the Poaceae family intensively studied as a source of lignocellulosic biomass to produce renewable energy. In recent years, important research efforts have been focused on improving the yields of crop species under water stress. Advances in functional genomics have been a major contribution to both the study and manipulation of abiotic stress in cereals as well as in forage species. These have been possible, in part, because of the increasing success in methods of grass genetic manipulation which have facilitated basic and applied research. The top four agricultural commodities by quantity are grass crops (sugarcane, maize, rice, wheat). Cow's milk, the sole animal product in the top 10 agricultural commodities by quantity, for the most part comes from animals fed by grasses. Primary production from agriculture, therefore, assumes an important role in the transition to increasingly sustainable food and industrial production

**1. Introduction** 

Zhang 2002; Fujita et al., 2006; Szalai et al., 2010).

**From Model Organisms to Forage Crops** 

Aimar D.1,4, Calafat M.1, Andrade A.M.2,3, Carassay L.1,2, Abdala G.I.2 and Molas M.L.1 *1Universidad Nacional de La Pampa, La Pampa,* 

*3Universidad Nacional de Río Cuarto, Río Cuarto,* 

*2Consejo Nacional de Investigaciones Científicas y Técnicas,* 

*4Agencia Nacional de Promoción Científica y Tecnológica,* 


### **Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops**

Aimar D.1,4, Calafat M.1, Andrade A.M.2,3,

Carassay L.1,2, Abdala G.I.2 and Molas M.L.1 *1Universidad Nacional de La Pampa, La Pampa, 2Consejo Nacional de Investigaciones Científicas y Técnicas, 3Universidad Nacional de Río Cuarto, Río Cuarto, 4Agencia Nacional de Promoción Científica y Tecnológica, Argentina* 

### **1. Introduction**

136 Plants and Environment

Teguo, WP.; Fauconneau, B.; Deffieux, G.; Huguet, F.; Vercauteren, J. & Merillon, JM. (1998).

van Der Heijden, R.; Jacobs, DI.; Snoeijer, W.; Hallard, D. & Verpoorte, R. (2004). The

Vasconsuelo, A. & Boland, R. (2007). Molecular aspects of the early stages of elicitation of

Versari, A.; Parpinello, GP.; Tornielli, GB.; Ferrarini, R. & Giulivo, C. (2001). Stilbene

Wang, JW. & Wu. JY. (2005). Nitric oxide is involved in methyl jasmonate induced defense

Wink, M. (2003). Evolution of secondary metabolites from an ecological and molecular

Wink, M. (2008). Plant secondary metabolism: Diversity, function and its evolution. *Natural Products Communications*, Vol.*3*, No.8, pp. 1205–1216, ISSN 1934-578X Woyengo, TA.; Ramprasath, VR. & Jones, PJH. (2009). Anticancer effects of phytosterols.

Yang, RL.; Yan, ZH. & Lu, Y. (2008). Cytotoxic Phenylpropanoids from Carrot. *Journal of* 

Yukimune, Y.; Tabata, H.; Higashi, Y. & Hara, Y. (1996). Methyl jasmonate-induced

Zamboni, A.; Gatto, P.; Cestaro, A.; Pilati, S.; Viola, R.; Mattivi, F.; Moser, C. & Velasco, R.

Zhao, J.; Zhu, W. & Hu, Q. (2001). Enhanced catharanthine production in *Catharanthus roseus*

Zhou, ML.; Shao, JR. & Tang, YX. (2009). Production and metabolic engineering of terpenoid

*Physiology*, Vol.46, No 6, (June 2005), pp. 923-930, ISSN 0032-0781.

*Chemistry*, Vol.11, No.5, (March 2004), pp. 607-628, ISSN 0929-8673

No.11, (October 2001), pp. 5531–5536, ISSN 0021-8561

(April 1998), pp. 655–657, ISSN 0163-3864

(June 2005), pp.283-333, ISSN 0734-9750

(April 2009), pp. 313–323, ISSN 0885-4513

No.15, (August 2000), pp. 221-1226, ISSN 0141-5492

875, ISSN 0168-9452

ISSN 0031-9422

3007

0021-8561

Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted from *Vitis vinifera* cell cultures. *Journal of Natural Products*, Vol.61, No.5,

*Catharanthus* alkaloids: pharmacognosy and biotechnology. *Current Medicinal* 

secondary metabolites in plants. *Plant Science,* Vol.172, No.5, (May 2007), pp. 861–

compounds and stilbene synthase expression during ripening, wilting, and UV treatment in grape cv. Corvina. *Journal of Agricultural and Food Chemistry,* Vol.49,

responses and secondary metabolism activities of *Taxus* cells. *Plant and Cell* 

phylogenetic perspective. *Phytochemistry,* Vol.64, No.1, (September 2003), pp. 3–19,

*European Journal of Clinical Nutrition*, Vol.63, (June 2009), pp. 813-820, ISSN 0954-

*Agricultural and Food Chemistry*, Vol.56, No.9, (April 2008), pp. 3024-3027, ISSN

overproduction of paclitaxel and baccatin III in *Taxu*s cell suspension cultures. *Nature Biotechnology*, Vol.14, No.9, (September 1996), pp. 1129-1132, ISSN 1087-0156

(2009). Grapevine cell early activation of specific responses to DIMEB, a resveratrol elicitor. *BMC Genomics*, Vol.10, No.363, (August 2009), pp 1-13, ISSN 1471-2164 Zhao, J.; Davis, LC. & Verpoorte, R. (2005). Elicitor signal transduction leading to

production of plant secondary metabolites. *Biotechnology Advances*, Vol.23, No.4,

cell cultures by combined elicitor treatment in shake flasks and bioreactors. *Enzyme and Microbial Technology,* Vol.28, No.7, (May 2001), pp. 673–681, ISSN 0141-0229 Zhao, J.; Zhu, WH.; Hu, Q. & He, XW. (2000). Improved alkaloid production in *Catharanthus* 

*roseus* suspension cell cultures by various chemicals. *Biotechnology Letters,* Vol.22,

indole alkaloids in cell cultures of the medicinal plant *Catharanthus roseus* (L.) G. Don (Madagascar periwinkle). *Biotechnology and Applied Biochemistry*, Vol.52, No.4, Among environmental factors, water availability is probably the most limiting for crop quality and productivity, comprising economical output and human food supply (Roche et al., 2009). Water deficit is a multidimensional stress affecting plants at various levels of their organization (Yordanov et al., 2000). Thus, the effects of stress are often manifested at morpho-physiological, biochemical and molecular level, such as inhibition of growth (Bahrani et al., 2010), accumulation of compatible organic solutes (Sánchez-Díaz et al., 2008; DaCosta and Huang 2009), changes in phytohormones endogenous contents (Perales et al., 2005; Seki et al., 2007; Huang, 2008; Dobra et al., 2010), modifications in expression of stress responsive-genes (Xiong and Yang 2003; Yamaguchi-Shinozaki and Shinozaki, 2005; Huang et al., 2008), among others. Some of these responses are directly triggered by the changing water status of the tissues while others are brought about by plant hormones (Chaves et al., 2003). In this sense, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) are involved in a complex signal-transduction network that coordinates growth and development with plant responses to the environment (Agrawal et al., 2002; Jiang and Zhang 2002; Fujita et al., 2006; Szalai et al., 2010).

The aim of this chapter is to present the results of an actual progression of water stress tolerance, its associated hormones and the crosstalk between them in *Panicum virgatum*  (switchgrass), a member of the Poaceae family intensively studied as a source of lignocellulosic biomass to produce renewable energy. In recent years, important research efforts have been focused on improving the yields of crop species under water stress. Advances in functional genomics have been a major contribution to both the study and manipulation of abiotic stress in cereals as well as in forage species. These have been possible, in part, because of the increasing success in methods of grass genetic manipulation which have facilitated basic and applied research. The top four agricultural commodities by quantity are grass crops (sugarcane, maize, rice, wheat). Cow's milk, the sole animal product in the top 10 agricultural commodities by quantity, for the most part comes from animals fed by grasses. Primary production from agriculture, therefore, assumes an important role in the transition to increasingly sustainable food and industrial production

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 139

embryo and seed development, acquisition of desiccation tolerance and dormancy, flowering and organogenesis (Finkelstein et al., 2002; Barrero et al., 2005; De Smet et al., 2006; Liang et al., 2007). ABA also promotes plant growth under non stressful condition and has shown to be essential for vegetative growth in several organs (Sharp et al., 2000; Spollen

Continuous synthesis, transport and degradation dynamically maintain ABA levels in plant cells. Therefore, plants control their developmental programs and stresses responses by

The molecular basis of ABA biosynthesis and catabolism were established by genetic and biochemical approaches (Seki, 2002; Yamaguchi-Shinozaki and Shinozaki, 2005). Based on these studies it has become clear that ABA is synthesized from zeazanthin, a C40 carotenoid. The conversion of zeaxanthin to xanthoxin, which is the C15 intermediates, is catalyzed in plastids by distinct enzyme: zeaxanthin epoxidase (ZEP) (Agrawal et al., 2001; Xiong et al., 2002), neoxanthin synthase (North et al., 2007), an unidentified epoxycarotenoid isomerase, and 9-cis-epoxycarotenoid dioxygenase (NCED) (Schwartz et al., 1997; Qin and Zeevaart 1999; Iuchi et al., 2001). In cytosol, the oxidation of xanthoxin produces abscisic aldehyde,

Catabolism of ABA can occur through different pathways, the nature of which often depends on the species, their developmental stage or tissue. There are at least two pathways for ABA catabolism, an oxidative pathway and conjugation (Kushiro et al., 2004; Nambara and Marion-Poll 2005). The most common oxidative pathway is initiated by oxidation of the 8'-hydroxy ABA (8'-OH ABA), which can reversibly cyclize to phaseic acid (PA) (Zaharia et al., 2005). This compound can then be reduced to the major product dihydrophaseic acid (DPA), with minor amounts of epi- dihydrophaseic acid (epi-DPA). The minor oxidation pathway includes the formation of 7'-hydroxy ABA (7'-OH ABA) and 9'-hydroxy ABA (9'- OH ABA). The latter can cyclize reversibly to neophaseic acid (neoPA) (Zhou et al., 2004). In addition, ABA and hydroxy ABA may be conjugated with glucose, thereby forming corresponding glucose esters at C-1 (ABA-GE) or glycosides at C-1' or C-4' (Zeevaart 1999;

ABA action is one of the most studied topics in abiotic stress response research (Hirayama and Shinozaki 2007; Wasilewska et al., 2008). An increase in ABA content in response to water-deficit stress may arise from an increase in ABA biosynthesis and/ or a decrease in ABA breakdown (reviewed by Cutler and Krochko, 1999; Zeevaart, 1999). In *Arabidopsis thaliana* seedlings, Huang et al. (2008) showed that drought enhanced both ABA biosynthesis and catabolism, resulting in an increase in ABA and catabolites. Likewise, drought-treated plants of *Laurus azorica* (Seub) showed an increase in leaf ABA concentrations respect to that of the control (Sánchez-Díaz et al., 2008). On the other hand, exogenous application of ABA enhances the tolerance of plants or plant cells to drought (Lu et al., 2009). In relation to endogenous ABA, different reports showed that drought tolerant cultivars have more ABA than susceptible ones (Perales et al., 2005; Veselov et al., 2008; Thameur et al., 2011). Nevertheless, the direct relation between stress tolerance and

In addition to the well established model of *Arabidopsis*, increments in endogenous ABA level under water stress are also reported in cereals and forage crops. For instance, increment in ABA contents under water stress in diverse developmental stages was reported in maize (Xin et al., 1997; Wang et al., 2008; Nyysar 2005), sorghum (Kannangara et al.,

which can be converted to ABA by aldehyde oxidase 3 (AAO3) (Seo et al., 2000).

et al., 2000; Cheng et al., 2002).

Oritani and Kiyota 2003).

increased ABA contents does not always exist.

modulating endogenous ABA levels (Schwartz et al., 2003).

methods. Grass crops are centrally important targets for biotechnological improvement for food and fuel production. In particular the exploitation of a currently untapped resource of grass biomass (primarily lignocellulosic cell walls) is of high interest for sustainable fuel production. Basic and applied research on the sequencing of the rice, sugarcane, maize and sorghum genome have provided an invaluable resource to infer gene localization in other grasses that have not been sequenced yet. In the mid term, increases in drought tolerance could be introgressed from tolerant genotypes using a marker-assisted breeding approach. The Poaceae is the fourth largest plant family in the world -with over 10000 species distributed widely across the earth- and has an extensive synteny among the genomes of its members. Hence, what we learn about one member of the family can enhance our understanding of the entire group and contribute to the improvement of grass crops in meeting the challenges of attaining a sustainable agriculture for feeding the world's population and for developing renewable supplies of fuel and industrial products (Baven et al., 2010).

### **2. Phytohormones and drought stress**

As sessile organisms, plants are only able to survive by their ability to build up fast and highly adapted responses to diverse environmental stresses, *e.g.*, drought, high salinity, and low temperature. Perception of these stress signals often results in the production of a huge arsenal of chemical compounds, among which a variety of hormones to adapt and respond to environmental challenges are included. Some of these compounds, such as the phytohormones are in a prominent position, playing important regulatory roles in plant physiology (Wasternack and Hause 2002; Chen et al., 2006; Browse, 2009a) affecting both developmental processes and responses to a wide range of abiotic and biotic stresses.

The key role of ABA, JA and SA as primary signals in the regulation of plant defense has been well established (Bari and Jones 2009; Pieterse et al., 2009). These hormones generate a signal transduction network that leads to a cascade of events responsible for the physiological adaptation of the plant to stress. It should be noted that the degree of drought tolerance varies with developmental stages in most plant species (El-Far and Allan 1995; Reddy et al., 2004; Rassaa et al., 2008). Experiments conducted to identify highly drought sensitive growth stages of sunflower showed that maximum reduction in yield occurred when drought was imposed during flowering (Karaata, 1991). In addition, drought during the vegetative phase of sunflower plants affects both final biological and economic yields (Agele 2003; Turhan and Baser 2004). In maize water deficit in the late developmental stage tends to reduce kernel size rather than number (Saini and Westgate, 2000; Boyer and Westgate, 2004). Similarly, Barney et al. (2009) evaluated fitness under stressful growing conditions to characterize the agronomic and ecological traits related to environmental tolerance of switchgrass and found that drought treatments (-4.0 and -11.0 MPa) reduced tiller length and number, leaf area, and biomass production by up to 80%.

The final outcome of stress response indicates that there is no single response pattern that is highly correlated with yield under all drought environments.

#### **2.1 Abscisic acid (ABA)**

ABA is well known hormone for its regulatory role in integrating environmental adversity with the developmental programs of plants (Chow and McCourt 2004; Christmann et al., 2005). Thus, it affects a wide range of processes at different developmental stages such as

methods. Grass crops are centrally important targets for biotechnological improvement for food and fuel production. In particular the exploitation of a currently untapped resource of grass biomass (primarily lignocellulosic cell walls) is of high interest for sustainable fuel production. Basic and applied research on the sequencing of the rice, sugarcane, maize and sorghum genome have provided an invaluable resource to infer gene localization in other grasses that have not been sequenced yet. In the mid term, increases in drought tolerance could be introgressed from tolerant genotypes using a marker-assisted breeding approach. The Poaceae is the fourth largest plant family in the world -with over 10000 species distributed widely across the earth- and has an extensive synteny among the genomes of its members. Hence, what we learn about one member of the family can enhance our understanding of the entire group and contribute to the improvement of grass crops in meeting the challenges of attaining a sustainable agriculture for feeding the world's population and for developing renewable supplies of fuel and industrial products (Baven et

As sessile organisms, plants are only able to survive by their ability to build up fast and highly adapted responses to diverse environmental stresses, *e.g.*, drought, high salinity, and low temperature. Perception of these stress signals often results in the production of a huge arsenal of chemical compounds, among which a variety of hormones to adapt and respond to environmental challenges are included. Some of these compounds, such as the phytohormones are in a prominent position, playing important regulatory roles in plant physiology (Wasternack and Hause 2002; Chen et al., 2006; Browse, 2009a) affecting both developmental

The key role of ABA, JA and SA as primary signals in the regulation of plant defense has been well established (Bari and Jones 2009; Pieterse et al., 2009). These hormones generate a signal transduction network that leads to a cascade of events responsible for the physiological adaptation of the plant to stress. It should be noted that the degree of drought tolerance varies with developmental stages in most plant species (El-Far and Allan 1995; Reddy et al., 2004; Rassaa et al., 2008). Experiments conducted to identify highly drought sensitive growth stages of sunflower showed that maximum reduction in yield occurred when drought was imposed during flowering (Karaata, 1991). In addition, drought during the vegetative phase of sunflower plants affects both final biological and economic yields (Agele 2003; Turhan and Baser 2004). In maize water deficit in the late developmental stage tends to reduce kernel size rather than number (Saini and Westgate, 2000; Boyer and Westgate, 2004). Similarly, Barney et al. (2009) evaluated fitness under stressful growing conditions to characterize the agronomic and ecological traits related to environmental tolerance of switchgrass and found that drought treatments (-4.0 and -11.0 MPa) reduced

The final outcome of stress response indicates that there is no single response pattern that is

ABA is well known hormone for its regulatory role in integrating environmental adversity with the developmental programs of plants (Chow and McCourt 2004; Christmann et al., 2005). Thus, it affects a wide range of processes at different developmental stages such as

al., 2010).

**2. Phytohormones and drought stress** 

processes and responses to a wide range of abiotic and biotic stresses.

tiller length and number, leaf area, and biomass production by up to 80%.

highly correlated with yield under all drought environments.

**2.1 Abscisic acid (ABA)** 

embryo and seed development, acquisition of desiccation tolerance and dormancy, flowering and organogenesis (Finkelstein et al., 2002; Barrero et al., 2005; De Smet et al., 2006; Liang et al., 2007). ABA also promotes plant growth under non stressful condition and has shown to be essential for vegetative growth in several organs (Sharp et al., 2000; Spollen et al., 2000; Cheng et al., 2002).

Continuous synthesis, transport and degradation dynamically maintain ABA levels in plant cells. Therefore, plants control their developmental programs and stresses responses by modulating endogenous ABA levels (Schwartz et al., 2003).

The molecular basis of ABA biosynthesis and catabolism were established by genetic and biochemical approaches (Seki, 2002; Yamaguchi-Shinozaki and Shinozaki, 2005). Based on these studies it has become clear that ABA is synthesized from zeazanthin, a C40 carotenoid. The conversion of zeaxanthin to xanthoxin, which is the C15 intermediates, is catalyzed in plastids by distinct enzyme: zeaxanthin epoxidase (ZEP) (Agrawal et al., 2001; Xiong et al., 2002), neoxanthin synthase (North et al., 2007), an unidentified epoxycarotenoid isomerase, and 9-cis-epoxycarotenoid dioxygenase (NCED) (Schwartz et al., 1997; Qin and Zeevaart 1999; Iuchi et al., 2001). In cytosol, the oxidation of xanthoxin produces abscisic aldehyde, which can be converted to ABA by aldehyde oxidase 3 (AAO3) (Seo et al., 2000).

Catabolism of ABA can occur through different pathways, the nature of which often depends on the species, their developmental stage or tissue. There are at least two pathways for ABA catabolism, an oxidative pathway and conjugation (Kushiro et al., 2004; Nambara and Marion-Poll 2005). The most common oxidative pathway is initiated by oxidation of the 8'-hydroxy ABA (8'-OH ABA), which can reversibly cyclize to phaseic acid (PA) (Zaharia et al., 2005). This compound can then be reduced to the major product dihydrophaseic acid (DPA), with minor amounts of epi- dihydrophaseic acid (epi-DPA). The minor oxidation pathway includes the formation of 7'-hydroxy ABA (7'-OH ABA) and 9'-hydroxy ABA (9'- OH ABA). The latter can cyclize reversibly to neophaseic acid (neoPA) (Zhou et al., 2004). In addition, ABA and hydroxy ABA may be conjugated with glucose, thereby forming corresponding glucose esters at C-1 (ABA-GE) or glycosides at C-1' or C-4' (Zeevaart 1999; Oritani and Kiyota 2003).

ABA action is one of the most studied topics in abiotic stress response research (Hirayama and Shinozaki 2007; Wasilewska et al., 2008). An increase in ABA content in response to water-deficit stress may arise from an increase in ABA biosynthesis and/ or a decrease in ABA breakdown (reviewed by Cutler and Krochko, 1999; Zeevaart, 1999). In *Arabidopsis thaliana* seedlings, Huang et al. (2008) showed that drought enhanced both ABA biosynthesis and catabolism, resulting in an increase in ABA and catabolites. Likewise, drought-treated plants of *Laurus azorica* (Seub) showed an increase in leaf ABA concentrations respect to that of the control (Sánchez-Díaz et al., 2008). On the other hand, exogenous application of ABA enhances the tolerance of plants or plant cells to drought (Lu et al., 2009). In relation to endogenous ABA, different reports showed that drought tolerant cultivars have more ABA than susceptible ones (Perales et al., 2005; Veselov et al., 2008; Thameur et al., 2011). Nevertheless, the direct relation between stress tolerance and increased ABA contents does not always exist.

In addition to the well established model of *Arabidopsis*, increments in endogenous ABA level under water stress are also reported in cereals and forage crops. For instance, increment in ABA contents under water stress in diverse developmental stages was reported in maize (Xin et al., 1997; Wang et al., 2008; Nyysar 2005), sorghum (Kannangara et al.,

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 141

water deprivation, while intermediate values were obtained in cvs. Rihane, Pakistan and

In alfalfa cvs. Longdong (strong drought-resistance) and BL-02-329 (weak droughtresistance) ABA contents were evaluated. Under water stress, the ABA content increased in leaves. In response to severe drought stress, the drought- resistant cv. Longdong adjusted better to growth rate reduction to ensure surviving and avoid water deficit damage (Han et

In addition, exogenous ABA was demonstrated to increase drought tolerance in some forage crops. For example, Shaoyun et al. (2009) studied the effect of exogenous ABA added on plant of bermudagrass cv. TifEagle. They evaluated the protective effect of ABA based on relative water content and found that every ABA treatments (e.g. 19, 38 and 57 µM) significantly decreased the mortality rate in drought conditions compared to control.

The increase in ABA endogenous level under drought induces the stomatal closure. This fact constitutes one of the first external symptoms of water deficit, and is recorded as the increase of stomatal resistance or the decrease of its inverse (stomatal conductance). Stomatal closure take place to minimize the water loss by transpiration, and ABA plays a fundamental role in this process. Thus, stomatal resistance is used as a reference to compare the intensity of water deficit in different species and growth conditions (Medrano et al., 2002). Guard cells continuously sense information from the surrounding environment, biotic and abiotic, as well as long distant signals coming from the roots. Stomatal closing under drought is a response to increasing levels of endogenous ABA synthesized in the roots as a result of water deprivation in the soil (Kim et al., 2010). Hence, decreasing of stomatal conductance under water stress is a wide-ranging response in plants. For instance, in kidney beam stomatal conductance diminishes rapidly after two days of drought, but it recovers the levels of well watered plants after two days of re-watering (Miyashita et al., 2005). In *Brachiaria decumbens* and *brizantha*, stomatal conductance significantly decreased after six

Another symptom of water deficit is the reduction in cell turgency, which in turn, limits cell expansion and growth. Drought tolerance of grasses is associated closely with their morphological and physiological traits, with varying degrees of reduction of them among the species. For example, water stress decreases plant height in most grass species (Pennypacker et al., 1990; Jiang et al., 1995; Berg and Zeng 2006). On the contrary, this stress

Bahrani et al. (2010) found that water stress constrained the total water use in ten forage species through a reduction in plant height, leaf water potential, leaf area and dry weight of roots. In corn, 160 lines (pure lines and hybrids) were evaluated in their tolerance to drought; one of the first detected symptoms was a reduction in plant height, with values ranging from 60 to 75 % (Dass et al., 2001). Similar effect was found in genotypes of wheat, where plant height showed a significant reduction under water stress (Shirazi et al., 2010). In our laboratory we investigated the response of *Panicum virgatum* cv. Greenville to water stress. Plants of 55 days old were grown in a growth chamber; water was withheld at the same time as stomatal conductance was monitored. After water withholding, a consistent drop in the conductance was detected (drought treatment) and plants were re-watered to evaluate their recovering after 12 and 24 h. Plant height, stomatal conductance, content of stress related hormones (ABA, JA, SA) were evaluated. Water stress negatively affected the

Treatment of 19 µM ABA showed the best protection against injury.

days of water deprivation (Carmona et al., 2003).

generally had no effects on the root: shoot ratio of the grasses.

Manel (Thameur et al., 2010).

al., 2008).

1983), wheat (Iqbal et al., 2010; Raziuddin et al., 2010), festuca (Abernethy and McManus 1998), barley (Thameur et al., 2010) and alfalfa (Han et al., 2008).

Plants of wheat and maize, representatives of C3 and C4 plants, respectively, were subjected to mild (−0.4MPa), moderate (−0.8MPa) and high (−1.5MPa) water stress levels induced by PEG-6000 for 7 days under controlled conditions. No significant change occurred in ABA content in roots and leaves of both species at mild stress level. Moderate stress resulted in higher accumulation of ABA in roots and leaves of maize as compared to wheat roots and leaves. At high stress level, ABA content increased in maize whereas wheat did not show any significant change. The differences were more pronounced between the leaves of the two species. These findings suggest a differential sensitivity of C3 and C4 plants to water stress. Higher ABA content in maize may also impose greater stomatal restrictions on these species to reduce water loss more effectively compared with wheat having lower ABA content (Nayysar, 2005).

In maize seedlings, Wang et al. (2008) assesed the inhibitory effect of ABA on the grain growth and reported that, at early stages, the endogenous ABA contents increased dramatically in leaves after 24 h of exposure to water stress, and then it remained high till the end. On the other hand, ABA content in seeds of wheat plants subjected to water deficit during grain filling showed variations. Water status parameters, ABA levels in flag leaf and grains, and grain yield were investigated in two drought tolerant (i.e. cv. MV Emese and cv. Plainsman V) and two drought-sensitive (i.e. cvs. GK E´let and Cappelle Desprez) wheat genotypes. In flag leaves, endogenous ABA levels increased significantly after the suspension of irrigation in all genotypes and remained high during anthesis; afterwards, it decreased markedly. In grains, ABA increased significantly in all genotypes exposed to water stress at 9 days post anthesis (DPA). Tolerant cultivars had higher ABA levels at 9 DPA and then it decreased rapidly toward maturity. By contrast, in sensitive cultivars ABA levels remained high until the end of grain filling period, which affected more negatively the grain yield of sensitive cultivars (Guoth et al., 2009).

Water stress effect and ABA levels were studied in sorghum cv. CSH8. A gradient of water stress was created among sorghum plants with a line-source sprinkler irrigation system and it was observed that leaf ABA levels increased with decreasing irrigation. ABA was very sensitive to stress, ranging over the irrigation gradient from 50 to 800 ng g-1 DW in the well irrigated and water stressed plants, respectively. This study shows that ABA synthesis in leaves begins with a water potential of -1.3 MPa. This threshold has been observed in several species in a variety of conditions. The increase in ABA levels also correlated with a marked decrease in plant height and leaf senescence (Kannangara et al., 1983).

In plants of *Festuca arundinacea* cv. Grasslands Roa drought was imposed through water deprivation. An increase in leaf ABA levels from a range of 5–30 ng g-1 FW in leaf tissue from water sufficient plants (control) to up to 200 ng g-1 FW in leaf tissue of stressed plant was observed. ABA concentration was correlated with soil moisture content and leaf water potential. The accumulation of ABA occurred after the soil moisture content had dropped below approx. 8% in pots of treatment. The maximum rate of ABA accumulation occurred between water potential values of -1.5 and -2.5 MPa. Under these conditions, leaf elongation ceased and there was an increase in proline levels (Abernethy et al., 1998).

In barley, the differences in responses among five genotypes (i.e. Ardahoui, Pakistan, Rihane, Manel ad Roho) were evaluated. Water stress induced a reduction in relative water content, as well as an increase in proline content and endogenous ABA in all genotypes. Drought tolerant cv. Ardhaoui had the highest increase in endogenous ABA (5-fold) after

1983), wheat (Iqbal et al., 2010; Raziuddin et al., 2010), festuca (Abernethy and McManus

Plants of wheat and maize, representatives of C3 and C4 plants, respectively, were subjected to mild (−0.4MPa), moderate (−0.8MPa) and high (−1.5MPa) water stress levels induced by PEG-6000 for 7 days under controlled conditions. No significant change occurred in ABA content in roots and leaves of both species at mild stress level. Moderate stress resulted in higher accumulation of ABA in roots and leaves of maize as compared to wheat roots and leaves. At high stress level, ABA content increased in maize whereas wheat did not show any significant change. The differences were more pronounced between the leaves of the two species. These findings suggest a differential sensitivity of C3 and C4 plants to water stress. Higher ABA content in maize may also impose greater stomatal restrictions on these species to reduce water loss more effectively compared with wheat having lower ABA

In maize seedlings, Wang et al. (2008) assesed the inhibitory effect of ABA on the grain growth and reported that, at early stages, the endogenous ABA contents increased dramatically in leaves after 24 h of exposure to water stress, and then it remained high till the end. On the other hand, ABA content in seeds of wheat plants subjected to water deficit during grain filling showed variations. Water status parameters, ABA levels in flag leaf and grains, and grain yield were investigated in two drought tolerant (i.e. cv. MV Emese and cv. Plainsman V) and two drought-sensitive (i.e. cvs. GK E´let and Cappelle Desprez) wheat genotypes. In flag leaves, endogenous ABA levels increased significantly after the suspension of irrigation in all genotypes and remained high during anthesis; afterwards, it decreased markedly. In grains, ABA increased significantly in all genotypes exposed to water stress at 9 days post anthesis (DPA). Tolerant cultivars had higher ABA levels at 9 DPA and then it decreased rapidly toward maturity. By contrast, in sensitive cultivars ABA levels remained high until the end of grain filling period, which affected more negatively

Water stress effect and ABA levels were studied in sorghum cv. CSH8. A gradient of water stress was created among sorghum plants with a line-source sprinkler irrigation system and it was observed that leaf ABA levels increased with decreasing irrigation. ABA was very sensitive to stress, ranging over the irrigation gradient from 50 to 800 ng g-1 DW in the well irrigated and water stressed plants, respectively. This study shows that ABA synthesis in leaves begins with a water potential of -1.3 MPa. This threshold has been observed in several species in a variety of conditions. The increase in ABA levels also correlated with a marked

In plants of *Festuca arundinacea* cv. Grasslands Roa drought was imposed through water deprivation. An increase in leaf ABA levels from a range of 5–30 ng g-1 FW in leaf tissue from water sufficient plants (control) to up to 200 ng g-1 FW in leaf tissue of stressed plant was observed. ABA concentration was correlated with soil moisture content and leaf water potential. The accumulation of ABA occurred after the soil moisture content had dropped below approx. 8% in pots of treatment. The maximum rate of ABA accumulation occurred between water potential values of -1.5 and -2.5 MPa. Under these conditions, leaf elongation

In barley, the differences in responses among five genotypes (i.e. Ardahoui, Pakistan, Rihane, Manel ad Roho) were evaluated. Water stress induced a reduction in relative water content, as well as an increase in proline content and endogenous ABA in all genotypes. Drought tolerant cv. Ardhaoui had the highest increase in endogenous ABA (5-fold) after

1998), barley (Thameur et al., 2010) and alfalfa (Han et al., 2008).

the grain yield of sensitive cultivars (Guoth et al., 2009).

decrease in plant height and leaf senescence (Kannangara et al., 1983).

ceased and there was an increase in proline levels (Abernethy et al., 1998).

content (Nayysar, 2005).

water deprivation, while intermediate values were obtained in cvs. Rihane, Pakistan and Manel (Thameur et al., 2010).

In alfalfa cvs. Longdong (strong drought-resistance) and BL-02-329 (weak droughtresistance) ABA contents were evaluated. Under water stress, the ABA content increased in leaves. In response to severe drought stress, the drought- resistant cv. Longdong adjusted better to growth rate reduction to ensure surviving and avoid water deficit damage (Han et al., 2008).

In addition, exogenous ABA was demonstrated to increase drought tolerance in some forage crops. For example, Shaoyun et al. (2009) studied the effect of exogenous ABA added on plant of bermudagrass cv. TifEagle. They evaluated the protective effect of ABA based on relative water content and found that every ABA treatments (e.g. 19, 38 and 57 µM) significantly decreased the mortality rate in drought conditions compared to control. Treatment of 19 µM ABA showed the best protection against injury.

The increase in ABA endogenous level under drought induces the stomatal closure. This fact constitutes one of the first external symptoms of water deficit, and is recorded as the increase of stomatal resistance or the decrease of its inverse (stomatal conductance). Stomatal closure take place to minimize the water loss by transpiration, and ABA plays a fundamental role in this process. Thus, stomatal resistance is used as a reference to compare the intensity of water deficit in different species and growth conditions (Medrano et al., 2002). Guard cells continuously sense information from the surrounding environment, biotic and abiotic, as well as long distant signals coming from the roots. Stomatal closing under drought is a response to increasing levels of endogenous ABA synthesized in the roots as a result of water deprivation in the soil (Kim et al., 2010). Hence, decreasing of stomatal conductance under water stress is a wide-ranging response in plants. For instance, in kidney beam stomatal conductance diminishes rapidly after two days of drought, but it recovers the levels of well watered plants after two days of re-watering (Miyashita et al., 2005). In *Brachiaria decumbens* and *brizantha*, stomatal conductance significantly decreased after six days of water deprivation (Carmona et al., 2003).

Another symptom of water deficit is the reduction in cell turgency, which in turn, limits cell expansion and growth. Drought tolerance of grasses is associated closely with their morphological and physiological traits, with varying degrees of reduction of them among the species. For example, water stress decreases plant height in most grass species (Pennypacker et al., 1990; Jiang et al., 1995; Berg and Zeng 2006). On the contrary, this stress generally had no effects on the root: shoot ratio of the grasses.

Bahrani et al. (2010) found that water stress constrained the total water use in ten forage species through a reduction in plant height, leaf water potential, leaf area and dry weight of roots. In corn, 160 lines (pure lines and hybrids) were evaluated in their tolerance to drought; one of the first detected symptoms was a reduction in plant height, with values ranging from 60 to 75 % (Dass et al., 2001). Similar effect was found in genotypes of wheat, where plant height showed a significant reduction under water stress (Shirazi et al., 2010).

In our laboratory we investigated the response of *Panicum virgatum* cv. Greenville to water stress. Plants of 55 days old were grown in a growth chamber; water was withheld at the same time as stomatal conductance was monitored. After water withholding, a consistent drop in the conductance was detected (drought treatment) and plants were re-watered to evaluate their recovering after 12 and 24 h. Plant height, stomatal conductance, content of stress related hormones (ABA, JA, SA) were evaluated. Water stress negatively affected the

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 143

Fig. 1. **A.** Content of ABA in leaves of Panicum virgatum cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Data are means and SEs of three replicates, P ≤ 0.05. **B.** Plant height C. Stomatal resistance (S.R.). Measurements were made with porometer Delta-T on the abaxial side of leaves. Black circle: control conditions. Gray square: Drought, RW 12 h and RW 24 h of re-watering. Data are means of twenty-four replicates with SEs. Values with the same letter are not significantly

different, P ≤ 0.05.

plant height and, after watering was restored (i.e. 24 h after re-watering) plant growth reached the control height (Fig. 1.B). In addition, the stomatal resistance drastically increased during the stress period and it gradually decreased to the control level at 24 h of re-watering (Fig. 1.C).

After five days under stress, endogenous ABA content increased 4.5 fold compared to the control (Fig. 1.A). After 12 h of rehydration ABA content decreased to 1.5 fold the control and, after 24 h, ABA content in treated and control plants were similar. This increment in ABA content under stress is associated with the increase of stomatal resistance. Once plants recoved, both ABA content and stomatal resistance decreased to the control level. These results are in agreement with reports from other plant species as we discussed earlier.

The first steps of ABA sensing and signaling during stomatal closure under drought is related to the localization of ABA receptors in the guard cells. Two of these ABA receptors reside inside the cell but a third was found on the cell surface (Liu et al., 2007). Therefore, plant cell could sense both extracellular and intracellular ABA concentrations. Under drought, an increasing in stomata closure occurs because of an increasing in the pH of sap. This fact suggests that extracellular ABA is sensed by guard cells via receptors on the plasma membrane (Schachtman and Goodger, 2008). In the last decade, hydrogen peroxide (H2O2) and nitric oxide (NO) have also been involved in the ABA-induced stomatal closure (Assmann 2003; Desikan et al., 2004; Bright et al., 2006).

It is well documented that, in response to biotic and abiotic stimuli, there is an increment in the reactive oxygen species (ROS). ROS are short-lived molecules produced through diverse cellular mechanisms in different cell compartments, e.g. chloroplast, peroxisomes, mitochondria (Cho et al., 2009). This overproduction of ROS is highly controlled by a versatile oxidative system that establishes the redox balance inside the cell. On the other side, increase of ROS under stress conditions act as a signal of warning that activates responses of acclimation and/ or defense. Particularly, it activates specific pathways where H2O2 is involved as a second messenger. ROS signaling is connected to ABA, flux of Ca+2 and sugars, and it is possible that they participate both up and downstream of pathways dependent of ABA in drought conditions (Kwak et al., 2006). In *Panicum virgatum*, ROS has been related to ABA signaling during germination (Sarath et al., 2007). Inhibition of germination imposed by ABA apparently requires both ROS and NO as intermediates in its action, where ROS produced by membrane-bound NADPH-oxidases responsive to ABA. In switchgrass seeds, externally supplied hydrogen peroxide restrain ABA-imposed inhibition of germination. Apart from this study on germination, no other report has involved ABA and ROS in switchgrass responses.

At molecular level, many transcription factors (TFs), such as dehydration-responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF), DREB2 and ABAresponsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) can be used to improve stress tolerance to abiotic stresses in various grasses. ABA is involved in transcriptional regulations of numerous drought responsive genes (Zhang et al., 2006). Some drought-inducible genes may be regulated by both the ABA-independent and the ABAdependent regulatory systems. For example, the promoter of a drought-, high salinity-, and cold- inducible gene, RD29A/COR78/LTI7, contains two major cis-acting elements (ABRE) and DRE/ C-RepeaT (CRT), both of which are involved in stress-inducible gene expression (Yamaguchi-Shinozaki and Shinozaki, 2005).

plant height and, after watering was restored (i.e. 24 h after re-watering) plant growth reached the control height (Fig. 1.B). In addition, the stomatal resistance drastically increased during the stress period and it gradually decreased to the control level at 24 h of

After five days under stress, endogenous ABA content increased 4.5 fold compared to the control (Fig. 1.A). After 12 h of rehydration ABA content decreased to 1.5 fold the control and, after 24 h, ABA content in treated and control plants were similar. This increment in ABA content under stress is associated with the increase of stomatal resistance. Once plants recoved, both ABA content and stomatal resistance decreased to the control level. These results are in agreement with reports from other plant species as we discussed

The first steps of ABA sensing and signaling during stomatal closure under drought is related to the localization of ABA receptors in the guard cells. Two of these ABA receptors reside inside the cell but a third was found on the cell surface (Liu et al., 2007). Therefore, plant cell could sense both extracellular and intracellular ABA concentrations. Under drought, an increasing in stomata closure occurs because of an increasing in the pH of sap. This fact suggests that extracellular ABA is sensed by guard cells via receptors on the plasma membrane (Schachtman and Goodger, 2008). In the last decade, hydrogen peroxide (H2O2) and nitric oxide (NO) have also been involved in the ABA-induced stomatal closure

It is well documented that, in response to biotic and abiotic stimuli, there is an increment in the reactive oxygen species (ROS). ROS are short-lived molecules produced through diverse cellular mechanisms in different cell compartments, e.g. chloroplast, peroxisomes, mitochondria (Cho et al., 2009). This overproduction of ROS is highly controlled by a versatile oxidative system that establishes the redox balance inside the cell. On the other side, increase of ROS under stress conditions act as a signal of warning that activates responses of acclimation and/ or defense. Particularly, it activates specific pathways where H2O2 is involved as a second messenger. ROS signaling is connected to ABA, flux of Ca+2 and sugars, and it is possible that they participate both up and downstream of pathways dependent of ABA in drought conditions (Kwak et al., 2006). In *Panicum virgatum*, ROS has been related to ABA signaling during germination (Sarath et al., 2007). Inhibition of germination imposed by ABA apparently requires both ROS and NO as intermediates in its action, where ROS produced by membrane-bound NADPH-oxidases responsive to ABA. In switchgrass seeds, externally supplied hydrogen peroxide restrain ABA-imposed inhibition of germination. Apart from this study on germination, no other report has involved ABA

At molecular level, many transcription factors (TFs), such as dehydration-responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF), DREB2 and ABAresponsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) can be used to improve stress tolerance to abiotic stresses in various grasses. ABA is involved in transcriptional regulations of numerous drought responsive genes (Zhang et al., 2006). Some drought-inducible genes may be regulated by both the ABA-independent and the ABAdependent regulatory systems. For example, the promoter of a drought-, high salinity-, and cold- inducible gene, RD29A/COR78/LTI7, contains two major cis-acting elements (ABRE) and DRE/ C-RepeaT (CRT), both of which are involved in stress-inducible gene expression

(Assmann 2003; Desikan et al., 2004; Bright et al., 2006).

and ROS in switchgrass responses.

(Yamaguchi-Shinozaki and Shinozaki, 2005).

re-watering (Fig. 1.C).

earlier.

Fig. 1. **A.** Content of ABA in leaves of Panicum virgatum cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Data are means and SEs of three replicates, P ≤ 0.05. **B.** Plant height C. Stomatal resistance (S.R.). Measurements were made with porometer Delta-T on the abaxial side of leaves. Black circle: control conditions. Gray square: Drought, RW 12 h and RW 24 h of re-watering. Data are means of twenty-four replicates with SEs. Values with the same letter are not significantly different, P ≤ 0.05.

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 145

chlorophyll content compared to untreated control. The SA treatment also provided a considerable protection to the enzyme nitrate reductase thereby maintaining the level of diverse proteins in leaves (Singh and Usha, 2003). In addition, the treatment of water stressed *Licopersicum esculentum* plants with SA low concentrations significantly enhances the photosynthetic parameters, membrane stability index, leaf water potential, activities of the enzymes nitrate reductase and carbonic anhydrase; thus improving tolerance to drought (Hayat et al., 2008). SA is also involved in the promotion of drought-induced leaf senescence in *Salvia officinalis* plants grown under drought in Mediterranean filed conditions (Abreu and Munne-Bosch 2008). In addition, SA applied exogenously was effective in providing resistance to the plants against the excessive water stress in cell suspensions from the fully

Exogenous application of SA and glycin-betaine (GB, a compatible osmotic solute) enhanced the yield of sunflower hybrids under different degrees of water stress. Under stress, diameter of the head (inflorescence), number of achene and seed oil content was reduced.

In plants exposed to abiotic stress (e.g. salinity and drought), the accumulation of ROS, such as superoxide radicals (O2-), hydroxyl (OH-), and H2O2 is induced. The increasing ROS levels in plants produce oxidative stress of lipids, proteins and nucleic acids, which, in turn, alter the redox homeostasis (Smirnoff, 1993). SA increases the activity of the oxidative enzymatic system as is the case of CAT and SOD. In plants of *B. juncea,* exogenous application of SA increased CAT and SOD activity. In the same line of evidence, Kadioglu et al. (2010)

Fig. 2. Content of SA in leaves of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Data are means of three replicates with SEs. Values with the same letter are not significantly different at P ≤

0.05.

However, applications of SA and GB improved these parameters (Hussain, 2008).

turgid leaves of *Sporobcdus stapfianus* (Ghasempour et al., 2001).

### **2.2 Salicylic acid (SA)**

SA is an endogenous regulator of growth involved in a broad range of physiologic and metabolic responses in plants (Hayat, 2010). During the last years, SA has been intensively studied as a signal molecule mediating local and systemic defense responses against pathogens. Currently, it has been reported that this compound plays also a role in plants responses to abiotic stresses, such as drought, low and high temperatures, heavy metals, and osmotic stress (Janda et al., 1999; Rao and Davis 1999; Molina et al., 2002, Nemeth et al., 2002; Munne-Bosch and Peñuelas 2003; Shi et al., 2008; Rivas-San Vicente and Plasencia 2011). SA was also shown to influence a number of physiological processes, including seed germination, seedling growth, fruit ripening, flowering, ion uptake and transport, photosynthesis rate, stomata conductance, biogenesis of chloroplast (Fariduddin et al., 2003; Khodary 2004; Hayat et al., 2005; Shakirova 2007).

There are two main routes for SA biosynthesis in plants (Shah 2003). Earlier studies suggested that SA is synthesized from phenylalanine via cinnamic acid. The decarboxylation of the side chain of cinnamic acid may generate benzoic acid, which may then undergo hydroxylation at the C-2 position forming SA (Yalpani et al., 1993 ; Ribnicky et al., 1998). The other pathway for the SA biosynthesis involves a 2-hydroxylation of cinnamic acid to o-coumaric which is then decarboxylated to salycilic acid (Alibert and Ranjeva 1971; 1972). Recent studies in *Arabidopsis* plants showed that there is another main route for SA biosynthesis taking place in the chloroplast, where SA is synthesized from chorismate via isochorismate (Wildermuth 2006; Mustafa et al., 2009). SA may be conjugated with a variety of molecules either by glycosylation or by esterification (Popova et al., 1997), and may also be metabolized to 2,3 dihydrobenzoic acid or 2,5 dihydrobenzoic acid (Billek and Schmook, 1977).

Recent results show that most abiotic stresses altered *in planta* SA endogenous contents, which also point to its involvement in stress signaling (Horváth et al., 2007). For example, endogenous SA increased in roots of barley plants under water stress. In addition, when plants were treated with SA before stress, the damaging effect of water deficit on the cell membrane in the leaves decreased, and an increase in ABA content was observed. Also, the proline level increased only in the wild species of *Hordeum spontaneum*. These results suggest that ABA and proline may contribute to the development of the antistress reactions, induced by SA (Bandurska and Stroinski, 2005). Previously, Munne- Bosch and Peñuelas (2003) reported that in *Phillyrea angustifolia* L. plants exposed to drought the SA level increased progressively to as much as 5-fold, and showed a strong negative correlation with the relative water content. During recovery, SA levels decreased, but remained slightly higher than those observed before drought. SA levels were positively correlated with those of tocopherol -also known as vitamin E acetate- during drought, but not during recovery. This result also indicates the possible role of endogenous SA in the induction of a protective mechanism during water stress.

Application of exogenous SA improves the plant performance under water, as reported by several authors. Low concentrations of exogenous SA provided tolerance against the damaging effects of drought in tomato and bean plants, whereas, higher concentrations did not show the same positive results (Senaratna et al., 2000). Enhanced tolerance to drought and dry matter accumulation was also observed in plants of wheat raised from grains soaked in acetyl salicylic acid aqueous solution (Hamada 1998; Hamada and Al-Hakimi 2001). Wheat seedlings subjected to drought and treated with SA exhibited higher moisture content and dry matter accumulation, carboxylase activity of Rubisco, SOD and total

SA is an endogenous regulator of growth involved in a broad range of physiologic and metabolic responses in plants (Hayat, 2010). During the last years, SA has been intensively studied as a signal molecule mediating local and systemic defense responses against pathogens. Currently, it has been reported that this compound plays also a role in plants responses to abiotic stresses, such as drought, low and high temperatures, heavy metals, and osmotic stress (Janda et al., 1999; Rao and Davis 1999; Molina et al., 2002, Nemeth et al., 2002; Munne-Bosch and Peñuelas 2003; Shi et al., 2008; Rivas-San Vicente and Plasencia 2011). SA was also shown to influence a number of physiological processes, including seed germination, seedling growth, fruit ripening, flowering, ion uptake and transport, photosynthesis rate, stomata conductance, biogenesis of chloroplast (Fariduddin et al., 2003;

There are two main routes for SA biosynthesis in plants (Shah 2003). Earlier studies suggested that SA is synthesized from phenylalanine via cinnamic acid. The decarboxylation of the side chain of cinnamic acid may generate benzoic acid, which may then undergo hydroxylation at the C-2 position forming SA (Yalpani et al., 1993 ; Ribnicky et al., 1998). The other pathway for the SA biosynthesis involves a 2-hydroxylation of cinnamic acid to o-coumaric which is then decarboxylated to salycilic acid (Alibert and Ranjeva 1971; 1972). Recent studies in *Arabidopsis* plants showed that there is another main route for SA biosynthesis taking place in the chloroplast, where SA is synthesized from chorismate via isochorismate (Wildermuth 2006; Mustafa et al., 2009). SA may be conjugated with a variety of molecules either by glycosylation or by esterification (Popova et al., 1997), and may also be metabolized to 2,3 dihydrobenzoic acid or 2,5

Recent results show that most abiotic stresses altered *in planta* SA endogenous contents, which also point to its involvement in stress signaling (Horváth et al., 2007). For example, endogenous SA increased in roots of barley plants under water stress. In addition, when plants were treated with SA before stress, the damaging effect of water deficit on the cell membrane in the leaves decreased, and an increase in ABA content was observed. Also, the proline level increased only in the wild species of *Hordeum spontaneum*. These results suggest that ABA and proline may contribute to the development of the antistress reactions, induced by SA (Bandurska and Stroinski, 2005). Previously, Munne- Bosch and Peñuelas (2003) reported that in *Phillyrea angustifolia* L. plants exposed to drought the SA level increased progressively to as much as 5-fold, and showed a strong negative correlation with the relative water content. During recovery, SA levels decreased, but remained slightly higher than those observed before drought. SA levels were positively correlated with those of tocopherol -also known as vitamin E acetate- during drought, but not during recovery. This result also indicates the possible role of endogenous SA in the induction of a protective

Application of exogenous SA improves the plant performance under water, as reported by several authors. Low concentrations of exogenous SA provided tolerance against the damaging effects of drought in tomato and bean plants, whereas, higher concentrations did not show the same positive results (Senaratna et al., 2000). Enhanced tolerance to drought and dry matter accumulation was also observed in plants of wheat raised from grains soaked in acetyl salicylic acid aqueous solution (Hamada 1998; Hamada and Al-Hakimi 2001). Wheat seedlings subjected to drought and treated with SA exhibited higher moisture content and dry matter accumulation, carboxylase activity of Rubisco, SOD and total

**2.2 Salicylic acid (SA)** 

Khodary 2004; Hayat et al., 2005; Shakirova 2007).

dihydrobenzoic acid (Billek and Schmook, 1977).

mechanism during water stress.

chlorophyll content compared to untreated control. The SA treatment also provided a considerable protection to the enzyme nitrate reductase thereby maintaining the level of diverse proteins in leaves (Singh and Usha, 2003). In addition, the treatment of water stressed *Licopersicum esculentum* plants with SA low concentrations significantly enhances the photosynthetic parameters, membrane stability index, leaf water potential, activities of the enzymes nitrate reductase and carbonic anhydrase; thus improving tolerance to drought (Hayat et al., 2008). SA is also involved in the promotion of drought-induced leaf senescence in *Salvia officinalis* plants grown under drought in Mediterranean filed conditions (Abreu and Munne-Bosch 2008). In addition, SA applied exogenously was effective in providing resistance to the plants against the excessive water stress in cell suspensions from the fully turgid leaves of *Sporobcdus stapfianus* (Ghasempour et al., 2001).

Exogenous application of SA and glycin-betaine (GB, a compatible osmotic solute) enhanced the yield of sunflower hybrids under different degrees of water stress. Under stress, diameter of the head (inflorescence), number of achene and seed oil content was reduced. However, applications of SA and GB improved these parameters (Hussain, 2008).

In plants exposed to abiotic stress (e.g. salinity and drought), the accumulation of ROS, such as superoxide radicals (O2-), hydroxyl (OH-), and H2O2 is induced. The increasing ROS levels in plants produce oxidative stress of lipids, proteins and nucleic acids, which, in turn, alter the redox homeostasis (Smirnoff, 1993). SA increases the activity of the oxidative enzymatic system as is the case of CAT and SOD. In plants of *B. juncea,* exogenous application of SA increased CAT and SOD activity. In the same line of evidence, Kadioglu et al. (2010)

Fig. 2. Content of SA in leaves of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Data are means of three replicates with SEs. Values with the same letter are not significantly different at P ≤ 0.05.

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 147

In *P. virgatum,* we found that endogenous SA contents decreased considerably during a moderate water stress treatment and after 24 h of rehydration the endogenous contents increased significantly (p ≤ 0.05, Figure 2). This decrease in SA is accompanied with important peak of ABA content (four-fold increase) during the stress treatment (Figure 1.C). It has been proposed that an antagonistic interaction between these two hormones in response to water stress naturally occurs in several species, probably as a result of sharing common intermediaries in the signaling cascade (Yasuda et al., 2008). In addition, the increase in SA content corresponds with a raise in SOD and CAT activities after plants were

Despite of SA participation in abiotic stress responses, its role is ambiguous. The stress tolerance imparted by SA appears to be dosis-dependent, since deficiency or very high SA contents increase the susceptibility. Hence, the role of SA under a certain level of moderate or severe stress might be different. It could possibly be a result of the interaction between ROS and SA down-stream signals, where redox regulations play a key role (Yuan and Lin,

JA, and its cyclic precursors and derivatives constitute a family of bioactive oxylipins that regulate plant development and responses to environmental cues (Turner et al., 2002; Devoto and Turner, 2003). This family of compounds is form by 12-oxophytodienoic acid (OPDA), methyl jasmonate (Me-JA), JA hydroxylated (11-OH-JA and 12-OH-JA), JA conjugated to some amino acids such as leucine (JA-leucine) and isoleucine (JA-Ile) as well as the glucoside and sulfate of 12-OH-JA (12-*O*-Glc-JA, 12-HSO4-JA), and collectively receive the name of jasmonates (JAs). These molecules are involved in a variety of processes related to plant development and survival, including direct and indirect defense responses (*e.g*., defense against insects and necrotrophic pathogens), secondary metabolism, reproductive processes (*e.g*., pollen maturation and anther dehiscence, ovule development), and fruit development, among others (Seo et al., 2001; Wasternack and Hause, 2002; Arimura et al., 2005; Liechti and Farmer, 2006; Wasternack, 2007). In addition, it is known that JA-related responses are directly associated with a reset downstream of gene expression in the

Vick and Zimmerman (1983) were the first authors to demonstrate the steps of the JA biosynthesis, and recently it was reviewed by Wasternack and Kombrink (2010). JA biosynthesis and signaling pathway have been extensively studied, mainly in dicots such as *Arabidopsis* and tomato, and to a lesser extent in some monocots (Kazan and Manners, 2008). JAs are produced from α-linolenic acid (α-LeA; C18:3) or hexadecatrienoic acid (C16:3) released from plastidial galactolipids by phospholipases. Following the oxidation of α-LeA by lipoxygenase (LOX) to 13(S)-hydroperoxyoctadecatrienoic acid (13(S)-HPOT), the first committed step of JA biosynthesis is conversion of the LOX product to the allene oxide 12,13(S)-epoxyoctadecatrienoic acid (12,13(S)-EOT) by allene oxide synthase (AOS). This unstable allylic epoxide can be enzymatically cyclized by allene oxide cyclase (AOC) to optically pure cis-(+)-12-oxophytodienoic acid (9S,13S)-OPDA), which is the last product of the plastid-localized part of the JA biosynthesis pathway. Translocation of OPDA into peroxisomes, where the subsequent part of the JA biosynthesis pathway occurs, is mediated by the ABC transporter COMATOSE and/or an ion-trapping mechanism (Theodoulou et al., 2005). The OPDA reduction is catalyzed by a peroxisomal OPDA reductase (OPR) to

rehydrated (Figure 3 A and B).

**2.3 Jasmonic acid (JA)** 

biosynthesis pathway (Thines et al., 2007).

2008).

reported that exogenous application of SA induced the activity of antioxidant enzymes at the same time that alleviates the water stress damage in the long run in plants of *Ctenanthe setosa*. In seedlings of wheat under water stress and supplemented with SA (1 mM), ABA (0,5 mM), Ca2+ (5 mM) and H2O2 (0,05 mM), the activity of SOD, CAT, ascorbate peroxidase (APX), and NADPH oxidase (Agarwal, 2005) was induced.

Fig. 3. **A.** Activity of superoxide dismutase (SOD) and **B.** catalase (CAT), on leaves of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of rewatering (RW 12 h and RW 24 h). Data are means of four replicates with SEs. Values with the same letter are not significantly different at P ≤ 0.05.

In *P. virgatum,* we found that endogenous SA contents decreased considerably during a moderate water stress treatment and after 24 h of rehydration the endogenous contents increased significantly (p ≤ 0.05, Figure 2). This decrease in SA is accompanied with important peak of ABA content (four-fold increase) during the stress treatment (Figure 1.C). It has been proposed that an antagonistic interaction between these two hormones in response to water stress naturally occurs in several species, probably as a result of sharing common intermediaries in the signaling cascade (Yasuda et al., 2008). In addition, the increase in SA content corresponds with a raise in SOD and CAT activities after plants were rehydrated (Figure 3 A and B).

Despite of SA participation in abiotic stress responses, its role is ambiguous. The stress tolerance imparted by SA appears to be dosis-dependent, since deficiency or very high SA contents increase the susceptibility. Hence, the role of SA under a certain level of moderate or severe stress might be different. It could possibly be a result of the interaction between ROS and SA down-stream signals, where redox regulations play a key role (Yuan and Lin, 2008).

### **2.3 Jasmonic acid (JA)**

146 Plants and Environment

reported that exogenous application of SA induced the activity of antioxidant enzymes at the same time that alleviates the water stress damage in the long run in plants of *Ctenanthe setosa*. In seedlings of wheat under water stress and supplemented with SA (1 mM), ABA (0,5 mM), Ca2+ (5 mM) and H2O2 (0,05 mM), the activity of SOD, CAT, ascorbate peroxidase

Fig. 3. **A.** Activity of superoxide dismutase (SOD) and **B.** catalase (CAT), on leaves of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of rewatering (RW 12 h and RW 24 h). Data are means of four replicates with SEs. Values with

the same letter are not significantly different at P ≤ 0.05.

(APX), and NADPH oxidase (Agarwal, 2005) was induced.

JA, and its cyclic precursors and derivatives constitute a family of bioactive oxylipins that regulate plant development and responses to environmental cues (Turner et al., 2002; Devoto and Turner, 2003). This family of compounds is form by 12-oxophytodienoic acid (OPDA), methyl jasmonate (Me-JA), JA hydroxylated (11-OH-JA and 12-OH-JA), JA conjugated to some amino acids such as leucine (JA-leucine) and isoleucine (JA-Ile) as well as the glucoside and sulfate of 12-OH-JA (12-*O*-Glc-JA, 12-HSO4-JA), and collectively receive the name of jasmonates (JAs). These molecules are involved in a variety of processes related to plant development and survival, including direct and indirect defense responses (*e.g*., defense against insects and necrotrophic pathogens), secondary metabolism, reproductive processes (*e.g*., pollen maturation and anther dehiscence, ovule development), and fruit development, among others (Seo et al., 2001; Wasternack and Hause, 2002; Arimura et al., 2005; Liechti and Farmer, 2006; Wasternack, 2007). In addition, it is known that JA-related responses are directly associated with a reset downstream of gene expression in the biosynthesis pathway (Thines et al., 2007).

Vick and Zimmerman (1983) were the first authors to demonstrate the steps of the JA biosynthesis, and recently it was reviewed by Wasternack and Kombrink (2010). JA biosynthesis and signaling pathway have been extensively studied, mainly in dicots such as *Arabidopsis* and tomato, and to a lesser extent in some monocots (Kazan and Manners, 2008). JAs are produced from α-linolenic acid (α-LeA; C18:3) or hexadecatrienoic acid (C16:3) released from plastidial galactolipids by phospholipases. Following the oxidation of α-LeA by lipoxygenase (LOX) to 13(S)-hydroperoxyoctadecatrienoic acid (13(S)-HPOT), the first committed step of JA biosynthesis is conversion of the LOX product to the allene oxide 12,13(S)-epoxyoctadecatrienoic acid (12,13(S)-EOT) by allene oxide synthase (AOS). This unstable allylic epoxide can be enzymatically cyclized by allene oxide cyclase (AOC) to optically pure cis-(+)-12-oxophytodienoic acid (9S,13S)-OPDA), which is the last product of the plastid-localized part of the JA biosynthesis pathway. Translocation of OPDA into peroxisomes, where the subsequent part of the JA biosynthesis pathway occurs, is mediated by the ABC transporter COMATOSE and/or an ion-trapping mechanism (Theodoulou et al., 2005). The OPDA reduction is catalyzed by a peroxisomal OPDA reductase (OPR) to

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 149

Exogenous application of JA or Me-JA increased antioxidative ability of plants under water stress (Wang, 1999; Bandurska et al., 2003). Along the same line, other studies also showed that JAs play an important role in signaling drought-induced antioxidant responses, including ascorbate metabolism (Li et al., 1998; Ai et al., 2008). For instance, in shoots and roots of maize seedlings treated with Paraquat, an herbicide, and exogenous concentrations of Me-JA (50 and 100 μM) the expression of genes corresponding to the anti-oxidative defense system was detected. Certainly, Me-JA promoted increased production of several anti-oxidative enzymes, including glutathione reductase, guaiacol peroxidase and ascorbate peroxidase, and it has been suggested that this increase may be due to up-regulation of genes controlling the synthesis of these enzymes, or by activation of diverse constitutive

To survive under various biotic and abiotic stresses, plants have developed complex mechanisms to perceive external signals, allowing them optimal response to the environment. ABA, SA, JA, and ethylene (ET) regulate protective responses of plants against both abiotic and biotic stresses via synergistic and antagonistic actions, which are referred to as signaling crosstalk (Fujita et al., 2006). Furthermore, ROS generation has been proposed as a pivotal process that is shared between abiotic and biotic responses (Apel and Hirt, 2004;

ABA has been extensively involved in responses to various abiotic stresses (*e.g*., drought, salinity, low temperature) and, on the other side, SA, JA and ET play a key role in responses to biotic stress upon pathogen infection. Several studies have indicated that plant responses to environmental stresses have some effects on their response to pathogens. In many cases, ABA acts as a negative regulator of disease resistance (Narusaka et al., 2004). For instance, the ABA-deficient tomato mutant *sitiens* has increased resistance to pathogens and application of exogenous ABA restored the susceptibility of *sitiens* mutants. The *sitiens* mutant has greater SA-mediated responses, suggesting that high ABA concentrations inhibit the SA-dependent defense response in tomato (Fujita et al., 2006). It has also been reported that ABA treatment suppresses the induction of SAR in *Arabidopsis*. The use of several mutants in combination with chemicals that inhibit and/or stimulate SA revealed that ABA suppressed the SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the JA/ET mediated signaling pathway. These data strongly suggest that an antagonistic crosstalk might occur at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses (Yasuda et al., 2008). This antagonistic interaction between ABA mediated abiotic stress signaling and disease resistance might simply suggest that plants developed strategies to simultaneously producing proteins that are involved in abiotic stress and disease resistance (Anderson et al., 2004). Since pathogen infection requires relatively humid conditions, a simultaneous exposure of plants to drought and necrotrophic pathogens attack is actually rare in nature. In fact, high humidity and temperature weaken the plant resistance to pathogen attack. Thus, the view that the ABA-mediated abiotic stress signaling potentially takes precedence over biotic stress signaling (Anderson et al., 2004) supports the notion that water stress threatens plant survival more significant than pathogen infection

genes (Norastehnia and Asghari, 2006).

Torres and Dangl, 2005).

does (Fujita et al., 2006).

**3. ABA, SA, JA and cross talk between each other** 

produced 3-oxo-2(2[Z]-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0). Then, three cycles of β-oxidation catalyzed by acyl-CoA oxidase (ACX), multifunctional protein (MFP), and L-3-ketoacyl-CoA thiolase (KAT) lead to jasmonoyl-CoA, from which a yet unknown thioesterase releases (+)-7-iso-JA ((3R,7S)-JA) that equilibrates to the more stable (-)-JA ((3R,7R)-JA).

The participation of JA in response to abiotic stress, such as drought and salinity, has been reported in several species. For instance, the treatment of barley leaves with sorbitol or mannitol (compatibles solutes to simulate water stress) increased JAs endogenous contents, followed by synthesis of jasmonate-induced proteins (JIPs, Lehmann et al., 1995). Other study showed that sorbitol treatment enhanced octadecanoids and JAs content, and this threshold was necessary and sufficient to initiate JA-responsive gene expression (Kramell et al., 2000). In addition, under water stress, endogenous JA content increased in maize root cells (Xin et al., 1997) and this compound was able to elicit betaine accumulation in pear leaves (Gao et al., 2004). Pedranzani et al. (2003) showed that tomato cultivars differing in salt tolerance differed in basal JA content. Steady-state amounts of JA and related compounds were higher in salttolerant cv. Pera compared to the salt-sensitive cv. Hellfrucht frühstamm. Moreover, studies in contrasting environments showed different basal JAs contents and patterns of response to water stress in two populations of *Pinus pinaster* Ait., perhaps as an adaptation to diverse ecological conditions (Pedranzani et al., 2007).

Studies performed in our laboratory with *Panicum virgatum* showed that, during the drought treatments, JA levels did not increase significantly compared to the control level. However, after watering was restored, contents of JA consistently increased and overcome the control (Figure 4).

Fig. 4. Content of JA in leaves of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Data are means of three replicates with SEs. Values with the same letter are not significantly different at P ≤ 0.05

produced 3-oxo-2(2[Z]-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0). Then, three cycles of β-oxidation catalyzed by acyl-CoA oxidase (ACX), multifunctional protein (MFP), and L-3-ketoacyl-CoA thiolase (KAT) lead to jasmonoyl-CoA, from which a yet unknown thioesterase releases (+)-7-iso-JA ((3R,7S)-JA) that equilibrates to the more stable (-)-JA

The participation of JA in response to abiotic stress, such as drought and salinity, has been reported in several species. For instance, the treatment of barley leaves with sorbitol or mannitol (compatibles solutes to simulate water stress) increased JAs endogenous contents, followed by synthesis of jasmonate-induced proteins (JIPs, Lehmann et al., 1995). Other study showed that sorbitol treatment enhanced octadecanoids and JAs content, and this threshold was necessary and sufficient to initiate JA-responsive gene expression (Kramell et al., 2000). In addition, under water stress, endogenous JA content increased in maize root cells (Xin et al., 1997) and this compound was able to elicit betaine accumulation in pear leaves (Gao et al., 2004). Pedranzani et al. (2003) showed that tomato cultivars differing in salt tolerance differed in basal JA content. Steady-state amounts of JA and related compounds were higher in salttolerant cv. Pera compared to the salt-sensitive cv. Hellfrucht frühstamm. Moreover, studies in contrasting environments showed different basal JAs contents and patterns of response to water stress in two populations of *Pinus pinaster* Ait., perhaps as an adaptation to diverse

Studies performed in our laboratory with *Panicum virgatum* showed that, during the drought treatments, JA levels did not increase significantly compared to the control level. However, after watering was restored, contents of JA consistently increased and overcome

Fig. 4. Content of JA in leaves of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Data are means of three replicates with SEs. Values with the same letter are not significantly different at P ≤ 0.05

((3R,7R)-JA).

ecological conditions (Pedranzani et al., 2007).

the control (Figure 4).

Exogenous application of JA or Me-JA increased antioxidative ability of plants under water stress (Wang, 1999; Bandurska et al., 2003). Along the same line, other studies also showed that JAs play an important role in signaling drought-induced antioxidant responses, including ascorbate metabolism (Li et al., 1998; Ai et al., 2008). For instance, in shoots and roots of maize seedlings treated with Paraquat, an herbicide, and exogenous concentrations of Me-JA (50 and 100 μM) the expression of genes corresponding to the anti-oxidative defense system was detected. Certainly, Me-JA promoted increased production of several anti-oxidative enzymes, including glutathione reductase, guaiacol peroxidase and ascorbate peroxidase, and it has been suggested that this increase may be due to up-regulation of genes controlling the synthesis of these enzymes, or by activation of diverse constitutive genes (Norastehnia and Asghari, 2006).

### **3. ABA, SA, JA and cross talk between each other**

To survive under various biotic and abiotic stresses, plants have developed complex mechanisms to perceive external signals, allowing them optimal response to the environment. ABA, SA, JA, and ethylene (ET) regulate protective responses of plants against both abiotic and biotic stresses via synergistic and antagonistic actions, which are referred to as signaling crosstalk (Fujita et al., 2006). Furthermore, ROS generation has been proposed as a pivotal process that is shared between abiotic and biotic responses (Apel and Hirt, 2004; Torres and Dangl, 2005).

ABA has been extensively involved in responses to various abiotic stresses (*e.g*., drought, salinity, low temperature) and, on the other side, SA, JA and ET play a key role in responses to biotic stress upon pathogen infection. Several studies have indicated that plant responses to environmental stresses have some effects on their response to pathogens. In many cases, ABA acts as a negative regulator of disease resistance (Narusaka et al., 2004). For instance, the ABA-deficient tomato mutant *sitiens* has increased resistance to pathogens and application of exogenous ABA restored the susceptibility of *sitiens* mutants. The *sitiens* mutant has greater SA-mediated responses, suggesting that high ABA concentrations inhibit the SA-dependent defense response in tomato (Fujita et al., 2006). It has also been reported that ABA treatment suppresses the induction of SAR in *Arabidopsis*. The use of several mutants in combination with chemicals that inhibit and/or stimulate SA revealed that ABA suppressed the SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the JA/ET mediated signaling pathway. These data strongly suggest that an antagonistic crosstalk might occur at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses (Yasuda et al., 2008). This antagonistic interaction between ABA mediated abiotic stress signaling and disease resistance might simply suggest that plants developed strategies to simultaneously producing proteins that are involved in abiotic stress and disease resistance (Anderson et al., 2004). Since pathogen infection requires relatively humid conditions, a simultaneous exposure of plants to drought and necrotrophic pathogens attack is actually rare in nature. In fact, high humidity and temperature weaken the plant resistance to pathogen attack. Thus, the view that the ABA-mediated abiotic stress signaling potentially takes precedence over biotic stress signaling (Anderson et al., 2004) supports the notion that water stress threatens plant survival more significant than pathogen infection does (Fujita et al., 2006).

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 151

suggest that the reduced growth in response to drought stress, as a developmental program for acclimation, is not switched on in the absence of JA signal perception. Thus, the down-regulation of JA biosynthesis to minimize the inhibitory effect of JA on plant growth as well as signaling pathways under prolonged drought can establish new

The crosstalk between JA and ABA might occur as they utilize a similar cascade of events to stimulate some responses (Harb et al., 2010; Fujita et al., 2006). Recent studies have revealed several molecules, including transcription factors and kinases, as promising candidates for common players that are involved in this crosstalk. The convergence points in JA and ABA stress signaling occurs, in part, by sharing some transcription factors. Transcription factor AtMYC2 plays a role in multiple hormone signaling pathways. Genetic analysis of the jasmonate-insensitive jin1 mutant revealed that JIN1 is allelic to AtMYC2, which was first identified as a transcriptional activator that is involved in the ABA mediated drought stress signaling pathway (Abe et al., 2003). The dehydration-inducible RD22 gene (involved in response to salt stress and response to desiccation) respond to both AtMYC2 and the R2R3MYB-type transcription factor. RD26 expression is induced by JA, hydrogen peroxide and pathogen infections, as well as by drought, high salinity and ABA treatment (Fujita et al., 2004; Harb et al., 2010; Fujita et al., 2010). In addition, protein phosphorylation and dephosphorylation significantly influence both the regulation of physiological morphology and gene expression associated with basic cellular activities in JA-dependent root growth and in AtMYC2 gene expression. The gene expression and kinase activity of OsMPK5 is also

induced by ABA, various abiotic stresses and pathogen infection (Xiong et al., 2003).

Participation of ABA and JAs in stomatal closing was studied in *Arabidopsis* wild type and mutants, ABA-insensitive (*ost1-2*), and Me-JA-insensitive mutants (*jar1-1*), in order to examine a crosstalk between ABA and Me-JA signal transduction. In that study, cytoplasmic pH changes and ROS production in response to ABA or Me-JA were used to assess the respective roles of these genes in ABA or Me-JA signaling pathways, leading to stomatal closure. The modulation of Ca2+ mediates the response, and it appears to be a common effect of ABA and Me-JA. The primary actions of ABA and Me-JA at the plasma membrane level appear to be different: while Me-JA targets the Ca2+ channels, ABA activates effectors in the plasma membrane (i.g. phospholipase C, D). However, both signal transduction pathways converge at level of intracellular Ca2+. The regulation of intracellular Ca2+ level, indeed, has a much greater dependence of Me-JA action than that of ABA (Blatt et al., 1993; McAinsh et

Similar interaction between ABA and JA signaling pathways has been observed in seed germination in *Arabidopsis*. In this case, seed germination of the JA-resistant1 (*jar1*) and JAinsensitive4 (*jin4*) mutants were more sensitive to ABA than its wild type (Staswick et al.,

Evidence of antagonistic interactions of ABA/JA was also found at the level of gene expression in *Arabidopsis* (Balbi and Devoto, 2007). Wild type and *coi1* plants were wounded or treated with Me-JA, and changes in the expression of 8200 genes were examined using microarrays. A survey of the genes that were repressed by Me-JA identified many genes that have been implicated in ABA and drought stress response. These include the ATHB-12 transcription factor, the bZIP-transcription factor ABF3, COR47 and LEA D113. The nitrate transporter NTP2 and three members of the aquaporin family of transporters were also repressed by Me-JA in a COI1-independent manner. These findings reinforce the role of JA

homeostasis during the acclimation process.

al., 1995; Suhita et al., 2004).

1992; Berger et al., 1996).

Likewise, a positive interaction between SA and ABA might occur in abiotic stress. The roles playing by free SA, conjugated SA, and ABA in thermo-tolerance induced by heat acclimation (38°C) were investigated. To evaluate their potential functions, three inhibitors of synthesis or activity were infiltrated into pea leaves prior to heat acclimation treatment. The results showed that the burst of free SA in response to heat acclimation could be attributed to the conversion of SA 2-*O*-D-glucose, the main conjugated form of SA, to free SA. Inhibition of ABA biosynthesis also resulted in a defect in the free SA peak during heat acclimation. Overall, these results suggest that exogenous SA and ABA may lead to the enhancement of thermo-tolerance (Liu et al., 2006).

Our study in *Panicum virgatum* adds evidence to ABA/SA association. Results in our laboratory show that under drought, the content of endogenous SA is lower than that of the control. However, after 12 h of re-watering SA content reach the control value, and after 24 h the contents are significantly higher than that of the control (p ≤ 0.05, Figure 2). On the other hand, an opposite trend is described in ABA (Figure 1.C), showing that when ABA reach its maximum, SA content is minimum. By the time that ABA recovers the control value, SA content significantly increases over the well-watered control.

Interaction between ABA and JA has been reported in salt stress response. Moons et al. (1997) compared the effects of exogenous ABA and JA in the rice seedlings response to salt stress. In view of the proposed roles for JA and Me-JA in plants exposed to waterlimiting stresses, changes in endogenous jasmonates -in particular MeJA content- were compared with the well established increase in endogenous ABA in plants subjected to salt stress. Salt shock (150 mM NaCl) induced a rapid increase in ABA content in roots of 10-day-old seedlings, reaching a maximum at 8 h of stress and decreasing to near control values after 12 h. On the contrary, Me-JA concentrations, showed a delayed and gradual increase of approximately 4-fold after 12 h of stress. This accumulation occurred when ABA levels were decreasing. In the same study, eight stress- induced proteins were compared for their ABA and/or JA response. In addition, the effect of JA, ABA, and salt stress on the transcript levels of three genes encoding pathogenic related proteins, a salt stress-responsive protein, and a group three LEA protein were analyzed. ABA and JA were found to exert antagonistic effects on the transcript and/or protein accumulation of two classes of salt stress-responsive genes.

In addition, in *Arabidopsis* it has been proposed that both ABA and JA participate in the responses to moderate drought (30% field capacity). Nevertheless, ABA and JA would be involved in different stages of the response, driving an acclimation process during growth through an extensive genetic reprogramming to finally reach a new homeostasis (Harb et al., 2010). These authors suggest that, during early stages of moderate drought, endogenous JA in combination with high ABA level is enough to stimulate the preparatory response needed for drought acclimation (e.g. stomatal closure and cell wall modification). JA is probably not required at high concentration under drought stress, and an increase in its concentration might negatively affect plant response to growth. Under moderate drought treatment, the response of *Arabidopsis* mutants *coi1* and *jin1* (both JA-insensitive) were found to be significantly resistant (or insensitive to drought stress). Compared to the wild type, biomass accumulation under drought did not differ from the well-watered control. These results are in agreement with studies showing that in *coi1* mutant the JA-mediated inhibition of seedling and root growth is suppressed (Xie et al., 1998). Harb et al. (2010)

Likewise, a positive interaction between SA and ABA might occur in abiotic stress. The roles playing by free SA, conjugated SA, and ABA in thermo-tolerance induced by heat acclimation (38°C) were investigated. To evaluate their potential functions, three inhibitors of synthesis or activity were infiltrated into pea leaves prior to heat acclimation treatment. The results showed that the burst of free SA in response to heat acclimation could be attributed to the conversion of SA 2-*O*-D-glucose, the main conjugated form of SA, to free SA. Inhibition of ABA biosynthesis also resulted in a defect in the free SA peak during heat acclimation. Overall, these results suggest that exogenous SA and ABA may lead to the

Our study in *Panicum virgatum* adds evidence to ABA/SA association. Results in our laboratory show that under drought, the content of endogenous SA is lower than that of the control. However, after 12 h of re-watering SA content reach the control value, and after 24 h the contents are significantly higher than that of the control (p ≤ 0.05, Figure 2). On the other hand, an opposite trend is described in ABA (Figure 1.C), showing that when ABA reach its maximum, SA content is minimum. By the time that ABA recovers the control value, SA

Interaction between ABA and JA has been reported in salt stress response. Moons et al. (1997) compared the effects of exogenous ABA and JA in the rice seedlings response to salt stress. In view of the proposed roles for JA and Me-JA in plants exposed to waterlimiting stresses, changes in endogenous jasmonates -in particular MeJA content- were compared with the well established increase in endogenous ABA in plants subjected to salt stress. Salt shock (150 mM NaCl) induced a rapid increase in ABA content in roots of 10-day-old seedlings, reaching a maximum at 8 h of stress and decreasing to near control values after 12 h. On the contrary, Me-JA concentrations, showed a delayed and gradual increase of approximately 4-fold after 12 h of stress. This accumulation occurred when ABA levels were decreasing. In the same study, eight stress- induced proteins were compared for their ABA and/or JA response. In addition, the effect of JA, ABA, and salt stress on the transcript levels of three genes encoding pathogenic related proteins, a salt stress-responsive protein, and a group three LEA protein were analyzed. ABA and JA were found to exert antagonistic effects on the transcript and/or protein accumulation of

In addition, in *Arabidopsis* it has been proposed that both ABA and JA participate in the responses to moderate drought (30% field capacity). Nevertheless, ABA and JA would be involved in different stages of the response, driving an acclimation process during growth through an extensive genetic reprogramming to finally reach a new homeostasis (Harb et al., 2010). These authors suggest that, during early stages of moderate drought, endogenous JA in combination with high ABA level is enough to stimulate the preparatory response needed for drought acclimation (e.g. stomatal closure and cell wall modification). JA is probably not required at high concentration under drought stress, and an increase in its concentration might negatively affect plant response to growth. Under moderate drought treatment, the response of *Arabidopsis* mutants *coi1* and *jin1* (both JA-insensitive) were found to be significantly resistant (or insensitive to drought stress). Compared to the wild type, biomass accumulation under drought did not differ from the well-watered control. These results are in agreement with studies showing that in *coi1* mutant the JA-mediated inhibition of seedling and root growth is suppressed (Xie et al., 1998). Harb et al. (2010)

enhancement of thermo-tolerance (Liu et al., 2006).

two classes of salt stress-responsive genes.

content significantly increases over the well-watered control.

suggest that the reduced growth in response to drought stress, as a developmental program for acclimation, is not switched on in the absence of JA signal perception. Thus, the down-regulation of JA biosynthesis to minimize the inhibitory effect of JA on plant growth as well as signaling pathways under prolonged drought can establish new homeostasis during the acclimation process.

The crosstalk between JA and ABA might occur as they utilize a similar cascade of events to stimulate some responses (Harb et al., 2010; Fujita et al., 2006). Recent studies have revealed several molecules, including transcription factors and kinases, as promising candidates for common players that are involved in this crosstalk. The convergence points in JA and ABA stress signaling occurs, in part, by sharing some transcription factors. Transcription factor AtMYC2 plays a role in multiple hormone signaling pathways. Genetic analysis of the jasmonate-insensitive jin1 mutant revealed that JIN1 is allelic to AtMYC2, which was first identified as a transcriptional activator that is involved in the ABA mediated drought stress signaling pathway (Abe et al., 2003). The dehydration-inducible RD22 gene (involved in response to salt stress and response to desiccation) respond to both AtMYC2 and the R2R3MYB-type transcription factor. RD26 expression is induced by JA, hydrogen peroxide and pathogen infections, as well as by drought, high salinity and ABA treatment (Fujita et al., 2004; Harb et al., 2010; Fujita et al., 2010). In addition, protein phosphorylation and dephosphorylation significantly influence both the regulation of physiological morphology and gene expression associated with basic cellular activities in JA-dependent root growth and in AtMYC2 gene expression. The gene expression and kinase activity of OsMPK5 is also induced by ABA, various abiotic stresses and pathogen infection (Xiong et al., 2003).

Participation of ABA and JAs in stomatal closing was studied in *Arabidopsis* wild type and mutants, ABA-insensitive (*ost1-2*), and Me-JA-insensitive mutants (*jar1-1*), in order to examine a crosstalk between ABA and Me-JA signal transduction. In that study, cytoplasmic pH changes and ROS production in response to ABA or Me-JA were used to assess the respective roles of these genes in ABA or Me-JA signaling pathways, leading to stomatal closure. The modulation of Ca2+ mediates the response, and it appears to be a common effect of ABA and Me-JA. The primary actions of ABA and Me-JA at the plasma membrane level appear to be different: while Me-JA targets the Ca2+ channels, ABA activates effectors in the plasma membrane (i.g. phospholipase C, D). However, both signal transduction pathways converge at level of intracellular Ca2+. The regulation of intracellular Ca2+ level, indeed, has a much greater dependence of Me-JA action than that of ABA (Blatt et al., 1993; McAinsh et al., 1995; Suhita et al., 2004).

Similar interaction between ABA and JA signaling pathways has been observed in seed germination in *Arabidopsis*. In this case, seed germination of the JA-resistant1 (*jar1*) and JAinsensitive4 (*jin4*) mutants were more sensitive to ABA than its wild type (Staswick et al., 1992; Berger et al., 1996).

Evidence of antagonistic interactions of ABA/JA was also found at the level of gene expression in *Arabidopsis* (Balbi and Devoto, 2007). Wild type and *coi1* plants were wounded or treated with Me-JA, and changes in the expression of 8200 genes were examined using microarrays. A survey of the genes that were repressed by Me-JA identified many genes that have been implicated in ABA and drought stress response. These include the ATHB-12 transcription factor, the bZIP-transcription factor ABF3, COR47 and LEA D113. The nitrate transporter NTP2 and three members of the aquaporin family of transporters were also repressed by Me-JA in a COI1-independent manner. These findings reinforce the role of JA

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 153

reached a new homeostasis status where SA-JA-ABA balance is different from the wellwatered control. Humidity produced by re-watering after a water stress could trigger a defense response to pathogen facing a potential attack. Or, it could reorganize the endogenous levels of plant hormones to reach a new homeostasis in acclimation to new environmental conditions (Fujita et al., 2006). Overall, this new hormonal status suggests the

Forage crops, which are grown to be utilized by grazing or harvesting as a whole crop, are essential for the successful operation of animal production systems. This fact is more relevant for ruminants which heavily depend upon forages for their health and for a costeffective and sustainable production . While forages are an economical source of nutrients for animal production, they also help conserve the soil integrity, water supply and air quality (Chaudhry, 2008). In the last years, forage species have been widely studied for nonforage purposes—especially for bioenergy. Grasses are a source of lignocellulosic biomass to generate biofuels and they belong to a group of plant species considered as second generation crops. Nowadays, second generation of biofuels have gained relevance since they do not directly compete with human nutrition, unlike first generation of biofuel crops. The incorporation of forage species to the production of bioenergy is expected to expand the amount of biofuel that can be produced sustainably by using biomass of non-food crops such as swithgrass, whole crop maize, miscanthus and cereals that bear little grain, among others (Inderwildi and King, 2009). However, one of the major concerns about these crops is the environmental impact. It is likely that the expansion of crops for bioenergy utilization occurs with greater intensity in natural ecosystems, often characterized by their fragility in soil stability and water content. Global climate change intensifies these challenges as current crops are poorly adapted to more uncertain and extreme climatic conditions. In this context, the study of plant responses to water deficit as a strategy for the optimization in the use of water is of remarkable importance to increase production without further damage to the environment. In this chapter, we presented our contribution to this topic through the study of drought tolerance in *Panicum virgatum*, a member of the Poaceae family intensively studied as a source of lignocellulosic biomass to produce renewable energy. The Poaceae, a family with numerous species important to human nutrition, shares an extensive similarity among its members; hence, the comprehension of the bases of water stress tolerance in *Panicum virgatum* will improve our understanding of the entire group. Providing food and energy in conditions that maintain the sustainability of resources is a challenge that must be addressed. Faced with a global energy crisis and the steadily growing world population, forage crops are a suitable alternative to meet current

The biological significance of crosstalk between signaling pathways operating under stress conditions as well as the mechanism that underlie this crosstalk are still unclear. At present, these pathways have become better resolved due to the development of new tools that allow for the exploration of the physiological, genetic, and biochemical foundation of such processes. The genomic, proteomic and metabolomic approach is now widely used in model plants and, to a lesser extent, in crop and forage plants. The growing interest in forage crops has promoted its study at the molecular level, making it promising to research the improvement of these species. To date, the complete genome sequences of four grass species

interplay among SA-JA-ABA in water stress responses in *P. virgatum*.

**4. Conclusions and perspectives** 

and future demands of food and energy.

in osmotic homeostasis and are complementary to the study of Armengaud et al. (2004). This author shows that transcript levels for the JA biosynthetic enzymes (i.e. lipoxygenase, allene oxide synthase, and allene oxide cyclase) as well as JA responsive genes (i.e. genes involved in storage of amino acids –VSP-, glucosinolate production -CYP79-, polyamine biosynthesis -ADC2-,and defense -PDF1.2) strongly increase during potassium starvation and quickly decreased after potassium resupply. These finding highlight the role of JA in nutrient signaling and stress management through a variety of physiological processes such as nutrient storage, recycling, and reallocation.

In our work, the experiments with *Panicum virgatum* show that endogenous JA content is not affected by a moderate water deficit, but such contents increase significantly after 24 h of rewatering. This trend is similar to the response observed in *Arabidopsis* during early stages of water and salt stress, where the contents of JA remain constant under drought and gradually recover after re-watering (Moons et al., 1997; Harb et al., 2010). Conversely, there is an increment in ABA levels under a moderate stress that corresponds with an increase in SOD and CAT activities (Figure 5). At the same time, the SA contents decreased, resembling an antagonistic interaction ABA/SA. After re-watering, ABA contents decreases at the same time as SA and JA endogenous contents display an increase. This last trend is accompanied by a rising in SOD and CAT activity during 24 h of plant recovering. Thus, recovered plants

Fig. 5. Model of hormonal response of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Abscisic acid(ABA), salicylic acid (SA), jasmonic acid (JA), catalase (CAT), superoxide dismutase (SOD).

reached a new homeostasis status where SA-JA-ABA balance is different from the wellwatered control. Humidity produced by re-watering after a water stress could trigger a defense response to pathogen facing a potential attack. Or, it could reorganize the endogenous levels of plant hormones to reach a new homeostasis in acclimation to new environmental conditions (Fujita et al., 2006). Overall, this new hormonal status suggests the interplay among SA-JA-ABA in water stress responses in *P. virgatum*.

### **4. Conclusions and perspectives**

152 Plants and Environment

in osmotic homeostasis and are complementary to the study of Armengaud et al. (2004). This author shows that transcript levels for the JA biosynthetic enzymes (i.e. lipoxygenase, allene oxide synthase, and allene oxide cyclase) as well as JA responsive genes (i.e. genes involved in storage of amino acids –VSP-, glucosinolate production -CYP79-, polyamine biosynthesis -ADC2-,and defense -PDF1.2) strongly increase during potassium starvation and quickly decreased after potassium resupply. These finding highlight the role of JA in nutrient signaling and stress management through a variety of physiological processes such

In our work, the experiments with *Panicum virgatum* show that endogenous JA content is not affected by a moderate water deficit, but such contents increase significantly after 24 h of rewatering. This trend is similar to the response observed in *Arabidopsis* during early stages of water and salt stress, where the contents of JA remain constant under drought and gradually recover after re-watering (Moons et al., 1997; Harb et al., 2010). Conversely, there is an increment in ABA levels under a moderate stress that corresponds with an increase in SOD and CAT activities (Figure 5). At the same time, the SA contents decreased, resembling an antagonistic interaction ABA/SA. After re-watering, ABA contents decreases at the same time as SA and JA endogenous contents display an increase. This last trend is accompanied by a rising in SOD and CAT activity during 24 h of plant recovering. Thus, recovered plants

Fig. 5. Model of hormonal response of *Panicum virgatum* cv. Greenville grown under drought (Drought) and after 12 and 24 h of re-watering (RW 12 h and RW 24 h). Abscisic acid(ABA), salicylic acid (SA), jasmonic acid (JA), catalase (CAT), superoxide dismutase

(SOD).

as nutrient storage, recycling, and reallocation.

Forage crops, which are grown to be utilized by grazing or harvesting as a whole crop, are essential for the successful operation of animal production systems. This fact is more relevant for ruminants which heavily depend upon forages for their health and for a costeffective and sustainable production . While forages are an economical source of nutrients for animal production, they also help conserve the soil integrity, water supply and air quality (Chaudhry, 2008). In the last years, forage species have been widely studied for nonforage purposes—especially for bioenergy. Grasses are a source of lignocellulosic biomass to generate biofuels and they belong to a group of plant species considered as second generation crops. Nowadays, second generation of biofuels have gained relevance since they do not directly compete with human nutrition, unlike first generation of biofuel crops. The incorporation of forage species to the production of bioenergy is expected to expand the amount of biofuel that can be produced sustainably by using biomass of non-food crops such as swithgrass, whole crop maize, miscanthus and cereals that bear little grain, among others (Inderwildi and King, 2009). However, one of the major concerns about these crops is the environmental impact. It is likely that the expansion of crops for bioenergy utilization occurs with greater intensity in natural ecosystems, often characterized by their fragility in soil stability and water content. Global climate change intensifies these challenges as current crops are poorly adapted to more uncertain and extreme climatic conditions. In this context, the study of plant responses to water deficit as a strategy for the optimization in the use of water is of remarkable importance to increase production without further damage to the environment. In this chapter, we presented our contribution to this topic through the study of drought tolerance in *Panicum virgatum*, a member of the Poaceae family intensively studied as a source of lignocellulosic biomass to produce renewable energy. The Poaceae, a family with numerous species important to human nutrition, shares an extensive similarity among its members; hence, the comprehension of the bases of water stress tolerance in *Panicum virgatum* will improve our understanding of the entire group. Providing food and energy in conditions that maintain the sustainability of resources is a challenge that must be addressed. Faced with a global energy crisis and the steadily growing world population, forage crops are a suitable alternative to meet current and future demands of food and energy.

The biological significance of crosstalk between signaling pathways operating under stress conditions as well as the mechanism that underlie this crosstalk are still unclear. At present, these pathways have become better resolved due to the development of new tools that allow for the exploration of the physiological, genetic, and biochemical foundation of such processes. The genomic, proteomic and metabolomic approach is now widely used in model plants and, to a lesser extent, in crop and forage plants. The growing interest in forage crops has promoted its study at the molecular level, making it promising to research the improvement of these species. To date, the complete genome sequences of four grass species

Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 155

expression and disease resistance in *Arabidopsis*. *Plant Cell.* 16: 3460-3479. Andrade A., Vigliocco A., Alemano S., Miersch O. & Abdala G. (2005). Endogenous

Apel K., & Hirt. H. (2004). Reactive oxygen species: metabolism, oxidative stress, and signal

Arimura G., Kost C. & Boland W. (2005). Herbivore-induced, indirect plant defences.

Armengaud P., Breitling R. & Amtmann A. (2004). The potassium-dependent transcriptome

Assmann S.M. (2003). Open stomata opens the door to ABA signalling in *Arabidopsis* guard

Bahrani M. J., Bahrami H. & Haghighi, A.A.K. (2010). Effect of water stress on ten forage

Balbi V. & Devoto A. (2007). Jasmonate signalling network in *Arabidopsis thaliana*: crucial regulatory nodes and new physiological scenarios. *New Phytol*. 177: 301-18. Bandurska H. & Stroiński A. (2005). The effect of salicylic acid on barley response to water

Bandurska H., Stroiński A. & Kubiś J. (2003). The effect of jasmonic acid on the accumulation

Bari R. & Jones J.D.G. (2009). Role of hormones in plant defense responses. *Plant Mol. Biol.*

Barney J.N. , Mann J.J., Kyser G.B., Blumwald E., Van Deynze A. & DiTomaso J.M. (2009)

Barrero J.M., Piqueras .P, Gonzalez-Guzman M., Serrano R., Rodriguez P.L., Ponce M.R. &

Berg L.V.D. & Zeng Y.J. (2006). Response of South African indigenous grass species to drought stress induced by polyethyleneglycol (PEG) 6000. *S Afr J Bot* 72: 284–286. Berger S., Bell E. & Mullet J.E. (1996). Two methyl jasmonate-insensitive mutants show

Billek G. & Schmook F.P. (1977). Zur biosynthese der gentisinaure. *Monatsh. Chem*. 98: 1651-

Blatt M.R. & Armstrong F. (1993). K+ channels of stomatal guard cells: abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH. *Planta.* 191: 330–341. Boyer J.S. & Westgate M.E. (2004). Grain yields with limited water. *J. Exp. Bot.* 55: 2385-2394. Bright J., Desikan R., Hancock J.T., Weir I.S. & Neill S.J. (2006). ABA-induced NO generation

deficit in two barley genotypes. *Acta Physiol. Plant*. 25: 279-285.

of ABA, proline and spermidine and its influence on membrane injury under water

Tolerance of switchgrass to extreme soil moisture stress: Ecological implications.

Micol J.L. (2005). A mutational analysis of the ABA1 gene of Arabidopsis thaliana highlights the involvement of ABA in vegetative development. *J Exp Bot*. 56: 2071–

altered expression of AtVsp in response to methyl jasmonate and wounding. *Plant* 

and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. *Plant J*.

transduction. *Annu. Rev. Plant Biol*. 55:373–399.

grasses native or introduced to Iran. *Grassl. Sci.* 56: 1–5.

*Biochim. Biophys. Acta.* 1734: 91-111.

cells. *Trends Plant Sci.* 5: 151-153.

deficit. *Acta Physiol. Plant.* 27: 379-386.

*Physiol*. 136:2556-2576.

69: 473-488.

2083.

1664.

45: 113–122.

*Plant Sci.* 177: 724-732.

*Physiol.* 111: 525–31.

318.

abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene

jasmonates and octadecanoids during germination and seedling development: their relation with hypersensitive tomato mutants to abiotic stress. *Seed Sci. Res.* 15: 309-

of *Arabidopsis* reveals a prominent role of jasmonic acid in nutrient signaling. *Plant* 

(i.e. maize, sorghum, rice and brachypodium) representing the three most economically important grass subfamilies have been analyzed. In the same line, the first pooid grass, *Brachypodium distachyon* (*Brachypodium*), has recently been sequenced completely and proposed as a new model that can contribute to grass crop improvement (Bevan et al., 2010). This knowledge can be directly applied to accelerate the domestication of wild grasses (e.g. Switchgrass and Miscanthus) that are promising biomass crops. Genomics and functional genomics resources are centrally important for this research as they also directly facilitate biotechnological and genetic improvement through plant breeding. This information along with a system-level approach will significantly increase our knowledge of grass biology in order to understand how biotic and abiotic environments influence crop yield. In the near future, the combination of these new technologies will help to unravel the complex interactions between plant hormones in forage crops.

### **5. References**


(i.e. maize, sorghum, rice and brachypodium) representing the three most economically important grass subfamilies have been analyzed. In the same line, the first pooid grass, *Brachypodium distachyon* (*Brachypodium*), has recently been sequenced completely and proposed as a new model that can contribute to grass crop improvement (Bevan et al., 2010). This knowledge can be directly applied to accelerate the domestication of wild grasses (e.g. Switchgrass and Miscanthus) that are promising biomass crops. Genomics and functional genomics resources are centrally important for this research as they also directly facilitate biotechnological and genetic improvement through plant breeding. This information along with a system-level approach will significantly increase our knowledge of grass biology in order to understand how biotic and abiotic environments influence crop yield. In the near future, the combination of these new technologies will help to unravel the complex

Abe H., Urao T., Ito T., Seki M., Shinozaki K. & Yamaguchi-Shinozaki K. (2003). *Arabidopsis*

Abernethy G.A. & McManus M.T. (1998). Biochemical responses to an imposed water deficit in mature leaf tissue of *Festuca arundinacea*. *Environ. Exp. Bot.* 40, pp. 17–28. Abreu M.E. & Munne-Bosch S. (2008). Salicylic acid may be involved in the regulation of

Agarwal S., Sairam R.K., Srivastava G.C., Tyagi A. & Meena R.C. (2005). Role of ABA,

Agele S.O. (2003). Sunflower responses to weather variations in rainy and dry, cropping

Agrawal G.K., Rakwal R., Jwa N.S., Han K.S. & Agrawal V.P. (2002). Molecular cloning and

Agrawal G.K., Yamazaki M., Kobayashi M., Hirochika R., Miyao A. & Hirochika H. (2001).

Ai L.,Li Z.H., Xie, Z.X., Tian, X.L., Eneji, A.E. & Duan, L.S. (2008). Coronatine alleviates

Alibert G. & Ranjeva R. (1971). Recharches sur les enzymes catalysant la biosyntheses des

Alibert G. & Ranjeva R. (1972). Recharches sur les enzymes catalysant la biosyntheses des

Anderson J.P., Badruzsaufari E., Schenk P.M., Manners J.M., Desmond O.J., Ehlert C.,

AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in

drought-induced leaf senescence in perennials: a case study in field-grown *Salvia* 

salicylic acid, calcium and hydrogen peroxide on antioxidant enzymes induction in

mRNA expression analysis of the first rice jasmonate biosynthetic pathway gene

Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene.

polyethylene glycol-induced water stress in two rice (Oryza sativa L.) cultivars. *J.* 

acid phenoliques chez Quarcus pedunculata (Ehrn): I- formation des series

acid phenoliques chez *Quarcus pedunculata* (Ehrn): II- localization intercelulaire de la phenyalanin mmonique-lyase, de la cinnamate 4-hydroxylase, et de la "benzoote

Maclean D.J., Ebert P.R. & Kazan K. (2004). Antagonistic interaction between

interactions between plant hormones in forage crops.

abscisic acid signaling. *Plant Cell* 15: 63-78.

wheat seedlings. *Plant Sci.* 169: 559-570.

*Plant Physiol.* 125: 1248-1257.

*Agron. Crop. Sci*. 194:360-368.

*officinalis* L. plants*. Environ. Exp. Bot.* 64: 105-112.

seasons in a tropical rainforest zone. *IJOB.* 32: 17-33.

cinnamique et benzoique*. FEBS Lett*. 19: 11-14.

synthase". *Biochem. Biophys*. Acta 279: 282-289.

allene oxide synthase. *Plant Physiol. Biochem.* 40: 771-782.

**5. References** 

abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in *Arabidopsis*. *Plant Cell.* 16: 3460-3479.


Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 157

Finkelstein R., Gampala S. & Rock C. (2002). Abscisic acid signaling in seeds and seedlings.

Fujita M., Fujita Y., Maruyama K., Seki M., Hiratsu K., Ohme-Takagi M., Tran L.S.,

Fujita M., Fujita Y., Noutoshi Y., Takahashi F., Narusaka Y., Yamaguchi-Shinozaki K. &

Gao X.P., Wang X.F., Lu Y.F., Zhang L.Y., Shen Y.Y., Liang Z. & Zhang D.P. (2004).

Ghasempour H.R., Anderson E.M. & Gaff D.F. (2001). Effects of growth substances on the

Guoth A., Tari I., Galle A., Csiszar J., Pecsvaradi A., L. Cseuz & Erdei L. (2009). Comparison

Hamada A.M. & Al-Hakimi A.M.A. (2001). Salicylic acid versus salinity-drought induced

Hamada A.M. (1998). Effects of exogenously added ascorbic acid, thiamin or aspirin on

Han R., Zhang Y., Tian H. & Lu X. (2008). Study on Changes of Endogenous Hormones in the Leaves of Alfalfa under Drought Stress. *Acta Agriculturae Boreali-Sinica.*  Harb A., Krishnan A., Ambavaram M.M. & Pereira A. (2010). Molecular and physiological

Hayat Q., Hayat S., Irfan M. & Ahmad A. (2010). Effect of exogenous salicylic acid under

Hayat S., Fariduddin Q., Ali B. & Ahmad A. (2005) Effect of salicylic acid on growth and

Hayat S., Hasan S.A., Fariduddin Q. & Ahmad A. (2008). Growth of tomato (*Lycopersicon esculentum*) in response to salicylic acid under water stress. *J. Plant Int.* 3: 297-304. Hirayama T. & Shinozaki K. (2007). Perception and transduction of abscisic acid signals:

Horváth E., Szalai G. & Janda T. (2007). Induction of abiotic stress tolerance by salicylic acid

Huang D., Wu W., Abrams S.R. & Cutler A.J. (2008). The relationship of drought-related

Yamaguchi-Shinozaki K. & Shinozaki K. (2004). A dehydrationinduced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway.

Shinozaki K. (2006). Crosstalk between abiotic and biotic stress responses: A current view from the points of convergence in the stress signaling networks. *Curr.* 

Jasmonic acid is involved in the water-stress-induced betaine accumulation in pear

protoplasmic drought tolerance of leave cells of the resurrection grass, *Sporobolus* 

of the drought stress responses of tolerant and sensitive wheat cultivars during grain lling: changes in ag leaf photosynthetic activity, ABA levels, and grain

photosynthesis and some related activities of drought-stressed wheat plants. In: *Photosynthesis: Mechanisms and Effects.* Garab G. (Ed.). Vol. 4, Kluwer Academic

analysis of drought stress in *Arabidopsis* reveals early responses leading to

keys to the function of the versatile plant hormone ABA. *Trends Plant Sci.* 12: 343-

gene expression in Arabidopsis thaliana to hormonal and environmental factors*. J.* 

*Plant Cell.* 14: S15-S45.

*Plant J.* 39: 863-876.

351.

*Opin. Plant Biol.* 9: 436-442.

leaves plant. *Plant Cell Environ.* 27: 497-507.

*stapfianus*. *Aust. J. Plant Physiol*. 28: 1115-1120.

stress on wheat seedlings. *Rostl. Vyr*. 47: 444-450.

Publishers, Dordrecht, pp 2581-2584. ISBN 0-7923-5545-8.

acclimation in plant growth. *Plant Physiol.* 154:1254-71.

signaling. *J. Plant Growth Regul.* 26: 290-300.

*Exp. Bot.* 11: 2991-3007.

changing environment: A review*. Environ. Exp. Bot.* 8: 14-25.

enzyme activities of wheat seedlings. *Acta Agron Hung*. 53:433–437.

yield. J *Plant Growth Regul.* 28:167–176


Browse J. (2009a). Jasmonate passes muster: a receptor and targets for the defense hormone.

Carmona M.I., Carlos T.L., Ramírez V.P., García de los Santos G. & Pérez C.B. (2003).

Chaves M.M., Maroco J.P. & Pereira J.S. (2003). Understanding plant responses to drought-

Chen S., Li J., Wang T., Polle A. & Hüttermann A. (2002). Osmotic stress and ion-specific

Cheng W.-H., Endo A., Zhou L., Penney J., Chen H.-C., Arroyo A. Leon P., Nambara E.,

Cho D., Shin D., Wook Jeon B. & Kwa J. M. (2009). ROS-Mediated ABA Signaling. *J. Plant* 

Chow B. & McCourt P. (2004). Hormone signalling from a developmental context. *J. Exp.* 

Christmann A., Hoffmann T., Teplova I., Grill E. & Müller A. (2005). Generation of active

Cutler A.J. & Krochko J.E. (1999). Formation and breakdown of ABA. *Trends Plant Sci.* 4: 472-

DaCosta M. & Huang B. (2009). Physiological adaptations of perennial grasses to drought

Dass S., Arora P., Kumari M. & Pal D. (2001). Morphological traits determining drought.

De Smet I., Zhang H., Inze D. & Beeckman T. (2006). A novel role for abscisic acid emerges

Desikan R., Cheung M.K., Bright J., Henson D., Hancock J.T. & Neill S.J. (2004). ABA

Devoto A. & Turner J.G. (2003). Regulation of Jasmonate mediated plant responses in

Dobra J., Motyka V., Dobrev P., Malbeck J., Prasil I.T., Haisel D., Gaudinova A., Havlova M.,

El-Far I.A. & A.Y. Allan. (1995). Responses of some wheat cultivars to sowing methods and drought at different stages of growth. *Assuit J. Agric. Sci.* 26: 267–277. Fariduddin Q., Hayat S. & Ahmad A. (2003). Salicylic acid influences net photosynthetic

tolerance in maize (*Zea mays l.*) Indian. *J. Agric. Res.* 35 : 190 – 193.

from underground. *Trends Plant Sci.* 11: 434–439.

*Arabidopsis*. *Ann. Bot.* 92: 329-337.

*juncea. Photosynthetica.* 41: 281-284.

*Physiol.* 167: 1360-1370.

from genes to the whole plants. *Funct. Plant Biol* 30: 239-264.

biosynthesis and functions. *Plant Cell.* 14: 2723-2743.

Drought resistance of *Brachiaria* spp. i. physiological aspects. *Rev. Fitotec. Mex*. 26:

affects on xylem abscisic acid and the relevance to salinity tolerance in poplar*. J.* 

Asami T., Seo M., Koshiba T. & Sheen J. (2002). A unique short-chain dehydrogenase/reductase in *Arabidopsis* glucose signaling and abscisic acid

pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis.

stress. In: *Perspectives in biophysical plant ecophysiology.* E.D. Barrera and W.K. Smith, Editors, Universidad Nacional Autónoma de México, México. 169–190. ISBN 987-0-

hydrogen peroxide and nitric oxide signaling in stomatal guard cells*. J. Exp. Bot.* 55:

Gubis J. & Vankova R. (2010). Comparison of hormonal response to heat, drought and combined stress in tobacco plants with elevated proline content. *J. Plant* 

rate, carboxylation efficiency, nitrate reductase activity and seed yield in *Brassica* 

*Annu. Rev. Plant Biol.* 60: 183-205.

*Plant Growth Regul.* 21: 224-233.

153-159.

*Biol*. 52:102–113.

*Bot.* 55:247–51.

578-00421-1.

205-212.

478.

*Plant Physiol.* 137: 209–219.


Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 159

Lehmann J., Atzorn R., Brückner C., Reinbothe S., Leopold J., Wasternack C. & Parthier B.

Li L., Van Staden J. & Jager A.K. (1998). Effects of plant growth regulators on the antioxidant

Liang Y., Mitchell D.M. & Harris J.M. (2007). Abscisic acid rescues the root meristem defects

Liu N.Y., Ko S.S., Yeh K.C. & Charng Y.Y. (2006). Isolation and characterization of tomato

Liu X.G., Yue Y.L., Li B., Nie Y.L., Li W., Wu W.H. & Ma L.G. (2007). A G protein-coupled

Lu S., Su W., Li H. & Guo Z. (2009). Abscisic acid improves drought tolerance of triploid

Lu S., Su W., Li H. & Guo Z. (2009). Abscisic acid improves drought tolerance of triploid

McAinsh M.R., Webb A.A.R, Taylor J.E. & Hetherington A.M. (1995). Stimulusinduced oscillations in guard cell cytosolic free calcium. *Plant Cell.* 7: 1207–1219. Medrano H., Escalona J.M., Bota J., Gulías J. & Flexas J. (2002). Regulation of photosynthesis

Miyashita K., Tanakamaru S., Maitani T. & Kimura K. (2005). Recovery responses of

Molina A., Bueno P., Marín M.C., Rodríguez-Rosales M.P., Belver A. & Venema K. (2002).

Moons A., Prinsen E., Bauw G. & Van Montagu. (1997). Antagonistic effects of abscisic acid

Munne-Bosch S. &. Peñuelas J. (2003). Photo- and antioxidative protection, and a role for

Mustafa N.R., Kim H.K., Choi Y.H., Erkelens C., Lefeber A.W.M., Spijksma G., Van der

Nambara E. & Marion-Poll A. (2005). Abscisic acid biosynthesis and catabolism. *Annu. Rev.* 

Narusaka Y., Narusaka M., Seki M., Umezawa T., Ishida J.,Nakajima M., Enju A. &

in osmotically stressed barley leaf segments. *Planta.* 197: 156-162.

of the *Medicago truncatula* latd mutant. *Dev Biol*. 304: 297–307. Liechti R. & Farmer E.E. (2006). Jasmonate biochemical pathway. *Sci. STKE* 322: 1-3.

Hsa32 encoding a novel heat-shock protein. *Plant Sci.* 170: 976–985.

*Regul*. 25:81-87.

*Science.* 315: 1712–1716.

*Plant Physiol. Biochem.* 47

*Phytol.* 156: 409–415.

plants. *Planta*. 217: 758–766.

*Plant Physiol. Mol. Biol.* 56: 165-185.

59.

*Plant Physiol. Biochem:* 47: 132–138.

reference parameter. *Ann Bot.* 89: 895-905.

drought stress*. Environ. Exper. Bot*. 53: 205-214.

Catharanthus roseus cells. *Phytochem.* 70: 532-539.

(1995). Accumulation of Jasmonate, abscisic acid, specific transcripts and proteins

system in seedlings of two maize cultivars subjected to water stress. *Plant Growth* 

receptor is a plasma membrane receptor for the plant hormone abscisic acid.

bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activities.

bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activities,

of C3 plants in response to progressive drought: stomatal conductance as a

photosynthesis, transpiration, and stomatal conductance in kidney bean following

Involvement of endogenous salicylic acid content, lipoxygenase and antioxidant enzyme activities in the response of tomato cell suspension cultures to NaCl. *New* 

and jasmonates on salt stress-inducible transcripts in rice roots. *Plant Cell.* 9: 2243-

salicylic acid during drought and recovery in field-grown *Phillyrea angustifolia*

Heijden R. & Verpoorte R. (2009). Biosynthesis of salicylic acid in fungus elicited

Shinozaki K. (2004). Crosstalk in the responses to abiotic and biotic stresses in


Hussain M., Malik M. A., Farooq M., Ashraf M. Y. & Cheema M. A. (2008). Improving

Iqbal S. & Bano A. (2010). Effect of Drought and Abscisic Acid Application on the Osmotic

Iuchi S., Kobayshi M., Taji T., Naramoto M., Seki M., Kato T., Tabata S., Kakubari Y.,

Janda T., Szalai G., Tari I. & Páldi E. (1999). Hydroponic treatment with salicylic acid

Jiang M. & Zhang J. 2002. Water stress-induced abscisic acid accumulation triggers the

Jiang Q.L., Rong X.V., Tang H. & Xu R. (1995). A study on drought tolerance in cool season

Kadioglu A., Saruhan N., Saglam A.,Terzi R. & Tuba A. (2010). Exogenous salicylic acid

Kannangara T., Seetharama N., Durley R.C. & Simpson G.M. (1983). Drought resistance of

Khodary S.F.A. (2004). Effect of salicylic acid on the growth, photosynthesis and carbohydrate metabolism in salt stressed maize plants. *Int. J. Agric. Biol.* 6: 5-8. Kim T.H., Böhmer M., Hu H., Nishimura N. & Schroeder J.I. (2010). Guard cell signal

Koch T., Bandemer K. & Boland W. (1997). Biosynthesis of *cis*-Jasmone: a pathway for the

Kramell R., Miersch O., Atzorn R., Parthier B. & Wasternack C. (2000). Octadecanoid-

Kwak J.M., Nguyen V. & Schroeder J.I. (2006). The role of reactive oxygen species in

across an irrigation gradient. *Canadian Journal of Plant Science.* 63:147-15 Karaata H., (1991). Water-production functions of sunflower under Krklareli conditions, No. 28. Journal of Atatürk Village Affair Research Institute, Krklareli, 92 pp. Kazan K. & Manners J.M. (2008). Jasmonate signaling: toward an integrated view. *Plant* 

Adjustment of Four Wheat Cultivars. *J. Chem. Soc. Pak.* 32:13-19.

antioxidant enzymes in maize leaves. *J. Exp Bot*. 53: 2401-2410.

turfgrasses. *Nigeria J Agric Forest Sci Te*chnol. 1: 17–20.

antioxidant system. *Plant Growth Regul.* 64: 27-37.

signaling. *Annu. Rev. Plant Biol.* 61: 561–591.

hormonal responses. *Plant Physiol.* 141:323-9.

phase*?. Helv. Chim. 80: 838–850.* 

23: 1647-1656.

in Sunflower*. J. Agron. Crop. Sc.* 194: 193–199.

Arabidopsis. *Plant J.* 27: 325-333.

*Physiol*. 146: 1459-1468.

180.

Drought Tolerance by Exogenous Application of Glycinebetaine and Salicylic Acid

Yamaguchi-Shinozaki K. & Shinozaki K. (2001). Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid, a key in abscisic acid biosynthesis in

decreases the effects of chilling injury in maize (*Zea mays L*.) plants. *Planta* 208:175–

increased generation of reactive species and up-regulates the activities of

alleviates effects of long term drought stress and delays leaf rolling by inducing

Sorghum bicolor: 6. Changes in endogenous growth regulators of plants grown

transduction network: Advances in understanding abscisic acid, CO2, and Ca2+

inactivation and the disposal of the plant stress hormone jasmonic acid to the gas

derived alteration of gene expression and the "oxylipin signature" in stressed barley leaves. Implications for different signaling pathways. *Plant Physiol*. 123: 177-187. Kushiro T., Okamoto M., Nakabayashi K., Yamagishi K., Kimatura S., Asami T., Hirai N.,

Koshiba T., Kamiya Y. & Nambara E. (2004). The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism. *EMBO* J.


Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 161

Ribnicky D.M., Shulaev V. & Raskin I. (1998). Intermediates of salicylic acid biosynthesis in

Rivas-San Vicente M. & Plasencia J. (2011). Salicylic acid beyond defence: its role in plant

Roche J., Hewezi T., Bouniols A. & Gentzbittel L. (2009). Real-time PCR monitoring of signal

Saini H.S. & Westgate M.E. (2000). Reproductive development in grain crops during

Sánchez-Díaz M., Tapia C. & Antolín M.C. (2008). Abscisic acid and drought response of

Sarath G., Hou G., Baird L.M. & Mitchell R.B. (2007). ABA, ROS and NO are key players

Schachtman D.P. & Goodger J.Q. (2008). Chemical root to shoot signaling under drought.

Schwartz S.H., Qin X. & Zeevaart J.A.D. (2003). Elucidation of the indirect pathway of

Schwartz S.H., Tan B.C., Gage D.A., Zeevaart J.A.D. & McCarty D.R. (1997). Specific oxidative cleavage of carotenoids by Vp14 of maize. *Science* 276: 1872-1874. Seki M., Narusaka M., Ishida J., Nanjo T., Fujita M., Oono Y., Kamiya A., Nakajima M., Enju

Seki M., Umezawa T., Urano K. & Shinozaki K. (2007). Regulatory metabolic networks in

Senaratna T., Touchell D., Bunn E. & Dixon K. (2000). Acetyl salicylic acid (aspirin) and

Seo H.S., Song J.T., Cheong J.J., Lee Y.-H., Lee Y.-W., Hwang I., Lee J.S. & Choi Y.D. (2001)

Seo M., Peeters A.J.M., Koiwai H., Oritani T., Marion-Poll A., Zeevart J.A.D., Koorneef M.,

Shah J. (2003). The salicylic acid loop in plant defense. *Curr. Opin. Plant. Biol*. 6: 365-371. Shakirova F.M. (2007). Role of hormonal system in the manifestation of growth promoting

evidence for an interaction with ethylene*. J. Exp. Bot.* 51: 1575-1584.

during switchgrass seed germination. *Plant Signal Behav.* 2: 492-493.

using a full-length cDNA microarray. *Plant J* 31:279–292.

plant responses. *Proc. Natl. Acad. Sci. USA.* 98:4788-93.

drought stress responses. *Curr. Opin. Plant Biol.* 10: 296-302

transduction related genes involved in water stress tolerance mechanism of

Canarian laurel forest tree species growing under controlled conditions. *Environ.* 

abscisic acid biosynthesis by mutants, genes, and enzymes. *Plant Physiol*. 131: 1591-

A., Sakurai T., Satou M., Akiyama K., Taji T. , Yamaguchi-Shinozaki K., Carninci P., Kawai J., Hayashizaki Y. & Shinozaki K. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold, and high-salinity stresses

salicylic acid induce multiples tress tolerance in bean and tomato plants. *Plant* 

Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated

Kamiya Y. & Koshiba T. (2000). The *Arabidopsis* aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves. *Proc. Natl.* 

and anti-stress action of salicylic acid. In: *Salicylic Acid. A Plant Hormone* (Eds.) Hayat S., Ahman A. Springer. ISBN 978-1-4020-5183-8. Dordrecht, Netherlands. Sharp R.E., LeNoble M.E., Else M.A., Thorne E.T. & Gherardi F. (2000). Endogenous ABA

maintains shoot growth in tomato independently of affects on plant water balance

growth and development. *J. Exp Bot.* doi: 10.1093/jxb/err031.

tobacco. *Plant Physiol.* 118: 565-572.

drought. *Adv. Agron.* 68: 59-96.

*Trends Plant Sci.* 13: 281–287.

*Growth Regul.* 30: 157-161.

*Acad. Sci. USA* 97: 12908-12913.

*Exp. Bot.* 64: 155-161.

1601.

sunflower. *Plant Physiol. Biochem.* 47: 139-145.

*Arabidopsis:* analysis of gene expression in cytochrome P450 gene superfamily by cDNA microarray. *Plant Mol. Biol.* 55: 327-342.


Nayyar H. & Gupta D. (2006). Differential sensitivity of C3 and C4 plants to water deficit

Nemeth M., Janda T., Horvath E. , Paldi E. & Szalai G. (2002). Exogenous salicylic acid

Norastehnia A. & Asghari M.N. (2006). Effects of methyl jasmonate on the enzymatic

North H.M., Almeida A.D., Boutin J.P., Frey A., To A., Botran L., Sotta B. & Marion-Poll A.

Oritani T. & Kiyota H. (2003). Biosynthesis and metabolism of abscisic acid and related

Pedranzani H., Sierra-de-Grado R., Vigliocco A., Otto Miersch O. & Guillermina Abdala G.

Pedranzani H., Racagni G., Alemano S., Miersch O., Ramírez I., Peña Cortés H., Machado-

Pennypacker B.W., Leath K.T., Stout W.L. & Hill R.R.Jr (1990). Technique for simulating

Perales L., Arbona B., Gómez-Cadenas A., Cornejo M.J. & Sanz A. (2005). A relationship

Pieterse C.M.J., Leon-Reyes A., Van der Ent S. & Van Wees S.C.M. (2009). Networking by small-molecule hormones in plant immunity. *Nat. Chem. Biol*. 5: 308–316. Popova L., Pancheva T. & Uzunova A. (1997). Salicylic acid: properties, biosynthesis and

Qin X.Q. & Zeevaart J.A.D. (1999). The 9-cis-epoxicarotenoid cleavage reaction is the key

Rao M.V. & Davis R.D. (1999). Ozone-induced cell death occurs via two distinct mechanism

Rassaa N., Ben Haj Salah H., Latiri,K. (2008). Thermal responses of Durum wheat Triticum

Raziuddin J., Bakht Swati Z.A., Shafi M., Farhat U. & Akmal M. (2010). Effect of cultivars

Reddy A.R., Chaitanya K.V. & Vivekanandan M. (2004). Drought-induced responses of

populations of *Pinus pinaster Ait*. *Plant Growth Regul*. 52: 111-112.

field drought stress in the greenhouse. *Agron J.* 82: 951–957.

cDNA microarray. *Plant Mol. Biol.* 55: 327-342.

compounds. *Nat. Prod. Rep.* 20: 414–425.

stress. *Plant Physiol. Biochem*. 43: 786-792.

*Sci.* USA 96: 15354-15361.

*Biologies* 331: 363–371.

1189–1202.

of jasmonic acid. *Plant Growth Regul*. 41: 149-158.

physiological role. Bulg. J. *Plant Physiol*. 23: 85-93.

in *Arabidopsis*: the role of salicylic acid. *Plant J.* 17: 603-614.

embryos of wheat (*Triticum aestivum L*). *Pak. J. Bot*. 42: 639-652.

113.

*Sci.* 162: 569-574.

*Sci*. 5: 17-23.

810-824.

*Arabidopsis:* analysis of gene expression in cytochrome P450 gene superfamily by

stress: association with oxidative stress and antioxidants. *Environ. Exp. Bot*. 58. 106–

increases polyamine content but may decrease drought tolerance in maize. *Plant* 

antioxidant defense system in maize seedlings subjected to Paraquat. *Asian J. Plant* 

(2007). The *Arabidopsis* ABA-deficient mutant *aba4* demonstrates that the major 32 route for stress-induced ABA accumulation is via neoxanthin isomers. *Plant J*. 50:

(2007). Cold and water stresses produce changes in endogenous jasmonates in two

Domenech E. & Abdala G. (2003). Salt tolerant tomato plants show increased levels

between tolerance to dehydration of rice lines and ability for ABA synthesis under

regulatory step of abscisic acid biosynthesis in water stress bean. *Proc. Natl. Acad.* 

durum to early water stress. Consequence on leaf and flower development. *C. R.* 

and culture medium on callus formation and plant regeneration from mature

photosynthesis and antioxidant metabolism in higher plants. *J. Plant Physiol.* 161:


Drought Tolerance and Stress Hormones: From Model Organisms to Forage Crops 163

Veselov, D.S., Sharipova, G.V., Veselov, S.U., Kudoyarova, G.R., 2008. The effects of NaCl

Vick B.A. & Zimmerman D.C. (1983). The biosynthesis of jasmonic acid: a physiological role

Wang C., Yang A., Yin H. & Zhang J. (2008). Inuence of water stress on endogenous

Wang S Y. (1999). Methyl jasmonate reduces water stress in strawberry. *Plant Growth Regul.*

Wasilewska A., Vlad F., Sirichandra C., Redko Y., Jammes F., Valon C., Frei dit Frey N. &

Wasternack C. & Hause B. (2002). Jasmonates and octadecanoids: signals in plant stress responses and development. *Prog. Nucleic Acid Res. Mol. Biol.* 72: 165-221. Wasternack C. & Kombrinck E. (2010). Jasmonates: Structural requirements for lipid-derived

Wasternack C. (2007). Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. *Ann. Bot.* 100: 681-697. Wildermuth M.C. (2006). Variations on a theme: synthesis and modifications of plant

Xie D.X., Feys B.F., James S., Nieto-Rostro M. & Turner J.G. (1998). COI1: an Arabidopsis

Xin Z.Y., Zhou X. & Pilet P.E. (1997). Level changes of jasmonic, abscisic and indole-3ylacetic acids in maize under desiccation stress. *J. Plant Physiol*. 151: 120-124. Xiong L. & Yang Y. (2003). Disease resistance and abiotic stress tolerance in rice are

Xiong L., Shumaker K.S. & Zhu J.K. (2002). Cell signaling during cold, drought and salt

Yalpani N., Leen J., Lawthon M.A. & Raskin I. (1993). Pathway of salicylic acid biosynthesis

Yalpani N., Schulz M., Davies M.P. & Balke N.E. (1992). Partial purification of an inducible

Yamaguchi-Shinozaki K. & Shinozaki K. (2005). Organization of cis-acting regulatory

Yasuda M., Ishikawa A., Jikumaru Y., Seki M., Umezawa T., Asami T., Maruyama-

acid-mediated abiotic stress response in *Arabidopsis. Plant Cell.* 20:1678-92. Yordanov I., Velikova V. & TsoneV. (2000). Plant response to drought, acclimatation and

in healthy and virus-inoculated tobacco. *Plant Physiol.* 103: 315-321.

for plant lipoxygenase. *Biochem. Biophys. Res. Commun*. 111: 470-77.

in drought tolerance. *J Plant Growth Regul.* 27, 380–386.

benzoic acids. *Curr. Opin. Plant Biol.* 9: 288-296.

kinase. *Plant Cell*. 15: 745-759.

stress*. Plant Cell*. 14: S165-S183.

*Plant Physiol.* 100: 457-463.

stree tolerance. *Photosynthetica.* 30: 171-186.

94.

*Biology*. 50: 427 –434

18: 127-134.

1: 198-217.

63-77.

treatment on water relations, growth, and ABA content in barley cultivars differing

hormone contents and cell damage of maize seedlings. *Journal of Integrative Plant* 

Leung J. (2008). An update on abscisic acid signaling in plants and more. *Mol. Plant.*

signals active in plant stress responses and development. *A.C.S. Chemical Biology.* 5:

gene required for jasmonate-regulated defense and fertility. *Science.* 280: 1091–1094.

inversely modulated by an abscisic acid-inducible mitogen-activated protein

uridine-5'.diphosphate glucose: salicylic acid glucosyltransferase from oat roots.

elements in osmotic- and cold-stress-responsive promoters. *Trends Plant Sci.* 10: 88-

Nakashita A., Kudo T., Shinozaki K., Yoshida S., & Nakashita H. (2008). Antagonistic interaction between systemic acquired resistance and the abscisic


Shi Q. & Zhu Z. (2008). Effects of exogenous salicylic acid on manganese toxicity, element contents and antioxidative system in cucumber. *Environ. Exper. Bot.* 63: 317-326. Shirazi M.U., Gyamfi J.A., Ram T., Bachiri H., Rasyid B. , Rehman A., Khan M.A., Mujtaba

Singh B. & Usha K. (2003). Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress*. Plant Growth Regul*. 39: 137-141. Smirnoff N. (1993). The role of active oxygen in the response of plants to water deficit and

Spollen W.G., LeNoble M.E., Sammuels T.D., Bernstein N. & Sharp R.E. (2000). Abscisisc

Suhita D., Raghavendra A.S., Kwak J.M. & Vavasseur A. (2004). Cytoplasmic alkalization

Szalai G., Horgosi S., Soós V., Majláth I., Balázs E., Janda T. (2010). Salicylic acid treatment of pea seeds induces its de novo synthesis*. J. Plant Physiol.* 168: 213-219. Thameur A., Ferchichi A. & López-Carbonell M. (2011). Quantification of free and

Thameur A., Ferchichi A. & López-Carbonell M. (2010). Quantification of free and

Theodoulou F.L., Job K., Slocombe S.P., Footitt S., Holdsworth M., Baker A., Larson T.R. &

Thines B., Katsir L., Melotto M., Niu Y., Mandaokar A., Liu G., Nomura K., He S.Y., Howe

Torres M.A., Jones J.D. & Dangl J.L. (2005). Pathogen-induced, NADPH oxidase-derived

Turhan H. & Baser I. (2004). In vitro and In vivo water stress in sunflower (*Helianthus annuus* 

Turner J.G., Ellis Ch. & Devoto A. (2002). The jasmonate signal pathway. *Plant Cell.* 14: 153-

Veselov D.S., Sharipova G.V., Veselov S.U. & Kudoyarova G.R. (2008). The effects of NaCl

acid-induced stomatal closure. *Plant Physiol.* 134: 1536-45.

water stress conditions. *S. Afr. J. Bot.* 77: 222-228.

water stress conditions. *S Afr J Bot*. 77: 222–228

into peroxisomes. *Plant Physiol.* 137: 835-840.

*Nat. Genet.* 37: 1130–1134.

*L.*). *Helia* 27: 227-236.

164.

complex during jasmonate signalling. *Nature.* 448: 661-665.

in drought tolerance. *J. Plant Growth Regul.* 27: 380-386.

potentials by restricting ethylene production. *Plant Physiol.* 122: 967-976. Staswick P.E., Su W. & Howell S.H. (1992). Methyl jasmonate inhibition of root growth and

*J. Bot*. 42: 3639-3644.

desiccation. *New Phytol*. 125: 27–58.

*Natl. Acad. Sci. U S A*. 89: 6837-40.

S.M., Ali M., Shreen Aisha & Mumtaz S. (2010). Selection of some suitable drought tolerant wheat genotypes using carbon isotopes discrimination (cid) technique. *Pak.* 

acid accumulation maintains maize primary roots elongation at low water

induction of a leaf protein are decreased in an *Arabidopsis thaliana* mutant. *Proc.* 

precedes reactive oxygen species production during methyl jasmonate- and abscisic

conjugated abscisic acid in five genotypes of barley (*Hordeum vulgare L.*) under

conjugated abscisic acid in five genotypes of barley (*Hordeum vulgare L*.) under

Graham I.A. (2005). Jasmonic acid levels are reduced in COMATOSE ATP-binding cassette transporter mutants. Implications for transport of jasmonate precursors

G.A. & Browse J. (2007). JAZ repressor proteins are targets of the SCF (COI1)

reactive oxygen intermediates suppress spread of cell death in *Arabidopsis thaliana.*

treatment on water relations, growth and ABA content in barley cultivars differing


**1. Introduction** 

have been grafted on.

**2. Iron chlorosis in plants** 

Agustí, 2003).

**7** 

*Spain* 

**Iron Stress in Citrus** 

Maria Angeles Forner-Giner and Gema Ancillo *Instituto Valenciano de Investigaciones Agrarias (IVIA)* 

Iron is an essential element for plant growth and development, since it is fundamental for the proper functioning of numerous metabolic and enzymatic processes. Calcareous soils with restricted iron availability for plants are commonly found in the Mediterranean basin where citrus are the major fruit crop. The genus *Citrus* and related rootstocks species are considered to be susceptible to iron chlorosis. Iron deficiency tolerance is determined by the rootstock so citrus trees display differences in susceptibility according to the rootstock they

Field trials have been carried out to select chlorosis-tolerant genotypes. Evaluation of growth and yield parameters may not be sufficient to rank citrus rootstocks according to their tolerance to iron chlorosis, and in addition, field trials take a long time to obtain results. Greenhouse screening tests are easier to implement than field trials in order to evaluate the tolerance. Several physiological parameters are measured in order to test the tolerance to iron deficiency. Nevertheless new screening techniques are needed to identify

The term "essential mineral nutrient" was initially proposed by Arnon & Stout (1939) and applies to all elements necessary for to develop life and reproduction of plants. A plant can complete its life cycle if it is supplied in sufficient quantity and every one of the minerals that are essential. There are two aspects to consider a nutrient as essential. The first one is that the requirement on the element must be specified and can not be replaced by another, and the second one is that the elements must have a direct influence on the metabolism of plants. The Fe is involved in important processes as photosynthesis, respiration, metabolism of proteins, fixation and nitrogen assimilation and nitrate reduction (Romera & Guardia, 1991). It is also a cofactor for enzymes (such as cytochrome oxidase, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) that catalyze biochemical reactions only, making it essential role in the growth and development of plants (Abbey, 1992; Larbi et al. 2006; Molassiotis et al. 2006;

Of the two oxidation states that iron may occur in the soil: ferric (Fe3 +) and ferrous (Fe2 +), it is accepted that the plant takes preferably the latter, for this plant is forced to reduce the predominant form of iron in aerobic soils (Fe3 +). This process is performed by a reductase enzyme located in the plasma membrane of the root (Bienfait 1985; Römheld 1987) . This enzyme reaches its maximum activity at pH between 4 and 5 (Schmidt & Bartels 1997). On

chlorosis-tolerant genotypes which can be used in breeding programs.


### **Iron Stress in Citrus**

Maria Angeles Forner-Giner and Gema Ancillo *Instituto Valenciano de Investigaciones Agrarias (IVIA) Spain* 

### **1. Introduction**

164 Plants and Environment

Yuan S. & Lin H.H. (2008). Role of salicylic acid in plant abiotic stress. *Z. Naturforsch C.*

Zaharia L.I., Walker-Simmon M., Rodríguez C. & Abrams S. (2005). Chemistry of abscisic acid, abscisic acid catabolites and analogs. *Plant Growth Regul.* 24: 274-284. Zeevaart J.A.D. (1999). Abscisic acid metabolism and its regulation. In: *Biochemistry and* 

Zhang J., Jia W., Yang J. & Ismail A.M. (2006). Role of ABA in integrating plant responses to

Zhou Z.S., Guo K., Elbaz A.A. & Yang Z.M. (2009). Salicylic acid alleviates mercury toxicity

drought and salt stress*. Field Crop Res.* 97: 111-119.

*Molecular Biology of Plant Hormones.* (Eds.), Hooykaas P.J.J., Hall M.A., Libbenga K.R. Elsevier Science. ISBN 10: 0-444-89825-5Amsterdam, The Netherlands, pp.

by preventing oxidative stress in roots of *Medicago sativa. Environ. Exp. Bot*. 65: 27-

63:313-20.

189–207.

34.

Iron is an essential element for plant growth and development, since it is fundamental for the proper functioning of numerous metabolic and enzymatic processes. Calcareous soils with restricted iron availability for plants are commonly found in the Mediterranean basin where citrus are the major fruit crop. The genus *Citrus* and related rootstocks species are considered to be susceptible to iron chlorosis. Iron deficiency tolerance is determined by the rootstock so citrus trees display differences in susceptibility according to the rootstock they have been grafted on.

Field trials have been carried out to select chlorosis-tolerant genotypes. Evaluation of growth and yield parameters may not be sufficient to rank citrus rootstocks according to their tolerance to iron chlorosis, and in addition, field trials take a long time to obtain results. Greenhouse screening tests are easier to implement than field trials in order to evaluate the tolerance. Several physiological parameters are measured in order to test the tolerance to iron deficiency. Nevertheless new screening techniques are needed to identify chlorosis-tolerant genotypes which can be used in breeding programs.

### **2. Iron chlorosis in plants**

The term "essential mineral nutrient" was initially proposed by Arnon & Stout (1939) and applies to all elements necessary for to develop life and reproduction of plants. A plant can complete its life cycle if it is supplied in sufficient quantity and every one of the minerals that are essential. There are two aspects to consider a nutrient as essential. The first one is that the requirement on the element must be specified and can not be replaced by another, and the second one is that the elements must have a direct influence on the metabolism of plants.

The Fe is involved in important processes as photosynthesis, respiration, metabolism of proteins, fixation and nitrogen assimilation and nitrate reduction (Romera & Guardia, 1991). It is also a cofactor for enzymes (such as cytochrome oxidase, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) that catalyze biochemical reactions only, making it essential role in the growth and development of plants (Abbey, 1992; Larbi et al. 2006; Molassiotis et al. 2006; Agustí, 2003).

Of the two oxidation states that iron may occur in the soil: ferric (Fe3 +) and ferrous (Fe2 +), it is accepted that the plant takes preferably the latter, for this plant is forced to reduce the predominant form of iron in aerobic soils (Fe3 +). This process is performed by a reductase enzyme located in the plasma membrane of the root (Bienfait 1985; Römheld 1987) . This enzyme reaches its maximum activity at pH between 4 and 5 (Schmidt & Bartels 1997). On

Iron Stress in Citrus 167

The reduction of the thylakoid membrane is accompanied by decreased concentrations of photosynthetic pigments (chlorophylls a and b and carotenoids) in the leaves of affected plants (Morales et al., 1990, 1994). The loss of leaf pigments sheet does not imply that diminish their ability to capture light energy (Terry & Zayed, 1995) due in part to the increase in the relationship leaf carotene / chlorophyll concentrations decline with leaf carotene deficiency Fe to a lesser degree than the chlorophylls (Terry, 1980, Morales et al., 1990, 1994, 2000). The characteristic yellow chlorotic leaves is a consequence of the imbalance between the contents of chlorophyll and carotenoids (Abbey, 1992, Terry &

Iron is also involved in chlorophyll synthesis (Miller et al., 1984) in different crops subject to conditions of deficiency, has shown an increase in the ratio of chlorophyll a: chlorophyll b (Abbey et al. 1989; Monge et al., 1987, Nishio et al., 1985). One explanation for this increase is that under conditions of iron deficiency in the field and there is full sunlight preferential photodestruction of chlorophyll b (Díez-Altar, 1959). Fe deficiency also increases the activity of chlorophyllase, which is involved in the degradation of chlorophyll in citrus fruits

Finally, iron is a constituent of many electron transporters (Terry & Abadía, 1986; Abbey, 1992, Terry & Zayed, 1995; Soldatini et al. 2000), so that iron deficiency is also reduced photosynthetic electron transport. These facts lead to a reduction in photosynthetic capacity of the plant that results in decreased levels of sugars, starch, certain amino acids and accumulation of others, thus altering the spectrum of proteins (Terry & Abadía, 1986; Abbey, 1992, Terry & Zayed, 1995) and enrichment in unsaturated lipids (Terry & Abadía, 1986; Abbey, 1992, Terry & Zayed, 1995), which alters the physiological functioning of the plant. The reduction in plant growth may be related to decreased photosynthetic capacity of chlorotic leaves. Both vegetative growth and production decrease with iron chlorosis (Hurley et al., 1986). The low capacity of the plant to translocate iron from old leaves, is manifested by the yellowing of young leaves , except their nerves remain green. These outbreaks are becoming less vigorous and its leaves, small, can fall prematurely, starting with the most apical (Agustí, 2003). One can also induce severe chlorosis reduced stem growth by inhibiting the formation of new leaves (Loue, 1993). Reducing the number and final size of the fruit, as well as total soluble solids content of the juice are also consequences

Many agricultural crops in arid and semiarid regions suffer chlorosis. Among the plants most affected are the citrus, planted in chalky soils often show signs of severe iron deficiency (Wallace, 1986). Iron chlorosis affects many biochemical, morphological and physiological parameters, and therefore their growth and development of plants (Larbi et

Iron deficiency in citrus tends to be observed during the months of winter and spring. And internervial yellowing of the leaves of young shoots are manifested, due to the inability of the plant to translocate iron from old leaves. The loss of pigmentation is caused by decreased chlorophyll content in chloroplasts (Marschner, 1995). This negatively affects the rate of photosynthesis and, therefore, the development of biomass (Abbey et al., 2004). The young shoots are becoming less vigorous and its leaves, small, can fall prematurely, starting

Zayed, 1995).

(Fernandez-Lopez et al., 1991).

that result from a deficiency of Fe (Agustí, 2003).

**3. Effects of iron chlorosis in citrus** 

al., 2006, Molassiotis et al., 2006, Agustí, 2003).

with the most apical (Agustí, 2003).

the other hand, extreme temperatures (significantly above or below 25 º C), pH values greater than 7.5 and the presence of heavy metals affects their activity (Lucena 2000).

Fe is involved in such important processes as photosynthesis, respiration, metabolism of proteins, fixation and nitrogen assimilation and nitrate reduction (Romera & Guard 1991). It is also a cofactor for enzymes (such as cytochrome oxidase, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) that catalyze biochemical reactions only, making it essential role in the growth and development of plants (Abbey 1992; Larbi et al. 2006; Molassiotis et al. 2006; Agustí 2003).

Chlorosis means lack of chlorophyll in a plant organ, resulting in a loss of green color. Chlorosis can be caused by both the supply deficit of essential elements for plant growth (Fe, manganese (Mn), magnesium (Mg), zinc (Zn), nitrogen (N), etc), water stress or pests, fungi, bacteria or viruses. Iron deficiency, also called iron chlorosis, is one of the most important abiotic stresses. The causes of iron deficiency are numerous and vary, highlighting the availability of iron and bicarbonate ion concentration in the middle of development and other factors. Iron deficiency is usually caused by an insufficient concentration of it in the soil, but the existence of several factors makes affect the solubility and mobility of the Fe. These factors may be a high pH insolubilice some compounds, there limestone and other components, redox potential, interaction between Fe and other nutrients, moisture, organic amendment, salinity, extreme temperatures, etc. (Tagliavini & Rombola 2001). The low availability of iron is increased in soils with the presence of high bicarbonate levels that make the pH is around 8 (Marchner & Römheld 1995), this pH the concentration of Fe quite low and therefore difficult to iron nutrition of the plant (Lucena et al. 2006a). The problem of iron chlorosis is widespread because the limestone soils occupy approximately 30% of the land surface (Chen et al. 1982). It is estimated that between 20 and 50% of fruit growing in Mediterranean areas deficient in iron (Jaeger et al. 2000).

Some higher plants, in the absence of Fe have developed a number of mechanisms to increase the availability of Fe in the soil solution. Strategy I plants increase the Fe-making by the excretion of reducing substances of low molecular weight, through the extrusion of protons by roots acidifying the rhizosphere, solubilizing nutrients not available in an alkaline medium (Jones 2000) and increasing the activity of a reductase associated with the plasma membrane of the root responsible for the reduction of Fe3 +. Other, as the formation of root hairs and transfer cells in the root occur before or simultaneously with the physiological responses, which suggests that structural alterations may be a prerequisite for the functioning of mechanisms for efficient Fe (Landsberg 1986).

In higher plants Fe is predominantly found in chloroplasts, thus iron deficiency affects almost exclusively to the chloroplast, while the other cellular organelles that contain iron, such as peroxisomes, endoplasmic reticulum, mitochondria, etc.. seems to remain unchanged (Platt-Aloia et al., 1983).

Most of the mobilized Fe in plants is a phosphoprotein in the form of iron, the fitoferritina. The chloroplasts can contain up to 80% iron plant as fitoferritina (Tiffin, 1972).

The effect of Fe deficiency results in decreased concentrations of photosynthetic pigments and other components of the thylakoid membrane (Morales et al., 1991, 1994). Iron-deficient plants have less chlorophyll per unit chloroplast, but the number of these does not decrease per unit leaf. However, reducing the amount of chlorophyll is accompanied by alteration of the structure and functions of the chloroplast, reducing both the number and degree of stacking of the thylakoid membranes (Spiller & Terry, 1980, Terry & Abadía, 1986; Terry & Zayed, 1995; Soldatini et al., 2000).

the other hand, extreme temperatures (significantly above or below 25 º C), pH values

Fe is involved in such important processes as photosynthesis, respiration, metabolism of proteins, fixation and nitrogen assimilation and nitrate reduction (Romera & Guard 1991). It is also a cofactor for enzymes (such as cytochrome oxidase, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) that catalyze biochemical reactions only, making it essential role in the growth and development of plants (Abbey 1992; Larbi et al. 2006; Molassiotis et al. 2006;

Chlorosis means lack of chlorophyll in a plant organ, resulting in a loss of green color. Chlorosis can be caused by both the supply deficit of essential elements for plant growth (Fe, manganese (Mn), magnesium (Mg), zinc (Zn), nitrogen (N), etc), water stress or pests, fungi, bacteria or viruses. Iron deficiency, also called iron chlorosis, is one of the most important abiotic stresses. The causes of iron deficiency are numerous and vary, highlighting the availability of iron and bicarbonate ion concentration in the middle of development and other factors. Iron deficiency is usually caused by an insufficient concentration of it in the soil, but the existence of several factors makes affect the solubility and mobility of the Fe. These factors may be a high pH insolubilice some compounds, there limestone and other components, redox potential, interaction between Fe and other nutrients, moisture, organic amendment, salinity, extreme temperatures, etc. (Tagliavini & Rombola 2001). The low availability of iron is increased in soils with the presence of high bicarbonate levels that make the pH is around 8 (Marchner & Römheld 1995), this pH the concentration of Fe quite low and therefore difficult to iron nutrition of the plant (Lucena et al. 2006a). The problem of iron chlorosis is widespread because the limestone soils occupy approximately 30% of the land surface (Chen et al. 1982). It is estimated that between 20 and

50% of fruit growing in Mediterranean areas deficient in iron (Jaeger et al. 2000).

the functioning of mechanisms for efficient Fe (Landsberg 1986).

unchanged (Platt-Aloia et al., 1983).

Zayed, 1995; Soldatini et al., 2000).

Some higher plants, in the absence of Fe have developed a number of mechanisms to increase the availability of Fe in the soil solution. Strategy I plants increase the Fe-making by the excretion of reducing substances of low molecular weight, through the extrusion of protons by roots acidifying the rhizosphere, solubilizing nutrients not available in an alkaline medium (Jones 2000) and increasing the activity of a reductase associated with the plasma membrane of the root responsible for the reduction of Fe3 +. Other, as the formation of root hairs and transfer cells in the root occur before or simultaneously with the physiological responses, which suggests that structural alterations may be a prerequisite for

In higher plants Fe is predominantly found in chloroplasts, thus iron deficiency affects almost exclusively to the chloroplast, while the other cellular organelles that contain iron, such as peroxisomes, endoplasmic reticulum, mitochondria, etc.. seems to remain

Most of the mobilized Fe in plants is a phosphoprotein in the form of iron, the fitoferritina.

The effect of Fe deficiency results in decreased concentrations of photosynthetic pigments and other components of the thylakoid membrane (Morales et al., 1991, 1994). Iron-deficient plants have less chlorophyll per unit chloroplast, but the number of these does not decrease per unit leaf. However, reducing the amount of chlorophyll is accompanied by alteration of the structure and functions of the chloroplast, reducing both the number and degree of stacking of the thylakoid membranes (Spiller & Terry, 1980, Terry & Abadía, 1986; Terry &

The chloroplasts can contain up to 80% iron plant as fitoferritina (Tiffin, 1972).

greater than 7.5 and the presence of heavy metals affects their activity (Lucena 2000).

Agustí 2003).

The reduction of the thylakoid membrane is accompanied by decreased concentrations of photosynthetic pigments (chlorophylls a and b and carotenoids) in the leaves of affected plants (Morales et al., 1990, 1994). The loss of leaf pigments sheet does not imply that diminish their ability to capture light energy (Terry & Zayed, 1995) due in part to the increase in the relationship leaf carotene / chlorophyll concentrations decline with leaf carotene deficiency Fe to a lesser degree than the chlorophylls (Terry, 1980, Morales et al., 1990, 1994, 2000). The characteristic yellow chlorotic leaves is a consequence of the imbalance between the contents of chlorophyll and carotenoids (Abbey, 1992, Terry & Zayed, 1995).

Iron is also involved in chlorophyll synthesis (Miller et al., 1984) in different crops subject to conditions of deficiency, has shown an increase in the ratio of chlorophyll a: chlorophyll b (Abbey et al. 1989; Monge et al., 1987, Nishio et al., 1985). One explanation for this increase is that under conditions of iron deficiency in the field and there is full sunlight preferential photodestruction of chlorophyll b (Díez-Altar, 1959). Fe deficiency also increases the activity of chlorophyllase, which is involved in the degradation of chlorophyll in citrus fruits (Fernandez-Lopez et al., 1991).

Finally, iron is a constituent of many electron transporters (Terry & Abadía, 1986; Abbey, 1992, Terry & Zayed, 1995; Soldatini et al. 2000), so that iron deficiency is also reduced photosynthetic electron transport. These facts lead to a reduction in photosynthetic capacity of the plant that results in decreased levels of sugars, starch, certain amino acids and accumulation of others, thus altering the spectrum of proteins (Terry & Abadía, 1986; Abbey, 1992, Terry & Zayed, 1995) and enrichment in unsaturated lipids (Terry & Abadía, 1986; Abbey, 1992, Terry & Zayed, 1995), which alters the physiological functioning of the plant. The reduction in plant growth may be related to decreased photosynthetic capacity of chlorotic leaves. Both vegetative growth and production decrease with iron chlorosis (Hurley et al., 1986). The low capacity of the plant to translocate iron from old leaves, is manifested by the yellowing of young leaves , except their nerves remain green. These outbreaks are becoming less vigorous and its leaves, small, can fall prematurely, starting with the most apical (Agustí, 2003). One can also induce severe chlorosis reduced stem growth by inhibiting the formation of new leaves (Loue, 1993). Reducing the number and final size of the fruit, as well as total soluble solids content of the juice are also consequences that result from a deficiency of Fe (Agustí, 2003).

### **3. Effects of iron chlorosis in citrus**

Many agricultural crops in arid and semiarid regions suffer chlorosis. Among the plants most affected are the citrus, planted in chalky soils often show signs of severe iron deficiency (Wallace, 1986). Iron chlorosis affects many biochemical, morphological and physiological parameters, and therefore their growth and development of plants (Larbi et al., 2006, Molassiotis et al., 2006, Agustí, 2003).

Iron deficiency in citrus tends to be observed during the months of winter and spring. And internervial yellowing of the leaves of young shoots are manifested, due to the inability of the plant to translocate iron from old leaves. The loss of pigmentation is caused by decreased chlorophyll content in chloroplasts (Marschner, 1995). This negatively affects the rate of photosynthesis and, therefore, the development of biomass (Abbey et al., 2004). The young shoots are becoming less vigorous and its leaves, small, can fall prematurely, starting with the most apical (Agustí, 2003).

Iron Stress in Citrus 169

sometimes leads to problems of gumming (Heras et al., 1976). The main disadvantages of high pressure fluid injections are its high cost, because we have to move to field a pressure

Land application of iron fertilizer is the most widely used practice, and is done once the chlorosis appears in the crop (Pestana et al., 2003). The iron is supplied to the soil as chelates. Chelates are organic substances containing Fe in stable molecules. The iron remains assimilable by plants and doesn´t suffer the insolubilization reactions of this type of soils. The most commonly used is chelated Fe (III)-EDDHA and is often added to irrigation water (Papastylianou et al., 1993). Zude et al. (1999) showed that application of chelated iron in the soil produces a greening of Fe-deficient leaves of citrus. The same authors also indicated that both the chlorophyll content and photosynthesis increase after the application of Fe

Citrus trees grown today are composed of two parts, the rootstock and the variety, the second grafted onto it first, so that together combine the best features possible. In the screening citrus for iron chlorosis, the selection of plant material has to be done primarily, being the rootstock the determinant of tolerance to chlorosis. The performance of citrus rootstocks with iron chlorosis is very variable. Thus, although development is not limited by low levels, elevated content in active lime limit their use. The sensitivity of a rootstocks to elevated levels of iron chlorosis has many symptoms, on which also affected by other factors such as soil rich in calcium carbonate, calcium, moisture, pH and the grafted variety (Agustí,

The Swingle citrumelo is very sensitive to iron chlorosis. The rootstocks P. *trifoliata*, Carrizo citrange and sweet orange are susceptible to lime-induced chlorosis, while the sour orange and Cleopatra mandarin are tolerant to this deficiency (Hamza et al., 1986, Castle, 1987; Treeby & Uren, 1993; Pestana et al., 2005). Cleopatra mandarin are tolerant to iron chlorosis, but grows slowly in the field. Besides the production of fruit is not very high and although

There is a citrus rootstock breeding program taking place in the IVIA (Valencia, Spain) with the objective of the search for new citrus rootstocks well adapted to calcareous soils. In this regard, a new hybrid is now available in Spanish citrus nurseries: Forner-Alcaide 5 (FA 5) (Cleopatra mandarin x P. *trifoliata*). This rootstock has been evaluated from an agronomic point of view in calcareous soils (Gonzalez-Mas et al., 2009; Llosá et al., 2009) and it is more tolerant than Carrizo citrange, and trees 'Navelina' grafted in FA 5 produce 40% more than the trees grafted on Carrizo citrange, with the smaller fruit but equal quality to those of trees

There is no a perfect rootstock. All citrus rootstocks, that are currently used in the world, have some limitations. Over the years, every citrus producing area has been selected those rootstocks best suited to their conditions. However, there are still many areas for which there is no pattern can solve all their problems properly. Currently, there is large number of studies aimed at looking for a methodology for assessing the tolerance to iron chlorosis of citrus rootstocks (Castle, 2010; Gonzalez-Mas et al., 2009; Llosá et al., 2009). So far, that assessment could only be done reliably in field tests. The fruit breeding rootstocks represents the most cost-effective and sustainable solution to the problem of iron deficiency

the fruit is of good quality, is smaller than that produced on other rootstocks.

grafted on Carrizo (Forner et al., 2003, Forner-Giner et al., 2003).

(Cianzio, 1995; Wirén, 2004).

generating equipment with an injection machine (Yoshikawa, 1988).

**4. Screening citrus rootstocks to iron chlorosis** 

chelates.

2003).

Fig. 1. Control and chlorotic leaves of citrange Carrizo.

In the case of a more serious deficiency, loss of pigmentation may also affect adult leaves, and the younger end completely yellow and devoid of chlorophyll. The fruits are also affected by severe iron deficiency. It reduces the number of fruits and their growth shortly after the set and these become yellow unexpected; Also decreases the concentration of juice soluble solids (Amorós, 1995; Agustí, 2003).

The treatment for iron chlorosis can be started before it appears, preventing the causes that lead. The best preventive practices include avoiding certain combinations plantations growing on soils unsuitable or use of tolerant cultivars. Once detected the symptoms of iron chlorosis trees can be treated with corrective methods to increase the availability of iron (Wallace, 1991), avoiding a loss of production (Pestana et al., 2001b). In cases of late diagnosis, will not be able to avoid the loss of annual production, but could be enhanced vegetative growth for the next harvest (Abbey et al., 2004). It has been described different types of treatments to correct iron chlorosis. Most important are foliar application, soil application and trunk injections. Foliar applications are very fast acting and can be less expensive than soil-applied treatments (Pestana et al., 2003), but have disadvantages of short-lived effects and cause phytotoxicity in certain cases (Wallace et al. 1984; Mortvedt, 1986), as well as cause the appearance of burns, defoliation, the greening in spots and no effect on the leaves that develop after treatment (Legaz et al., 1992, García et al., 1998; Fernández & Ebert, 2005). Thus foliar treatments are effective only in situations of symptoms of mild to moderate chlorosis (Rombolá et al., 2000). Moreover, Pestana et al. (2001a) indicated that foliar application of FeSO4 in citrus avoid losses in production and quality of fruits caused by it. The same authors found that several foliar applications are able to relieve iron chlorosis in field trials. The application of ferrous sulfate was more effective in increasing the size and quality of the fruits that the Fe (III)-chelate. Pestana et al. (2001b) also indicated that the addition of sulphuric acid produced a small increase in the concentration of chlorophyll and Fe in the leaves, without causing any effect on the size and quality of fruit. The same authors concluded that foliar application can reduce production losses and quality of fruits caused by iron deficiency.

Injections into the trunk and branches consist of introducing iron compounds either in solid or in solution, by drilling into the wood. This type of treatment is recommended for mature trees in which has a big trunk diameter. Both the solid and the liquid injection under high pressure have the advantage of having a great effect and great persistence of treatment, usually two or more years (Hurley et al., 1986, Yoshikawa, 1988). The major drawback to injections of solid type is the need for a large number of holes around the trunk, which

In the case of a more serious deficiency, loss of pigmentation may also affect adult leaves, and the younger end completely yellow and devoid of chlorophyll. The fruits are also affected by severe iron deficiency. It reduces the number of fruits and their growth shortly after the set and these become yellow unexpected; Also decreases the concentration of juice

The treatment for iron chlorosis can be started before it appears, preventing the causes that lead. The best preventive practices include avoiding certain combinations plantations growing on soils unsuitable or use of tolerant cultivars. Once detected the symptoms of iron chlorosis trees can be treated with corrective methods to increase the availability of iron (Wallace, 1991), avoiding a loss of production (Pestana et al., 2001b). In cases of late diagnosis, will not be able to avoid the loss of annual production, but could be enhanced vegetative growth for the next harvest (Abbey et al., 2004). It has been described different types of treatments to correct iron chlorosis. Most important are foliar application, soil application and trunk injections. Foliar applications are very fast acting and can be less expensive than soil-applied treatments (Pestana et al., 2003), but have disadvantages of short-lived effects and cause phytotoxicity in certain cases (Wallace et al. 1984; Mortvedt, 1986), as well as cause the appearance of burns, defoliation, the greening in spots and no effect on the leaves that develop after treatment (Legaz et al., 1992, García et al., 1998; Fernández & Ebert, 2005). Thus foliar treatments are effective only in situations of symptoms of mild to moderate chlorosis (Rombolá et al., 2000). Moreover, Pestana et al. (2001a) indicated that foliar application of FeSO4 in citrus avoid losses in production and quality of fruits caused by it. The same authors found that several foliar applications are able to relieve iron chlorosis in field trials. The application of ferrous sulfate was more effective in increasing the size and quality of the fruits that the Fe (III)-chelate. Pestana et al. (2001b) also indicated that the addition of sulphuric acid produced a small increase in the concentration of chlorophyll and Fe in the leaves, without causing any effect on the size and quality of fruit. The same authors concluded that foliar application can reduce production

Injections into the trunk and branches consist of introducing iron compounds either in solid or in solution, by drilling into the wood. This type of treatment is recommended for mature trees in which has a big trunk diameter. Both the solid and the liquid injection under high pressure have the advantage of having a great effect and great persistence of treatment, usually two or more years (Hurley et al., 1986, Yoshikawa, 1988). The major drawback to injections of solid type is the need for a large number of holes around the trunk, which

Fig. 1. Control and chlorotic leaves of citrange Carrizo.

losses and quality of fruits caused by iron deficiency.

soluble solids (Amorós, 1995; Agustí, 2003).

sometimes leads to problems of gumming (Heras et al., 1976). The main disadvantages of high pressure fluid injections are its high cost, because we have to move to field a pressure generating equipment with an injection machine (Yoshikawa, 1988).

Land application of iron fertilizer is the most widely used practice, and is done once the chlorosis appears in the crop (Pestana et al., 2003). The iron is supplied to the soil as chelates. Chelates are organic substances containing Fe in stable molecules. The iron remains assimilable by plants and doesn´t suffer the insolubilization reactions of this type of soils. The most commonly used is chelated Fe (III)-EDDHA and is often added to irrigation water (Papastylianou et al., 1993). Zude et al. (1999) showed that application of chelated iron in the soil produces a greening of Fe-deficient leaves of citrus. The same authors also indicated that both the chlorophyll content and photosynthesis increase after the application of Fe chelates.

### **4. Screening citrus rootstocks to iron chlorosis**

Citrus trees grown today are composed of two parts, the rootstock and the variety, the second grafted onto it first, so that together combine the best features possible. In the screening citrus for iron chlorosis, the selection of plant material has to be done primarily, being the rootstock the determinant of tolerance to chlorosis. The performance of citrus rootstocks with iron chlorosis is very variable. Thus, although development is not limited by low levels, elevated content in active lime limit their use. The sensitivity of a rootstocks to elevated levels of iron chlorosis has many symptoms, on which also affected by other factors such as soil rich in calcium carbonate, calcium, moisture, pH and the grafted variety (Agustí, 2003).

The Swingle citrumelo is very sensitive to iron chlorosis. The rootstocks P. *trifoliata*, Carrizo citrange and sweet orange are susceptible to lime-induced chlorosis, while the sour orange and Cleopatra mandarin are tolerant to this deficiency (Hamza et al., 1986, Castle, 1987; Treeby & Uren, 1993; Pestana et al., 2005). Cleopatra mandarin are tolerant to iron chlorosis, but grows slowly in the field. Besides the production of fruit is not very high and although the fruit is of good quality, is smaller than that produced on other rootstocks.

There is a citrus rootstock breeding program taking place in the IVIA (Valencia, Spain) with the objective of the search for new citrus rootstocks well adapted to calcareous soils. In this regard, a new hybrid is now available in Spanish citrus nurseries: Forner-Alcaide 5 (FA 5) (Cleopatra mandarin x P. *trifoliata*). This rootstock has been evaluated from an agronomic point of view in calcareous soils (Gonzalez-Mas et al., 2009; Llosá et al., 2009) and it is more tolerant than Carrizo citrange, and trees 'Navelina' grafted in FA 5 produce 40% more than the trees grafted on Carrizo citrange, with the smaller fruit but equal quality to those of trees grafted on Carrizo (Forner et al., 2003, Forner-Giner et al., 2003).

There is no a perfect rootstock. All citrus rootstocks, that are currently used in the world, have some limitations. Over the years, every citrus producing area has been selected those rootstocks best suited to their conditions. However, there are still many areas for which there is no pattern can solve all their problems properly. Currently, there is large number of studies aimed at looking for a methodology for assessing the tolerance to iron chlorosis of citrus rootstocks (Castle, 2010; Gonzalez-Mas et al., 2009; Llosá et al., 2009). So far, that assessment could only be done reliably in field tests. The fruit breeding rootstocks represents the most cost-effective and sustainable solution to the problem of iron deficiency (Cianzio, 1995; Wirén, 2004).

Iron Stress in Citrus 171

its enzymatic activity and binds to RNA of genes involved in Fe homeostasis, altering their expression (Hentze & Kuh 1996). In Fe-sufficient conditions, during the first half of fruit development, the decrease in the mitochondrial aconitase activity could play a role in the citric acid accumulation in the vacuole of the sac cells of the fruit (Sadka et al. 2000). During the second half of fruit development, the activity of the cytosolic aconitase increases, playing a role in acid decline. Shlizerman et al. (2007) studied the relation between the aconitase activity and the citric acid content that takes place after Fe deprivation. Cytosolic aconitase is more susceptible than mitochondrial one to Fe shortage. They demonstrate that only cytosolic activity was affected by Fe limitation whereas mitochondrial one was not. The reduction in the activity of the cytosolyc aconitase results in a decrease of the citrate catabolism, what account for the increase in pulp acidity of citrus fruit detected in trees grown in Fe deficiency. The authors observed that the application of Fe treatments during fruit maturation can reduce the acid content of the fruit juice what is an important practical question as in many scions as sweet oranges or clementines, high acid contents reduces the quality of the mature fruits delaying the harvest. However no data exist about this acid

content decline is due to an activation of the cytosolic aconitase activity in the fruits.

Nisi & Zocchi, 2000).

Organic acid accumulation has in addition been interpreted in terms of the pH-stat theory (Sakano, 1998). The pH of the root cytosol and vacuole increases a consequence of the apoplast acidification that occurs in response to Fe deficiency (Espen et al., 2000). This argument is supported by the fact that Fe deficiency stress correlates with and increase in Phospoenolpyruvate carboxylase (PEPC) activity, resulting in an increased nonphotosynthetic carbon fixation and a net carbon fluxes toward organic acid production (De

The adaptative changes in strategy I plants include morphological changes in the root of Fe.deficient plants. Such morphological changes have been reported for other plants as Arabidopsis, *Ficus* or sunflower (Römheld & Marschner, 1981, Rosenfield et al., 1991, Schmidt et al. 2000). Typical morphological changes included additional cell division in the rhizodermis layer and enhanced formation of root hairs. In citrus, there is currently no

Fig. 3. Root morphology of Fe-sufficient and Fe-deficient *Poncirus trifoliata* plants

Fig. 2. Differences between a tolerant (left) and susceptible (right) citrus rootstock

### **5. Molecular mechanisms underlying iron deficiency in citrus**

The molecular components that are involved in the Fe deficiency response have been poorly identified and only in isolated cases. Mechanisms by which plants adapt to Fe deficiency have been frequently described in grasses (Strategy II) and other plants (strategy I) (Moog & Brueggemann, 1994). Strategy I (which is the one followed by *Citrus*) relies on increasing iron solubility by inducing membrane-bound Fe(III)-chelate reductases that reduce Fe(III) to Fe(II), which is more soluble and is subsequently taken up by specific transporters (Chaney et al. 1972).

In citrus, little is known about the behaviour at a molecular level of of the Fe(III)-chelate reductase in response to Fe starvation. Fe(III)-chelate reductase activity is much more increased in roots of *Citrus junos* (tolerant to iron chlorosis) than in *Poncirus trifoliate* (susceptible to iron chlorosis). Ling et al. (2002) founded that increase of Fe(III)-chelate reductase activity was about twenty fold whereas *P. trifoliate* was stimulated to increase only about threefold at the same time and under the same conditions. The result suggest that the increase in the enzyme activity under Fe stress was an important reason for the tolerance of the *Citrus junos* rootstock to Fe deficiency.

It has been reported a correlation between the development of Fe(III)-chelate reductase activity, acidification of the rhizosphere, and the accumulation of organic acids as citrate and malate in roots of *Capsicum annum* L. (Landsberg, 1986). In citrus, Fe(III)-chelate reductase activity and rhizosphere acidification have been reported to be induced in roots of several rootstocks as *Citrus volkameriana*, *Citrus taiwanica* and *Citrus junos* by Fe-shortage (Chouliaras et al. 2004). An increase in citric acid content in parallel with an aconitase activity decrease have also been reported in roots and fruit vesicles and calli of C*itrus Lemon* under Fe deprivation (Shlizerman et al., 2007). Aconitase catalyses the conversion of citrate into isocitrate, requiring Fe for its activity. Cytosolic aconitase represents a regulatory link between Fe homeostasis and organic acid metabolism. Under Fe shortage the enzyme loses

Fig. 2. Differences between a tolerant (left) and susceptible (right) citrus rootstock

The molecular components that are involved in the Fe deficiency response have been poorly identified and only in isolated cases. Mechanisms by which plants adapt to Fe deficiency have been frequently described in grasses (Strategy II) and other plants (strategy I) (Moog & Brueggemann, 1994). Strategy I (which is the one followed by *Citrus*) relies on increasing iron solubility by inducing membrane-bound Fe(III)-chelate reductases that reduce Fe(III) to Fe(II), which is more soluble and is subsequently taken up by specific transporters (Chaney

In citrus, little is known about the behaviour at a molecular level of of the Fe(III)-chelate reductase in response to Fe starvation. Fe(III)-chelate reductase activity is much more increased in roots of *Citrus junos* (tolerant to iron chlorosis) than in *Poncirus trifoliate* (susceptible to iron chlorosis). Ling et al. (2002) founded that increase of Fe(III)-chelate reductase activity was about twenty fold whereas *P. trifoliate* was stimulated to increase only about threefold at the same time and under the same conditions. The result suggest that the increase in the enzyme activity under Fe stress was an important reason for the tolerance of

It has been reported a correlation between the development of Fe(III)-chelate reductase activity, acidification of the rhizosphere, and the accumulation of organic acids as citrate and malate in roots of *Capsicum annum* L. (Landsberg, 1986). In citrus, Fe(III)-chelate reductase activity and rhizosphere acidification have been reported to be induced in roots of several rootstocks as *Citrus volkameriana*, *Citrus taiwanica* and *Citrus junos* by Fe-shortage (Chouliaras et al. 2004). An increase in citric acid content in parallel with an aconitase activity decrease have also been reported in roots and fruit vesicles and calli of C*itrus Lemon* under Fe deprivation (Shlizerman et al., 2007). Aconitase catalyses the conversion of citrate into isocitrate, requiring Fe for its activity. Cytosolic aconitase represents a regulatory link between Fe homeostasis and organic acid metabolism. Under Fe shortage the enzyme loses

**5. Molecular mechanisms underlying iron deficiency in citrus** 

et al. 1972).

the *Citrus junos* rootstock to Fe deficiency.

its enzymatic activity and binds to RNA of genes involved in Fe homeostasis, altering their expression (Hentze & Kuh 1996). In Fe-sufficient conditions, during the first half of fruit development, the decrease in the mitochondrial aconitase activity could play a role in the citric acid accumulation in the vacuole of the sac cells of the fruit (Sadka et al. 2000). During the second half of fruit development, the activity of the cytosolic aconitase increases, playing a role in acid decline. Shlizerman et al. (2007) studied the relation between the aconitase activity and the citric acid content that takes place after Fe deprivation. Cytosolic aconitase is more susceptible than mitochondrial one to Fe shortage. They demonstrate that only cytosolic activity was affected by Fe limitation whereas mitochondrial one was not. The reduction in the activity of the cytosolyc aconitase results in a decrease of the citrate catabolism, what account for the increase in pulp acidity of citrus fruit detected in trees grown in Fe deficiency. The authors observed that the application of Fe treatments during fruit maturation can reduce the acid content of the fruit juice what is an important practical question as in many scions as sweet oranges or clementines, high acid contents reduces the quality of the mature fruits delaying the harvest. However no data exist about this acid content decline is due to an activation of the cytosolic aconitase activity in the fruits.

Organic acid accumulation has in addition been interpreted in terms of the pH-stat theory (Sakano, 1998). The pH of the root cytosol and vacuole increases a consequence of the apoplast acidification that occurs in response to Fe deficiency (Espen et al., 2000). This argument is supported by the fact that Fe deficiency stress correlates with and increase in Phospoenolpyruvate carboxylase (PEPC) activity, resulting in an increased nonphotosynthetic carbon fixation and a net carbon fluxes toward organic acid production (De Nisi & Zocchi, 2000).

The adaptative changes in strategy I plants include morphological changes in the root of Fe.deficient plants. Such morphological changes have been reported for other plants as Arabidopsis, *Ficus* or sunflower (Römheld & Marschner, 1981, Rosenfield et al., 1991, Schmidt et al. 2000). Typical morphological changes included additional cell division in the rhizodermis layer and enhanced formation of root hairs. In citrus, there is currently no

Fig. 3. Root morphology of Fe-sufficient and Fe-deficient *Poncirus trifoliata* plants

Iron Stress in Citrus 173

Fig. 5. Calcofluor staining in roots of *Poncirus trifoliate* treated with (control) or without Fe-

The composition of the cell wall and the cross-linking of the different polymers that form it, most probably determine the wall mechanical properties. So that, one may hypothesize that this changes may occur because the plasticity of the cell wall there must be probably important for plant acclimation to iron deficiency (as well as to other environmental conditions). However, whether this is a mechanism to improve adaptation to iron shortage or it is a final consequence of the stress produced by the iron deficiency is still unknown and

As for other plants, some authors have found an important decrease in soluble and ionically-bound-to-cell-wall peroxidase activities in citrus in response to Fe-deficiency (Chouliaras et al. 2004, Forner-Giner et al. 2010). As to ionically-bound-to-cell-wall peroxidases are implied in lignin metabolisms and several cell wall modifications are taking place in response to iron deficiency, one may infer that this component is being also altered during the response process. However no changes in lignin accumulation could be

Fig. 6. Phoroglucinol staining in roots of *Poncirus trifoliate* treated with (control) or without

EDDHA (iron deficiency)

Fe-EDDHA (iron deficiency)

it will have to be elucidated in the future.

report about morphological changes in the root caused by iron deficiency. In the susceptible rootstock *Poncirus trifoliata*, Forner-Giner et al. (unpublished data) have not observed morphological changes in roots due to iron starvation (Fig 1). Changes occurring in citrus roots of susceptible plants are mainly at a molecular level. Forner-Giner et al. (2010), in a genomic survey, found that within the genes that were differentially expressed in the plant in response to iron deficiency, a large group had to do with cell wall (Table 1).


Table 1. Cell wall related gene that are overspressed in response to Fe-deficiency

By specific staining for each component, they demonstrated that the cell wall becomes thinner in *Poncirus* plants under iron starvation and they also highlight that pectin and xyloglucan component changed in these plants. As it is shown in figure 2, ruthenium red staining (specific for pectin) revealed the thinning of the cell wall in the Fe deficient plants.

Fig. 4. Ruthenium red staining in roots of *Poncirus trifoliate* treated with (control) or without Fe-EDDHA (iron deficiency)

Moreover, calcofluor (a dye specific for various beta-D-glucans including Xyloglucans) stained the cell wall of the roots of more intensely than those of control plants (fig. 3).

report about morphological changes in the root caused by iron deficiency. In the susceptible rootstock *Poncirus trifoliata*, Forner-Giner et al. (unpublished data) have not observed morphological changes in roots due to iron starvation (Fig 1). Changes occurring in citrus roots of susceptible plants are mainly at a molecular level. Forner-Giner et al. (2010), in a genomic survey, found that within the genes that were differentially expressed in the plant

**Gene ID Best** *Arabidopsis* **BLAST hit Cell Wall Component** 

cuticle

in response to iron deficiency, a large group had to do with cell wall (Table 1).

C31502B08 Calmodulin-regulated Ca(2+)-pump pectin C05811H06 polygalacturonase (pectinase) pectin

known as PEN1

C05133B06 endo-xyloglucan transferase xyloglucan C19009B12 xyloglucan endotransglucosylase xyloglucan

Table 1. Cell wall related gene that are overspressed in response to Fe-deficiency

By specific staining for each component, they demonstrated that the cell wall becomes thinner in *Poncirus* plants under iron starvation and they also highlight that pectin and xyloglucan component changed in these plants. As it is shown in figure 2, ruthenium red staining (specific for pectin) revealed the thinning of the cell wall in the Fe deficient

Fig. 4. Ruthenium red staining in roots of *Poncirus trifoliate* treated with (control) or without

Moreover, calcofluor (a dye specific for various beta-D-glucans including Xyloglucans)

stained the cell wall of the roots of more intensely than those of control plants (fig. 3).

C05140C08 SYR1, Syntaxin Related Protein 1, also

plants.

Fe-EDDHA (iron deficiency)

Fig. 5. Calcofluor staining in roots of *Poncirus trifoliate* treated with (control) or without Fe-EDDHA (iron deficiency)

The composition of the cell wall and the cross-linking of the different polymers that form it, most probably determine the wall mechanical properties. So that, one may hypothesize that this changes may occur because the plasticity of the cell wall there must be probably important for plant acclimation to iron deficiency (as well as to other environmental conditions). However, whether this is a mechanism to improve adaptation to iron shortage or it is a final consequence of the stress produced by the iron deficiency is still unknown and it will have to be elucidated in the future.

As for other plants, some authors have found an important decrease in soluble and ionically-bound-to-cell-wall peroxidase activities in citrus in response to Fe-deficiency (Chouliaras et al. 2004, Forner-Giner et al. 2010). As to ionically-bound-to-cell-wall peroxidases are implied in lignin metabolisms and several cell wall modifications are taking place in response to iron deficiency, one may infer that this component is being also altered during the response process. However no changes in lignin accumulation could be

Fig. 6. Phoroglucinol staining in roots of *Poncirus trifoliate* treated with (control) or without Fe-EDDHA (iron deficiency)

Iron Stress in Citrus 175

information, the continued development of resources promises to yield many more insights in the future. Information gained from studies of this type may allow the development of

MA Forner-Giner and G Ancillo are recipient of a contract from Conselleria de Agricultura, Pesca y Alimentación (Generalitat Valenciana, Spain) under Proy\_IVIA09/05 and Proy\_IVIA09/03 respectively. The authors would like to thank Prof. L Navarro and E Primo

Abadìa J, Lopez-Millan A, Rombola A, & Abadìa A (2002). Organic acids and Fe deficiency:

Castle, W.S. (1987). Citrus rootstocks. En: Rootstocks for Fruits Crops. pp. 361-369. Rom R.C.

Castle, WS, Nunnallee, J., & Manthey JA.(2009). Screening citrus rootstocks and related

Chaney, R.L., Brown, J.C., & Tiffin, L.O. (1972). Obligatory reduction of ferric chelates in

Chen, Y., Barak, P. (1982). Iron nutrition of plants in calcareous soils. *Advances in agronomy*.

Chouliaras V., Dimassi K., Therios I., Molassiotis A., & Diamantidis G. (2004). Root-reducing

Cianzio, S.R. (1995). Strategies for the genetic improvement of Fe efficiency in plants. In:

De Nisi P., & Zocchi G.(2000). Phosphoenolpyruvate carboxylase in cucumber (*Cucumis* 

Díez-Altarés, M. (1959). Fotodescomposición de clorofila en casos de deficiencia inducida de

Dunand, C., Cre`vecoeur, M., & Penel, C. (2007). Distribution of superoxide and hydrogen

Espen L, Dell'Orto M, De Nisi P, & Zocchi G. (2000) Metabolic responses in cucumber

Fernández, V., & Ebert, G. (2005). Foliar iron fertilization: a critical review. *J. Plant Nutr.* 

Characterization of ferric reducing activity in roots of Fe deficient Phaseolus

selections in soil and solution culture for tolerance to low-iron stress*. Hortscience*

capacity, rhizosphere acidification, peroxidase and catalase activities and nutrient levels of *Citrus taiwanica* and *C. volkameriana* seedlings, under Fe deprivation

Abadía J. (Ed.). Iron Nutrition in Soils and Plants. Kluwer Academic Publishers,

*sativus* L.) roots under iron deficiency: activity and kinetic characterization. *J Exp* 

peroxide in Arabidopsis root and their influence on root development: possible

(*Cucumis sativus* L.) roots under Fe-deficiency: a 31P-nuclear magnetic resonance in-

Amorós, M. (1995). Producción de agrios: 286. Ediciones Mundi-Prensa. Madrid, Spain. Bienfait, H.F., Bino, R.J., Van Der Bliek, A.M., Duivenvoorden, J.F., & Fontaine, J. M. (1983).

citrus plants that are capable of growth on soils that are iron-deficient.

**7. Acknowledgment** 

**8. References** 

for critical reading of the manuscript.

44(3): 638-645.

35: 217-240.

a review. *Plant Soil* 241: 75–86.

vulgaris, Physiol. plant. 59: 196.

conditions. *Agronomie* 24:1-6.

*Bot.*; 51:1903–1909.

28:2113-2124.

Dordrecht, Netherlands. 119-125.

vivo study. *Planta*, 210:985–992.

Agustí, M.( 2003). Citricultura. Ed. Mundi-Prensa. Madrid. Spain.

& Carlson R.F. (eds). John Wiley & Sons, New York.

iron uptake by soybean. *Plant Phys* 50:208-213.

hierro. Ann. Estac. Exp. Aula Dei (Zaragoza). 6: 1-80.

interaction with peroxidases. *New Phytol*. 174, 332–341.

appreciated (fig 4) when root sections of control and Fe-deficient plants of *P. trifoliate* were stained with phoroglucinol (a specific dye that stains lignified tissue red and leaves everything else unstained) and visualised under the microscopy (Forner-Giner & Ancillo, unpublished).

Peroxidase activity is also responsible of the conversion of H2O2 into water by oxidizing various hydrogen donor molecules such as phenolic compounds and auxin metabolites (Dunand et al. 2007). As in other plants, in citrus, the Fe-related decline of peroxidase activity coincide with a significant decline in catalase activity (Chouliaras et al. 2004, Forner-Giner et al. 2010), which is also involved in H2O2 metabolism. In maize, the decrease in these enzymatic activities entails an increase in the concentration of H2O2 and superoxide anion radical (O2-) that causes oxidative stress in Fe- deficient plants (Tewari et al. 2005). No direct evidences of the H2O2 increase have been published in citrus, but Forner-Giner et al. (2010) reported the induction, in response to Fe-deficiency, of a NADPH thioredoxin reductase C which is involved in reduction of H2O2. All together, these results suggest that the reduction in the level of catalase and peroxidase activities in iron-deficient citrus plants produce an increase of H2O2 and other ROS that could lead to oxidative stress and, as reported for other plants (Perez-Ruiz et al., 2006) the induction of NADPH thioredoxin reductase C may be part of the response of the plant to prevent damage by the oxidative stress caused by iron deficiency. Chouliaras et al. (2004) founded that peroxidase and catalase activities were higher in the case *Citrus taiwanica* ( a tolerant rootstock) rather than in *Citrus volkameriana* (a non-tolerant one). They propose that the ability of plants to synthesize more peroxidase and catalase could be a feature associated with tolerance to Fe deficiency.

### **6. Future research and conclusion**

Iron is an essential nutrient playing critical roles in life-sustaining processes. Due to its ability to gain and lose electrons, iron works as a cofactor for enzymes involved in a wide variety of oxidation-reduction reactions (as photosynthesis, respiration, hormone synthesis, and DNA synthesis, etc). This essential role made of iron an absolutely required nutrient, and its deficiency causes iron chlorosis which seriously constraints the normal development of the plant. Iron chlorosis is a widespread problem especially for regions where the bioavailability of iron in soil is low. Usual remediation strategies consist of amending iron to soil, which is an expensive practice. Thus genetically improved chlorosis resistant rootstocks and new cultivar/rootstock combinations offer the best solution to iron chlorosis and is one of the most important lines of investigation needed to prevent this nutritional problem nowadays. However tolerant rootstocks are difficult to develop when not available and mean a long-term approach. Therefore, there is a need for new methods to diagnose and correct this nutritional disorder.

The use of microarray techniques revealed changes in gene expression level due to Fe deficiency and has allowed insights into the transcriptional regulation of some functions. These studies have extended our knowledge of citrus response to iron deficiency. These experiments have identified candidate genes and processes for further experimentation to increase our understanding of citrus response to iron deficiency stress.

It is likely that more extensive microarray analysis, coupled with a suitable annotation of the citrus genome, which has recently been completely sequenced, will prove invaluable in future studies of iron-stress responses in plants. Thus, while the use of functional genomic approaches to study iron-stress responses in plants already has yielded important

appreciated (fig 4) when root sections of control and Fe-deficient plants of *P. trifoliate* were stained with phoroglucinol (a specific dye that stains lignified tissue red and leaves everything else unstained) and visualised under the microscopy (Forner-Giner & Ancillo,

Peroxidase activity is also responsible of the conversion of H2O2 into water by oxidizing various hydrogen donor molecules such as phenolic compounds and auxin metabolites (Dunand et al. 2007). As in other plants, in citrus, the Fe-related decline of peroxidase activity coincide with a significant decline in catalase activity (Chouliaras et al. 2004, Forner-Giner et al. 2010), which is also involved in H2O2 metabolism. In maize, the decrease in these enzymatic activities entails an increase in the concentration of H2O2 and superoxide anion radical (O2-) that causes oxidative stress in Fe- deficient plants (Tewari et al. 2005). No direct evidences of the H2O2 increase have been published in citrus, but Forner-Giner et al. (2010) reported the induction, in response to Fe-deficiency, of a NADPH thioredoxin reductase C which is involved in reduction of H2O2. All together, these results suggest that the reduction in the level of catalase and peroxidase activities in iron-deficient citrus plants produce an increase of H2O2 and other ROS that could lead to oxidative stress and, as reported for other plants (Perez-Ruiz et al., 2006) the induction of NADPH thioredoxin reductase C may be part of the response of the plant to prevent damage by the oxidative stress caused by iron deficiency. Chouliaras et al. (2004) founded that peroxidase and catalase activities were higher in the case *Citrus taiwanica* ( a tolerant rootstock) rather than in *Citrus volkameriana* (a non-tolerant one). They propose that the ability of plants to synthesize more peroxidase and

Iron is an essential nutrient playing critical roles in life-sustaining processes. Due to its ability to gain and lose electrons, iron works as a cofactor for enzymes involved in a wide variety of oxidation-reduction reactions (as photosynthesis, respiration, hormone synthesis, and DNA synthesis, etc). This essential role made of iron an absolutely required nutrient, and its deficiency causes iron chlorosis which seriously constraints the normal development of the plant. Iron chlorosis is a widespread problem especially for regions where the bioavailability of iron in soil is low. Usual remediation strategies consist of amending iron to soil, which is an expensive practice. Thus genetically improved chlorosis resistant rootstocks and new cultivar/rootstock combinations offer the best solution to iron chlorosis and is one of the most important lines of investigation needed to prevent this nutritional problem nowadays. However tolerant rootstocks are difficult to develop when not available and mean a long-term approach. Therefore, there is a need for new methods to diagnose and

The use of microarray techniques revealed changes in gene expression level due to Fe deficiency and has allowed insights into the transcriptional regulation of some functions. These studies have extended our knowledge of citrus response to iron deficiency. These experiments have identified candidate genes and processes for further experimentation to

It is likely that more extensive microarray analysis, coupled with a suitable annotation of the citrus genome, which has recently been completely sequenced, will prove invaluable in future studies of iron-stress responses in plants. Thus, while the use of functional genomic approaches to study iron-stress responses in plants already has yielded important

catalase could be a feature associated with tolerance to Fe deficiency.

increase our understanding of citrus response to iron deficiency stress.

**6. Future research and conclusion** 

correct this nutritional disorder.

unpublished).

information, the continued development of resources promises to yield many more insights in the future. Information gained from studies of this type may allow the development of citrus plants that are capable of growth on soils that are iron-deficient.

### **7. Acknowledgment**

MA Forner-Giner and G Ancillo are recipient of a contract from Conselleria de Agricultura, Pesca y Alimentación (Generalitat Valenciana, Spain) under Proy\_IVIA09/05 and Proy\_IVIA09/03 respectively. The authors would like to thank Prof. L Navarro and E Primo for critical reading of the manuscript.

### **8. References**


Iron Stress in Citrus 177

Marschner, H. (1995). Mineral Nutrition of Higher Plants. Academic Press, London, UK

Marchner H, & Römheld V.(1995). Strategies of plants for adquisition of iron. In: Abadía J

Molassiotis, A., Tanou, G., Diamantidis, G., Patakas, A., & Therios, I. (2006). Effects of 4-

rootstocks differing in Fe deficiency tolerance. *J. Plant Physiol*. 163: 176-185. Moog PR, & Brüggemann W. (1994). Iron reductase systems on the plant plasma membrane:

Morales, F., Abadía, A., & Abadía, J.( 1990). Characterizacion of the xanthophyll cycle and

Morales, F., Abadía, A., & Abadía, J. (1991). Chlorophyll fluorescence and photon yield of

Morales, F., Abadía, A., Belkhodja, R., & Abadía, J. (1994). Iron deficiency-induced changes

Morales, F., Belkhoja, R.L., Abadía, A., & Abadía, J. (2000). Photosystem II efficiency and

Mortvedt, J.J. (1986). Grain sorghum response to banded acid-type fertilizers in iron

Nishio, J.N., Abadía, J., & Terry, N. (1985). Chlorophyll proteins and electron transport

Papastylianou, I. (1993). Timing and rate of iron chelate application to correct chlorosis in

Pérez-Ruiz J. M., González M.C., Spínola M. C., Sandalio, L. M. & Cejudo F. J. (2009) The

Pestana, M., David, M., De Varennes, A., Abadía, J., Araújo, E., & Faria, E.A.( 2001a).

Pestana, M., Correia, P.J., De Varennes, A., Abadía, J., Araújo, E., & Faria, E.A. (2001b). The

Pestana, M., Varennes, A., Araújo, E., & Faria, E.A. (2003). Diagnosis and correction of iron

Pestana, M., Varennes, A., Abadía, J., & Faria, E.A. (2005). Differential tolerance to iron deficiency of citrus rootstocks grown in nutrient solution. *Sci. Hort*. 104: 25-36. Platt-Aloia, K.A., Thomson, W.W., & Terry, N. 1983. Changes in plastid untraestructure during iron nutrition mediated chloroplast development. *Protoplasma.* 114: 85-92. Rombolà, A.D., Brüggemann, W., Tagliavini, M., Marangoni, B., & Moog, P.R. 2000. Iron

chlorosis in fruit trees: a review. *Food, Agric. & Environ*. 1: 46-51.

(Ed.). Iron Nutrition in Soils and Plants. Kluwer Academic Publishers, Dordrecht,

month Fe deficiency exposure on Fe reduction mechanism, photosyntetic gas exchange, chlorophyll fluorescence and antioxidant defense in two peach

other photosynthetic pigmento changes induced by iron deficiency in sugar beet

oxygen evolution in iron-deficiency sugar beet (Beta vulgaris L.) leaves. *Plant* 

in the photosynthetic pigmento composition of field-grown pear (Pyrus communis

mechanism of energy dissipation in iron-deficient, field grown pear trees (Pyrus

during iron nutrition-mediated chloroplast development. *Plant Physiol.* 78: 296-299.

cuaternary structure of NADPH thioredoxin reductase C is redox sensitive. *Mol.* 

Responses of "Newhall" orange trees to iron deficiency in hydroponics: Effects on leaf chlorophyll, photosynthetic efficiency, and root ferric chelate reductase

use of floral analysis to diagnose the nutritional status of orange trees. *J. Plant Nutr*.

source affects Fe reduction and re-greening of kiwifruit (*Actinidia deliciosa*) leaves. *J.* 

(2ºEd.). 862.

Netherlands. 375-388.

*Physiol.* 97: 886-893.

*Plant* 2:457-467

24, 1913-1823.

a review. *Plant Soil*. 165:241–260.

(Beta vulgaris L.). *Plant Physiol.* 94: 607-613.

L.) leaves. *Plant, Cells&Environment* 17: 1153-1160.

communis L.). *Photosyn. Res.* 63: 9-21.

peanut. *J. of Plant Nutr.* 16: 1193-1203.

activity. *J. Plant Nutr.* 24: 1609-1620.

*of Plant Nutr.* 23: 1751-1765.

deficient soil. *J. Plant Nutr.* 11: 1297-1310.


Fernández-López, J.A., Almela, L., López-Roca, J.M, & Alcaraz, C. (1991). Iron deficiency in

Forner, J.B., Forner-Giner, M.A., & Alcaide A. (2003). Forner-Alcaide 5 and Forner-Alcaide 13: Two new Citrus rootstocks released in Spain. Hort. Sci. 38: 629-630. Forner-Giner, M.A., Alcaide A., Primo-Millo E., & Forner, J.B. (2003). Performance of "Navelina" orange on 14 rootstocks in Northern Valencia (Spain). Sci. Hort. 98: 223 -232. Forner-Giner, MA., Llosá, MJ., Carrasco, J. L., Perez-Amador, M. A., Navarro, L., & Ancillo,

García, P., Abadía, J., & Abadía, A. (1998). Tratamientos foliares para la corrección de la

González-Mas MC., Llosá MJ., Quijano A., & Forner-Giner MA. (2009). Rootstock effects on leaf photosynthesis in Navelina trees grown in calcareous soil. *Hortscience* 44(2) 280-283. Hentze MW, & Kuh LC (1996) Molecular control of vertebrate Fe metabolism: mRNA-based

Heras, L., Sanz, M., & Montañés, L. (1976). Corrección de la clorosis férrica en melocotonero

Hurley, A.K., Walser, R.H., Davis, T.D., & Barney, D.L. (1986). Net photosynthesis and

Jaegger, B., Goldbach, H., & Sommer, K.. (2000). Release from lime induced iron chlorosis by cultan in fruit trees and its characterisation by analysis. *Acta Hort.* 531:107-113. Jones, B.J. (2000). Hydroponics, a Practial Guide for the Soilles Grower. St. Lucie Press.

Landsberg E-C (1986) Function of rhizodermal transfer cells in the FE stress response

Larbi, A., Abadía, A., Abadía, J., & Morales, F.(2006). Down co-regulation of light

Legaz, Z., Serna, M.D., Primo-Millo, E., & Martin, B. (1992). Leaf spray end soil application

Ling, LI, Yan-Hua, F., Xiao-Ying, L., Yan, P. & Ze-Yang, Z. (2002). Expression of ferric

Llosá MJ., Bermejo A., Cano A., & Forner-Giner MA. The citrus rootstocks Cleopatra

Lucena J.J. (2000). Effects of bicarbonate, nitrate and other environmental factors on iron

Lucena, J.J., Romera, F.J., Rojas, C., García, M.J., Morales, M., Montilla, I., Alacántara, E., &

mechanism of *Capsicum annuum* L. *Plant Physiol*., 82: 511–517.

Loué, A. 1993. Oligélements en Agriculture. SCPA-Nathan, Luçon. France. 557.

deficiency chlorosis. A review. *J. Plant Nutr.* 23: 1591-1606

different environments. *Photosynth. Res*. 89: 113-126.

activity. J. Plant Nutr. 14: 1133-1144.

*trifoliata* (L.) Raf.. *J Exp Bot* 61: 483-490.

clorosis férrica. Geórgica. 6: 27-31.

Anales de Aula Dei. 13: 261-289.

*Sci USA,* 93: 8175–8182.

*HortSci*. 21: 1029-1031

Citriculture. 2: 616-617.

*Integrative Plant Biology* 44(7): 771-774.

U.S.A. 42-44.

citrus lemon: effects on photochlorophyllase synthetic pigments and chlorophyllase

G. (2010). Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock *Poncirus* 

regulatory circuits operated by Fe, nitric oxide, and oxidative stress. *Proc Natl Acad* 

y su repercusión sobre el contenido mineral, relaciones nutritivas y rendimientos.

chlorophyll, and foliar iron in apple trees after injection with ferrous sulphate.

absorption, photochemistry, and carboxylation in Fe-deficient plants growing in

of Fe-chelates to Navelina orange trees. Proceedings of the International Society of

chelate reductase gene in *Citrus junos* and *Poncirus trifoliata* Tissues. *Journal of* 

mandarin, Poncirus trifoliata, Forner-Alcaide 5 and Forner-Alcaide 13 vary in susceptibility to iron deficiency chlorosis*. J. Am. Pomological Soc*. 63(4) 160-167.

Pérez-Vicente, R.( 2006). Bicarbonate blocks the expression of several genes involved in the physiological responses to Fe-deficiency of strategy I plants. 13th International Symposium of Iron Nutrition and Interactions in Plants. Montpellier. France. 92.


**8** 

Changhe Zhang1,2 et al.\*

*1Portugal 2China* 

*Apartado 1013, 5001-801 Vila Real* 

**Response, Tolerance and Adaptation to Abiotic** 

**Stress of Olive, Grapevine and Chestnut in the** 

Hot, dry summers and mild to cool, wet winters are the characters of the Mediterranean climate. Drought, extreme temperatures and extreme irradiation (UVs) often concomitantly in some cases also together with salinity, significantly affect the growth, yield and quality of

Olive (*Olea europaea* L), grapevine (*Vitis vinifera* L) and sweet chestnut (*Castanea sativa*) are the most important woody crops in the Mediterranean among others. The olive tree and vineyard are familiar features of the Mediterranean landscape. In some mountain regions, these features are accompanied by the orchards of chestnut. Olive oil and wine are important products in that region. In some regions, such as Italy, Turkey, Spain, Portugal, and Greece, chestnut is one of the most important fruit products as well. Olive oil, grape and wine are a traditional icon of the Mediterranean diet. Enjoying the plentiful indigenous plant products, especially wine,

Olive oil is the main source of fat in the Mediterranean diet and one of those basic ingredients essential to life in the Mediterranean. It may also protect against heart disease, stroke, and certain cancers. The vine and wine are among the most important symbols of

José Gomes-Laranjo1, Carlos M. Correia1, José M. Moutinho-Pereira1, Berta M. Carvalho Gonçalves1,

*1Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of Biology and Environment, University of Trás-os-Montes and Alto Douro (UTAD), Apartado 1013, 5001-801* 

*3CECAV/Department of Chimestry, University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila* 

olive oil and chestnut, is part of the Mediterranean civilization.

Eunice L. V. A. Bacelar1, Francisco P. Peixoto3 and Victor Galhano1

**1. Introduction** 

 \*

*Vila Real, Portugal* 

*Real, Portugal* 

the Mediterranean crops.

**Mediterranean Region: Role of Abscisic** 

 **Acid, Nitric Oxide and MicroRNAs** 

 *University of Trás-os-Montes and Alto Douro (UTAD)* 

*2School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074* 

*1Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of Biology and Environment* 


## **Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs**

Changhe Zhang1,2 et al.\*

*1Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of Biology and Environment University of Trás-os-Montes and Alto Douro (UTAD) Apartado 1013, 5001-801 Vila Real 2School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 1Portugal 2China* 

### **1. Introduction**

178 Plants and Environment

Romera, F.J., Alcántara, E., & De La Guardia., M.D. (1991). Characterization of the tolerance

Römheld V, Marschner H (1981). Iron deficiency stress induced morphological and physiological changes in root tips of sunflower. *Physiol. Plant.* 53(3): 354-360 Römheld, V. (1987). Existence of two difference strategies for the acquisition of iron in

Rosenfield CL, Reed DW, & Kent MW. (1991). Dependency of iron reduction on

Sakano K.(1998). Revision of biochemical pH-stat: involvement of alternative pathway

Schmidt, W., & Bartels, M. (1997.) Topography of the NADH-linked ferric chelate reductase

Schmidt W, Tittel J, & Schikora A. (2000). Role of hormones in the induction of iron deficiency responses in Arabidopsis roots. *Plant Physiol*. 122(4):1109-18. Shlizerman L, Marsh K, Blumwald E, & Sadka A. (2007). Iron-shortage-induced increase in

Soldatini, G., Tognini, M., Castagna, A., Baldan, B., & Ranieri, A. (2000). Alterations in

Spiller, S.C., & Terry, N. (1980). Limiting factors in photosynthesis. II. Iron stress diminishes

Tagliavini, M., & Rombolà, A.D. (2001). Iron deficiency and chlorosis in orchard and

Terry, N. (1980). Limiting factors in photosynthesis I. Use of iron stress to control

Treeby, M., & Uren, N. (1993). Iron deficiency stress responses amongst Citrus rootstocks. Z*.* 

Wallace, A. (1991). Rational approaches to control of iron deficiency other than plant

Wirén, N.V. (2004). Progress in research on iron nutrition and interactions in plants. *Soil* 

Terry, N., & Abadía, J. (1986). Function of iron chloroplasts. *J. of Plant Nutr*. 9(3-7): 609-646. Terry, N., & Zayed, A.M. (1995). Physiology and biochemistry of leaves under iron

vineyard ecosystems. *European Journal of Agronomy*. 15: 71-92.

photochemical capacity in vivo. *Plant Physiol.* 65: 114-120.

Publishers. Dordrecht. ISBN: 0-7923-2900-7: 283-294.

of bicarbonate and phosphate. *Plant Soil*. 130: 115-119.

metabolism. *Plant Cell Physiol*. 39:467–473.

vesicles and calli. *Physiol Plant*. 131(1):72-9.

*of Plant Nutrition.* 23: 1717-1732

*Physiol.* 65: 121-125.

*Pflanzenernahr* 156:75-81.

*Science and Plant Nutr*. 50(7): 955-964.

Netherlands.

95:1120–1124.

Stuttgart. Germany. 58.

D.van der Helm, J.B. Neiland. VCH-Verlag. Weinheim. 353-374

to iron chlorosis in different peach rootstocks grown in nutrient solution. In: Effect

higher plants. Iron transport in microbes, plant and animals. G. Winkelmann.

development of a unique root morphology in *Ficus benjamina* L. *Plant Physiol*.

in plasma membrane from Plantago roots. Abstracts of the IX International Symposium on iron nutrition and interactions in plants. University of Hoheneim.

citric acid content and reduction of cytosolic aconitase activity in Citrus fruit

thylakoid membrane composition induced by iron starvation in sunflower plants. *J.* 

photochemical capacity by reducing the number of photosynthetic units. *Plant* 

deficiency.In Iron Nutrition in Soils and Plants. (J. Abadía ed.). Kluwer Academic

breeding and choice of resistant cultivars. pp. 324-330. In Iron nutrition and interactions in plants. Y. Chen, Y. Hadar (Eds.). Kluwer Academic Publishers. Hot, dry summers and mild to cool, wet winters are the characters of the Mediterranean climate. Drought, extreme temperatures and extreme irradiation (UVs) often concomitantly in some cases also together with salinity, significantly affect the growth, yield and quality of the Mediterranean crops.

Olive (*Olea europaea* L), grapevine (*Vitis vinifera* L) and sweet chestnut (*Castanea sativa*) are the most important woody crops in the Mediterranean among others. The olive tree and vineyard are familiar features of the Mediterranean landscape. In some mountain regions, these features are accompanied by the orchards of chestnut. Olive oil and wine are important products in that region. In some regions, such as Italy, Turkey, Spain, Portugal, and Greece, chestnut is one of the most important fruit products as well. Olive oil, grape and wine are a traditional icon of the Mediterranean diet. Enjoying the plentiful indigenous plant products, especially wine, olive oil and chestnut, is part of the Mediterranean civilization.

Olive oil is the main source of fat in the Mediterranean diet and one of those basic ingredients essential to life in the Mediterranean. It may also protect against heart disease, stroke, and certain cancers. The vine and wine are among the most important symbols of

<sup>\*</sup> José Gomes-Laranjo1, Carlos M. Correia1, José M. Moutinho-Pereira1, Berta M. Carvalho Gonçalves1, Eunice L. V. A. Bacelar1, Francisco P. Peixoto3 and Victor Galhano1

*<sup>1</sup>Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of Biology and Environment, University of Trás-os-Montes and Alto Douro (UTAD), Apartado 1013, 5001-801 Vila Real, Portugal* 

*<sup>3</sup>CECAV/Department of Chimestry, University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal* 

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

exchange when transpiration is restricted by stomatal closure.

reduce the transpiration of the leaves.

olive leaves (Karabourniotis et al., 1994).

& Ferreres, 2005).

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 181

mm year1 (Bongi & Palliotti, 1994). Abd-El-Rahman et al. (1966) measured the water content of olive leaves at saturation, finding a value, 1.59 g water g1 dry weight, extremely low compared with other species growing in the same environment (5.77 g water g1 dry weight for fig, 5.85 g water g1 dry weight for grape). There are many mechanisms by which it resists to more or less extended drought periods but some differences among olive cultivars have been observed concerning their ability for adaptation and production under drought

Olive culture has prospered under rainfed conditions in Mediterranean environments because the tree is capable of acceptable yield while subjected to the characteristic prolonged summer water shortage. Olive achieves this result with physiological, biochemical and morpho-anatomical responses that reduce water loss and maintain water uptake at high plant water status as drought commences (drought avoidance), and with others that tolerate dehydration at low plant water status as the drought deepens (drought tolerance) (Connor

Olive leaves are well designed to control water loss. Morphological characteristics allow minimum radiation load and maximum heat exchange while the physiological responses of stomata to leaf water status and atmospheric humidity provide effective control of transpiration (Fernández et al., 1997; Loreto & Sharkey, 1990). Leaves minimise radiation load by small size, a dominantly vertical display (Mariscal et al., 2000) that is further aided by paraheliotropic movement under water stress (Natali et al., 1999) (Fig. 1A), a dense packing of the mesophyll layers (Bongi et al., 1987) and high reflectivity by a thick cuticle and epicuticular wax layers (Leon & Bukovac, 1978) (Fig. 1B). This combination of morphological features restricts temperature increase in leaves with small latent heat

Stomata are small and dense and occur only on the abaxial surface, under dense layers of peltate trichomes (or peltate scales) (Fig. 1C). The peltate trichomes reflect the sunlight and

An interesting characteristic in the anatomy of olive leaf is the presence of a complicated, dense network of filiform sclereids that are of idioblast nature (Karabourniotis et al., 1994) (Fig. 1D). This entangled network follows two major distribution patterns: the "subepidermal layer" consisting of the "T"-shaped sclereids extending between the adaxial epidermis and the palisade layer, and the branched the ῾polymorphic sclereids that transverse the spongy mesophyll layers, producing a chaotic pattern. Sclereids act like synthetic optical fibres and, besides other functions, may contribute to the improvement of the light microenvironment within the mesophyll of the thick and compact sclerophyllous

It has been reported that olive leaves formed under water stress are more able to control transpiration, being smaller and thicker and having more dense and smaller stomata (Bosabalidis & Kofidis, 2002; Chartzoulakis et al., 1999b). However, Lo Gullo and Salleo (1988) observed that despite all this protection against water loss, leaves of the wild olive

A drought avoidance response not displayed by olive is the development of a deep rooting system (Bongi & Palliotti, 1994). However, the extensive root system of olive tree seems to be designed for absorbing the water of the light and intermittent rainfall usual in its habitat (Fernández & Moreno, 1999). Most of the main roots grow more or less in parallel to the soil surface, and the highest root density is found close to the trunk, although the volume explored by the roots can easily extend beyond the canopy projection (Fernández & Moreno,

tree (*O. oleaster*) underwent a substantial water loss under conditions of water stress.

conditions (Bacelar et al., 2004; Bosabalidis & Kofidis, 2002; Chartzoulakis et al., 1999b).

societies that have emerged around the shores of the Mediterranean (Stanislawski, 1970). In most Mediterranean countries such as Portugal, France, Italy, Greece and Spain—wine is more than just a beverage; it is an integral part of meals and an essential aspect of social gatherings.

The European chestnut species *C. sativa* has been cultivated in the Mediterranean region for both fruit and timber for dozens of centuries. Sweet chestnut provided staple food with nutritious and health properties for people in the Mediterranean for centuries especially in the mountains and used to be called the 'bread-tree' (Avanzato, 2009). Chestnuts are delicious and healthy foods, containing many highly valuable carbohydrates and phytochemicals, and no cholesterol and low fat. It is an ingredient in many traditional recipes.

Plant abiotic stresses and response of plants to these stresses have been extensively studied. In this chapter, we have summarized the recent advance in the response, tolerance and adaptation of these Mediterranean woody crops to the environmental stresses especially drought and extreme temperatures imposed by the typical Mediterranean climate, and the underlying mechanism. At molecular level, plants share some common pathways involved in different abiotic stress responses. Different forms of abiotic stresses may lead to similar responses in plants during the stress; likewise, different kinds of stresses have also been found to trigger responses in similar sets signalling molecules. The perception of stresses and the consequent adaptation by plants include physiological, molecular and biochemical changes in plants which largely depend on factors such as severity of stress, plant developmental stage and their genotype (Agarwal & Zhu, 2005). After the perception of a signal by plants, immediately there will be generated secondary signals which are normally nonprotein molecules, including membrane ion (K+ and Ca2+) flux, inositol phosphates (IPs), reactive oxygen species (ROS), and nitric oxide (NO). Each of these can activate plant mitogen-activated protein kinase (MAPK) and Ca2+-dependant protein kinase (CDPK) and activation of protein phosphatases. These early events lead to hormone accumulation, particularly abscisic acid (ABA), salicylic acid (SA) and brassinosteroid hormonal, the synthesis of heat shock proteins, activation of antioxidant enzymes and synthesis of low molecular antioxidants and compatible solutes and membrane lipid peroxidation, followed by changes in transpiration, gas exchange, respiration, and growth, resulting in stress adaptation. MicroRNAs (miRNAs) also participate in stress adaption response in plants. In this chapter we concentrate in the role of ABA, NO and miRNAs in the abiotic stress response and adaptation. Finally, the progress in genetic modification targeting improved abiotic stress tolerance of these plant species is reviewed.

### **2. Morpho-anatomical, physiological, and biochemical response and adaption**

### **2.1 Olive capacity to withstand arid environments**

Olive is a perennial, long-lived, evergreen tree of subtropical origin (Bongi & Palliotti, 1994) that, in the Mediterranean, flowers in mid-to-late spring. This adaptation allows olive to escape the deleterious effects of cold on flowering and fruit set but serves to increase reliance on a range of avoidance and tolerance mechanisms that maintain internal water status and metabolic activity during the hot, dry summers (Connor, 2005).

Olive tree is well known to be very resistant to drought (Bacelar et al., 2009; Bacelar et al., 2006; Connor, 2005; Fernández & Moreno, 1999; Giorio et al., 1999; Tognetti et al., 2004). Furthermore, it has been postulated that the minimum water requirement for olive is 200

societies that have emerged around the shores of the Mediterranean (Stanislawski, 1970). In most Mediterranean countries such as Portugal, France, Italy, Greece and Spain—wine is more than just a beverage; it is an integral part of meals and an essential aspect of social

The European chestnut species *C. sativa* has been cultivated in the Mediterranean region for both fruit and timber for dozens of centuries. Sweet chestnut provided staple food with nutritious and health properties for people in the Mediterranean for centuries especially in the mountains and used to be called the 'bread-tree' (Avanzato, 2009). Chestnuts are delicious and healthy foods, containing many highly valuable carbohydrates and phytochemicals, and no cholesterol and low fat. It is an ingredient in many traditional

Plant abiotic stresses and response of plants to these stresses have been extensively studied. In this chapter, we have summarized the recent advance in the response, tolerance and adaptation of these Mediterranean woody crops to the environmental stresses especially drought and extreme temperatures imposed by the typical Mediterranean climate, and the underlying mechanism. At molecular level, plants share some common pathways involved in different abiotic stress responses. Different forms of abiotic stresses may lead to similar responses in plants during the stress; likewise, different kinds of stresses have also been found to trigger responses in similar sets signalling molecules. The perception of stresses and the consequent adaptation by plants include physiological, molecular and biochemical changes in plants which largely depend on factors such as severity of stress, plant developmental stage and their genotype (Agarwal & Zhu, 2005). After the perception of a signal by plants, immediately there will be generated secondary signals which are normally nonprotein molecules, including membrane ion (K+ and Ca2+) flux, inositol phosphates (IPs), reactive oxygen species (ROS), and nitric oxide (NO). Each of these can activate plant mitogen-activated protein kinase (MAPK) and Ca2+-dependant protein kinase (CDPK) and activation of protein phosphatases. These early events lead to hormone accumulation, particularly abscisic acid (ABA), salicylic acid (SA) and brassinosteroid hormonal, the synthesis of heat shock proteins, activation of antioxidant enzymes and synthesis of low molecular antioxidants and compatible solutes and membrane lipid peroxidation, followed by changes in transpiration, gas exchange, respiration, and growth, resulting in stress adaptation. MicroRNAs (miRNAs) also participate in stress adaption response in plants. In this chapter we concentrate in the role of ABA, NO and miRNAs in the abiotic stress response and adaptation. Finally, the progress in genetic modification targeting improved

abiotic stress tolerance of these plant species is reviewed.

**2.1 Olive capacity to withstand arid environments** 

**2. Morpho-anatomical, physiological, and biochemical response and** 

status and metabolic activity during the hot, dry summers (Connor, 2005).

Olive is a perennial, long-lived, evergreen tree of subtropical origin (Bongi & Palliotti, 1994) that, in the Mediterranean, flowers in mid-to-late spring. This adaptation allows olive to escape the deleterious effects of cold on flowering and fruit set but serves to increase reliance on a range of avoidance and tolerance mechanisms that maintain internal water

Olive tree is well known to be very resistant to drought (Bacelar et al., 2009; Bacelar et al., 2006; Connor, 2005; Fernández & Moreno, 1999; Giorio et al., 1999; Tognetti et al., 2004). Furthermore, it has been postulated that the minimum water requirement for olive is 200

gatherings.

recipes.

**adaption** 

mm year1 (Bongi & Palliotti, 1994). Abd-El-Rahman et al. (1966) measured the water content of olive leaves at saturation, finding a value, 1.59 g water g1 dry weight, extremely low compared with other species growing in the same environment (5.77 g water g1 dry weight for fig, 5.85 g water g1 dry weight for grape). There are many mechanisms by which it resists to more or less extended drought periods but some differences among olive cultivars have been observed concerning their ability for adaptation and production under drought conditions (Bacelar et al., 2004; Bosabalidis & Kofidis, 2002; Chartzoulakis et al., 1999b).

Olive culture has prospered under rainfed conditions in Mediterranean environments because the tree is capable of acceptable yield while subjected to the characteristic prolonged summer water shortage. Olive achieves this result with physiological, biochemical and morpho-anatomical responses that reduce water loss and maintain water uptake at high plant water status as drought commences (drought avoidance), and with others that tolerate dehydration at low plant water status as the drought deepens (drought tolerance) (Connor & Ferreres, 2005).

Olive leaves are well designed to control water loss. Morphological characteristics allow minimum radiation load and maximum heat exchange while the physiological responses of stomata to leaf water status and atmospheric humidity provide effective control of transpiration (Fernández et al., 1997; Loreto & Sharkey, 1990). Leaves minimise radiation load by small size, a dominantly vertical display (Mariscal et al., 2000) that is further aided by paraheliotropic movement under water stress (Natali et al., 1999) (Fig. 1A), a dense packing of the mesophyll layers (Bongi et al., 1987) and high reflectivity by a thick cuticle and epicuticular wax layers (Leon & Bukovac, 1978) (Fig. 1B). This combination of morphological features restricts temperature increase in leaves with small latent heat exchange when transpiration is restricted by stomatal closure.

Stomata are small and dense and occur only on the abaxial surface, under dense layers of peltate trichomes (or peltate scales) (Fig. 1C). The peltate trichomes reflect the sunlight and reduce the transpiration of the leaves.

An interesting characteristic in the anatomy of olive leaf is the presence of a complicated, dense network of filiform sclereids that are of idioblast nature (Karabourniotis et al., 1994) (Fig. 1D). This entangled network follows two major distribution patterns: the "subepidermal layer" consisting of the "T"-shaped sclereids extending between the adaxial epidermis and the palisade layer, and the branched the ῾polymorphic sclereids that transverse the spongy mesophyll layers, producing a chaotic pattern. Sclereids act like synthetic optical fibres and, besides other functions, may contribute to the improvement of the light microenvironment within the mesophyll of the thick and compact sclerophyllous olive leaves (Karabourniotis et al., 1994).

It has been reported that olive leaves formed under water stress are more able to control transpiration, being smaller and thicker and having more dense and smaller stomata (Bosabalidis & Kofidis, 2002; Chartzoulakis et al., 1999b). However, Lo Gullo and Salleo (1988) observed that despite all this protection against water loss, leaves of the wild olive tree (*O. oleaster*) underwent a substantial water loss under conditions of water stress.

A drought avoidance response not displayed by olive is the development of a deep rooting system (Bongi & Palliotti, 1994). However, the extensive root system of olive tree seems to be designed for absorbing the water of the light and intermittent rainfall usual in its habitat (Fernández & Moreno, 1999). Most of the main roots grow more or less in parallel to the soil surface, and the highest root density is found close to the trunk, although the volume explored by the roots can easily extend beyond the canopy projection (Fernández & Moreno,

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

Tognetti et al., 2002).

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 183

of olive xylem is a feature that seems to play an important role in the tree's water relations. Salleo and Lo Gullo (1993) observed losses of about 10% of hydraulic conductivity in 1-yearold twigs of young *O. oleaster* trees, when these became stressed, due to xylem cavitation. One consequence of this is that olive trees prevent excessive water loss on days of high water demand by closing their stomata soon after midmorning (Fernández et al., 1997). During periods of water stress, olive tree typically experience reductions in transpiration, stomatal conductance and net photosynthesis (Giorio et al., 1999). Nevertheless, environmental and physiological factors do not affect H2O and CO2 exchange to the same extent, resulting in possible variations in water use efficiency in this species (Xiloyannis et al., 1988). Meanwhile, some differences in gas exchange responses to water stress between olive cultivars have been observed in previous experiments (Chartzoulakis et al., 1999a;

In moderate drought conditions, olive plants stop shoot growth but not photosynthetic activity and transpiration. This allows the continued production of assimilates as well as their accumulation in the various plant parts, in particular in the root system, creating a

Olive tolerates drought by maintaining turgor through osmotic adjustment and changes in cell wall elasticity (Connor, 2005). Active and passive osmotic adjustment plays an important role in maintaining cell turgor and leaf activities which depend on it (Xiloyannis et al., 1999). Mannitol and glucose play a major part in the osmotic adjustment of olive leaves (Cataldi et al., 2000). In addition, the osmotic adjustment observed in the root system allows maintenance of cell turgor, avoiding or delaying the separation of roots from soil particles (Xiloyannis et al., 1999). The accumulation of proline under drought stress in both leaves and roots of 2-year-old *O. europaea* (cv. Coratina) plants (Sofo et al., 2004b) also

Under field conditions, particularly in the Mediterranean regions, water stress is often accompanied by other environmental constraints, such as steep leaf-to-air water vapour gradients, and high irradiance and temperature (Osório et al., 2006). Measurements have revealed non-stomatal limitations to photosynthesis consistent with photoinhibition in olive leaves exposed to high irradiance (Angelopoulos et al., 1996). The synergic action of high irradiance level and water stress reduces the capacity of the photosynthetic systems to utilize incident radiation, leading to a higher degree of photodamage (Bacelar et al., 2007; Sofo et al., 2004a). The increase of malondialdehyde content and lipoxygenase activity, two markers of oxidative damage, observed by Sofo et al., (2004b) in both leaf and root tissues of olive plants during the progressive increment of drought stress, indicates that water deficit induces lipid peroxidation. This result suggests that higher activities of some antioxidant enzymes and non-enzymatic antioxidants are required for a better protection against

Most of the world Wine Regions, such as the Douro Region in Portugal, has a Mediterranean climate with a strong continental influence. In these regions the rainfall is mainly concentrated in the winter months and the springs and summers are characterized by exceedingly hot and dry. In these conditions grapevines are often subjected to periods of severe drought associated with strong light and high temperature (Chaves et al., 2002). Consequently, the vineyard experiences irreparable damage on physiology behaviour and

higher root/leaf ratio compared to well-watered plants (Xiloyannis et al., 1999).

indicates a possible role of proline in drought tolerance.

**2.2 Vine's response, tolerance and adaptation to abiotic stress** 

oxidative stress related to water deficit.

1999). This rooting habit is probably the result of sensitivity to hypoxia and may allow for efficient water absorption (Bongi & Palliotti, 1994; Fernández & Moreno, 1999). A high portion of the root is of small diameter, which also favours the absorption capacity. Absorption by olive roots is also enhanced by high potential gradients between roots and soil caused by osmotic adjustment (Fernández & Moreno, 1999).

Fig. 1. Olive protections at leaf level against water loss and excessive irradiance. (A) Paraheliotropic movement under water stress; (B) Dense packing of the mesophyll layers and thick cuticle and epicuticular wax layers (optical micrograph); (C) Dense trichome layer of abaxial surface protecting the stomata (SEM micrograph); (D) Dense network of sclereids (optical micrograph, cross-polarized light).

The olive is a diffuse-porous tree having a dense wood with abundant fibers and little parenchyma (Fernández & Moreno, 1999). The large amount of fibers, which makes olive wood so hard, accounts for the low vessel lumina of the species in comparison with other diffuse-porous Mediterranean plants. Salleo et al. (1985) observed that the vessel lumina, when expressed as percentage of the total xylem cross-sectional area, was half that measured in other Mediterranean species such as *V. vinifera*. The low hydraulic conductivity

1999). This rooting habit is probably the result of sensitivity to hypoxia and may allow for efficient water absorption (Bongi & Palliotti, 1994; Fernández & Moreno, 1999). A high portion of the root is of small diameter, which also favours the absorption capacity. Absorption by olive roots is also enhanced by high potential gradients between roots and

Fig. 1. Olive protections at leaf level against water loss and excessive irradiance. (A) Paraheliotropic movement under water stress; (B) Dense packing of the mesophyll layers and thick cuticle and epicuticular wax layers (optical micrograph); (C) Dense trichome layer of abaxial surface protecting the stomata (SEM micrograph); (D) Dense network of sclereids

**100 μm D**

The olive is a diffuse-porous tree having a dense wood with abundant fibers and little parenchyma (Fernández & Moreno, 1999). The large amount of fibers, which makes olive wood so hard, accounts for the low vessel lumina of the species in comparison with other diffuse-porous Mediterranean plants. Salleo et al. (1985) observed that the vessel lumina, when expressed as percentage of the total xylem cross-sectional area, was half that measured in other Mediterranean species such as *V. vinifera*. The low hydraulic conductivity

**C 100 μm** 

**B 100 μm** 

(optical micrograph, cross-polarized light).

**A** 

soil caused by osmotic adjustment (Fernández & Moreno, 1999).

of olive xylem is a feature that seems to play an important role in the tree's water relations. Salleo and Lo Gullo (1993) observed losses of about 10% of hydraulic conductivity in 1-yearold twigs of young *O. oleaster* trees, when these became stressed, due to xylem cavitation. One consequence of this is that olive trees prevent excessive water loss on days of high water demand by closing their stomata soon after midmorning (Fernández et al., 1997).

During periods of water stress, olive tree typically experience reductions in transpiration, stomatal conductance and net photosynthesis (Giorio et al., 1999). Nevertheless, environmental and physiological factors do not affect H2O and CO2 exchange to the same extent, resulting in possible variations in water use efficiency in this species (Xiloyannis et al., 1988). Meanwhile, some differences in gas exchange responses to water stress between olive cultivars have been observed in previous experiments (Chartzoulakis et al., 1999a; Tognetti et al., 2002).

In moderate drought conditions, olive plants stop shoot growth but not photosynthetic activity and transpiration. This allows the continued production of assimilates as well as their accumulation in the various plant parts, in particular in the root system, creating a higher root/leaf ratio compared to well-watered plants (Xiloyannis et al., 1999).

Olive tolerates drought by maintaining turgor through osmotic adjustment and changes in cell wall elasticity (Connor, 2005). Active and passive osmotic adjustment plays an important role in maintaining cell turgor and leaf activities which depend on it (Xiloyannis et al., 1999). Mannitol and glucose play a major part in the osmotic adjustment of olive leaves (Cataldi et al., 2000). In addition, the osmotic adjustment observed in the root system allows maintenance of cell turgor, avoiding or delaying the separation of roots from soil particles (Xiloyannis et al., 1999). The accumulation of proline under drought stress in both leaves and roots of 2-year-old *O. europaea* (cv. Coratina) plants (Sofo et al., 2004b) also indicates a possible role of proline in drought tolerance.

Under field conditions, particularly in the Mediterranean regions, water stress is often accompanied by other environmental constraints, such as steep leaf-to-air water vapour gradients, and high irradiance and temperature (Osório et al., 2006). Measurements have revealed non-stomatal limitations to photosynthesis consistent with photoinhibition in olive leaves exposed to high irradiance (Angelopoulos et al., 1996). The synergic action of high irradiance level and water stress reduces the capacity of the photosynthetic systems to utilize incident radiation, leading to a higher degree of photodamage (Bacelar et al., 2007; Sofo et al., 2004a). The increase of malondialdehyde content and lipoxygenase activity, two markers of oxidative damage, observed by Sofo et al., (2004b) in both leaf and root tissues of olive plants during the progressive increment of drought stress, indicates that water deficit induces lipid peroxidation. This result suggests that higher activities of some antioxidant enzymes and non-enzymatic antioxidants are required for a better protection against oxidative stress related to water deficit.

### **2.2 Vine's response, tolerance and adaptation to abiotic stress**

Most of the world Wine Regions, such as the Douro Region in Portugal, has a Mediterranean climate with a strong continental influence. In these regions the rainfall is mainly concentrated in the winter months and the springs and summers are characterized by exceedingly hot and dry. In these conditions grapevines are often subjected to periods of severe drought associated with strong light and high temperature (Chaves et al., 2002). Consequently, the vineyard experiences irreparable damage on physiology behaviour and

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

soil preparation are one way to achieving these mitigation objectives.

**3. Limitations of European chestnut growth at low latitudes** 

United Kingdom, northern Germany, Poland and Ukraine.

Matthews, 1988).

alterations.

(Patakas & Noitsakis, 1999).

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 185

roots and/or by reducing water loss. The formation of a deeper and dense root system by rootstock depends on the interaction of its genetic characteristics and is usually an effective strategy for grapevine to capture more water in periods of lower water availability (Palliotti et al., 2000). In this context, the selection of rootstock with these characteristics and a good

One of the most widespread mechanisms to reduce grapevine water loss is achieved through lower vigor and/or partial senescence of leaves (Chaves, 1991). The increase in stomatal conductance mediated by the ABA concentration is another mechanism developed for the same purpose, especially in the periods of the day with deficits of higher vapor pressure (Iacono et al., 1998). The prevention of photoinhibition and overheating of the leaves in consequence of the lower leaf transpiration can also be undertaken by changing the

The grapevine adaptations to dry and hot habitats seem to be strengthened by changes that occur at the level of vascular system, particularly the reduction in xylem section, which induce a significant decrease in hydraulic conductivity and thus minimize the susceptibility of these vessels to the phenomenon of cavitation (Lovisolo & Schubert, 1998; Schultz &

The active accumulation of soluble sugars and other low molecular compounds is responsible for lower osmotic potential, allowing the cell turgor maintained as much as possible, with positive values. This process, known as osmotic adjustment, has been shown in vines gradually subjected to water stress, either in leaves (Düring, 1984) or in roots (Düring & Dry, 1995). Under prolonged drought, a decrease of 4 to 5 bars in osmotic potential, mainly more evident and rapid in young leaves than in adult leaves (Düring, 1984). The capacity for lowering the osmotic potential might be the dominant strategy for better restricting the leaf water losses in grapevines growing under water stress conditions

European chestnut (*Castanea sativa* Mill.) is characterized as a mesophilic species (Cortizo et al., 1996). Plants from this species are moderately thermophilic and well adapted to ecosystems with a yearly mean temperature ranging between 8 ºC and 15 ºC and monthly mean temperatures during 6 months over 10 ºC. Unfortunatly, nowadays, chestnut tree growth shows some constrains which might be partially attributed to the climatic

In Europe, the chestnut is widespread. The Azores archipelago (25º - 31º W) is the most Occidental point for *C. sativa* and the Canary Islands is the most Southern point (27º - 29º N). Towards the north, chestnut fruit production reaches 52º N latitude to the south of the

It is found at sea level in some littoral areas above 39º N latitude, as such the northern Iberian peninsula, north of Italy and Middle Eastern Greece due to sea influence. Below this latitude, still in the littoral areas, adequated climatic conditions for chestnut are found in higher altitudes, as it happens in Sierra Nevada (1500 m a.s.l., Granada, Southern Spain), Teide Mountain (2000 m a.s.l., Santa Cruz Tenerife, Canary Islands) or in Etna mountain (2000 m a.s.l., Sicily Island, Southern Italy). In the interior part of Europe, under continental climatic influence, chestnut only can grow above 500 m a.s.l. being the maximal altitude 1100 m a.s.l. in the highest mountains of Trás-os-Montes (Northeast of Portugal) or even to

leaf angle, e.g., from 53° to 80° (angle between the blade and petiole) (Smart, 1974).

yield attributes. The implementation of cultural strategies, which aims a better adaptation to these conditions, is a major goal, especially in the current scenario of global climate change (IPCC 2007).

### **2.2.1 Abiotic stress response**

Under summer stress and as the first limitation, the photosynthetic productivity is limited by the stomatal closure, either in response to a large decrease in leaf water potential or due to an increase in atmospheric vapour pressure deficit. Several studies undertaken in the Douro Region clearly have shown that grapevines growing under severe summer stress experience a significant decline in productivity, mostly owing to stomatal limitations to photosynthesis (Moutinho-Pereira et al., 2004).

Grapevine cultivars differ in the degree of control exerted by stomata under conditions of water limitation. While some varieties are genetically programmed to react to early signs of dryness in the air and/or soil, others may have greater difficulties in stomatal regulation (Moutinho-Pereira et al., 2007). For instance, under water stress conditions the water use efficiency and the correlation between net photosynthesis rate and stomatal conductance are significantly higher in 'Riesling' than that in 'Silvaner' (Düring, 1987). The ABA concentration, arriving from roots to leaves, is directly implicated in this behaviour (Correia et al., 1995). On the other hand, in grapevine the stomata response to ABA concentration is not uniform across the leaf surface. According to Düring (1992), this behaviour is related with the heterobaric anatomy (patchiness) of vine leaves, which makes the gas diffusion difficult in the intercellular spaces of the mesophyll and is responsible for non-uniform aperture of stomata over the leaf surface.

The photosynthetic apparatus is generally tolerant to water stress. However, if the imposition of dehydration of mesophyll cells is moderate but continued or severe but brief, a metabolic adjustment takes place through metabolic pathways, mainly related with RuBP regeneration and Rubisco activity (Medrano et al., 2002).

The structural integrity of chloroplasts and the photochemical reactions and electron transport chain do not seem to be much affected by low water potentials. Only the thickness of thylakoid lamellae seems to decrease (Chaves, 1991). In fact, Flexas et al. (1998) and Escalona et al. (1999) found that in its natural environment and under water stress the vine only developed a few signs of down-regulation of the photochemical activity. However, in Mediterranean climate, water stress is usually associated with many clear and hot days, which, in a synergistic action, leads to a significant down-regulation or photoinhibition of the photosynthetic apparatus (Osório et al., 1995). Under these conditions, the vineyard experiences irreparable damage. Frequently, some leaves display irreversible photoinhibition and chlorosis, followed by necrosis and leading to low grapevine water-use efficiency (Moutinho-Pereira et al., 2003). The air temperature increase will accelerate the grapevine phenology, leading to a reduction in the vegetative and reproductive period (Seguin & Cortazar, 2005).

### **2.2.2 Tolerance and adaptation**

The ability of the vineyards to grow and produce satisfactorily in severe summer stress conditions depends on the development of morphological and physiological mechanisms, which allows them to retard the level of dehydration that is detrimental to cellular metabolism. In general, this is achieved through an improvement in water absorption by

yield attributes. The implementation of cultural strategies, which aims a better adaptation to these conditions, is a major goal, especially in the current scenario of global climate change

Under summer stress and as the first limitation, the photosynthetic productivity is limited by the stomatal closure, either in response to a large decrease in leaf water potential or due to an increase in atmospheric vapour pressure deficit. Several studies undertaken in the Douro Region clearly have shown that grapevines growing under severe summer stress experience a significant decline in productivity, mostly owing to stomatal limitations to

Grapevine cultivars differ in the degree of control exerted by stomata under conditions of water limitation. While some varieties are genetically programmed to react to early signs of dryness in the air and/or soil, others may have greater difficulties in stomatal regulation (Moutinho-Pereira et al., 2007). For instance, under water stress conditions the water use efficiency and the correlation between net photosynthesis rate and stomatal conductance are significantly higher in 'Riesling' than that in 'Silvaner' (Düring, 1987). The ABA concentration, arriving from roots to leaves, is directly implicated in this behaviour (Correia et al., 1995). On the other hand, in grapevine the stomata response to ABA concentration is not uniform across the leaf surface. According to Düring (1992), this behaviour is related with the heterobaric anatomy (patchiness) of vine leaves, which makes the gas diffusion difficult in the intercellular spaces of the mesophyll and is responsible for non-uniform

The photosynthetic apparatus is generally tolerant to water stress. However, if the imposition of dehydration of mesophyll cells is moderate but continued or severe but brief, a metabolic adjustment takes place through metabolic pathways, mainly related with RuBP

The structural integrity of chloroplasts and the photochemical reactions and electron transport chain do not seem to be much affected by low water potentials. Only the thickness of thylakoid lamellae seems to decrease (Chaves, 1991). In fact, Flexas et al. (1998) and Escalona et al. (1999) found that in its natural environment and under water stress the vine only developed a few signs of down-regulation of the photochemical activity. However, in Mediterranean climate, water stress is usually associated with many clear and hot days, which, in a synergistic action, leads to a significant down-regulation or photoinhibition of the photosynthetic apparatus (Osório et al., 1995). Under these conditions, the vineyard experiences irreparable damage. Frequently, some leaves display irreversible photoinhibition and chlorosis, followed by necrosis and leading to low grapevine water-use efficiency (Moutinho-Pereira et al., 2003). The air temperature increase will accelerate the grapevine phenology, leading to a reduction in the vegetative and reproductive period

The ability of the vineyards to grow and produce satisfactorily in severe summer stress conditions depends on the development of morphological and physiological mechanisms, which allows them to retard the level of dehydration that is detrimental to cellular metabolism. In general, this is achieved through an improvement in water absorption by

(IPCC 2007).

**2.2.1 Abiotic stress response** 

photosynthesis (Moutinho-Pereira et al., 2004).

aperture of stomata over the leaf surface.

(Seguin & Cortazar, 2005).

**2.2.2 Tolerance and adaptation** 

regeneration and Rubisco activity (Medrano et al., 2002).

roots and/or by reducing water loss. The formation of a deeper and dense root system by rootstock depends on the interaction of its genetic characteristics and is usually an effective strategy for grapevine to capture more water in periods of lower water availability (Palliotti et al., 2000). In this context, the selection of rootstock with these characteristics and a good soil preparation are one way to achieving these mitigation objectives.

One of the most widespread mechanisms to reduce grapevine water loss is achieved through lower vigor and/or partial senescence of leaves (Chaves, 1991). The increase in stomatal conductance mediated by the ABA concentration is another mechanism developed for the same purpose, especially in the periods of the day with deficits of higher vapor pressure (Iacono et al., 1998). The prevention of photoinhibition and overheating of the leaves in consequence of the lower leaf transpiration can also be undertaken by changing the leaf angle, e.g., from 53° to 80° (angle between the blade and petiole) (Smart, 1974).

The grapevine adaptations to dry and hot habitats seem to be strengthened by changes that occur at the level of vascular system, particularly the reduction in xylem section, which induce a significant decrease in hydraulic conductivity and thus minimize the susceptibility of these vessels to the phenomenon of cavitation (Lovisolo & Schubert, 1998; Schultz & Matthews, 1988).

The active accumulation of soluble sugars and other low molecular compounds is responsible for lower osmotic potential, allowing the cell turgor maintained as much as possible, with positive values. This process, known as osmotic adjustment, has been shown in vines gradually subjected to water stress, either in leaves (Düring, 1984) or in roots (Düring & Dry, 1995). Under prolonged drought, a decrease of 4 to 5 bars in osmotic potential, mainly more evident and rapid in young leaves than in adult leaves (Düring, 1984). The capacity for lowering the osmotic potential might be the dominant strategy for better restricting the leaf water losses in grapevines growing under water stress conditions (Patakas & Noitsakis, 1999).

### **3. Limitations of European chestnut growth at low latitudes**

European chestnut (*Castanea sativa* Mill.) is characterized as a mesophilic species (Cortizo et al., 1996). Plants from this species are moderately thermophilic and well adapted to ecosystems with a yearly mean temperature ranging between 8 ºC and 15 ºC and monthly mean temperatures during 6 months over 10 ºC. Unfortunatly, nowadays, chestnut tree growth shows some constrains which might be partially attributed to the climatic alterations.

In Europe, the chestnut is widespread. The Azores archipelago (25º - 31º W) is the most Occidental point for *C. sativa* and the Canary Islands is the most Southern point (27º - 29º N). Towards the north, chestnut fruit production reaches 52º N latitude to the south of the United Kingdom, northern Germany, Poland and Ukraine.

It is found at sea level in some littoral areas above 39º N latitude, as such the northern Iberian peninsula, north of Italy and Middle Eastern Greece due to sea influence. Below this latitude, still in the littoral areas, adequated climatic conditions for chestnut are found in higher altitudes, as it happens in Sierra Nevada (1500 m a.s.l., Granada, Southern Spain), Teide Mountain (2000 m a.s.l., Santa Cruz Tenerife, Canary Islands) or in Etna mountain (2000 m a.s.l., Sicily Island, Southern Italy). In the interior part of Europe, under continental climatic influence, chestnut only can grow above 500 m a.s.l. being the maximal altitude 1100 m a.s.l. in the highest mountains of Trás-os-Montes (Northeast of Portugal) or even to

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

predawn leaf water potential in the range of -0.6 to -0.9 MPa.

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 187

In the interior regions of Europe, where chestnut grows under continental climatic influence, altitude is decisive to define adequated climatic conditions. As the Fig. 3 shows, photosynthesis increases with altitude, and inversly with temperature. So, maximal rates can be found above 800 m a.s.l. where temperature down to 25 – 22 ºC, the range of optimal temperature as has been refered above. In the lowest altitudes, photosynthesis rate decreases around 40%, indicating that chestnuts under these climatic conditions, nowadays start to suffer from abiotic stresses, mainly due to the heat stress. Regarding internal water balance, Martins et al., (2010) have demonstrated that adult trees can be saved from water stress, since they can continuosly absorb water from deep soil layers and so preserving

Fig. 3. Variation of mean daily photosynthesis rate (A, closed symbols) in leaves from chestnut (var. Judia) and air temperature (open symbols) as a function of altitude.

differences, but are simply phenotypic adaptations to different climatic conditions.

Additionally, the European chestnut ecotypes coming from wet sites are more locally adapted and less plastic than those from dry sites and hence more vulnerable to the climate changes (Villani et al., 2010). Five gene pools have been determined in Europe: three in Greece, one on the northwestern coast of the Iberian Peninsula and a large gene pool covering the rest of the Mediterranean basin (Martin et al., 2010; Mattioni et al., 2008). The existence of some adaptative variation among populations from extreme conditions is proposed (Fernández-López et al., 2005): populations from Greece initiate growth earlier followed by those from South Italy and South Spain, while ecotypes from north Spain and Italy initiate later. A significant genetic variation between north and south Iberian ecotypes has also been confirmed (Fernández-López et al., 2005). The expected global climate changes are a great challenge for forest tree breeders. Eriksson et al., (2005) stablished a xerothermic index to characterize each one of those ecotype´s local origin and they found a negative correlation between it and plant growth at both 25ºC and 32ºC, but a positive correlation with carbon isotope discrimination, suggesting a large additive coefficient of variation for growth traits. This variation confers to the species good possibilities to respond genetically via natural or artificial selection to enviromental change. Dinis et al., (2011) working with plants from the portuguese Judia variety, have also concluded that the morphological and phenological differences among ecotypes are not only related to the small genetic

1800 m in Caucasus Mountains, the former altitudes corresponding to the ancient orchards and the highest altitudes to the newest plantations (Gomes-Laranjo et al., 2005; Pereira-Lorenzo et al., 2010).

Below 600 m a.s.l. climate is hotter and dryer than the adequate conditions for chestnut, corresponding to a transition altitude, vineyards, olive tree and almond being now the main crops. Contrarely, above 1100 m a.s.l. climate is colder and wetter, and vegetative cycle is shorter than that needed for fruit production. So, typical climate is a continental temperate type, with mean annual values of sunlight and precipitation, 2400 to 2600 h and 600 to 1200 mm, with the total amount of temperature from lowest and highest altitude orchards ranging between 2800 ºD and 3400 ºD, respectivly (Gomes-Laranjo et al., 2007). Degree-days (°D) represent the amount of heat required, between the lower and upper thresholds (where T0 = 6.0 ºC, see Fig. 2) for an organism to develop from one point to another in its life cycle (Cesaraccio et al., 2001; Zalom et al., 1983). For overall Portuguese varieties, half rate of photosynthesis (A50) is found when temperature reachs 11ºC (T50m) and 38ºC (T50M), being the optimal value around 24ºC (Gomes-Laranjo et al., 2007). In relation to limitant radiation intensities, results suggest that it is a dimlight species, since 75% of maximal photosynthetic rate is found at 900 µmol.m-2.s-1, which corresponds almost at half full sunlight intensity, and A50 is at 400 µmol.m-2.s-1. So, European chestnut could be indicated to be included in restoration programes for the European forest, since adult trees save most of light in the top of their canopies and only low intensity will attain soil level. Identical conclusions have been drawn by Joesting et al., (2009) in relation to American chestnut (*C. dentata* (Marsh.) Borkh) with the aim to restore chestnut populations in eastern deciduous forest from Appalachian mountains.

Fig. 2. Threshold temperature (left) and radiation (right) for photosynthesis rates in chestnut leaves. Study was done with 13 Portuguese varieties in Trás-os-Montes Region during 6 years. Concerning temperature study, T0 represents the temperature value for vegetative zero growth, T50M and T50m the values that induce half rate (A50) of the maximal photosynthesis (A100). Values were obtained according to second polynomial curve, y = - 0.0253x2 + 1.2349x - 6.2532, R² = 0.1094. In relation to the radiation study, A100, A75 and A50, mean the maximal, 75% and half of the maximal value of photosynthesis rate, respectively, being PAR50 and PAR75 the respetive values of photosynthetic active radiation (PAR). These values were calculated from logarithmic equation, y = 2.9628ln(x) - 12.62, R² = 0.5335 (n=8852).

1800 m in Caucasus Mountains, the former altitudes corresponding to the ancient orchards and the highest altitudes to the newest plantations (Gomes-Laranjo et al., 2005; Pereira-

Below 600 m a.s.l. climate is hotter and dryer than the adequate conditions for chestnut, corresponding to a transition altitude, vineyards, olive tree and almond being now the main crops. Contrarely, above 1100 m a.s.l. climate is colder and wetter, and vegetative cycle is shorter than that needed for fruit production. So, typical climate is a continental temperate type, with mean annual values of sunlight and precipitation, 2400 to 2600 h and 600 to 1200 mm, with the total amount of temperature from lowest and highest altitude orchards ranging between 2800 ºD and 3400 ºD, respectivly (Gomes-Laranjo et al., 2007). Degree-days (°D) represent the amount of heat required, between the lower and upper thresholds (where T0 = 6.0 ºC, see Fig. 2) for an organism to develop from one point to another in its life cycle (Cesaraccio et al., 2001; Zalom et al., 1983). For overall Portuguese varieties, half rate of photosynthesis (A50) is found when temperature reachs 11ºC (T50m) and 38ºC (T50M), being the optimal value around 24ºC (Gomes-Laranjo et al., 2007). In relation to limitant radiation intensities, results suggest that it is a dimlight species, since 75% of maximal photosynthetic rate is found at 900 µmol.m-2.s-1, which corresponds almost at half full sunlight intensity, and A50 is at 400 µmol.m-2.s-1. So, European chestnut could be indicated to be included in restoration programes for the European forest, since adult trees save most of light in the top of their canopies and only low intensity will attain soil level. Identical conclusions have been drawn by Joesting et al., (2009) in relation to American chestnut (*C. dentata* (Marsh.) Borkh) with the aim to restore chestnut populations in eastern deciduous forest from Appalachian

**A100**

**A50**

Fig. 2. Threshold temperature (left) and radiation (right) for photosynthesis rates in chestnut leaves. Study was done with 13 Portuguese varieties in Trás-os-Montes Region during 6 years. Concerning temperature study, T0 represents the temperature value for vegetative

photosynthesis (A100). Values were obtained according to second polynomial curve, y = - 0.0253x2 + 1.2349x - 6.2532, R² = 0.1094. In relation to the radiation study, A100, A75 and A50, mean the maximal, 75% and half of the maximal value of photosynthesis rate, respectively, being PAR50 and PAR75 the respetive values of photosynthetic active radiation (PAR). These values were calculated from logarithmic equation, y = 2.9628ln(x) - 12.62, R² = 0.5335

zero growth, T50M and T50m the values that induce half rate (A50) of the maximal


A (molCO2.m-2.s-1)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

**A100 A75 A50**

PAR (µmol.m-2.s-1)

**PAR PAR75 <sup>50</sup>**

Lorenzo et al., 2010).

mountains.

(n=8852).

A (molCO2-m-2.s-1)

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Temperature (ºC)

**T50M <sup>T</sup> T100 50m T0**

In the interior regions of Europe, where chestnut grows under continental climatic influence, altitude is decisive to define adequated climatic conditions. As the Fig. 3 shows, photosynthesis increases with altitude, and inversly with temperature. So, maximal rates can be found above 800 m a.s.l. where temperature down to 25 – 22 ºC, the range of optimal temperature as has been refered above. In the lowest altitudes, photosynthesis rate decreases around 40%, indicating that chestnuts under these climatic conditions, nowadays start to suffer from abiotic stresses, mainly due to the heat stress. Regarding internal water balance, Martins et al., (2010) have demonstrated that adult trees can be saved from water stress, since they can continuosly absorb water from deep soil layers and so preserving predawn leaf water potential in the range of -0.6 to -0.9 MPa.

Fig. 3. Variation of mean daily photosynthesis rate (A, closed symbols) in leaves from chestnut (var. Judia) and air temperature (open symbols) as a function of altitude.

Additionally, the European chestnut ecotypes coming from wet sites are more locally adapted and less plastic than those from dry sites and hence more vulnerable to the climate changes (Villani et al., 2010). Five gene pools have been determined in Europe: three in Greece, one on the northwestern coast of the Iberian Peninsula and a large gene pool covering the rest of the Mediterranean basin (Martin et al., 2010; Mattioni et al., 2008).

The existence of some adaptative variation among populations from extreme conditions is proposed (Fernández-López et al., 2005): populations from Greece initiate growth earlier followed by those from South Italy and South Spain, while ecotypes from north Spain and Italy initiate later. A significant genetic variation between north and south Iberian ecotypes has also been confirmed (Fernández-López et al., 2005). The expected global climate changes are a great challenge for forest tree breeders. Eriksson et al., (2005) stablished a xerothermic index to characterize each one of those ecotype´s local origin and they found a negative correlation between it and plant growth at both 25ºC and 32ºC, but a positive correlation with carbon isotope discrimination, suggesting a large additive coefficient of variation for growth traits. This variation confers to the species good possibilities to respond genetically via natural or artificial selection to enviromental change. Dinis et al., (2011) working with plants from the portuguese Judia variety, have also concluded that the morphological and phenological differences among ecotypes are not only related to the small genetic differences, but are simply phenotypic adaptations to different climatic conditions.

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

ABA signaling pathway in guard cells (Acharya & Assmann, 2009).

(Chaves et al., 2010).

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 189

stimuli. Environment parameters, such as dry soil conditions, temperature stress, relative atmospheric humidity, flooding, photoperiod, light intensity, salinity and wounding, as well as internal factors serving as developmental cues, affect ABA concentration in leaves and other plant organs. ABA regulates transpiration and water loss via stomatal closure (Rock et al., 2010). It is also noteworthy that nitric oxide (NO) functions as a second messenger in the

Cramer et al., (2007) have demonstrated that a large number of transcripts involved in ABA metabolism or responsive to ABA are increased with water deficit or salinity over 16 days, suggesting that ABA plays a critical role in grape abiotic stress responses. Water deficit induced increases in ABA concentrations in the xylem sap and leaves of grapevine and changes in stomatal conductance are well correlated with ABA concentrations of the xylem sap (Pou et al., 2008; Soar et al., 2004). ABA also influences hydraulic conductance, aquaporin gene expression and embolism repair in grapevines (Cramer, 2010; Lovisolo et al., 2010). It is hypothesised that in the isohydric grapevine cultivar Grenache, the drought-induced root ABA biosynthesis increases apoplastic concentration because of a concomitance of events: an increase in suberisation of apoplastic barriers causes a reduction in water conductivity which is not compensated by aquaporin-mediated water transport (Lovisolo et al., 2010). Kaldenhoff et al., (2008) suggest an ABA-aquaporin interaction in the repair of grapevine embolism and in the aquaporin activation during water stress. The root and shoot ABA-mediated responses to water stress conditions, or, more generally, to abiotic stresses, are relevant to vine yield and productivity (Lovisolo et al., 2010). As described earlier by Keller, (2005), water stress influences ABA accumulation at the root, shoot and leaf level, and also affects berry quality. However, a connection between ABA and berry quality (sugar composition during fruit development) has not yet been clarified. Grapevine is among the first plant species in which a direct role of ABA in stomatal closure is demonstrated (Lovisolo et al., 2010). In effect, in different grapevine genotypes, during the gradual imposition of soil water stress (non-irrigation) or partial root drying, negative correlations are often observed between stomatal conductance, and either xylem or leaf tissue ABA contents (Lovisolo et al., 2010). These authors have pointed that ABA synthesis in grapewine shoots and leaves increases in response to soil water stress, which implies that some other root-based biochemical signal may trigger this response. Also, further work is required to clearly understand the role of hydraulics on stomatal regulation in grapevine (Lovisolo et al., 2010), in spite of that it is leaf ABA and not whole-plant hydraulic conductivity that determines stomatal conductance. Nevertheless, the primary role of a root-to-shoot hydraulic signal is generally followed by an increased ABA biosynthesis in the shoot that regulates stomata and leaf growth

Vvrd22, a dehydration-responsive gene has been recently isolated and cloned from grapevine of the Cabernet Sauvignon variety (Hanana et al., 2008). It is constitutively expressed at a low level in all analyzed tissues, not only responsible for drought stress but

Gene expression of the ABA and ethylene pathways is particularly increased by stress compared with other hormone pathways and is negatively correlated with stem water potentials (Cramer, 2010). Using transcript and metabolite profiling, Deluc et al., (2009) have shown that water deficit has significant impacts on the metabolism of grape berries. Water deficit affects the metabolism of ABA in the grapewine cultivars Cabernet Sauvignon and Chardonnay in different ways: it increases ABA concentrations in Cabernet Sauvignon

also responsible for salt stress. ABA induces Vvrd22 expression, even at a low level.

Lowest altitudes and so, highest temperatures, seem to induce sun characteristics in leaves, demonstrated by low chlorophyll amount (Chl), since thermoinhibition might speed light saturation of the photosynthetic process (Dinis et al., 2011). On the other side, leaves present high Chla/b and low Chl/Car ratios are consistent with their acquired tolerance to warm and sunny conditions (Gomes-Laranjo et al., 2006; Pearcy, 1998). Chla is the main photosystem I pigment, which is located in exposed thyalakoid membranes, and carotenoids have the chlorophyll protection function against photoinhibition (DemmigAdams & Adams, 1996). Moreover, increase in Chla/Chlb suggests higher proportion of stacking thylakoid membranes, which in turn might induce higher photosynthesis rates, if any stress factor imposes (Anderson et al., 1988).


Table 1. Determination of photosynthetic pigment content (n=10), fatty acid composition (n=3) and malonic aldehyde (n=3) in chestnut chloroplast (var. Judia) (Gomes-Laranjo et al., 2005) isolated from leaves collected in the range of altitudes between 450 and 1050 m a.s.l

Altitude and consequently air temperature, also affects the thylakoid fatty acid composition (Table 1). In highest altitude locals, the unsaturation index is highest and inversely in the lowest ones that is the lowest. This adjustment is very important since hotness induces more fluidity in the membrane fatty acids and by this way, they must be more saturated in order to be more stable and consequently forming stable thylakoid membranes (Murata & Siegenthaler, 1998). In the Portuguese varieties, Judia, Longal and Aveleira, the most heat tolerant variety Aveleira has the lowest unsaturated fatty acid index (158.5) and viceversa Judia the least heat tolerant has the highest fatty acid index (175.1) (Gomes-Laranjo et al., 2006).

Additionally, deacrease in thiobarbituric reactive species (MDA) and ADP-Fe peroxidation in the leaves suggest the lower in peroxidation susceptibility the higher altitude (Table1).

### **4. Abiotic stress signalling**

### **4.1 The role of abscisic acid (ABA)**

Here we briefly introduce some of the recent research on the regulation of ABA levels in the aforementioned Mediterranean plants. ABA signal transduction will not be discussed in detail since the studies related to these plants are still scarce or almost inexistent, which undoubtedly open new frontiers for future investigations.

ABA, a phytohormone that plays a fundamental role in abiotic stress adaptation, is a small sequiterpenoid (C15) that also plays important roles in plant growth and development, as well as in response and tolerance to dehydration. ABA is central in regulating the plant response to a variety of abiotic stressful conditions *e.g.,* drought, salt and osmotic stress (Marion-Poll & Leung, 2006). Environmental parameters are known to affect ABA and water status, which in turn affect physiological processes in plants (Kitsaki & Drossopoulos, 2005). ABA is related to both, long- and short-term responses of the plant to several environmental

Lowest altitudes and so, highest temperatures, seem to induce sun characteristics in leaves, demonstrated by low chlorophyll amount (Chl), since thermoinhibition might speed light saturation of the photosynthetic process (Dinis et al., 2011). On the other side, leaves present high Chla/b and low Chl/Car ratios are consistent with their acquired tolerance to warm and sunny conditions (Gomes-Laranjo et al., 2006; Pearcy, 1998). Chla is the main photosystem I pigment, which is located in exposed thyalakoid membranes, and carotenoids have the chlorophyll protection function against photoinhibition (DemmigAdams & Adams, 1996). Moreover, increase in Chla/Chlb suggests higher proportion of stacking thylakoid membranes, which in turn might induce higher photosynthesis rates, if any stress factor

**Altitude Chla/b PI UI Malonic aldehyde x10-4 (mM)** (s.l.m) Saturated (%) unsaturated (%) **Control ADP-Fe** 1050 121.5 b 3.12 c 4.8 a 27.0 73.0 111.5 171.3 1.35 3.88 900 145.9 a 3.10 c 5.0 a 32.4 67.6 97.5 154.2 1.67 4.57 700 99.1 c 3.30 b 4.4 b 38.2 61.8 119.2 156.3 2.00 4.91 600 143.9 a 3.40 b 4.6 b 33.1 66.9 47.5 146.1 1.90 4.53 450 80.9 d 3.60 a 3.9 c 43.9 56.1 79.6 121.2 3.12 5.36

Table 1. Determination of photosynthetic pigment content (n=10), fatty acid composition (n=3) and malonic aldehyde (n=3) in chestnut chloroplast (var. Judia) (Gomes-Laranjo et al., 2005) isolated from leaves collected in the range of altitudes between 450 and 1050 m a.s.l

Altitude and consequently air temperature, also affects the thylakoid fatty acid composition (Table 1). In highest altitude locals, the unsaturation index is highest and inversely in the lowest ones that is the lowest. This adjustment is very important since hotness induces more fluidity in the membrane fatty acids and by this way, they must be more saturated in order to be more stable and consequently forming stable thylakoid membranes (Murata & Siegenthaler, 1998). In the Portuguese varieties, Judia, Longal and Aveleira, the most heat tolerant variety Aveleira has the lowest unsaturated fatty acid index (158.5) and viceversa Judia the least heat tolerant has the highest fatty acid index

Additionally, deacrease in thiobarbituric reactive species (MDA) and ADP-Fe peroxidation in the leaves suggest the lower in peroxidation susceptibility the higher altitude (Table1).

Here we briefly introduce some of the recent research on the regulation of ABA levels in the aforementioned Mediterranean plants. ABA signal transduction will not be discussed in detail since the studies related to these plants are still scarce or almost inexistent, which

ABA, a phytohormone that plays a fundamental role in abiotic stress adaptation, is a small sequiterpenoid (C15) that also plays important roles in plant growth and development, as well as in response and tolerance to dehydration. ABA is central in regulating the plant response to a variety of abiotic stressful conditions *e.g.,* drought, salt and osmotic stress (Marion-Poll & Leung, 2006). Environmental parameters are known to affect ABA and water status, which in turn affect physiological processes in plants (Kitsaki & Drossopoulos, 2005). ABA is related to both, long- and short-term responses of the plant to several environmental

**Chl/Car Total fatty acid**

imposes (Anderson et al., 1988).

(175.1) (Gomes-Laranjo et al., 2006).

**4. Abiotic stress signalling** 

**4.1 The role of abscisic acid (ABA)** 

undoubtedly open new frontiers for future investigations.

**Chltot** mg.cm-2 stimuli. Environment parameters, such as dry soil conditions, temperature stress, relative atmospheric humidity, flooding, photoperiod, light intensity, salinity and wounding, as well as internal factors serving as developmental cues, affect ABA concentration in leaves and other plant organs. ABA regulates transpiration and water loss via stomatal closure (Rock et al., 2010). It is also noteworthy that nitric oxide (NO) functions as a second messenger in the ABA signaling pathway in guard cells (Acharya & Assmann, 2009).

Cramer et al., (2007) have demonstrated that a large number of transcripts involved in ABA metabolism or responsive to ABA are increased with water deficit or salinity over 16 days, suggesting that ABA plays a critical role in grape abiotic stress responses. Water deficit induced increases in ABA concentrations in the xylem sap and leaves of grapevine and changes in stomatal conductance are well correlated with ABA concentrations of the xylem sap (Pou et al., 2008; Soar et al., 2004). ABA also influences hydraulic conductance, aquaporin gene expression and embolism repair in grapevines (Cramer, 2010; Lovisolo et al., 2010). It is hypothesised that in the isohydric grapevine cultivar Grenache, the drought-induced root ABA biosynthesis increases apoplastic concentration because of a concomitance of events: an increase in suberisation of apoplastic barriers causes a reduction in water conductivity which is not compensated by aquaporin-mediated water transport (Lovisolo et al., 2010). Kaldenhoff et al., (2008) suggest an ABA-aquaporin interaction in the repair of grapevine embolism and in the aquaporin activation during water stress. The root and shoot ABA-mediated responses to water stress conditions, or, more generally, to abiotic stresses, are relevant to vine yield and productivity (Lovisolo et al., 2010). As described earlier by Keller, (2005), water stress influences ABA accumulation at the root, shoot and leaf level, and also affects berry quality. However, a connection between ABA and berry quality (sugar composition during fruit development) has not yet been clarified. Grapevine is among the first plant species in which a direct role of ABA in stomatal closure is demonstrated (Lovisolo et al., 2010). In effect, in different grapevine genotypes, during the gradual imposition of soil water stress (non-irrigation) or partial root drying, negative correlations are often observed between stomatal conductance, and either xylem or leaf tissue ABA contents (Lovisolo et al., 2010). These authors have pointed that ABA synthesis in grapewine shoots and leaves increases in response to soil water stress, which implies that some other root-based biochemical signal may trigger this response. Also, further work is required to clearly understand the role of hydraulics on stomatal regulation in grapevine (Lovisolo et al., 2010), in spite of that it is leaf ABA and not whole-plant hydraulic conductivity that determines stomatal conductance. Nevertheless, the primary role of a root-to-shoot hydraulic signal is generally followed by an increased ABA biosynthesis in the shoot that regulates stomata and leaf growth (Chaves et al., 2010).

Vvrd22, a dehydration-responsive gene has been recently isolated and cloned from grapevine of the Cabernet Sauvignon variety (Hanana et al., 2008). It is constitutively expressed at a low level in all analyzed tissues, not only responsible for drought stress but also responsible for salt stress. ABA induces Vvrd22 expression, even at a low level.

Gene expression of the ABA and ethylene pathways is particularly increased by stress compared with other hormone pathways and is negatively correlated with stem water potentials (Cramer, 2010). Using transcript and metabolite profiling, Deluc et al., (2009) have shown that water deficit has significant impacts on the metabolism of grape berries. Water deficit affects the metabolism of ABA in the grapewine cultivars Cabernet Sauvignon and Chardonnay in different ways: it increases ABA concentrations in Cabernet Sauvignon

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

(http://microrna.sanger.ac.uk) (Kozomara & Griffiths-Jones, 2011).

**4.3.2 Progress in the study of grapevine miRNA on abiotic stress** 

cellular identity (Makeyev & Maniatis, 2008).

methods (Meyers et al., 2008).

± 42 (Y.D. Lu et al., 2008).

**4.3 The role of MicroRNAs** 

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 191

MicroRNAs (miRNAs) are a newly identified class of 21-24 nucleotide (nt) (predominantly 21 nt) in length, endogenous non-protein-coding short RNAs (sRNAs) in animals, plants and viruses. They are derived from 70- to 500-nt long single stranded primary transcripts (pri-miRNAs), which are transcribed from miRNA genes (*MIR* genes) by RNA polymerase II, by the action of RNase III-like enzymes DICER-LIKE1 (DCL1) or DCL4 (Liu et al., 2009). The mature miRNA is loaded to the RNA induced silencing complex (RISC) to guide the complex to the target mRNAs (containing a stretch of perfect or near perfect complementary sequence). miRNAs have been considered one of the most important regulatory molecules, which regulate gene expression at the post-transcriptional levels via targeting mRNAs for direct cleavage of mRNAs, repressing mRNA translation, or small RNA-directed transcriptional silencing (Jones-Rhoades et al., 2006). Recently, the identified number of conserved and non-conserved species-specific plant miRNAs is rising at an accelerated speed by the newly developed deep sequencing technologies. The latest information on plant miRNAs can be obtained from the miRBase database maintained by Sanger Institute

MicroRNAs play crucial roles in essential biological processes, including developmental timing, stem cell differentiation, signaling transduction, human disease, and cancer (Couzin, 2008). In plant, miRNAs play a pivotal role in many aspects, such as organ development, phase change, signal transduction, and response to environmental stress (Shukla et al., 2008; Zhang et al., 2007). Many miRNAs are expressed in a cell- or tissue-specific manner during development of organisms and may contribute to the establishment and/or maintenance of

Laboratory molecular cloning and computational prediction of miRNA genes based on the conservation of sequence and secondary structure are two methods of plant miRNA study. Historically, most plant miRNA genes have been discovered by one or both of the two

MicroRNA study concerning the Mediterranean species is rare and almost all from vine. Recently, the grapevine genome of a highly homozygous genotype (Jaillon et al., 2007) and of a heterozygous variety (Velasco et al., 2007) has been published by two independent groups, respectively. These genome data provide a solid support for the study of sRNAbased regulatory networks in grapevine. By a computational-based BLAST search of sequences using *Arabidopsis* miRNAs' genes as references Jaillon et al. identified 164 miRNA genes with a medium size of 103.5 bp and total of 0.002 Mb in the homozygous grape genome (2007); Velasco et al. (2007) identified 143 miRNA genes representing 28 families in the heterozygous genome. They predicted 28 conserved and non-conserved miRNAs in grapevine. A total of 81 potential miRNAs have been computationally predicted; the length of miRNA precursors in grapevine varies from 68 to 207 nucleotides, with an average of 117

Recently, attention has been paid on the role of miRNA in plant abiotic stress mediation, indicating that miRNAs participate in regulating various abiotic stress response, such as drought (Kantar et al., 2011; Xu et al., 2010), salt (Ding et al., 2009), cold (Zhou et al., 2008), heat (S.F. Lu et al., 2008), (see reviews by Phillips et al., (2007) and Shukla et al., (2008)). In

**4.3.1 MicroRNAs are ubiquitous gene regulators at post-transcriptional levels** 

berries, but doesn't in Chardonnay berries. Effectively, Cramer (2010) has recently emphasized that many of the grapevine responses to osmotic stress appear to be transcriptionally regulated, but proteomic studies indicate that there are post-translational controls as well. Also, metabolite profiling has revealed that accumulation of amino acids and polyamines (PAs) is dependent on ABA production, suggesting the integration of ABA signaling to accumulation of protective molecules (Toumi et al., 2010). The ABA signaling pathway integrates PAs and amine oxidases (AOs) in order to regulate the generation of hydrogen peroxide (H2O2), which signals further stress responses of the programmed cell death (PCD) syndrome. ABA enhances PA accumulation in grapes and, at the same time, induces the PA oxidation pathway, thus originating secondary protective effects *e.g.,* the stomata closure. Furthermore, PA's catabolism caused by enhanced expression of AOs, which is induced by ABA, generated H2O2 which correlated with the levels of peroxidases and phenolics during vascular differentiation (Paschalidis et al., 2009).

The olive tree tends to acclimatize to prolonged hot-dry periods by reducing the level of ABA at the end of summer, in spite of the low water potential (Kitsaki & Drossopoulos, 2005). During winter periods, leaf ABA content remains low, while water potential values are at their highest level. Concerning ABA content, young olive leaves to be more sensitive to most environmental parameters than old ones. Significant differences in water-stressinduced ABA accumulation have been observed between two *O. europea* cultivars, thus reflecting the degree of stress experienced (Guerfel et al., 2009). The drought tolerant cultivar 'Chemlali' accumulates lower levels of ABA in their leaves to regulate the stomatal control in response to water stress compared to the drought sensitive cultivar 'Chetoui', which accumulates ABA in large amounts. ABA in nutrient medium originates different olive carbohydrate spectra regarding abiotic stress type (salinity or low temperature) (Rejšková et al., 2007).

### **4.2 The role of nitric oxide (NO)**

Reactive nitrogen species (RNS) are a family of reactive molecules derived from nitric oxide (•NO; hereafter called NO). The major RNS in the plant cell is NO (Besson-Bard et al., 2008). NO has the hability to cross cell membranes and can thereby transmit signals to other cells. A biologically important reaction of NO is *S-*nitrosylation, converting thiol groups (including cysteine residues in proteins) in *S-*nitrosothiols (RSNO). NO has an important function in numerous cell signalling processes, regulating cell growth, the hypersensitive response, the closure of stomata, plant response to stressors such as drought, high or low temperature, salinity, heavy metals and oxidative stress (Besson-Bard et al., 2008), and also has defense functions (Neill et al., 2008).

Concerning the Mediterranean species, the studies about NO are very scarce. In olive plants, salinity produces a 40% reduction in leaf fresh weight, induces oxidative stress and a dramatical increase in proteins that undergo tyrosine nitration (Valderrama et al., 2007). The specific NOS activity in olive leaves is dependent on L-arginine, NADPH and calcium (Valderrama et al., 2007). Salt stress induces an increase in the L-arginine-dependent production of NO, total RSNO and several proteins that undergo tyrosine nitration, thus functioning as good markers of nitrosative stress. Additionally, the vascular tissues could play an important function in the redistribution of NO-derived forms during nitrosative stress and in signalling-related processes. NO are produced in the olive reproductive organs in a stage- and tissue- specific manner (Zafra et al., 2010).

### **4.3 The role of MicroRNAs**

190 Plants and Environment

berries, but doesn't in Chardonnay berries. Effectively, Cramer (2010) has recently emphasized that many of the grapevine responses to osmotic stress appear to be transcriptionally regulated, but proteomic studies indicate that there are post-translational controls as well. Also, metabolite profiling has revealed that accumulation of amino acids and polyamines (PAs) is dependent on ABA production, suggesting the integration of ABA signaling to accumulation of protective molecules (Toumi et al., 2010). The ABA signaling pathway integrates PAs and amine oxidases (AOs) in order to regulate the generation of hydrogen peroxide (H2O2), which signals further stress responses of the programmed cell death (PCD) syndrome. ABA enhances PA accumulation in grapes and, at the same time, induces the PA oxidation pathway, thus originating secondary protective effects *e.g.,* the stomata closure. Furthermore, PA's catabolism caused by enhanced expression of AOs, which is induced by ABA, generated H2O2 which correlated with the levels of peroxidases and phenolics during vascular differentiation (Paschalidis

The olive tree tends to acclimatize to prolonged hot-dry periods by reducing the level of ABA at the end of summer, in spite of the low water potential (Kitsaki & Drossopoulos, 2005). During winter periods, leaf ABA content remains low, while water potential values are at their highest level. Concerning ABA content, young olive leaves to be more sensitive to most environmental parameters than old ones. Significant differences in water-stressinduced ABA accumulation have been observed between two *O. europea* cultivars, thus reflecting the degree of stress experienced (Guerfel et al., 2009). The drought tolerant cultivar 'Chemlali' accumulates lower levels of ABA in their leaves to regulate the stomatal control in response to water stress compared to the drought sensitive cultivar 'Chetoui', which accumulates ABA in large amounts. ABA in nutrient medium originates different olive carbohydrate spectra regarding abiotic stress type (salinity or low temperature)

Reactive nitrogen species (RNS) are a family of reactive molecules derived from nitric oxide (•NO; hereafter called NO). The major RNS in the plant cell is NO (Besson-Bard et al., 2008). NO has the hability to cross cell membranes and can thereby transmit signals to other cells. A biologically important reaction of NO is *S-*nitrosylation, converting thiol groups (including cysteine residues in proteins) in *S-*nitrosothiols (RSNO). NO has an important function in numerous cell signalling processes, regulating cell growth, the hypersensitive response, the closure of stomata, plant response to stressors such as drought, high or low temperature, salinity, heavy metals and oxidative stress (Besson-Bard et al., 2008), and also

Concerning the Mediterranean species, the studies about NO are very scarce. In olive plants, salinity produces a 40% reduction in leaf fresh weight, induces oxidative stress and a dramatical increase in proteins that undergo tyrosine nitration (Valderrama et al., 2007). The specific NOS activity in olive leaves is dependent on L-arginine, NADPH and calcium (Valderrama et al., 2007). Salt stress induces an increase in the L-arginine-dependent production of NO, total RSNO and several proteins that undergo tyrosine nitration, thus functioning as good markers of nitrosative stress. Additionally, the vascular tissues could play an important function in the redistribution of NO-derived forms during nitrosative stress and in signalling-related processes. NO are produced in the olive reproductive organs

et al., 2009).

(Rejšková et al., 2007).

**4.2 The role of nitric oxide (NO)** 

has defense functions (Neill et al., 2008).

in a stage- and tissue- specific manner (Zafra et al., 2010).

### **4.3.1 MicroRNAs are ubiquitous gene regulators at post-transcriptional levels**

MicroRNAs (miRNAs) are a newly identified class of 21-24 nucleotide (nt) (predominantly 21 nt) in length, endogenous non-protein-coding short RNAs (sRNAs) in animals, plants and viruses. They are derived from 70- to 500-nt long single stranded primary transcripts (pri-miRNAs), which are transcribed from miRNA genes (*MIR* genes) by RNA polymerase II, by the action of RNase III-like enzymes DICER-LIKE1 (DCL1) or DCL4 (Liu et al., 2009). The mature miRNA is loaded to the RNA induced silencing complex (RISC) to guide the complex to the target mRNAs (containing a stretch of perfect or near perfect complementary sequence). miRNAs have been considered one of the most important regulatory molecules, which regulate gene expression at the post-transcriptional levels via targeting mRNAs for direct cleavage of mRNAs, repressing mRNA translation, or small RNA-directed transcriptional silencing (Jones-Rhoades et al., 2006). Recently, the identified number of conserved and non-conserved species-specific plant miRNAs is rising at an accelerated speed by the newly developed deep sequencing technologies. The latest information on plant miRNAs can be obtained from the miRBase database maintained by Sanger Institute (http://microrna.sanger.ac.uk) (Kozomara & Griffiths-Jones, 2011).

MicroRNAs play crucial roles in essential biological processes, including developmental timing, stem cell differentiation, signaling transduction, human disease, and cancer (Couzin, 2008). In plant, miRNAs play a pivotal role in many aspects, such as organ development, phase change, signal transduction, and response to environmental stress (Shukla et al., 2008; Zhang et al., 2007). Many miRNAs are expressed in a cell- or tissue-specific manner during development of organisms and may contribute to the establishment and/or maintenance of cellular identity (Makeyev & Maniatis, 2008).

Laboratory molecular cloning and computational prediction of miRNA genes based on the conservation of sequence and secondary structure are two methods of plant miRNA study. Historically, most plant miRNA genes have been discovered by one or both of the two methods (Meyers et al., 2008).

MicroRNA study concerning the Mediterranean species is rare and almost all from vine. Recently, the grapevine genome of a highly homozygous genotype (Jaillon et al., 2007) and of a heterozygous variety (Velasco et al., 2007) has been published by two independent groups, respectively. These genome data provide a solid support for the study of sRNAbased regulatory networks in grapevine. By a computational-based BLAST search of sequences using *Arabidopsis* miRNAs' genes as references Jaillon et al. identified 164 miRNA genes with a medium size of 103.5 bp and total of 0.002 Mb in the homozygous grape genome (2007); Velasco et al. (2007) identified 143 miRNA genes representing 28 families in the heterozygous genome. They predicted 28 conserved and non-conserved miRNAs in grapevine. A total of 81 potential miRNAs have been computationally predicted; the length of miRNA precursors in grapevine varies from 68 to 207 nucleotides, with an average of 117 ± 42 (Y.D. Lu et al., 2008).

### **4.3.2 Progress in the study of grapevine miRNA on abiotic stress**

Recently, attention has been paid on the role of miRNA in plant abiotic stress mediation, indicating that miRNAs participate in regulating various abiotic stress response, such as drought (Kantar et al., 2011; Xu et al., 2010), salt (Ding et al., 2009), cold (Zhou et al., 2008), heat (S.F. Lu et al., 2008), (see reviews by Phillips et al., (2007) and Shukla et al., (2008)). In

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

ath-miR156 and vvi-miR157 share the same target SPL(Y.D. Lu et al., 2008).

et al., 2010), which may explain the absence of cleaved targets.

computationally predicted in grapevine (Wei et al., 2008).

plant breeding methods.

Bohnert, 2000; Mittler & Blumwald, 2010).

**4.3.2.3 miRNAs and nutrient deficiency stress** 

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 193

miR1867, miR474, miR398, miR1450, miR1881, miR894, miR156, and miR1432 are upregulated in *Triticum dicoccoides* under drought stress (Kantar et al., 2011). It is expected that miRNAs that shut down processes involved in normal metabolism and growth are upregulated during drought stress, in order to conserve water and protect the cell. One good example would be miR156, which downregulates transcription factors involved in development and flowering. miR169g and miR169c have been computationally identified (Y.D. Lu et al., 2008), and 25 miR169 members (miR169a-y) have been validated in grapevine (Mica et al., 2010). miR393 has also been predicated to be involved in signalling pathways by regulating transport TIR1 (Y.D. Lu et al., 2008). Experimental work has shown that miR393 is expressed at a higher level in inflorescences than in tendrils in grapevine (Pantaleo et al., 2010). miR156 has been computationally identified (Velasco et al., 2007) and cloned (Pantaleo et al., 2010) in vine, respectively. In another independent work, it is predicted that

MicroRNA399 regulates phosphate stress responses. Upon Pi starvation, increased miR399 expression represses the expression of ubiquitin-conjugating E2 enzyme (UBC24) and consequently the repression of Pi uptake is alleviated in Arabidopsis (Aung et al., 2006). Where in grapevine miR399 is predicated to target AF2 (Y.D. Lu et al., 2008). miRNA399 is more highly expressed in roots (Mica et al., 2010) and at a very low level in leaves (Pantaleo

MiR395 is involved in sulphur accumulation and alocation by targeting both ATP sulfurylases and the sulfate transporter AST68 (SULTR2;1). During sulfate limitation, expression of miR395 is significantly up-regulated (Liang et al., 2010). In grapevine miR395 is predicted to target mRNAs coding for ATP sulphurylases (Y.D. Lu et al., 2008). The expression of miR395 family is higher at leaf than at tendrils, inflorescences and berry (Mica

In addition, several UV stress responding miRNAs and their target genes have been

Several plant breeding approaches will likely be needed to improve the abiotic stress tolerance and maintain optimum yield levels of the Mediterranean crops in field conditions. The main method of crop improvement continues to be the conventional plant breeding through sexual hybridization, sometimes combined with classical cytogenetic techniques (Roy et al., 2011). Conventional breeding and marker assisted selection are being used to develop cultivars more tolerant to abiotic stress. However, these methods are time and resource consuming and germplasm dependent. On the other hand, improvement of stress tolerance by genetic engineering overcomes the bottlenecks of

Recently, efforts have been devoted to identifying potential target genes for use in genetic engineering for crop abiotic stress tolerance (Cushman & Bohnert, 2000). These include specific heat shock proteins, ion transporters, water transporters (aquaporins), as well as signalling components, such as, MAP kinases, Ca2+-dependent protein kinases, transcription factors, like, DREB, CBF and Myb, and enzymes of plant hormone metabolism (Cushman &

et al., 2010); and that is higher at tendrils than in inflorescences (Pantaleo et al., 2010).

**5. Genetic modifications targeting improved plant abiotic stress** 

plants, miRNAs target regulatory proteins such as transcription factors, suggesting that miRNAs are master regulators (Phillips et al., 2007; Shukla et al., 2008). Stress-induced or upregulated miRNAs target negative regulators of stress responses or positive regulators of processes that are inhibited by stresses (e.g., cell division and expansion). Alternatively, stress downregulated miRNAs could repress the expression of positive regulators and/or stress upregulated genes. The existence of stress-related elements in miRNA promoter regions provides further evidence supporting its role in abiotic stress (Liu et al., 2008).

For the experimental analysis of miRNAs and other sRNAs, the first and most important step is the isolation of high-quality total RNA. High-quality RNA extraction from grapevine and other Mediterranean woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA. A rapid and effective cetyltrimethylammonium bromide (CTAB)-based method for RNA extraction from different tissues of grapevine and other woody plants including olive and chestnut has been reported (Gambino et al., 2008). Eighteen miRNA were computationally predicted to be responsible for abiotic stress in grape (Wei, 2009). Here we will summarize the identified and validated miRNAs of grapevine on abiotic stress.

### **4.3.2.1 miR398 and oxidative stress**

miR398 down-regulates two closely related Cu/Zn-Superoxide Dismutase genes: cytosolic CSD1 and plastidic CSD2 that can detoxify superoxide radicals (Sunkar et al., 2007; Sunkar et al., 2006). It is expressed in a spatial- and temporal-specific manner under normal growth conditions finely tuning the expression of CSD1 and CSD2 transcripts and in turn regulating the levels of superoxide or other ROS required for signalling. miR398 expression is downregulated transcriptionally by oxidative stress, and this downregulation is important for posttranscriptional CSD1 and CSD2 mRNA accumulation and oxidative stress tolerance. Computational prediction reveals that the miR398 family is represented by three members (MIR398a, MIR398b, and MIR398c) in grapevine (Y.D. Lu et al., 2008) and these members have been recently validated by deep sequencing analysis (Mica et al., 2010; Pantaleo et al., 2010). Among them, miR398b is at least 100 fold higher expressed in root than either leaf or early inflorescences. Furthermore, transgenic Arabidopsis plants overexpressing a miR398 resistant form of CSD2 accumulate more CSD2 mRNA than plants overexpressing a regular CSD2 and are consequently much more tolerant to high light, heavy metals and other oxidative stressors (Sunkar et al., 2006), which strongly demonstrates the role of miR398 in plant abiotic stress.

### **4.3.2.2 miRNAs and water deficit stress**

A few miRNAs have been identified to regulate plant drought stress. Two drought-induced miRNAs, miR169g and miR-393, have been validated by microarray analysis in rice plants upon drought stress (Zhao et al., 2007). miR169g is induced more prominent in roots than in shoots. Two ABA-independent dehydration-responsive elements (DREs) exist in the upstream of the promoter region of the MIR169g gene, supporting its role in plant water deficit (Zhao et al., 2007). miRNA169a/c are found to be drought downregulated in *Arabidopsis thaliana* (Li et al., 2008). miR393 is a plant miRNA thought to regulate expression of mRNAs encoding the F-box auxin receptor, including transport inhibitor response1 (TIR1), which in turn targets AUX/IAA proteins for proteolysis by SCF E3 ubiquitin ligases in an auxin-dependent manner and is necessary for auxin-induced growth processes*.* Thus, miR393-mediated inhibition of TIR1 would down-regulate auxin signalling and seedling growth under abiotic stress conditions and further relate to drought stress. In addition, miR1867, miR474, miR398, miR1450, miR1881, miR894, miR156, and miR1432 are upregulated in *Triticum dicoccoides* under drought stress (Kantar et al., 2011). It is expected that miRNAs that shut down processes involved in normal metabolism and growth are upregulated during drought stress, in order to conserve water and protect the cell. One good example would be miR156, which downregulates transcription factors involved in development and flowering. miR169g and miR169c have been computationally identified (Y.D. Lu et al., 2008), and 25 miR169 members (miR169a-y) have been validated in grapevine (Mica et al., 2010). miR393 has also been predicated to be involved in signalling pathways by regulating transport TIR1 (Y.D. Lu et al., 2008). Experimental work has shown that miR393 is expressed at a higher level in inflorescences than in tendrils in grapevine (Pantaleo et al., 2010). miR156 has been computationally identified (Velasco et al., 2007) and cloned (Pantaleo et al., 2010) in vine, respectively. In another independent work, it is predicted that ath-miR156 and vvi-miR157 share the same target SPL(Y.D. Lu et al., 2008).

### **4.3.2.3 miRNAs and nutrient deficiency stress**

192 Plants and Environment

plants, miRNAs target regulatory proteins such as transcription factors, suggesting that miRNAs are master regulators (Phillips et al., 2007; Shukla et al., 2008). Stress-induced or upregulated miRNAs target negative regulators of stress responses or positive regulators of processes that are inhibited by stresses (e.g., cell division and expansion). Alternatively, stress downregulated miRNAs could repress the expression of positive regulators and/or stress upregulated genes. The existence of stress-related elements in miRNA promoter regions provides further evidence supporting its role in abiotic stress (Liu et al., 2008). For the experimental analysis of miRNAs and other sRNAs, the first and most important step is the isolation of high-quality total RNA. High-quality RNA extraction from grapevine and other Mediterranean woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA. A rapid and effective cetyltrimethylammonium bromide (CTAB)-based method for RNA extraction from different tissues of grapevine and other woody plants including olive and chestnut has been reported (Gambino et al., 2008). Eighteen miRNA were computationally predicted to be responsible for abiotic stress in grape (Wei, 2009). Here we

will summarize the identified and validated miRNAs of grapevine on abiotic stress.

miR398 down-regulates two closely related Cu/Zn-Superoxide Dismutase genes: cytosolic CSD1 and plastidic CSD2 that can detoxify superoxide radicals (Sunkar et al., 2007; Sunkar et al., 2006). It is expressed in a spatial- and temporal-specific manner under normal growth conditions finely tuning the expression of CSD1 and CSD2 transcripts and in turn regulating the levels of superoxide or other ROS required for signalling. miR398 expression is downregulated transcriptionally by oxidative stress, and this downregulation is important for posttranscriptional CSD1 and CSD2 mRNA accumulation and oxidative stress tolerance. Computational prediction reveals that the miR398 family is represented by three members (MIR398a, MIR398b, and MIR398c) in grapevine (Y.D. Lu et al., 2008) and these members have been recently validated by deep sequencing analysis (Mica et al., 2010; Pantaleo et al., 2010). Among them, miR398b is at least 100 fold higher expressed in root than either leaf or early inflorescences. Furthermore, transgenic Arabidopsis plants overexpressing a miR398 resistant form of CSD2 accumulate more CSD2 mRNA than plants overexpressing a regular CSD2 and are consequently much more tolerant to high light, heavy metals and other oxidative stressors (Sunkar et al., 2006), which strongly demonstrates the role of miR398 in

A few miRNAs have been identified to regulate plant drought stress. Two drought-induced miRNAs, miR169g and miR-393, have been validated by microarray analysis in rice plants upon drought stress (Zhao et al., 2007). miR169g is induced more prominent in roots than in shoots. Two ABA-independent dehydration-responsive elements (DREs) exist in the upstream of the promoter region of the MIR169g gene, supporting its role in plant water deficit (Zhao et al., 2007). miRNA169a/c are found to be drought downregulated in *Arabidopsis thaliana* (Li et al., 2008). miR393 is a plant miRNA thought to regulate expression of mRNAs encoding the F-box auxin receptor, including transport inhibitor response1 (TIR1), which in turn targets AUX/IAA proteins for proteolysis by SCF E3 ubiquitin ligases in an auxin-dependent manner and is necessary for auxin-induced growth processes*.* Thus, miR393-mediated inhibition of TIR1 would down-regulate auxin signalling and seedling growth under abiotic stress conditions and further relate to drought stress. In addition,

**4.3.2.1 miR398 and oxidative stress** 

plant abiotic stress.

**4.3.2.2 miRNAs and water deficit stress** 

MicroRNA399 regulates phosphate stress responses. Upon Pi starvation, increased miR399 expression represses the expression of ubiquitin-conjugating E2 enzyme (UBC24) and consequently the repression of Pi uptake is alleviated in Arabidopsis (Aung et al., 2006). Where in grapevine miR399 is predicated to target AF2 (Y.D. Lu et al., 2008). miRNA399 is more highly expressed in roots (Mica et al., 2010) and at a very low level in leaves (Pantaleo et al., 2010), which may explain the absence of cleaved targets.

MiR395 is involved in sulphur accumulation and alocation by targeting both ATP sulfurylases and the sulfate transporter AST68 (SULTR2;1). During sulfate limitation, expression of miR395 is significantly up-regulated (Liang et al., 2010). In grapevine miR395 is predicted to target mRNAs coding for ATP sulphurylases (Y.D. Lu et al., 2008). The expression of miR395 family is higher at leaf than at tendrils, inflorescences and berry (Mica et al., 2010); and that is higher at tendrils than in inflorescences (Pantaleo et al., 2010).

In addition, several UV stress responding miRNAs and their target genes have been computationally predicted in grapevine (Wei et al., 2008).

### **5. Genetic modifications targeting improved plant abiotic stress**

Several plant breeding approaches will likely be needed to improve the abiotic stress tolerance and maintain optimum yield levels of the Mediterranean crops in field conditions. The main method of crop improvement continues to be the conventional plant breeding through sexual hybridization, sometimes combined with classical cytogenetic techniques (Roy et al., 2011). Conventional breeding and marker assisted selection are being used to develop cultivars more tolerant to abiotic stress. However, these methods are time and resource consuming and germplasm dependent. On the other hand, improvement of stress tolerance by genetic engineering overcomes the bottlenecks of plant breeding methods.

Recently, efforts have been devoted to identifying potential target genes for use in genetic engineering for crop abiotic stress tolerance (Cushman & Bohnert, 2000). These include specific heat shock proteins, ion transporters, water transporters (aquaporins), as well as signalling components, such as, MAP kinases, Ca2+-dependent protein kinases, transcription factors, like, DREB, CBF and Myb, and enzymes of plant hormone metabolism (Cushman & Bohnert, 2000; Mittler & Blumwald, 2010).

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

expression in transformation systems (Drosopoulou et al., 2009).

**6. Conclusions and future prospects** 

**7. References** 

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 195

High temperature tolerance has been genetically engineered in plants mainly by overexpressing the heat shock protein (HSP) genes or indirectly by altering levels of heat shock transcription factor proteins (Singh & Grover, 2008). HSP70 has been cloned and characterized from olive tree (Drosopoulou et al., 2009). The presence and organization of many typical binding sites for the Heat Shock and GAGA factors in the sequence suggest that the promoter of this gene is highly heat-inducible and could be used for conditional

Modern plant breeding involves novel technical approaches, and gene transfer is undoubtedly a powerful tool. However, genetic engineering does not always result in efficient transgene expression. Several cases have been reported, where transgene copy

Olive tree and grapevine have evolved fine adaptation mechanism to drought, heat and high irradiation at morphological, anatomical, physiological and biochemical levels. Low altitude (consequently high air temperature) restricts the distribution of chestnut in Europe. An abiotic stress may initiate multiple signaling pathways in Mediterranean plants. Because ABA is involved in abiotic stress signaling, revealing how ABA is perceived certainly will help reveal how stress signals are sensed. The study of the newly identified signal molecules NO and miRNA on the Mediterranean crops is just emerging. The related data concerning chestnut is still absent. The elucidation of the interaction and crosstalk among NO, ROS and ABA, and their relation with miRNAs in regulating plant abiotic stress will give a novel panorama of the abiotic stress signaling networks. Introducing the most important genes involved in tolerance to the various abiotic stresses into sensitive Mediterranean species will

number does not correlate with the level of transgene expression (Gelvin, 2003).

allow the coordinated expression of these genes to improve abiotic stress tolerance.

1-4051-2238-2, ISBN-13: 978-14051-2238-2, Oxford

305-327, (Apr 2005), ISSN, 1380-3743

Abd-El-Rahman, A.A.; Shalaby, A.F. & Balegh, M. (1966). Water economy of olive under desert conditions. *Flora*, Vol. 156, No., pp. 202–219, 1966), ISSN, 0367-2530 Acharya, B. & Assmann, S. (2009). Hormone interactions in stomatal function. *Plant Molecular Biology*, Vol. 69, No. 4, pp. 451-462, (Mar 2009), ISSN, 0167-4412 Agarwal, M. & Zhu, J.k. (2005) Integration of abiotic stress signaling pathways. In: *Plant* 

Altpeter, F.; Baisakh, N.; Beachy, R.; Bock, R.; Capell, T.; Christou, P.; Daniell, H.; Datta, K.;

Anderson, J.M.; Chow, W.S. & Goodchild, D.J. (1988) Thylakoid membrane organisation in

*Botany*, Vol. 47, No. 301, pp. 1093-1100, (Aug 1996), ISSN, 0022-0957

*abiotic stress*, Jenks MA, Hasegawa PM (eds), Blackwell Publishing, ISBN, ISBN-10:

Datta, S.; Dix, P.J. and others (2005). Particle bombardment and the genetic enhancement of crops: myths and realities. *Molecular Breeding*, Vol. 15, No. 3, pp.

sun/shade acclimation. In: *Ecology of photosynthesis in sun and shade*, Evans JR, Caemmerer Sv, III WWA (eds), pp. 11-26, CSIRO ISBN, 0643048235 Melbourne Angelopoulos, K.; Dichio, B. & Xiloyannis, C. (1996). Inhibition of photosynthesis in olive

trees (*Olea europaea* L.) during water stress and rewatering. *Journal of Experimental* 

The use of mutation techniques in *Arabidopsis* to obtain knock out and up-regulated mutants, and the elucidation of stress defence mechanisms in yeast and humans, where these mechanism are highly conserved in eukaryotes, has also made a major contribution (Cassells & Doyle, 2003). Publication of the genome draft sequence of two grapevine genotypes (Jaillon et al., 2007; Velasco et al., 2007) offers new perspectives on genomic research in grapevine as well as in other trees species (Gambino et al., 2009).

Plant response to drought stress is quite complex, and is associated with a large number of physiological and biochemical changes. Some of those changes, such as osmotic stress adjustment, ABA accumulation, and root morphology, are known to be controlled by multiple genes (Khan et al., 2009; Lilley et al., 1996). One promising genetic path is the mapping of quantitative trait loci (QTL) that relate performance and yield to abiotic stress factors (Collins et al., 2008). Recently, QTLs for downy mildew resistance have been localized in the genetic linkage maps of two interspecific grape crosses (Moreira et al., 2011).

Besides Agrobacteria-mediated gene transformation, another advantageous transformation method is the particle bombardment, and indeed the only one available for many species (Altpeter et al., 2005). Although there is widespread belief that particle bombardment generates large, multi-copy loci prone to instability and silencing, refinements of the technology to produce clean transgene loci have demonstrated clearly that this is not the case, and that particle bombardment has many advantages for the production of commercial transgenic plants that perform well in the field and comply with all relevant regulatory processes (Altpeter et al., 2005). Protocol for olive somatic embryos genetic transformation by particle bombardment has been recently established (Perez-Barranco et al., 2009).

The induction of stress tolerance through engineering for over-expression of transcriptional factor genes is emerging as an attractive proposition (Yang et al., 2009). Transcription factors are regulatory proteins that modulate gene expression through sequence-specific DNA binding and/or protein– protein interactions. They are capable of acting as switches of the regulatory cascade by activating or repressing transcription of target genes. In plants, the Crepeat (CRT)-binding factor/dehydration-responsive element (DRE) binding protein 1 (CBF/DREB1) transcription factors control an important pathway for increased freezing and drought tolerance. Three CBF/DREB1-like genes, CBF 1-3, have been cloned from both freezing-tolerant wild grape (*V. riparia*) and freezing-sensitive cultivated grape (*V. vinifera*) (Xiao et al., 2008). The transgenic grapevine over-expressing DREB1b has been proved to have a significantly improved resistance to cold stress (Jin et al., 2009).

The grapevine WRKY transcription factor has 66% and 58% identity at the DNA and amino acid sequence levels, respectively, with Arabidopsis AtWRKY11 genes, and has been therefore designated VvWRKY11 (Liu et al., 2011). Transgenic Arabidopsis seedlings over expressing VvWRKY11 show higher tolerance to water stress induced by mannitol than wild-type plants. It is expected that a rapid progress in the development of stress tolerance genotypes in grapevines will be achieved in the near future, because there are large genetic resources for grapevine and there are a large number of high-throughput genomic tools available to conduct functional genomic analyses (Cramer, 2010).

cDNAs from *O. europaea* related to the aquaporin (AQP) gene family have been isolated and characterized (Secchi et al., 2007). The transcript levels of each AQP gene diminished strongly in plants submitted to drought. The down-regulation of AQP genes may result in reduced membrane water permeability and may limit loss of cellular water during periods of water stress (Secchi et al., 2007).

High temperature tolerance has been genetically engineered in plants mainly by overexpressing the heat shock protein (HSP) genes or indirectly by altering levels of heat shock transcription factor proteins (Singh & Grover, 2008). HSP70 has been cloned and characterized from olive tree (Drosopoulou et al., 2009). The presence and organization of many typical binding sites for the Heat Shock and GAGA factors in the sequence suggest that the promoter of this gene is highly heat-inducible and could be used for conditional expression in transformation systems (Drosopoulou et al., 2009).

Modern plant breeding involves novel technical approaches, and gene transfer is undoubtedly a powerful tool. However, genetic engineering does not always result in efficient transgene expression. Several cases have been reported, where transgene copy number does not correlate with the level of transgene expression (Gelvin, 2003).

### **6. Conclusions and future prospects**

Olive tree and grapevine have evolved fine adaptation mechanism to drought, heat and high irradiation at morphological, anatomical, physiological and biochemical levels. Low altitude (consequently high air temperature) restricts the distribution of chestnut in Europe. An abiotic stress may initiate multiple signaling pathways in Mediterranean plants. Because ABA is involved in abiotic stress signaling, revealing how ABA is perceived certainly will help reveal how stress signals are sensed. The study of the newly identified signal molecules NO and miRNA on the Mediterranean crops is just emerging. The related data concerning chestnut is still absent. The elucidation of the interaction and crosstalk among NO, ROS and ABA, and their relation with miRNAs in regulating plant abiotic stress will give a novel panorama of the abiotic stress signaling networks. Introducing the most important genes involved in tolerance to the various abiotic stresses into sensitive Mediterranean species will allow the coordinated expression of these genes to improve abiotic stress tolerance.

### **7. References**

194 Plants and Environment

The use of mutation techniques in *Arabidopsis* to obtain knock out and up-regulated mutants, and the elucidation of stress defence mechanisms in yeast and humans, where these mechanism are highly conserved in eukaryotes, has also made a major contribution (Cassells & Doyle, 2003). Publication of the genome draft sequence of two grapevine genotypes (Jaillon et al., 2007; Velasco et al., 2007) offers new perspectives on genomic

Plant response to drought stress is quite complex, and is associated with a large number of physiological and biochemical changes. Some of those changes, such as osmotic stress adjustment, ABA accumulation, and root morphology, are known to be controlled by multiple genes (Khan et al., 2009; Lilley et al., 1996). One promising genetic path is the mapping of quantitative trait loci (QTL) that relate performance and yield to abiotic stress factors (Collins et al., 2008). Recently, QTLs for downy mildew resistance have been localized in the genetic linkage maps of two interspecific grape crosses (Moreira et al., 2011). Besides Agrobacteria-mediated gene transformation, another advantageous transformation method is the particle bombardment, and indeed the only one available for many species (Altpeter et al., 2005). Although there is widespread belief that particle bombardment generates large, multi-copy loci prone to instability and silencing, refinements of the technology to produce clean transgene loci have demonstrated clearly that this is not the case, and that particle bombardment has many advantages for the production of commercial transgenic plants that perform well in the field and comply with all relevant regulatory processes (Altpeter et al., 2005). Protocol for olive somatic embryos genetic transformation

by particle bombardment has been recently established (Perez-Barranco et al., 2009).

have a significantly improved resistance to cold stress (Jin et al., 2009).

available to conduct functional genomic analyses (Cramer, 2010).

of water stress (Secchi et al., 2007).

The induction of stress tolerance through engineering for over-expression of transcriptional factor genes is emerging as an attractive proposition (Yang et al., 2009). Transcription factors are regulatory proteins that modulate gene expression through sequence-specific DNA binding and/or protein– protein interactions. They are capable of acting as switches of the regulatory cascade by activating or repressing transcription of target genes. In plants, the Crepeat (CRT)-binding factor/dehydration-responsive element (DRE) binding protein 1 (CBF/DREB1) transcription factors control an important pathway for increased freezing and drought tolerance. Three CBF/DREB1-like genes, CBF 1-3, have been cloned from both freezing-tolerant wild grape (*V. riparia*) and freezing-sensitive cultivated grape (*V. vinifera*) (Xiao et al., 2008). The transgenic grapevine over-expressing DREB1b has been proved to

The grapevine WRKY transcription factor has 66% and 58% identity at the DNA and amino acid sequence levels, respectively, with Arabidopsis AtWRKY11 genes, and has been therefore designated VvWRKY11 (Liu et al., 2011). Transgenic Arabidopsis seedlings over expressing VvWRKY11 show higher tolerance to water stress induced by mannitol than wild-type plants. It is expected that a rapid progress in the development of stress tolerance genotypes in grapevines will be achieved in the near future, because there are large genetic resources for grapevine and there are a large number of high-throughput genomic tools

cDNAs from *O. europaea* related to the aquaporin (AQP) gene family have been isolated and characterized (Secchi et al., 2007). The transcript levels of each AQP gene diminished strongly in plants submitted to drought. The down-regulation of AQP genes may result in reduced membrane water permeability and may limit loss of cellular water during periods

research in grapevine as well as in other trees species (Gambino et al., 2009).


Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

1999b), ISSN, 0098-8472

(Jun 2002), ISSN, 0305-7364

(May 2010), ISSN, 0305-7364

486, (Jun 2008), ISSN, 0032-0889

ISSN, 0004-9409

1995), ISSN, 0140-7791

ISSN, 1322-7130

793X

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 197

Chartzoulakis, K.; Patakas, A. & Bosabalidis, A. (1999a). Comparative study on gas

*Biosciences*, Vol. 54, No. 9-10, pp. 688-692, (Sep-Oct 1999a), ISSN, 0939-5075 Chartzoulakis, K.; Patakas, A. & Bosabalidis, A.M. (1999b). Changes in water relations,

Chaves, M.M. (1991). Effects of water deficits on carbon assimilation. *Journal of Experimental* 

Chaves, M.M.; Pereira, J.S.; Maroco, J.; Rodrigues, M.L.; Ricardo, C.P.P.; Osorio, M.L.;

Chaves, M.M.; Zarrouk, O.; Francisco, R.; Costa, J.M.; Santos, T.; Regalado, A.P.; Rodrigues,

Collins, N.C.; Tardieu, F. & Tuberosa, R. (2008). Quantitative trait loci and crop performance

Connor, D.J. (2005). Adaptation of olive (Olea europaea L.) to water-limited environments.

Connor, D.J. & Ferreres, E. (2005) The physiology of adaptation and yield expression in

Correia, M.J.; Pereira, J.S.; Chaves, M.M.; Rodrigues, M.L. & Pacheco, C.A. (1995). ABA

Couzin, J. (2008). MicroRNAs make big impression in disease after disease. *Science*, Vol. 319, No. 5871, pp. 1782-4, (Mar 28 2008), ISSN, 0036-8075 (print), 1095-9203 (online) Cramer, G.R. (2010). Abiotic stress and plant responses from the whole vine to the genes.

Cramer, G.R.; Ergül, A.; Grimplet, J.; Tillett, R.L.; Tattersall, E.A.R.; Bohlman, M.C.; Vincent,

Cushman, J.C. & Bohnert, H.J. (2000). Genomic approaches to plant stress tolerance. *Current Opinion in Plant Biology*, Vol. 3, No. 2, pp. 117-124, (Apr 2000), ISSN, 1369-5266 Deluc, L.G.; Quilici, D.R.; Decendit, A.; Grimplet, J.; Wheatley, M.D.; Schlauch, K.A.;

229, John Wiley & Sons, ISBN, 0-471-66694-7, New Jersey

Cortizo, E.V.; Madriñan, M.L.V. & Madriñán, F.J.V. (1996) *El Castaño*. Edilesa Léon

*Botany*, Vol. 42, No. 234, pp. 1-16, (Jan 1991), ISSN, 0022-0957

exchange, water relations and leaf anatomy of two olive cultivars grown under well-irrigated and drought conditions. *Zeitschrift Fur Naturforschung C-a Journal of* 

photosynthesis and leaf anatomy induced by intermittent drought in two olive cultivars. *Environmental and Experimental Botany*, Vol. 42, No. 2, pp. 113-120, (Oct

Carvalho, I.; Faria, T. & Pinheiro, C. (2002). How plants cope with water stress in the field. Photosynthesis and growth. *Annals of Botany*, Vol. 89, No., pp. 907-916,

M.L. & Lopes, C.M. (2010). Grapevine under deficit irrigation: hints from physiological and molecular data. *Annals of Botany*, Vol. 105, No. 5, pp. 661-676,

under abiotic stress: Where do we stand? *Plant Physiology*, Vol. 147, No. 2, pp. 469-

*Australian Journal of Agricultural Research*, Vol. 56, No. 11, pp. 1181-1189, 2005),

olive. In: *Horticultural Reviws* Darnell R, Ferguson I, Hokanson SC (eds), pp. 155-

xylem concentrations determine maximum daily Lleaf conductance of field-grown *Vitis vinifera* L plants. *Plant Cell and Environment*, Vol. 18, No. 5, pp. 511-521, (May

*Australian Journal of Grape and Wine Research*, Vol. 16, No. Sp. Iss. 1, pp. 86-93, 2010),

D.; Sonderegger, J.; Evans, J.; Osborne, C. and others (2007). Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles. *Functional & Integrative Genomics*, Vol. 7, No. 2, pp. 111-134, (Apr 2007), ISSN, 1438-

Mérillon, J.M.; Cushman, J.C. & Cramer, G.R. (2009). Water deficit alters


Aung, K.; Lin, S.I.; Wu, C.C.; Huang, Y.T.; Su, C.L. & Chiou, T.J. (2006). pho2, a phosphate

*Plant Physiology*, Vol. 141, No. 3, pp. 1000-1011, (Jul 2006), ISSN, 0032-0889 Avanzato, D. (2009) *Following Chestnut Footprints (Castanea spp.) – Cultivation and Culture,* 

Bacelar, E.A.; Correia, C.M.; Moutinho-Pereira, J.M.; Gonçalves, B.C.; Lopes, J.I. & Torres-

Bacelar, E.A.; Moutinho-Pereira, J.M.; Gonçalves, B.C.; Lopes, J.I. & Correia, C.M. (2009).

Bacelar, E.A.; Santos, D.L.; Moutinho-Pereira, J.M.; Lopes, J.I.; Gonçalves, B.C.; Ferreira, T.C.

Bongi, G.; Mencuccini, M. & Fontanazza, G. (1987). Photosynthesis of olive leaves: effect of

Bongi, G. & Palliotti, A. (1994) Olive. In: *Handbook of Environmental Physiology of Fruit Crops:* 

Bosabalidis, A.M. & Kofidis, G. (2002). Comparative effects of drought stress on leaf

Cassells, A.C. & Doyle, B.M. (2003). Genetic engineering and mutation breeding for

Cataldi, T.R.I.; Margiotta, G.; Iasi, L.; Di Chio, B.; Xiloyannis, C. & Bufo, S.A. (2000).

Cesaraccio, C.; Spano, D.; Duce, P. & Snyder, R.L. (2001). An improved model for

*Biometeorology*, Vol. 45, No. 4, pp. 161-169, (Nov 2001), 0020-7128

No. 16, pp. 3902-3907, (Aug 15 2000), ISSN, 0003-2700

*Plant and Soil*, Vol. 292, No. 1-2, pp. 1-12, (Mar 2007), ISSN, 0032-079X Besson-Bard, A.; Pugin, A. & Wendehenne, D. (2008). New insights into nitric oxide

6605-632-9, Leuven 1

pp. 233-239, (Feb 2004), ISSN, 0829-318X

605, (Mar 2006), ISSN, 0168-9452

pp. 143-148, (Jan 1987), ISSN, 0003-1062

0849301759, Boca Raton, Florida

2002), ISSN, 0168-9452

1310-4586

ISSN, 1040-2519

overaccumulator, is caused by a nonsense mutation in a MicroRNA399 target gene.

*Folklore and History, Tradition and Uses. Editor Avanzato, D.* . ISHS, ISBN, 978-90-

Pereira, J.M.G. (2004). Sclerophylly and leaf anatomical traits of five field-grown olive cultivars growing under drought conditions. *Tree Physiology*, Vol. 24, No. 2,

Physiological responses of different olive genotypes to drought conditions. *Acta Physiologiae Plantarum*, Vol. 31, No. 3, pp. 611-621, (May 2009), ISSN, 0137-5881 Bacelar, E.A.; Santos, D.L.; Moutinho-Pereira, J.M.; Gonçalves, B.C.; Ferreira, H.F. & Correia,

C.M. (2006). Immediate responses and adaptative strategies of three olive cultivars under contrasting water availability regimes: changes on structure and chemical composition of foliage and oxidative damage. *Plant Science*, Vol. 170, No. 3, pp. 596-

& Correia, C.M. (2007). Physiological behaviour, oxidative damage and antioxidative protection of olive trees grown under different irrigation regimes.

signaling in plants. *Annual Review of Plant Biology*, Vol. 59, No., pp. 21-39, 2008),

light flux density, leaf age, temperature, peltates, and H2O vapor pressure deficit on gas exchange. *Journal of the American Society for Horticultural Science*, Vol. 112, No. 1,

*Temperate Crops*, Schaffer B, Andersen PC (eds), pp. 165–187, CRC Press, ISBN, 978-

anatomy of two olive cultivars. *Plant Science*, Vol. 163, No. 2, pp. 375-379, (Aug

tolerance to abiotic and biotic stresses: science, technology and safety. *Bulgarian Journal of Plant Physiology* Vol., No. 2003 Special Issue pp. pp: 52-82, 2003), ISSN,

Determination of sugar compounds in olive plant extracts by anion-exchange chromatography with pulsed amperometric detection. *Analytical Chemistry*, Vol. 72,

determining degree-day values from daily temperature data. *International Journal of* 


Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

No. 3, pp. 305-320, (Oct 2009), ISSN, 1380-3743

0098-8472

ISSN, 0567-7572

ISSN, 0018-5345

825-831, (Jul 2009), ISSN, 0137-5881

No. 8, pp. 569-578, (Aug 2008), ISSN, 1631-0691

No. 1-2, pp. 27-39, (Jun 16 1998), 0304-4238

pp. 16-37 2003), ISSN, 10922172 (print ),10985557 (online)

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 199

Gambino, G.; Chitarra, W.; Maghuly, F.; Laimer, M.; Boccacci, P.; Marinoni, D.T. &

Gambino, G.; Perrone, I. & Gribaudo, I. (2008). A rapid and effective method for rna

Gelvin, B.S. (2003). Agrobacterium-mediated plant transformation: the Biology behind the

Giorio, P.; Sorrentino, G. & d'Andria, R. (1999). Stomatal behaviour, leaf water status and

Gomes-Laranjo, J.; Coutinho, J.P.; Peixoto, F. & Araujo-Alves, J. (2007) Ecologia do

Gomes-Laranjo, J.; Coutinho, J.P.; Peixoto, F. & Torres-Pereira, J. (2005). Acclimation of

Gomes-Laranjo, J.; Peixoto, F.; Sang, H. & Torres-Pereira, J. (2006). Study of the temperature

Guerfel, M.; Beis, A.; Zotos, T.; Boujnah, D.; Zarrouk, M. & Patakas, A. (2009). Differences in

Hanana, M.; Deluc, L.; Fouquet, R.; Daldoul, S.; Leon, C.; Barrieu, F.; Ghorbel, A.; Mliki, A.

Iacono, F.; Buccella, A. & Peterlunger, E. (1998). Water stress and rootstock influence on leaf

Jaillon, O.; Aury, J.M.; Noel, B.; Policriti, A.; Clepet, C.; Casagrande, A.; Choisne, N.;

Jin, W.M.; Dong, J.; Hu, Y.L.; Lin, Z.P.; Xu, X.F. & Han, Z.H. (2009). Improved cold-resistant

Joesting, H.M.; McCarthy, B.C. & Brown, K.J. (2009). Determining the shade tolerance of

Vol. 449, No. 7161, pp. 463-U5, (Sep 27 2007), ISSN, 0028-0836

CG (eds), pp. 109-149, UTAD, ISBN, 978-972-669-844-9, Vila real

*Physiology*, Vol. 163, No. 9, pp. 945-955, 2006), ISBN, 0176-1617

Gribaudo, I. (2009). Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation. *Molecular Breeding*, Vol. 24,

extraction from different tissues of grapevine and other woody plants. *Phytochemical Analysis*, Vol. 19, No. 6, pp. 520-525, (Nov-Dec 2008), ISSN, 0958-0344

"Gene-Jockeying" tool. *Microbiology and Molecular Biology Reviews*, Vol. 67, No. 1,

photosynthetic response in field-grown olive trees under water deficit. *Environmental and Experimental Botany*, Vol. 42, No. 2, pp. 95-104, (Oct 1999), ISSN,

castanheiro. In: *Castanheiros*, Gomes-Laranjo J, Ferreira-Cardoso J, Portela E, Abreu

chloroplasts from north and south exposed canopy sectors of chestnut trees (Castanea sativa Mill.). *Acta Horticulturae*, Vol. 693, No., pp. 279-284, (Oct 2005),

effect in three chestnut (Castanea sativa Mill.) cultivars' behaviour. *Journal of Plant* 

abscisic acid concentration in roots and leaves of two young Olive (*Olea europaea* L.) cultivars in response to water deficit. *Acta Physiologiae Plantarum*, Vol. 31, No. 4, pp.

& Hamdi, S. (2008). Identification and characterization of 'rd22' dehydration responsive gene in grapevine (Vitis vinifera L.). *Comptes Rendus Biologies*, Vol. 331,

gas exchange of grafted and ungrafted grapevines. *Scientia Horticulturae*, Vol. 75,

Aubourg, S.; Vitulo, N.; Jubin, C. and others (2007). The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. *Nature*,

performance in transgenic grape (*Vitis vinifera L.*) overexpressing cold-inducible transcription factors AtDREB1b. *Hortscience*, Vol. 44, No. 1, pp. 35-39, (Feb 2009),

American chestnut using morphological and physiological leaf parameters. *Forest Ecology and Management*, Vol. 257, No. 1, pp. 280-286, (Jan 20 2009), ISBN, 0378-1127

differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay. *Bmc Genomics*, Vol. 10, No. 212, pp., (May 8 2009), ISSN, 1471-2164


DemmigAdams, B. & Adams, W.W. (1996). Xanthophyll cycle and light stress in nature:

Ding, D.; Zhang, L.F.; Wang, H.; Liu, Z.J.; Zhang, Z.X. & Zheng, Y.L. (2009). Differential

Dinis, L.T.; Peixoto, F.; Pinto, T.; Costa, R.; Bennett, R.N. & Gomes-Laranjo, J. (2011). Study

Drosopoulou, E.; Chrysopoulou, A.; Nikita, V. & Mavragani-Tsipidou, P. (2009). The heat

Düring, H. (1984). Evidence for osmotic adjustment to drought in grapevines (*Vitis vinifera*

Düring, H. (1992). Low air humidity causes nonuniform stomatal closure in heterobaric leaves of Vitis species. *Vitis*, Vol. 31, No. 1, pp. 1-7, (Mar 1992), ISSN, 0042-7500 Düring, H. (1987). Stomatal responses to alterations of soil and air humidity in grapevines.

Düring, H. & Dry, P.R. (1995). Osmoregulation in water-stressed roots - Responses of leaf

Eriksson, G.; Jonson, A.; Laureti, M. & Pliura, A. (2005). Genetic variation in drought

Escalona, J.M.; Flexas, J. & Medrano, H. (1999). Stomatal and non-stomatal limitations of

Fernández-López, J.; Zas, R.; Diaz, R.; Aravanopoulos, F.A.; Alizoti, P.G.; Botta, R.; Mellano,

Fernández, J.E. & Moreno, F. (1999) Water use by the olive tree. In: *Water Use in Crop* 

Fernández, J.E.; Moreno, F.; Giron, I.F. & Blazquez, O.M. (1997). Stomatal control of water

Flexas, J.; Escalona, J.M. & Medrano, H. (1998). Down-regulation of photosynthesis by

*Physiology*, Vol. 25, No. 8, pp. 893-900, 1998), ISSN, 0310-7841

*Plant Physiology*, Vol. 26, No. 5, pp. 421-433, 1999), ISSN, 0310-7841

conductance and photosynthesis. *Vitis*, Vol. 34, No. 1, pp. 15-17, (Mar 1995), ISSN,

response of *Castanea sativa* Mill. seedlings. *Acta Horticulturae*, Vol. 693, No., pp. 247-

photosynthesis under water stress in field-grown grapevines. *Australian Journal of* 

M.G.; Villani, F.; Cherbini, M. & Eriksson, G. (2005). Geographic variability among extreme European wild chestnut populations. *Acta Horticulturae*, Vol. 693, No., pp.

*Production*, Kirkham MB (eds), pp. 101-162, The Haworth Press, ISBN, 1-56022-068-

use in olive tree leaves. *Plant and Soil*, Vol. 190, No. 2, pp. 179-192, (Mar 1997), ISSN,

drought under field conditions in grapevine leaves. *Australian Journal of Plant* 

*Genome*, Vol. 52, No. 2, pp. 210-214, (Feb 2009), ISSN, 0831-2796

L). *Vitis*, Vol. 23, No. 1, pp. 1-10, 1984), ISSN, 0042-7500

*Vitis*, Vol. 26, No. 1, pp. 9-18, (Mar 1987), ISSN, 0042-7500

212, pp., (May 8 2009), ISSN, 1471-2164

3, pp. 110-120, 2011), ISSN, 0098-8472

0042-7500

0032-079X

254, 2005), ISBN, 0567-7572

181-186, 2005), ISBN, 0567-7572

6, Binghamton, New York

198, No. 3, pp. 460-470, (Mar 1996), ISSN, 0032-0935

Vol. 103, No. 1, pp. 29-38, (Jan 2009), ISSN, 0305-7364

differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay. *Bmc Genomics*, Vol. 10, No.

Uniform response to excess direct sunlight among higher plant species. *Planta*, Vol.

expression of miRNAs in response to salt stress in maize roots. *Annals of Botany*,

of morphological and phenological diversity in chestnut trees ([`]Judia' variety) as a function of temperature sum. *Environmental and Experimental Botany*, Vol. 70, No. 2-

shock 70 genes of the olive pest Bactrocera oleae: genomic organization and molecular characterization of a transcription unit and its proximal promoter region.


Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

No. 2, pp. 98-116, 2010), ISSN, 445-4408

693-700, (Apr 1998), ISSN, 0022-0957

pp. 183-197, (Feb 5 2000), ISSN, 0168-1923

Vol. 4, No. 3, pp. 563-574, (Jul 2008), ISSN, 1614-2942

(print), 1095-9203 (online)

2010), ISSN, 1614-2942

(Jun 2002), ISSN, 0305-7364

0829-318X

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 201

Liu, Q.P.; Feng, Y. & Zhu, Z.J. (2009). Dicer-like (DCL) proteins in plants. *Functional & Integrative Genomics*, Vol. 9, No. 3, pp. 277-286, (Aug 2009), ISSN, 1438-793X Lo Gullo, M.A. & Salleo, S. (1988). Different strategies of drought resistance in three

*New Phytologist*, Vol. 108, No. 3, pp. 267-276, (Mar 1988), ISSN, 0028-646X Loreto, F. & Sharkey, T.D. (1990). Low humidity can cause uneven photosynthesis in olive

Lovisolo, C.; Perrone, I.; Carra, A.; Ferrandino, A.; Flexas, J.; Medrano, H. & Schubert, A.

Lovisolo, C. & Schubert, A. (1998). Effects of water stress on vessel size and xylem hydraulic

Lu, S.F.; Sun, Y.H. & Chiang, V.L. (2008). Stress-responsive microRNAs in Populus. *Plant* 

Lu, Y.D.; Gan, Q.H.; Chi, X.Y. & Qin, S. (2008). Identification and Characterization of

Makeyev, E.V. & Maniatis, T. (2008). Multilevel regulation of gene expression by

Marion-Poll, A. & Leung, J. (2006) Abscisic acid synthesis, metabolism and signal

Martin, M.A.; Mattioni, C.; Cherubini, M.; Taurchini, D. & Villani, F. (2010). Genetic

Martins, A.; Raimundo, F.; Borges, O.; Linhares, I.; Sousa, V.; Coutinho, J.P.; Gomes-Laranjo,

Medrano, H.; Escalona, J.M.; Bota, J.; Gulias, J. & Flexas, J. (2002). Regulation of

Blackwell Publishing, ISBN, 978-14051-3887-1, Oxford, UK; Ames, Iowa Mariscal, M.J.; Orgaz, F. & Villalobos, F.J. (2000). Modelling and measurement of radiation

*Journal*, Vol. 55, No. 1, pp. 131-151, (Jul 2008), ISSN, 0960-7412

*China*, Vol. 7, No. 8, pp. 929-943, (August 2008), ISBN, 1671-2927

Mediterranean sclerophyllous trees growing in the same environmental conditions.

(*Olea europea* L.) leaves. *Tree Physiology*, Vol. 6, No. 4, pp. 409-415, (Dec 1990), ISSN,

(2010). Drought-induced changes in development and function of grapevine (*Vitis*  spp.) organs and in their hydraulic and non-hydraulic interactions at the wholeplant level: a physiological and molecular update. *Functional Plant Biology*, Vol. 37,

conductivity in *Vitis vinifera* L. *Journal of Experimental Botany*, Vol. 49, No. 321, pp.

MicroRNAs and their targets in grapevine (*Vitis vinifera*). *Agricultural Sciences in* 

microRNAs. *Science*, Vol. 319, No. 5871, pp. 1789-90, (Mar 28 2008), ISSN, 0036-8075

transduction. In: *Plant Hormone Signaling*, Hedden P, Thomas SG (eds), pp. 1-35,

interception by olive canopies. *Agricultural and Forest Meteorology*, Vol. 100, No. 2-3,

diversity in European chestnut populations by means of genomic and genic microsatellite markers. *Tree Genetics & Genomes*, Vol. 6, No. 5, pp. 735-744, (Oct

J. & Madeira, M. (2010). Effects of soil management practices and irrigation on plant water relations and productivity of chestnut stands under Mediterranean conditions. *Plant and Soil*, Vol. 327, No. 1-2, pp. 57-70, (Feb 2010), ISBN, 0032-079X Mattioni, C.; Cherubini, M.; Micheli, E.; Villani, F. & Bucci, G. (2008). Role of domestication

in shaping Castanea sativa genetic variation in Europe. *Tree Genetics & Genomes*,

photosynthesis of C-3 plants in response to progressive drought: Stomatal conductance as a reference parameter. *Annals of Botany*, Vol. 89, No., pp. 895-905,


Jones-Rhoades, M.W.; Bartel, D.P. & Bartel, B. (2006). MicroRNAs and their regulatory roles

Kaldenhoff, R.; Ribas-Carbo, M.; Flexas, J.; Lovisolo, C.; Heckwolf, M. & Uehlein, N. (2008).

Kantar, M.; Lucas, S.J. & Budak, H. (2011). miRNA expression patterns of *Triticum dicoccoides*

Karabourniotis, G.; Papastergiou, N.; Kabanopoulou, E. & Fasseas, C. (1994). Foliar sclereids

Khan, M.S.; Yu, X.; Kikuchi, A.; Asahina, M. & Watanabe, K.N. (2009). Genetic engineering

Kozomara, A. & Griffiths-Jones, S. (2011). miRBase: integrating microRNA annotation and

Leon, J.M. & Bukovac, M.J. (1978). Cuticle development and surface morphology of olive

Li, W.X.; Oono, Y.; Zhu, J.H.; He, X.J.; Wu, J.M.; Iida, K.; Lu, X.Y.; Cui, X.P.; Jin, H.L. & Zhu,

Liang, G.; Yang, F.X. & Yu, D.Q. (2010). MicroRNA395 mediates regulation of sulfate

Lilley, J.M.; Ludlow, M.M.; McCouch, S.R. & OToole, J.C. (1996). Locating QTL for osmotic

Liu, H.H.; Tian, X.; Li, Y.J.; Wu, C.A. & Zheng, C.C. (2008). Microarray-based analysis of

Liu, H.Y.; Yang, W.L.; Liu, D.C.; Han, Y.P.; Zhang, A.M. & Li, S.H. (2011). Ectopic expression

*Cell*, Vol. 20, No. 8, pp. 2238-2251, (Aug 2008), ISSN, 1040-4651

*Society*, Vol. 14, No. 5, pp. 836-843, (May 2008), ISSN, 1355-8382

pp. 1046-1057, (Jun 2010), ISSN, 0960-7412

No. 302, pp. 1427-1436, (Sep 1996), ISSN, 0022-0957

*Biotechnology*, Vol. 26, No. 1, pp. 125-134, (Mar 25 2009), ISSN, 1342-4580 Kitsaki, C.K. & Drossopoulos, J.B. (2005). Environmental effect on ABA concentration and

*and Viticulture*, Vol. 56, No. 3, pp. 267-283, 2005), ISSN, 0002-9254

No. 5, pp. 658-666, (May 2008), ISSN: 0140-7791

2519

ISSN, 0032-0935

(Aug 2005), ISSN, 0098-8472

2011), ISSN, 0305-1048

2011), ISSN, 0301-4851

0003-1062

in plants. *Annual Review of Plant Biology*, Vol. 57, No., pp. 19-53, 2006), ISSN, 1040-

Aquaporins and plant water balance. *Plant, Cell and Environment*, Vol. Vol. 31, No.

in response to shock drought stress. *Planta*, Vol. 233, No. 3, pp. 471-484, (Mar 2011),

of *Olea europaea* may function as optical fibers. *Canadian Journal of Botany-Revue Canadienne De Botanique*, Vol. 72, No. 3, pp. 330-336, (Mar 1994), ISSN, 0008-4026 Keller, M. (2005). Deficit irrigation and vine mineral nutrition. *American Journal of Enology* 

of glycine betaine biosynthesis to enhance abiotic stress tolerance in plants. *Plant* 

water potential in olive leaves (*Olea europaea* L. cv "Koroneiki") under non-irrigated field conditions. *Environmental and Experimental Botany*, Vol. 54, No. 1, pp. 77-89,

deep-sequencing data. *Nucleic Acids Research*, Vol. 39, No., pp. D152-D157, (Jan

leaves with reference to penetration of foliar-applied chemicals. *Journal of the American Society for Horticultural Science*, Vol. 103, No. 4, pp. 465-472, 1978), ISSN,

J.K. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. *Plant* 

accumulation and allocation in Arabidopsis thaliana. *Plant Journal*, Vol. 62, No. 6,

adjustment and dehydration tolerance in rice. *Journal of Experimental Botany*, Vol. 47,

stress-regulated microRNAs in *Arabidopsis thaliana*. *RNA-a Publication of the RNA* 

of a grapevine transcription factor VvWRKY11 contributes to osmotic stress tolerance in Arabidopsis. *Molecular Biology Reports*, Vol. 38, No. 1, pp. 417-427, (Jan


Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

ISSN, 1365-313X (online)

Cambridge

481-2304-9, Dordrecht, The Netherlands

154, No. 5-6, pp. 767-774, (May 1999), ISSN, 0176-1617

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 203

Palliotti, A.; Cartechini, A. & Ferranti, F. (2000). Morpho-anatomical and physiological

Pantaleo, V.; Szittya, G.; Moxon, S.; Miozzi, L.; Moulton, V.; Dalmay, T. & Burgyan, J. (2010).

Paschalidis, K.A.; Moschou, P.N.; Aziz, A.; Toumi, I. & Roubelakis-Angelakis, K.A. (2009)

Patakas, A. & Noitsakis, B. (1999). Mechanisms involved in diurnal changes of osmotic

Pearcy, R.W. (1998) Acclimation to sun and shade. In: *Photosynthesis. A comprehensive treatise*,

Pereira-Lorenzo, S.; Costa, R.; Ramos-Cabrer, A.; Ribeiro, C.; da Silva, M.; Manzano, G. &

Perez-Barranco, G.; Torreblanca, R.; Padilla, I.M.G.; Sanchez-Romero, C.; Pliego-Alfaro, F. &

*and Organ Culture*, Vol. 97, No. 3, pp. 243-251, (Jun 2009), ISSN, 0167-6857 Phillips, J.R.; Dalmay, T. & Bartels, D. (2007). The role of small RNAs in abiotic stress. *Febs Letters*, Vol. 581, No. 19, pp. 3592-3597, (Jul 31 2007), ISSN, 0014-5793 Pou, A.; Flexas, J.; Alsina, M.D.; Bota, J.; Carambula, C.; de Herralde, F.; Galmes, J.; Lovisolo,

Rejšková, A.; Patková, L.; Stodůlková, E. & Lipavská, H. (2007). The effect of abiotic stresses

Roy, B.; Noren, S.K.; Mandal, A.B. & Basu, A.K. (2011). Genetic engineering for Abiotic

Salleo, S. & Lo Gullo, M.A. (1993) Drought resistance strategies and vulnerability to

*& Genomes*, Vol. 6, No. 5, pp. 701-715, 2010), ISSN, 1614-2942

Vol. 134, No. 2, pp. 313-323, (Oct 2008), ISSN, 0031-9317

ISBN, 978-90-481-3111-2, Dordrecht, The Netherlands

2011), ISSN, eISSN: 1682-2978, pISSN: 1682-296x

*Viticulture*, Vol. 51, No. 2, pp. 122-130, (n.d. 2000), ISSN, 0002-9254

characteristics of primary and lateral shoot leaves of Cabernet Franc and Trebbiano Toscano grapevines under two irradiance regimes. *American Journal of Enology and* 

Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis. *Plant J*, Vol. 62, No. 6, pp. 960-76, (Jun 1 2010),

Polyamines in grapevine: An update. In: *Grapevine Molecular Physiology & Biotechnology*, Roubelakis-Angelakis KA (eds), pp. 207-228, Springer, ISBN, 978-90-

potential in grapevines under drought conditions. *Journal of Plant Physiology*, Vol.

Raghavendra AS (eds), pp. 250-263, Cambridge University Press, 0-521-57000-X,

Barreneche, T. (2010). Variation in grafted European chestnut and hybrids by microsatellites reveals two main origins in the Iberian Peninsula. *Tree Genetics* 

Mercado, J.A. (2009). Studies on genetic transformation of olive (*Olea europaea* L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for nptII selection; II. Transient transformation via particle bombardment. *Plant Cell Tissue* 

C.; Jimenez, M.; Ribas-Carbo, M. and others (2008). Adjustments of water use efficiency by stomatal regulation during drought and recovery in the droughtadapted *Vitis* hybrid Richter-110 (*V. berlandieri* x *V. rupestris*). *Physiologia Plantarum*,

on carbohydrate status of olive shoots (*Olea europaea* L.) under in vitro conditions. *Journal of Plant Physiology*, Vol. 164, No. 2, pp. 174-184, (Feb 2007), ISSN, 0176-1617 Rock, C.D.; Sakata, Y. & Quatrano, R.S. (2010) Stress signaling I: The role of abscisic acid

(ABA). In: *Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic Foundation*, Pareek A, Sopory SK, Bohnert HJ, Govindjee (eds), pp. 33-73, Springer,

stress tolerance in agricultural crops. *Biotechnology*, Vol. 10, No. 1, pp. 1-22, (n.d.

cavitation in some Mediterranean sclerophyllous trees. In: *Water Transport in Plants* 


Meyers, B.C.; Axtell, M.J.; Bartel, B.; Bartel, D.P.; Baulcombe, D.; Bowman, J.L.; Cao, X.;

Mica, E.; Piccolo, V.; Delledonne, M.; Ferrarini, A.; Pezzotti, M.; Casati, C.; Del Fabbro, C.;

Mittler, R. & Blumwald, E. (2010). Genetic engineering for Mmodern agriculture: Challenges

Moreira, F.M.; Madini, A.; Marino, R.; Zulini, L.; Stefanini, M.; Velasco, R.; Kozma, P. &

*Genetics & Genomes*, Vol. 7, No. 1, pp. 153-167, (Feb 2011), ISSN, 1614-2942 Moutinho-Pereira, J.; Magalhaes, N.; Goncalves, B.; Bacelar, E.; Brito, M. & Correia, C.

Moutinho-Pereira, J.M.; Correia, C.M.; Goncalves, B.M.; Bacelar, E.A. & Torres-Pereira, J.M.

Moutinho-Pereira, J.M.; Magalhães, N.; Correia, C.M. & Torres-Pereira, J.M. (2003). Effects of

Murata, N. & Siegenthaler, P.-A. (1998) Lipids in photosynthesis: An overview. In: *Lipids in* 

Natali, S.; Bignami, C.; Cammilli, C. & Muganu, M. (1999). Effect of water stress on leaf

Neill, S.; Barros, R.; Bright, J.; Desikan, R.; Hancock, J.; Harrison, J.; Morris, P.; Ribeiro, D. &

*Experimental Botany*, Vol. 59, No. 2, pp. 165-176, (Feb 2008), ISSN, 0022-0957 Osório, M.; Breia, E.; Rodrigues, A.; Osório, J.; Le Roux, X.; Daudet, F.A.; Ferreira, I. &

Osório, M.L.; Osório, J.; Pereira, J.S. & Chaves, M.M. (1995) Responses of photosynthesis to

1-20, Kluwer Academic Press, ISBN, 9780792351733 Dordrecht

*Botany*, Vol. 55, No. 3, pp. 235-247, (Mar 2006), ISSN, 0098-8472

Kluwer Academic Publishers, ISBN, 0-9723-3860-X, London

1040-4651 (print); 1532-298X (online)

(Feb 12 2010), ISSN, 1471-2164

ISSN, 1543-5008

2007), ISSN, 0300-3604

1999), ISSN, 0567-7572

0300-3604

0394-0438

Carrington, J.C.; Chen, X.; Green, P.J. and others (2008). Criteria for annotation of plant MicroRNAs. *Plant Cell*, Vol. 20, No. 12, pp. 3186-3190, (Dec 19 2008), ISSN,

Valle, G.; Policriti, A.; Morgante, M. and others (2010). High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera. *Bmc Genomics*, Vol. 11, No., pp. -,

and perspectives. *Annual Review of Plant Biology*, Vol. 61, No., pp. 443-462, 2010),

Grando, M.S. (2011). Genetic linkage maps of two interspecific grape crosses (*Vitis* spp.) used to localize quantitative trait loci for downy mildew resistance. *Tree* 

(2007). Gas exchange and water relations of three *Vitis vinifera* L. cultivars growing under Mediterranean climate. *Photosynthetica*, Vol. 45, No. 2, pp. 202-207, (June

(2004). Leaf gas exchange and water relations of grapevines grown in three different conditions. *Photosynthetica*, Vol. 42, No. 1, pp. 81-86, (March 2004), ISSN,

NW-SE row orientation on grapevine physiology under Mediterranean field conditions *Agricoltura Mediterranea*, Vol. 133, No. 3-4, pp. 218-225, (n.d. 2003), ISSN

*photosynthesis: structure, function and genetics*, Siegenthaler P-A, Murata N (eds), pp.

movement in olive cultivars. *Acta Horticulturae*, Vol. 474, No., pp. 445-448, (Apr

Wilson, I. (2008). Nitric oxide, stomatal closure, and abiotic stress. *Journal of* 

Chaves, M.M. (2006). Limitations to carbon assimilation by mild drought in nectarine trees growing under field conditions. *Environmental and Experimental* 

water stress under field conditions in grapevines are dependent on irradiance and temperature. In: *Photosynthesis: from light to biosphere*, Mathis P (eds), pp. 669-672,


Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine

pp. 519-525, (January 8 2010), ISSN, 0176-1617

Vol. 2, No. 12, pp. e1326, (Dec 19 2007), ISSN, 1932-6203

228-232, ISBN, 0769535593, Macau, China, 20-22 Feb, 2009

ISSN, 0014-5793

24 June 2008

1988), ISSN, 0394-6169

7791

and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs 205

Tognetti, R.; d'Andria, R.; Morelli, G.; Calandrelli, D. & Fragnito, F. (2004). Irrigation effects

Valderrama, R.; Corpas, F.J.; Carreras, A.; Fernández-Ocaña, A.; Chaki, M.; Luque, F.;

Velasco, R.; Zharkikh, A.; Troggio, M.; Cartwright, D.A.; Cestaro, A.; Pruss, D.; Pindo, M.;

Villani, F.; Mattioni, C.; Cherubini, M.; Lauteri, M. & Martin, M. (2010). An integrated

Wei, Z.L. (2009). Computational prediction of abiotic stress responsible MicroRNAs in *Vitis* 

Wei, Z.L.; Tian, Z.H.; Jiao, C.Z. & Dong, L. (2008). Computational prediction of UV-

Xiao, H.; Tattersall, E.A.R.; Siddiqua, M.K.; Cramer, G.R. & Nassuth, A. (2008). CBF4 is a

Xiloyannis, C.; Dichio, B.; Nuzzo, V. & Celano, G. (1999). Defence strategies of olive against water stress. *Acta Horticulturae*, Vol. 474, No., pp. 423-426, 1999), ISSN, 0567-7572 Xiloyannis, C.; Pezzarossa, B.; Jorba, J. & Angelini, P. (1988). Effects of soil water content on

Xu, C.; Yang, R.F.; Li, W.C. & Fu, F.L. (2010). Identification of 21 microRNAs in maize and

Yang, O.; Popova, O.V.; Suthoff, U.; Luking, I.; Dietz, K.J. & Golldack, D. (2009). The

Zafra, A.; Rodríguez-García, M.I. & Alché, J.D. (2010). Cellular localization of ROS and NO

Vol. 9, No. 30, pp. 4741-4753, (Jul 26 2010), ISSN, 1684-5315

2, pp. 45-55, (May 1 2009), ISSN, 0378-1119

No. 36, pp. 1-14, (Feb 24 2010), ISSN, 1471-2229

*Horticulturae*, Vol. 866, No., pp. 91-95, (June 2010), ISSN, 0567-7572

on daily and seasonal variations of trunk sap flow and leaf water relations in olive trees. *Plant and Soil*, Vol. 263, No. 1-2, pp. 249-264, (Jun 2004), ISSN, 0032-079X Toumi, I.; Moschou, P.N.; Paschalidis, K.A.; Bouamama, B.; Ben Salem-fnayou, A.; Ghorbel,

A.W.; Mliki, A. & Roubelakis-Angelakis, K.A. (2010). Abscisic acid signals reorientation of polyamine metabolism to orchestrate stress responses via the polyamine exodus pathway in grapevine. *Journal of Plant Physiology*, Vol. 167, No. 7,

Gómez-Rodríguez, M.V.; Colmenero-Varea, P.; del Rio, L.A. & Barroso, J.B. (2007). Nitrosative stress in plants. *Febs Letters*, Vol. 581, No. 3, pp. 453-461, (Feb 6 2007),

FitzGerald, L.M.; Vezzulli, S.; Reid, J. and others (2007). A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. *Plos One*,

approach in assess the genetic and adaptative variation in *Castanea sativa* Mill. *Acta* 

*vinifera* genome, *2009 International Conference on Electronic Computer Technology*, pp.

responsible MicroRNA genes in *Vitis vinifera* genome, *2008 International Conference on BioMedical Engineering and Informatics*, pp. ISBN, 978-0-7695-3118-2 Sanya, China,

unique member of the CBF transcription factor family of *Vitis vinifera* and *Vitis riparia*. *Plant Cell and Environment*, Vol. 31, No. 1, pp. 1-10, (Jan 2008), ISSN, 0140-

gas exchange in olive trees. *Advances in Horticultural Science*, Vol. 2, No. 2, pp. 58-63,

their differential expression under drought stress. *African Journal of Biotechnology*,

Arabidopsis basic leucine zipper transcription factor AtbZIP24 regulates complex transcriptional networks involved in abiotic stress resistance. *Gene*, Vol. 436, No. 1-

in olive reproductive tissues during flower development. *Bmc Plant Biology*, Vol. 10,

*under Stress Conditions*, Borghetti M, Grace J, Raschi A (eds), pp. 71-113, Cambridge University Press, ISBN, 0-521-44219-2, Cambridge


Salleo, S.; Lo Gullo, M.A. & Oliveri, F. (1985). Hydraulic parameters measured in 1-year-old

*Experimental Botany*, Vol. 36, No. 162, pp. 1-11, (n.d. 1985), ISSN, 0022-0957 Schultz, H.R. & Matthews, M.A. (1988). Resistance to water transport in shoots of *Vitis* 

Secchi, F.; Lovisolo, C.; Uehlein, N.; Kaldenhoff, R. & Schubert, A. (2007). Isolation and

Seguin, B. & Cortazar, I.G. (2005). Climate Warming: consequences for Viticulture and the

Shukla, L.I.; Chinnusamy, V. & Sunkar, R. (2008). The role of microRNAs and other

Singh, A. & Grover, A. (2008). Genetic engineering for heat tolerance in plants. *Physiology* 

Smart, R.E. (1974). Photosynthesis by grapevines canopies. *Journal of Applied Ecology*, Vol. 11 No. 3, pp. 997-1006, (Dec 1974 1974), ISSN, 00218901, E-ISSN 13652664 Soar, C.J.; Speirs, J.; Maffei, S.M. & Loveys, B.R. (2004). Gradients in stomatal conductance,

Sofo, A.; Dichio, B.; Xiloyannis, C. & Masia, A. (2004a). Effects of different irradiance levels

Sofo, A.; Dichio, B.; Xiloyannis, C. & Masia, A. (2004b). Lipoxygenase activity and proline

Sunkar, R.; Kapoor, A. & Zhu, J.K. (2006). Posttranscriptional induction of two Cu/Zn

Tognetti, R.; Costagli, G.; Minnocci, A. & Gucci, R. (2002). Stomatal behaviour and water use

University Press, ISBN, 0-521-44219-2, Cambridge

3, pp. 718-724, (Nov 1988), ISSN, 0032-0889

31, No. 6, pp. 659-669, 2004), ISSN, 1445-4408

No. 7, pp. 301-309, (Jul 2007), 1360-1385

2051-2065, (Aug 2006), ISSN, 1040-4651

2, pp. 90-97, (n.d. 2002), ISSN, 0394-0438

ISSN, 0567-7572

(Print) 0974-0430 (Online)

9399

Vol. 225, No. 2, pp. 381-392, (Jan 2007), ISSN, 0032-0935

*under Stress Conditions*, Borghetti M, Grace J, Raschi A (eds), pp. 71-113, Cambridge

twigs of some Mediterranean species with diffuse-porous wood: Changes in hydraulic conductivity and their possible functional significance. *Journal of* 

*vinifera* L - Relation to growth at low water potential. *Plant Physiology*, Vol. 88, No.

functional characterization of three aquaporins from olive (*Olea europaea* L.). *Planta*,

notion of 'terroirs' in Europe. *Acta Horticulturae*, Vol. 689, No., pp. 61-71, (n.d. 2005),

endogenous small RNAs in plant stress responses. *Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms*, Vol. 1779, No. 11, pp. 743-748, (Nov 2008), ISSN, 1874-

*and Molecular Biology of Plants*, Vol. 14, No. 1-2, pp. 155-166, (Apr 2008), 0971-5894

xylem sap ABA and bulk leaf ABA along canes of *Vitis vinifera* cv. Shiraz: molecular and physiological studies investigating their source. *Functional Plant Biology*, Vol.

on some antioxidant enzymes and on malondialdehyde content during rewatering in olive tree. *Plant Science*, Vol. 166, No. 2, pp. 293-302, (Feb 2004a), ISSN, 0168-9452

accumulation in leaves and roots of olive trees in response to drought stress. *Physiologia Plantarum*, Vol. 121, No. 1, pp. 58-65, (May 2004b), ISSN, 0031-9317 Stanislawski, D. (1970) *Landscapes of Bacchus: the Vine in Portugal. pp.Austin Texas: University of Texas Press*. University of Texas Press, ISBN, 9780292700109 Austin Texas Sunkar, R.; Chinnusamy, V.; Zhu, J.H. & Zhu, J.K. (2007). Small RNAs as big players in plant

abiotic stress responses and nutrient deprivation. *Trends in Plant Science*, Vol. 12,

superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. *Plant Cell*, Vol. 18, No. 8, pp.

efficiency in two cultivars of *Olea europaea* L. *Agricoltura Mediterranea*, Vol. 132, No.


**9** 

*China* 

**Molecular and Genetic Analysis of Abiotic** 

Abiotic stress, brought about by salinity, drought, extreme temperatures and oxidative stress are serious threats to agriculture and result in a huge reduction of production. Drought and salinity are becoming major threats throughout the world. Abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and development (Wang et al., 2001). In order to survive from these harsh stresses, forage plants have developed precise and complicated tolerance

Under serious threat, forage plants can change the shape of the leaves and roots to decrease the water loss. Some plants have evolved special structures, such as salt glands to excrete salt. At the physiological level, respiratory, photosynthesis metabolism and osmotic adjustments etc. all change in order to resist stress (Chaves, 1991; Sheng, 2010; Yang et al., 2007). In fact, all of these changes are related to gene expression. The complex plant response to abiotic stress involves many genes and molecular mechanisms. In the past several decades, multiple genes responding to drought, salt, low-temperature and oxidative stress have been identified. These genes are divided into two groups (Shinozaki et al., 2003). The first group functions to directly protect the plant against stress, involving key enzymes for osmolyte biosynthesis, LEA (late embryogenesis abundant) proteins, detoxification enzymes and enzymes involved in many metabolic processes. The other group consists of contained protein factors involved in the regulation of signal transduction, including various transcription factors, proteins kinases, protein phosphatases, enzymes involved in phospholipid metabolism, and other signaling molecules (Yamaguchi-Shinozaki & Shinozaki, 2006). Genes have been used extensively to improve the stress-tolerance in crop and forage crops. The investigation of stress-tolerance genes will increase our knowledge of tolerance mechanisms, which could in turn be used to promote improvements in forage

Plants often produce a visible response to certain types of environmental stress. Under drought stress, the leaf morphology changes in order to retain water and increase the water use efficiency in forage crops plants. In general, plants decrease the leaf area to limit water loss (Turner, 1979). Nobel investigated the relationship between leaf structure and water use

mechanisms at the morphological, physiological and molecular levels.

**2. The morphological response to abiotic stress** 

**1. Introduction** 

crop plants tolerance.

**Stress Resistance of Forage Crops** 

Xuemin Wang, Hongwen Gao, Zan Wang and Jun Li

*Institute of Animal Science, Chinese Academy* 

 *of Agricultural Science* 


## **Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops**

Xuemin Wang, Hongwen Gao, Zan Wang and Jun Li *Institute of Animal Science, Chinese Academy of Agricultural Science China* 

### **1. Introduction**

206 Plants and Environment

Zalom, F.G.; Goodell, P.B.; Wilson, L.T.; Barnett, W.W. & Bentley, W.J. (1983) *Degree days: The calculation and use of heat units in pest management*. University of California Zhang, B.H.; Wang, Q.L. & Pan, X.P. (2007). MicroRNAs and their regulatory roles in

Zhao, B.T.; Liang, R.Q.; Ge, L.F.; Li, W.; Xiao, H.S.; Lin, H.X.; Ruan, K.C. & Jin, Y.X. (2007).

Zhou, X.F.; Wang, G.D.; Sutoh, K.; Zhu, J.K. & Zhang, W.X. (2008). Identification of cold-

2007), ISSN, 0021-9541

1874-9399

animals and plants. *Journal of Cellular Physiology*, Vol. 210, No. 2, pp. 279-289, (Feb

Identification of drought-induced microRNAs in rice. *Biochemical and Biophysical Research Communications*, Vol. 354, No. 2, pp. 585-590, (Mar 9 2007), ISSN, 0006-291X

inducible microRNAs in plants by transcriptome analysis. *Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms*, Vol. 1779, No. 11, pp. 780-788, (Nov 2008), ISSN,

> Abiotic stress, brought about by salinity, drought, extreme temperatures and oxidative stress are serious threats to agriculture and result in a huge reduction of production. Drought and salinity are becoming major threats throughout the world. Abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and development (Wang et al., 2001). In order to survive from these harsh stresses, forage plants have developed precise and complicated tolerance mechanisms at the morphological, physiological and molecular levels.

> Under serious threat, forage plants can change the shape of the leaves and roots to decrease the water loss. Some plants have evolved special structures, such as salt glands to excrete salt. At the physiological level, respiratory, photosynthesis metabolism and osmotic adjustments etc. all change in order to resist stress (Chaves, 1991; Sheng, 2010; Yang et al., 2007). In fact, all of these changes are related to gene expression. The complex plant response to abiotic stress involves many genes and molecular mechanisms. In the past several decades, multiple genes responding to drought, salt, low-temperature and oxidative stress have been identified. These genes are divided into two groups (Shinozaki et al., 2003). The first group functions to directly protect the plant against stress, involving key enzymes for osmolyte biosynthesis, LEA (late embryogenesis abundant) proteins, detoxification enzymes and enzymes involved in many metabolic processes. The other group consists of contained protein factors involved in the regulation of signal transduction, including various transcription factors, proteins kinases, protein phosphatases, enzymes involved in phospholipid metabolism, and other signaling molecules (Yamaguchi-Shinozaki & Shinozaki, 2006). Genes have been used extensively to improve the stress-tolerance in crop and forage crops. The investigation of stress-tolerance genes will increase our knowledge of tolerance mechanisms, which could in turn be used to promote improvements in forage crop plants tolerance.

### **2. The morphological response to abiotic stress**

Plants often produce a visible response to certain types of environmental stress. Under drought stress, the leaf morphology changes in order to retain water and increase the water use efficiency in forage crops plants. In general, plants decrease the leaf area to limit water loss (Turner, 1979). Nobel investigated the relationship between leaf structure and water use

Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 209

*bisquamulatus* (Geerts *et al*., 1998). Osmotic adjustment measurements can be used to select drought-tolerant cultivars (Morgan,1983). The extent of osmotic adjustment was higher in buffalograss and zoysiagrass (*Zoysia japonica* Steud) with a better drought tolerance than tall

Osmotically active solutes include amino acids (proline), sugars (e.g.,sucrose, fructans), polyols (e.g., mannitol), and organic ions(e.g.,potassium, sodium) (Chaves et al., 2003). Those solutes are associated not only with turgor maintenance, but also with the maintenance of membrane and protein structures and protection against oxidative damage (Crowe et al., 1992; Hoekstra et al., 2001). OA for creeping bentgrass and velvet bentgrass is associated with the accumulation of water soluble carbohydrates during the early period of drought and increases in proline content following prolonged a period; however, inorganic ions were not found to relate OA in these species (DaCosta & Huang, 2006). In alfalfa (*Medicago sativa* L.), salt stress induces a large increase in the amino acid and carbohydrate pools. Amongst the amino acids, proline shows the largest increase in roots, cytosol, and bbacteroides. Its accumulation is reflected in an osmoregulatory mechanism not only in roots but also in nodule tissue. The concentration of the carbohydrate pinitol is also increased significantly (Fougere et al., 1991). In many other forage and turfgrasses, glycinebetaine and proline makes a significant contribution to OA under abiotic stress

Dormancy also is a mechanism by which forage crop plants become quiescent during prolonged environmental stress, especially drought. Grasses temporarily slow the growth of meristem to avoid drought damage and to allow survival (McWilliam, 1968). Poaceae forage plants, such as *Poa scabrella* (Laude, 1953), *Poa bullbosa* (Volaire *et al*., 2001), and some populations of forage grasses such as *Dactylis glomerata* 'Kasbah' (Norton *et al*., 2006), all

The plant responses to abiotic stress involves many genes and molecular mechanisms and stress-associated genes, proteins and metabolites from a complex regulatory network. A large number of genes have been found to be associated with abiotic stress (Jin et al., 2010; Kang et al., 2010; Shinozaki and Yamaguchi-Shinozaki 2000; Thomashow 1999). Genes induced during stress conditions function not only in protecting cells from stress by producing important metabolic proteins, but also in regulating signal transduction in the stress response (Yamaguchi-Shinozaki & Shinozaki, 2006). These gene products are divided into two groups (Shinozaki et al., 2003; Yamaguchi-Shinozaki & Shinozaki, 2006). The first group functions in the direct protection of the plant against stress and includes key enzymes for osmolyte biosynthesis, LEA (late embryogenesis abundant) proteins, detoxification enzymes and enzymes involved in many metabolic processes. The second group contains protein factors involved in further regulation of signal transduction, including various transcription factors, protein kinases, protein phosphatases, enzymes involved in phospholipid metabolism, and

Transcription factor genes play important roles in stress survival by serving as master regulators of sets of downstream stress-responsive genes. Transcription factors regulate

fescure (*Festuca arundinacea* Shreb.) (Qian & Fry, 1997).

**3.2 Dormancy is another countermeasure to survive from stresses** 

**4. The molecular and genetic response to stress** 

other signaling molecules (Yamaguchi-Shinozaki & Shinozaki , 2006).

(Girousse *et al*., 1996; Marcum, 1994).

exhibit summer dormancy.

**4.1 Transcriptional factors** 

efficiency under water deficit, and found that if other conditions are invariant. The mesophyll cells become smaller per unit area under water deficit, conversely the larger the area of mesophyll cells, the higher is the water use efficiency (Noble, 1980). In an earlier study, tall fescue and turfgrasses were seen to rely primarily on a deep and extensive root system for drought tolerance because the longer root system had greater volume and surface area in contact with the soil, facilitating water and nutrient uptake under drought stress (Qian et al., 1997).

Facing salinity stresses, plants evolved special structures to survive. *Mesembryanthemum crystallinum* is a salt-secreting plant and possesses epidermal bladder cells in its aerial parts, which store Na+ (Adams et al., 1998). While *Tamarix aphylla* uses a salt gland to excrete salt (Thomason et al., 1969).

Annual plants can escape drought by maturing before stress becomes severe. Some Fescue grass cultivars avoid drought stress through changes in leaf and root morphology (reducing the transpiration surface area and closing stomata) and probably through osmotic adjustment maintain sufficient turgor pressure in the growing zone for leaf elongation (Wang & Burghrara, 2008).

### **3. The physiological response to abiotic stress**

Serious environmental threats stimulates forage and turf grassess to produce a physiological response. Photosynthesis and cell growth are the primary processes which are affected by stress (Chaves, 1991; Munns, et al., 2006). Under such stress, the rate of photosynthesis and assimilation of products of forage crops decreases remarkably, resulting in a slow down of growth. In a study of eight grass forage plants, chlorophyll content was found to decrease with increased water stress (Yang et al., 2007). Low-temperature has been found to decrease the chlorophyll content in *Poa pratensis* L.qinghai, *Roegneria thoroldiana* and *Elymus nutans*. The major reason for this is that the stress decreaseds the stability of the chloroplast and then destroyed them (Yan et al., 2007). Generally, the degree of decreasing chlorophyll content correlates with the degree of damage to the forage crop plants, so that the chlorophyll level can be used as a guide to stress tolerance in these plants.

Under a variety stresses, the respiration rate of forage plants becomes unstable. For example, the respiration rate dramatically decrease after suffering from freezing, heat, highsalinity or flooding. While after drought or chilling, the respiration rate increase initially, then sharply decreases (Sheng, 2010). The respiratory metabolism pathway also alters under stress. The Pentose Phosphate (PPP) pathway increases under drought and mechanical damage conditions in forage crop plants (Sheng, 2010).

### **3.1 Osmotic adjustment in response to abiotic stress**

Different types of stress often produce interrelated effects and induce similar cellular damage. A general phenomenon is cell dehydration. Under the water deficit condition, plants sustain normal physiological processes through osmotic adjustment (OA). Osmotic adjustment is a major trait associated with maintenance of high cell turgor potential and water retention in response to dehydration stress (Hare et al., 1998; Ingram & Bartels, 1996). Osmotic adjustment can result in turgor maintenance, thereby sustaining cell elongation and leaf expansion as water deficits develop. Osmotic adjustment has been correlated with drought and salt tolerance in various forage and turfgrass species, including tall fescue (White et al., 1992), bermudagrass, buffalograss [Bouteloua dactyloides (Nutt.) Columbus] (Qian & Fry, 1997), *Cenchrus ciliaris* (Wilson & Ludlow, 1983), *Andropogon gayanus* var.

efficiency under water deficit, and found that if other conditions are invariant. The mesophyll cells become smaller per unit area under water deficit, conversely the larger the area of mesophyll cells, the higher is the water use efficiency (Noble, 1980). In an earlier study, tall fescue and turfgrasses were seen to rely primarily on a deep and extensive root system for drought tolerance because the longer root system had greater volume and surface area in contact with the soil, facilitating water and nutrient uptake under drought

Facing salinity stresses, plants evolved special structures to survive. *Mesembryanthemum crystallinum* is a salt-secreting plant and possesses epidermal bladder cells in its aerial parts, which store Na+ (Adams et al., 1998). While *Tamarix aphylla* uses a salt gland to excrete salt

Annual plants can escape drought by maturing before stress becomes severe. Some Fescue grass cultivars avoid drought stress through changes in leaf and root morphology (reducing the transpiration surface area and closing stomata) and probably through osmotic adjustment maintain sufficient turgor pressure in the growing zone for leaf elongation

Serious environmental threats stimulates forage and turf grassess to produce a physiological response. Photosynthesis and cell growth are the primary processes which are affected by stress (Chaves, 1991; Munns, et al., 2006). Under such stress, the rate of photosynthesis and assimilation of products of forage crops decreases remarkably, resulting in a slow down of growth. In a study of eight grass forage plants, chlorophyll content was found to decrease with increased water stress (Yang et al., 2007). Low-temperature has been found to decrease the chlorophyll content in *Poa pratensis* L.qinghai, *Roegneria thoroldiana* and *Elymus nutans*. The major reason for this is that the stress decreaseds the stability of the chloroplast and then destroyed them (Yan et al., 2007). Generally, the degree of decreasing chlorophyll content correlates with the degree of damage to the forage crop plants, so that the

Under a variety stresses, the respiration rate of forage plants becomes unstable. For example, the respiration rate dramatically decrease after suffering from freezing, heat, highsalinity or flooding. While after drought or chilling, the respiration rate increase initially, then sharply decreases (Sheng, 2010). The respiratory metabolism pathway also alters under stress. The Pentose Phosphate (PPP) pathway increases under drought and mechanical

Different types of stress often produce interrelated effects and induce similar cellular damage. A general phenomenon is cell dehydration. Under the water deficit condition, plants sustain normal physiological processes through osmotic adjustment (OA). Osmotic adjustment is a major trait associated with maintenance of high cell turgor potential and water retention in response to dehydration stress (Hare et al., 1998; Ingram & Bartels, 1996). Osmotic adjustment can result in turgor maintenance, thereby sustaining cell elongation and leaf expansion as water deficits develop. Osmotic adjustment has been correlated with drought and salt tolerance in various forage and turfgrass species, including tall fescue (White et al., 1992), bermudagrass, buffalograss [Bouteloua dactyloides (Nutt.) Columbus] (Qian & Fry, 1997), *Cenchrus ciliaris* (Wilson & Ludlow, 1983), *Andropogon gayanus* var.

chlorophyll level can be used as a guide to stress tolerance in these plants.

stress (Qian et al., 1997).

(Thomason et al., 1969).

(Wang & Burghrara, 2008).

**3. The physiological response to abiotic stress** 

damage conditions in forage crop plants (Sheng, 2010).

**3.1 Osmotic adjustment in response to abiotic stress** 

*bisquamulatus* (Geerts *et al*., 1998). Osmotic adjustment measurements can be used to select drought-tolerant cultivars (Morgan,1983). The extent of osmotic adjustment was higher in buffalograss and zoysiagrass (*Zoysia japonica* Steud) with a better drought tolerance than tall fescure (*Festuca arundinacea* Shreb.) (Qian & Fry, 1997).

Osmotically active solutes include amino acids (proline), sugars (e.g.,sucrose, fructans), polyols (e.g., mannitol), and organic ions(e.g.,potassium, sodium) (Chaves et al., 2003). Those solutes are associated not only with turgor maintenance, but also with the maintenance of membrane and protein structures and protection against oxidative damage (Crowe et al., 1992; Hoekstra et al., 2001). OA for creeping bentgrass and velvet bentgrass is associated with the accumulation of water soluble carbohydrates during the early period of drought and increases in proline content following prolonged a period; however, inorganic ions were not found to relate OA in these species (DaCosta & Huang, 2006). In alfalfa (*Medicago sativa* L.), salt stress induces a large increase in the amino acid and carbohydrate pools. Amongst the amino acids, proline shows the largest increase in roots, cytosol, and bbacteroides. Its accumulation is reflected in an osmoregulatory mechanism not only in roots but also in nodule tissue. The concentration of the carbohydrate pinitol is also increased significantly (Fougere et al., 1991). In many other forage and turfgrasses, glycinebetaine and proline makes a significant contribution to OA under abiotic stress (Girousse *et al*., 1996; Marcum, 1994).

### **3.2 Dormancy is another countermeasure to survive from stresses**

Dormancy also is a mechanism by which forage crop plants become quiescent during prolonged environmental stress, especially drought. Grasses temporarily slow the growth of meristem to avoid drought damage and to allow survival (McWilliam, 1968). Poaceae forage plants, such as *Poa scabrella* (Laude, 1953), *Poa bullbosa* (Volaire *et al*., 2001), and some populations of forage grasses such as *Dactylis glomerata* 'Kasbah' (Norton *et al*., 2006), all exhibit summer dormancy.

### **4. The molecular and genetic response to stress**

The plant responses to abiotic stress involves many genes and molecular mechanisms and stress-associated genes, proteins and metabolites from a complex regulatory network.

A large number of genes have been found to be associated with abiotic stress (Jin et al., 2010; Kang et al., 2010; Shinozaki and Yamaguchi-Shinozaki 2000; Thomashow 1999). Genes induced during stress conditions function not only in protecting cells from stress by producing important metabolic proteins, but also in regulating signal transduction in the stress response (Yamaguchi-Shinozaki & Shinozaki, 2006). These gene products are divided into two groups (Shinozaki et al., 2003; Yamaguchi-Shinozaki & Shinozaki, 2006). The first group functions in the direct protection of the plant against stress and includes key enzymes for osmolyte biosynthesis, LEA (late embryogenesis abundant) proteins, detoxification enzymes and enzymes involved in many metabolic processes. The second group contains protein factors involved in further regulation of signal transduction, including various transcription factors, protein kinases, protein phosphatases, enzymes involved in phospholipid metabolism, and other signaling molecules (Yamaguchi-Shinozaki & Shinozaki , 2006).

### **4.1 Transcriptional factors**

Transcription factor genes play important roles in stress survival by serving as master regulators of sets of downstream stress-responsive genes. Transcription factors regulate

Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 211

a Germination rates of WT (control) and transgenic lines (CkDBF) grown on MS medium(1), or MS medium supplemented with 200mM NaCl (2) or 250mM mannitol (3) (n=100, each experiment was repeated three times). b Primary root growth of WT and transgenic lines tobacco seedlings (CkDBF) under normal condition (1), treated with150mM mannitol (2), 250mMmannitol (3), 100mM NaCl (4), or 200mMNaCl (5)(n=20). c Expression analysis of downstream genes NtERD10A, NtERD10B, NtERD10C, NtERD10D and NtZfp in transgenic tobacco (CkDBF) by using real-time PCR. Genes were amplified with specific primers. The ACTIN gene was used to normalize samples. Experiments were repeated three times. d Primary root growth of transgenic lines (CkDREB) and WT tobacco seedlings under normal condition(1), treated with 100 mM NaCl (2), 200 mM NaCl(3), 150 mM mannitol (4) or 250 mM mannitol (5) (n = 20). e Germination rate of WT(control) and transgenic lines seeded on MS media (1), or MS media supplemented with200 mM NaCl (2) or 250mM mannitol (3). Each experiment was repeated

Fig. 1. Enhanced stress tolerance of transgenic tobacco carrying DREB transcription factor

three times.

genes from *Caragana korshinskii*.

downstream gene expression via binding to specific elements (cis-elements) in target genes and consequently, enhance stress tolerance in plants (Chen & Zhu, 2004; Yamaguchi-Shinozaki & Shinozaki, 2006).

Transcriptional control of the expression of stress-responsive genes is a crucial part of the plant response to a range of abiotic stresses (Singh et al., 2002). Several hundred types of transcription factors have been isolated from higher plants. Important families of stressresponsive transcription factors include AP2/EFR, basic-domain leucine-zipper (bZIP), MYC/B, WRKY, Zinc finger, MADS, NAC (Liu et al., 1999; Yamaguchi-Shinozaki & Shinozaki ,2006).

In the Arabidopsis genome, 145 DREB/ERF related proteins are classified into five groups the AP-2 subfamily, RAV subfamily, DREB subfamily, ERF subfamily, and others (Sakuma et al., 2002). Many AP2/EREBP transcription factor genes have been isolated from a variety forage crop plants (Chen et al., 2009; Niu et al., 2010; Wang et al., 2010; Wang et al., 2011; Xiong & Fei, 2006). In *Medicago falcate*, MfDREB1 and MfDREB1s encode an AP2/EREBP type transcription factor and are multi-copy genes. Also they are induced by low temperature stress, although hardly induced at all under salt and drought conditions (Niu et al., 2010). *LpCBF3*, encoding the transcription factor DREB1/CBF, is isolated form perennial ryegrass. *LpCBF3* is induced by cold stress, but not by abscisic acid (ABA), drought and salinity. Overexpression of *LpCBF3* in Arabidopsis was found to induce CBF3 target genes and enhance freezing tolerance by measuring electrolyte leakage (Xiong & Fei, 2006). *LpCBF3* can induce downstream gene expression in cold-tolerant perennial ryegrass accessions without cold treatment, but cannot be activated in cold-sensitive perennial ryegrass accessions. Also cold treatment can induce the downstream genes of CBF3 expression in these accessions. Overexpression of *LpCBF3* with a 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance (Zhao & Bughrara, 2008). *HsDREB1A* is isolated from xeric, wild barley in bahiagrass. *HsDREB1A* introduced into bahiagrass under the *HVA1s* promoter from barley, enhanced tolerance under severe salt stress and severe dehydration stress (James et al., 2008). The WXP1 gene from *Medicago truncatula* containing a AP2 domain was induced by cold, ABA and drought treatment mainly in shoot tissues. Over-expression of WXP1 in alfalfa not only induced a number of wax-related genes, but also significantly increased wax accumulation.Transgenic lines were found to enhance drought tolerance and quick recovery after re-watering (Zhang et al., 2005).

In our previous studies, we isolated and identified the AP2/EREBP genes from several forage crops, including *Caragana korshinskii*, *Galegae orientalis* (Chen et al., 2009; Wang et al., 2010; Wang et al., 2011) and *Ceratoides arborescens* (unpublished data).

*CkDBF*, a DREB like gene isolated from *C. korshinskii*, was confirmed as a transcription factor by one-hybrid experiments and located to the nucleus. CkDBF is induced by high salt, dehydration, low temperature and abscisic acid (ABA). Over-expression of *CkDBF* in transgenic tobacco induces the expression of downstream stress-responsive genes and increases tolerance under high salinity and osmotic stress (Wang et al., 2010) (figure 1). CkDREB, which contains a conserved AP2/ERF domain, is also isolated from *C. korshinskii*. It was located in the nucleus, and had a DRE element-binding activity and transcriptional activation ability. The expression of *CkDREB* is induced by a variety abiotic stress types including high salt, dehydration and low temperature. The over-expression of CkDREB in tobacco was found to enhance the tolerance for high salinity and mannitol stress by measuring the germination rate of seeds and primary root lengths (figure 1). The over-expression of *CkDREB* induces abiotic stress-response genes containing a DRE element in their promoters. These results show that CkDREB is involved in the regulation of stress-response signals (Wang et al., 2011).

downstream gene expression via binding to specific elements (cis-elements) in target genes and consequently, enhance stress tolerance in plants (Chen & Zhu, 2004; Yamaguchi-

Transcriptional control of the expression of stress-responsive genes is a crucial part of the plant response to a range of abiotic stresses (Singh et al., 2002). Several hundred types of transcription factors have been isolated from higher plants. Important families of stressresponsive transcription factors include AP2/EFR, basic-domain leucine-zipper (bZIP), MYC/B, WRKY, Zinc finger, MADS, NAC (Liu et al., 1999; Yamaguchi-Shinozaki &

In the Arabidopsis genome, 145 DREB/ERF related proteins are classified into five groups the AP-2 subfamily, RAV subfamily, DREB subfamily, ERF subfamily, and others (Sakuma et al., 2002). Many AP2/EREBP transcription factor genes have been isolated from a variety forage crop plants (Chen et al., 2009; Niu et al., 2010; Wang et al., 2010; Wang et al., 2011; Xiong & Fei, 2006). In *Medicago falcate*, MfDREB1 and MfDREB1s encode an AP2/EREBP type transcription factor and are multi-copy genes. Also they are induced by low temperature stress, although hardly induced at all under salt and drought conditions (Niu et al., 2010). *LpCBF3*, encoding the transcription factor DREB1/CBF, is isolated form perennial ryegrass. *LpCBF3* is induced by cold stress, but not by abscisic acid (ABA), drought and salinity. Overexpression of *LpCBF3* in Arabidopsis was found to induce CBF3 target genes and enhance freezing tolerance by measuring electrolyte leakage (Xiong & Fei, 2006). *LpCBF3* can induce downstream gene expression in cold-tolerant perennial ryegrass accessions without cold treatment, but cannot be activated in cold-sensitive perennial ryegrass accessions. Also cold treatment can induce the downstream genes of CBF3 expression in these accessions. Overexpression of *LpCBF3* with a 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance (Zhao & Bughrara, 2008). *HsDREB1A* is isolated from xeric, wild barley in bahiagrass. *HsDREB1A* introduced into bahiagrass under the *HVA1s* promoter from barley, enhanced tolerance under severe salt stress and severe dehydration stress (James et al., 2008). The WXP1 gene from *Medicago truncatula* containing a AP2 domain was induced by cold, ABA and drought treatment mainly in shoot tissues. Over-expression of WXP1 in alfalfa not only induced a number of wax-related genes, but also significantly increased wax accumulation.Transgenic lines were found to enhance drought tolerance and quick recovery

In our previous studies, we isolated and identified the AP2/EREBP genes from several forage crops, including *Caragana korshinskii*, *Galegae orientalis* (Chen et al., 2009; Wang et al.,

*CkDBF*, a DREB like gene isolated from *C. korshinskii*, was confirmed as a transcription factor by one-hybrid experiments and located to the nucleus. CkDBF is induced by high salt, dehydration, low temperature and abscisic acid (ABA). Over-expression of *CkDBF* in transgenic tobacco induces the expression of downstream stress-responsive genes and increases tolerance under high salinity and osmotic stress (Wang et al., 2010) (figure 1). CkDREB, which contains a conserved AP2/ERF domain, is also isolated from *C. korshinskii*. It was located in the nucleus, and had a DRE element-binding activity and transcriptional activation ability. The expression of *CkDREB* is induced by a variety abiotic stress types including high salt, dehydration and low temperature. The over-expression of CkDREB in tobacco was found to enhance the tolerance for high salinity and mannitol stress by measuring the germination rate of seeds and primary root lengths (figure 1). The over-expression of *CkDREB* induces abiotic stress-response genes containing a DRE element in their promoters. These results show that CkDREB is involved in

2010; Wang et al., 2011) and *Ceratoides arborescens* (unpublished data).

the regulation of stress-response signals (Wang et al., 2011).

Shinozaki & Shinozaki, 2006).

after re-watering (Zhang et al., 2005).

Shinozaki ,2006).

a Germination rates of WT (control) and transgenic lines (CkDBF) grown on MS medium(1), or MS medium supplemented with 200mM NaCl (2) or 250mM mannitol (3) (n=100, each experiment was repeated three times). b Primary root growth of WT and transgenic lines tobacco seedlings (CkDBF) under normal condition (1), treated with150mM mannitol (2), 250mMmannitol (3), 100mM NaCl (4), or 200mMNaCl (5)(n=20). c Expression analysis of downstream genes NtERD10A, NtERD10B, NtERD10C, NtERD10D and NtZfp in transgenic tobacco (CkDBF) by using real-time PCR. Genes were amplified with specific primers. The ACTIN gene was used to normalize samples. Experiments were repeated three times. d Primary root growth of transgenic lines (CkDREB) and WT tobacco seedlings under normal condition(1), treated with 100 mM NaCl (2), 200 mM NaCl(3), 150 mM mannitol (4) or 250 mM mannitol (5) (n = 20). e Germination rate of WT(control) and transgenic lines seeded on MS media (1), or MS media supplemented with200 mM NaCl (2) or 250mM mannitol (3). Each experiment was repeated three times.

Fig. 1. Enhanced stress tolerance of transgenic tobacco carrying DREB transcription factor genes from *Caragana korshinskii*.

Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 213

In *Medicago truncatula*, the HD-Zip 1 transcription factor HB1 is expressed in primary and lateral root meristerms and is induced by salt stress and constitutive expression of HB1 in

These studies taken together demonstrate that transcription factors play an important role in

In stress-tolerant plants, many genes are involved in the synthesis of osmoprotectants. Osmoregulation is believed to be the best strategy for abiotic stress tolerance, especially if an osmoregulatory gene can be triggered in response to drought, salinity or high temperature (Bhatnagar-Mathur et al., 2008). Osmotically active solutes include amino acids (e.g., proline, glycine betaine), sugars (e.g., sucrose, fructans), polyols (e.g.,mannitol), and organic ions(e.g., potassium, sodium) (Chaves et al.,2003). These active solutes accumulate in a large number of plant species under environmental stress conditions (Ashraf & Foolad, 2007;

Proline plays a vital role in plants, especially in abiotic stress conditions (Ashraf & Foolad, 2007; Aubert et al., 1999; Schat et al., 1997). Many genes are involved in the synthesis and degradation of proline under a variety of stress conditions such as salt, drought and metal toxicity etc. A role for P5CS (△1-pyrroline-5-carboxylate synthase) in the proline biosynthetic pathway under stress conditions, has been emphasized in the last two decades (Kishor et al., 1995; Zhu et al., 1998). In alfalfa, the expression levels of MtP5CS1 in the different plant organs closely correlated with proline levels but transcript abundance was not affected under osmotic stress condition; was seen to significantly accumulate although only in shoots under osmotic stress conditions (Armengaud et al., 2004). BADH (betaine aldehyde dehydrogenase) is a key enzyme in the biosynthesis of glycine betaine and a BADH gene, which originated from *Atriplex hortensis,* was transformed into alfalfa and was found to

Sugars also play an essential role in osmotic adjustment. Trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from yeast, driven by the rd29A promoter, were transformed into alfalfa. The transgenic lines led to increased plant biomass and tolerance under drought, freezing, salt and heat stress conditions (Suárez & Iturriaga, 2009). In alfalfa leaves, sucrose phosphate synthase (SPS) and sucrose synthase (SS) were examined to explore sucrose metabolism under cold (5°C) and heat stress conditions and it was found that(33°C) genes expression was significantly changed in the cold but not the

Putrescine aminopropyltransferase (PAPT) enzyme, highly specific for putrescine as the initial substrate, can yield multiple polyamine products which are associated with osmotic stress. PAPT activity from alfalfa was found to be mainly located in the meristematic shoot tip and floral bud tisseues. A scheme was proposed to comprehensively illustrate the role of PAPT in biosynthesis of several common and unusual polyamines in alfalfa (Bagga et al.,

In most of the aerobic organisms, elimination of ROS (reactive oxygen species) is needed under environment stress conditions. In order to control the level of ROS and protect the cells from oxidative injury, plants have developed a complex antioxidant defense system that includes various enzymes and non-enzymatic metabolites (Vranova et al., 2002). Key

*M. truncatula* roots alters their architecture (Ariel et al., 2010).

increase the salt tolerance of the transgenic plants (Liu et al., 2011).

the acquisition of stress tolerance in forage crops.

**4.2 Osmoregulatory genes** 

Chen & Murate, 2011; Mansour, 1998).

heat condition (Mo et al., 2011).

**4.3 Detoxifying genes** 

1997).

Previous studies with DREB transcriptional factor genes focused on DREB-1 and 2 types, which generally play important regulation roles. However, as an A-6 type DREB gene, *CkDBF* responds to a variety abiotic stress types , and the finding that over-expression could enhance the multiple stress-tolerance in transgenic plants indicates that the A-6 type factor also plays an essential role in transcriptional regulation, especially in forage crops.

In addition to DREB-type genes, other AP2/EREBP genes have also been investigated in forage crop plants. GoRAV, which was isolated from *Galegae orientalis*, belongs to the RAV family and has two AP2 and B3-like distinct DNA-binding domains. GoRAV is induced by cold, drought, high salinity and ABA (Chen et al., 2009).

Much researchs has been carried out with the aim of improving the stress-tolerance of forage crops through transgenic modification. Thus, introducing the Arabidopsis DREB1A/CBF3 gene into *Lolium perenne* can increase drpught and freezing stress tolerance, the increased stress tolerance being associated with increased activities of antioxidant enzymes (Li et al., 2011). In another experiment, GmDREB1 isolated from soybean, was introduced into alfalfa under the control of Arabidopsis Rd29A promoter and the transgenic lines induced by salt and showed high tolerance (Jin et al., 2010). Also the Arabidopsis *HARDY* gene, belonging to the stress-related AP2/ERF super family of transcription factors, was transformed into *Trifolium alexandrinum* L. By measuring fresh and dry weight, transpiration and sodium uptake in the transgenic lines and wild type, it was found that over-expression of *HARDY* improves drought and salt tolerance in transgenic plants (Abogadallah et al., 2011).

The zinc-finger motifs, which are classified based on the arrangement of Zinc-binding amino acids, are present in many transcription factors and play critical roles in interactions with other molecules (Chao et al., 2009; Sun et al., 2010). A number of zinc-finger transcription factors have been implicated in important biological processes and stress-tolerance regulation.

*MsZFN*, encoding a zinc-finger protein, was isolated from alfalfa. *MsZFN* is located in the nucleus, and is significantly induced by salt and reach a maximum level at 30 min (Chao et al. 2009). The Alfin1 gene from alfalfa which encodes a putative Zinc finger motif is specifically expressed in roots (Bastola et al., 1998; Winicov, 1993). The Alfin1 protein binds DNA in a sequence-specific manner in vitro, and can also bind to fragments of MsPRP2, which is considered to be root-specific and accumulates in alfalfa roots under a salt environment (Bastola et al., 1998). Over-expression of Alfin1 under the 35S promoter enhanced expression of the endogenous MsPRP2 gene in alfalfa and improved salinity tolerance (Winicov & Bastola, 1999). Alfin1 has been proposed as a root growth regulator, and transgene lines have increased in root growth under normal and saline conditions in alfalfa (Winicov, 2000). MtSAP1 (*Medicago truncatula* stress-associated protein1) encodes a zinc-finger domains, and It's expression is increased embryos during desiccation, and decreased significantly during the first hours of imbibing. MtSAP1 protein accumulated in the embryo axis under cold, hypoxia, ABA and desiccation related stress, but its expression is not notably changed under mild drought stress. RNAi studies showed that MtSAP1 storage proteins are very important for the success of germination (Gimeno-Gilles et al., 2011).

Other transcription factors also had been investigated. *AtHDG11* encodes a protein classified as a homeodomain-leucine zipper transcription factor number. Over-expression *AtHDG11* in tall fescue, with four 35S enhancers significantly enhance tolerance to drought and salt stress. The enhanced stress tolerance is associated with a more extensive root system, a lower level of malondialdehyde, a higher level of proline and superoxide dismutase (SOD) and catalase (CAT) (Cao et al., 2009).

In *Medicago truncatula*, the HD-Zip 1 transcription factor HB1 is expressed in primary and lateral root meristerms and is induced by salt stress and constitutive expression of HB1 in *M. truncatula* roots alters their architecture (Ariel et al., 2010).

These studies taken together demonstrate that transcription factors play an important role in the acquisition of stress tolerance in forage crops.

### **4.2 Osmoregulatory genes**

212 Plants and Environment

Previous studies with DREB transcriptional factor genes focused on DREB-1 and 2 types, which generally play important regulation roles. However, as an A-6 type DREB gene, *CkDBF* responds to a variety abiotic stress types , and the finding that over-expression could enhance the multiple stress-tolerance in transgenic plants indicates that the A-6 type factor

In addition to DREB-type genes, other AP2/EREBP genes have also been investigated in forage crop plants. GoRAV, which was isolated from *Galegae orientalis*, belongs to the RAV family and has two AP2 and B3-like distinct DNA-binding domains. GoRAV is induced by

Much researchs has been carried out with the aim of improving the stress-tolerance of forage crops through transgenic modification. Thus, introducing the Arabidopsis DREB1A/CBF3 gene into *Lolium perenne* can increase drpught and freezing stress tolerance, the increased stress tolerance being associated with increased activities of antioxidant enzymes (Li et al., 2011). In another experiment, GmDREB1 isolated from soybean, was introduced into alfalfa under the control of Arabidopsis Rd29A promoter and the transgenic lines induced by salt and showed high tolerance (Jin et al., 2010). Also the Arabidopsis *HARDY* gene, belonging to the stress-related AP2/ERF super family of transcription factors, was transformed into *Trifolium alexandrinum* L. By measuring fresh and dry weight, transpiration and sodium uptake in the transgenic lines and wild type, it was found that over-expression of *HARDY* improves drought and salt tolerance in transgenic plants

The zinc-finger motifs, which are classified based on the arrangement of Zinc-binding amino acids, are present in many transcription factors and play critical roles in interactions with other molecules (Chao et al., 2009; Sun et al., 2010). A number of zinc-finger transcription factors

*MsZFN*, encoding a zinc-finger protein, was isolated from alfalfa. *MsZFN* is located in the nucleus, and is significantly induced by salt and reach a maximum level at 30 min (Chao et al. 2009). The Alfin1 gene from alfalfa which encodes a putative Zinc finger motif is specifically expressed in roots (Bastola et al., 1998; Winicov, 1993). The Alfin1 protein binds DNA in a sequence-specific manner in vitro, and can also bind to fragments of MsPRP2, which is considered to be root-specific and accumulates in alfalfa roots under a salt environment (Bastola et al., 1998). Over-expression of Alfin1 under the 35S promoter enhanced expression of the endogenous MsPRP2 gene in alfalfa and improved salinity tolerance (Winicov & Bastola, 1999). Alfin1 has been proposed as a root growth regulator, and transgene lines have increased in root growth under normal and saline conditions in alfalfa (Winicov, 2000). MtSAP1 (*Medicago truncatula* stress-associated protein1) encodes a zinc-finger domains, and It's expression is increased embryos during desiccation, and decreased significantly during the first hours of imbibing. MtSAP1 protein accumulated in the embryo axis under cold, hypoxia, ABA and desiccation related stress, but its expression is not notably changed under mild drought stress. RNAi studies showed that MtSAP1 storage proteins are very important for the

Other transcription factors also had been investigated. *AtHDG11* encodes a protein classified as a homeodomain-leucine zipper transcription factor number. Over-expression *AtHDG11* in tall fescue, with four 35S enhancers significantly enhance tolerance to drought and salt stress. The enhanced stress tolerance is associated with a more extensive root system, a lower level of malondialdehyde, a higher level of proline and superoxide dismutase (SOD)

have been implicated in important biological processes and stress-tolerance regulation.

also plays an essential role in transcriptional regulation, especially in forage crops.

cold, drought, high salinity and ABA (Chen et al., 2009).

success of germination (Gimeno-Gilles et al., 2011).

and catalase (CAT) (Cao et al., 2009).

(Abogadallah et al., 2011).

In stress-tolerant plants, many genes are involved in the synthesis of osmoprotectants. Osmoregulation is believed to be the best strategy for abiotic stress tolerance, especially if an osmoregulatory gene can be triggered in response to drought, salinity or high temperature (Bhatnagar-Mathur et al., 2008). Osmotically active solutes include amino acids (e.g., proline, glycine betaine), sugars (e.g., sucrose, fructans), polyols (e.g.,mannitol), and organic ions(e.g., potassium, sodium) (Chaves et al.,2003). These active solutes accumulate in a large number of plant species under environmental stress conditions (Ashraf & Foolad, 2007; Chen & Murate, 2011; Mansour, 1998).

Proline plays a vital role in plants, especially in abiotic stress conditions (Ashraf & Foolad, 2007; Aubert et al., 1999; Schat et al., 1997). Many genes are involved in the synthesis and degradation of proline under a variety of stress conditions such as salt, drought and metal toxicity etc. A role for P5CS (△1-pyrroline-5-carboxylate synthase) in the proline biosynthetic pathway under stress conditions, has been emphasized in the last two decades (Kishor et al., 1995; Zhu et al., 1998). In alfalfa, the expression levels of MtP5CS1 in the different plant organs closely correlated with proline levels but transcript abundance was not affected under osmotic stress condition; was seen to significantly accumulate although only in shoots under osmotic stress conditions (Armengaud et al., 2004). BADH (betaine aldehyde dehydrogenase) is a key enzyme in the biosynthesis of glycine betaine and a BADH gene, which originated from *Atriplex hortensis,* was transformed into alfalfa and was found to increase the salt tolerance of the transgenic plants (Liu et al., 2011).

Sugars also play an essential role in osmotic adjustment. Trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from yeast, driven by the rd29A promoter, were transformed into alfalfa. The transgenic lines led to increased plant biomass and tolerance under drought, freezing, salt and heat stress conditions (Suárez & Iturriaga, 2009). In alfalfa leaves, sucrose phosphate synthase (SPS) and sucrose synthase (SS) were examined to explore sucrose metabolism under cold (5°C) and heat stress conditions and it was found that(33°C) genes expression was significantly changed in the cold but not the heat condition (Mo et al., 2011).

Putrescine aminopropyltransferase (PAPT) enzyme, highly specific for putrescine as the initial substrate, can yield multiple polyamine products which are associated with osmotic stress. PAPT activity from alfalfa was found to be mainly located in the meristematic shoot tip and floral bud tisseues. A scheme was proposed to comprehensively illustrate the role of PAPT in biosynthesis of several common and unusual polyamines in alfalfa (Bagga et al., 1997).

### **4.3 Detoxifying genes**

In most of the aerobic organisms, elimination of ROS (reactive oxygen species) is needed under environment stress conditions. In order to control the level of ROS and protect the cells from oxidative injury, plants have developed a complex antioxidant defense system that includes various enzymes and non-enzymatic metabolites (Vranova et al., 2002). Key

Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 215

Targeting the detoxification pathway is an appropriate approach for producing plants with multiple stress-tolerance traits (Bartels et al., 2001). It is expected that with increased understanding of this pathway, a breeding forage crop will be produced with multiple

Plant cells sense stress through signaling pathways and transmit the signal to cellular machinery activating an adaptive response essential for plant survival. Molecular and biochemical studies suggest that abiotic stress signaling in plants involves receptor-coupled phosphorylation, phosphoinositol-induced Ca2+ changes, mitogen-activated protein kinase cascades and transcriptional activation of stress-responsive genes. In addition, protein posttranslational modifications and adapter or scaffold-mediated protein-protein interactions

In alfalfa, P44MMK4 kinase was specifically activated under drought and cold treatment conditions, but not induced by high salt concentrations or heat shock. Under ABA treatment, MMK4 transcription levels and p44MMK4 kinase were not increased or activated (Jonak et al., 1996). In another study SIMK, an alfalfa mitogen-activated protein kinase (MAPK), was found to be highly regulated by salt stress, in terms of its levels and subcellular localization in roots (Baln ka et al., 2000). SIMK is a member of the family of MAPKs(mitogen-activated protein kinases) which are involved in transducing a variety of extracellular signals. It is transiently activated by NaCl, KCl, sorbitol, and in alfalfa reaches maximal activity between 8 and 16 min before a slow inactivation. Unlike other MAPKs in most mammalian and yeast cells, SIMK has a constituive nuclear localization and the activation is not correlated with nucleo-cytoplasmic

The Msapk1 gene, harbouring a unique ankyrin repeat, can be detected in almost every organ of alfalfa. It is found to be induced in the roots of alfalfa upon osmotic stress (Chinchilla et al., 2003). Also MsCPK3, a calmodulin-like domain protein kinase (CPK), was identified in alfalfa. The expression of MsCPK3 is activated by 2,4-Dichlorophenoxyacetic acid (2,4-D), ABA and NaCl but not by kinetin, ABA or salt treatment. Measurement of the recombinant protein activity showed that MsCPK3 involved in auxin and stress related Ca2+

The AnnMs2 gene, an annexin-like protein from alfalfa, is expressed in various tissues especially in buds, flowers and roots. It is activated in cells or tissues under osmotic stress or by ABA. The recombinant AnnMs2 protein is able to bind to phospholipids in the presence of Ca2+ and immunofluorescence studies showed that it is mainly localized in the

Another signaling element active oxygen species (AOS), can act as ubiquitous signal molecule in plants. It is a central component in the stress response and it's level determines

The gene *Srl*k, from *M. trnucatula*, a leucine-rich repeat RLK (receptor-like protein kinases) is rapidly induced by salt stress in roots. The gene expression study and a *Srl*k promoter-βglucuronidase (GUS) fusion location experiment suggested that Srlk is activated in the root epidermis. Through studies using RNAi and Srlk-TILLING mutants, Srlk would appear to be involved in the regulation of the adaptation of *M.truncatula* roots to salt stress (De

Heme oxygenase is the rate-limiting enzyme in the breakdown of heme changing into carbon monoxide (CO), iron etc (Shekhawat & Verma, 2010) and it plays a vital role in stress

are also important in abiotic stress signal transduction (Xiong & Zhu, 2001).

stress-tolerance.

**4.4 Signal transduction** 

translocation (Munnik et al., 1999).

nuclear (Kovács et al., 1998).

Lorenzo et al., 2009).

signalling pathways (Davletova et al., 2001).

the type of response (Vranova et al. , 2002).

enzymes for detoxifying ROS in plants include superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (Yang et al., 2006).

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms (Halliwell & Gutteridge, 1999; Scandalios, 1997). Until now, four types of SODs have been identified. Copper-zinc SOD is the most importance one, which is closely related to resistance to stress in plants (Feng et al., 2005; Song et al., 2006; Wang et al., 2005). PS-CuZn SOD from *Polygonum sibiricum* , which encodes a copper-zinc SOD, is a constitutively expressed gene and has different expression modes in different organs under salinity-alkalinity stress conditions (Qu et al., 2010). Our team isolated a copper-zinc SOD from *Galega orientails*. Expression of this gene, induced by drought and salt stress, indicated that the copper-zinc gene is involved in stress signaling (unpublished). Copper-zinc SOD can be divided into two forms, one is cytosolic and the other is associated with chloroplast isoenzymes (Sheri et al., 1996). Subcellular analysis showed that Cu-Zn SOD of *G.orientails* locates in chloroplast (unpublished).

Transgenic plants with over-expression of the SOD gene in alfalfa were found to be able to resist, and to possess markedly enhanced antioxidant capacities (Bryan et al., 1993; Bryan et al., 2000). The Mn-SOD gene from *Nicotiana plumbaginifolia* is introduced into alfalfa using two different plasmid vectors for targeting to mitochondria or chloroplast. The transgenic lines had enhanced SOD activity and increased re-growth after freezing stress (McKersie et al., 1993). Another study showed that the introduction of Mn-SOD improved survival, vigor and yield over three years in a natural field environment (McKersie et al., 1996). Further observations with many different transgenic plants in both laboratory and field evaluations show may that over-expression of Mn-SOD improve the winter survival and dry-matter yield, although some lines showed the converse. Although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing tolerance of the cells in the taproot or crown of transgenic alfalfa (McKersie et al., 1999). In another study, a Fe-SOD gene from Arabidopsis, with a chloroplast transit peptide, was over-expressed in alfalfa under the cauliflower mosaic virus 35S promoter. The transgenic alfalfa show a higher superoxide-scavenging capacity and winter survival. The Fe-SOD activity is found to have a close relationship with winter survival, but not with the oxidative stress tolerance and shoot dry matter production in a 2 year trial. The higher winter survival may stem from reducing secondary injury symptoms and enhancing recovery after stress (McKersie, et al. 2000). In a further investigation of transgenic MnSOD in mitochondria of leaves and nodules, MnSOD in the Chloroplasts and FeSOD in the Chloroplasts; it was shown that transgenic lines had a 20% higher photosynthetic activity than the parental line under mild water stress conditions, however there were no major differences between the untransformed and the transformed alfalfa for most parameters examined under a water stress environment (Rubio et al., 2002). To explore two SOD transgenes influencing the SOD stress-tolerance mechanisms, the F1 progeny was generated through a sexual cross of a hemizygous Mit-MnSOD alfalfa and a hemizygous Chl-MnSOD alfalfa. The results showed that the F1 progeny with the two genes inserted had increased total SOD activity and significantly higher storage organ biomass compared with the non-transgene siblings, but had a lower biomass production compared to siblings having only one transgene (Samis et al., 2002).

Catalase is a unique hydrogen peroxide-scavenging enzyme. A catalase gene *Facat1* is isolated from *Festuca arundinacea* Schreb. Facat1 is up-regulated in cold and salt stress treated leaves, and reached an expression peak at 2 and 4 h, respectively. However, under ABA and drought treatment conditions, the expression was down-regulated (Yang et al., 2006).

Targeting the detoxification pathway is an appropriate approach for producing plants with multiple stress-tolerance traits (Bartels et al., 2001). It is expected that with increased understanding of this pathway, a breeding forage crop will be produced with multiple stress-tolerance.

### **4.4 Signal transduction**

214 Plants and Environment

enzymes for detoxifying ROS in plants include superoxide dismutase (SOD), ascorbate

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms (Halliwell & Gutteridge, 1999; Scandalios, 1997). Until now, four types of SODs have been identified. Copper-zinc SOD is the most importance one, which is closely related to resistance to stress in plants (Feng et al., 2005; Song et al., 2006; Wang et al., 2005). PS-CuZn SOD from *Polygonum sibiricum* , which encodes a copper-zinc SOD, is a constitutively expressed gene and has different expression modes in different organs under salinity-alkalinity stress conditions (Qu et al., 2010). Our team isolated a copper-zinc SOD from *Galega orientails*. Expression of this gene, induced by drought and salt stress, indicated that the copper-zinc gene is involved in stress signaling (unpublished). Copper-zinc SOD can be divided into two forms, one is cytosolic and the other is associated with chloroplast isoenzymes (Sheri et al., 1996). Subcellular analysis showed that Cu-Zn

Transgenic plants with over-expression of the SOD gene in alfalfa were found to be able to resist, and to possess markedly enhanced antioxidant capacities (Bryan et al., 1993; Bryan et al., 2000). The Mn-SOD gene from *Nicotiana plumbaginifolia* is introduced into alfalfa using two different plasmid vectors for targeting to mitochondria or chloroplast. The transgenic lines had enhanced SOD activity and increased re-growth after freezing stress (McKersie et al., 1993). Another study showed that the introduction of Mn-SOD improved survival, vigor and yield over three years in a natural field environment (McKersie et al., 1996). Further observations with many different transgenic plants in both laboratory and field evaluations show may that over-expression of Mn-SOD improve the winter survival and dry-matter yield, although some lines showed the converse. Although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing tolerance of the cells in the taproot or crown of transgenic alfalfa (McKersie et al., 1999). In another study, a Fe-SOD gene from Arabidopsis, with a chloroplast transit peptide, was over-expressed in alfalfa under the cauliflower mosaic virus 35S promoter. The transgenic alfalfa show a higher superoxide-scavenging capacity and winter survival. The Fe-SOD activity is found to have a close relationship with winter survival, but not with the oxidative stress tolerance and shoot dry matter production in a 2 year trial. The higher winter survival may stem from reducing secondary injury symptoms and enhancing recovery after stress (McKersie, et al. 2000). In a further investigation of transgenic MnSOD in mitochondria of leaves and nodules, MnSOD in the Chloroplasts and FeSOD in the Chloroplasts; it was shown that transgenic lines had a 20% higher photosynthetic activity than the parental line under mild water stress conditions, however there were no major differences between the untransformed and the transformed alfalfa for most parameters examined under a water stress environment (Rubio et al., 2002). To explore two SOD transgenes influencing the SOD stress-tolerance mechanisms, the F1 progeny was generated through a sexual cross of a hemizygous Mit-MnSOD alfalfa and a hemizygous Chl-MnSOD alfalfa. The results showed that the F1 progeny with the two genes inserted had increased total SOD activity and significantly higher storage organ biomass compared with the non-transgene siblings, but had a lower biomass production compared to siblings having only

Catalase is a unique hydrogen peroxide-scavenging enzyme. A catalase gene *Facat1* is isolated from *Festuca arundinacea* Schreb. Facat1 is up-regulated in cold and salt stress treated leaves, and reached an expression peak at 2 and 4 h, respectively. However, under ABA and drought

treatment conditions, the expression was down-regulated (Yang et al., 2006).

peroxidase (APX) and catalase (Yang et al., 2006).

SOD of *G.orientails* locates in chloroplast (unpublished).

one transgene (Samis et al., 2002).

Plant cells sense stress through signaling pathways and transmit the signal to cellular machinery activating an adaptive response essential for plant survival. Molecular and biochemical studies suggest that abiotic stress signaling in plants involves receptor-coupled phosphorylation, phosphoinositol-induced Ca2+ changes, mitogen-activated protein kinase cascades and transcriptional activation of stress-responsive genes. In addition, protein posttranslational modifications and adapter or scaffold-mediated protein-protein interactions are also important in abiotic stress signal transduction (Xiong & Zhu, 2001).

In alfalfa, P44MMK4 kinase was specifically activated under drought and cold treatment conditions, but not induced by high salt concentrations or heat shock. Under ABA treatment, MMK4 transcription levels and p44MMK4 kinase were not increased or activated (Jonak et al., 1996). In another study SIMK, an alfalfa mitogen-activated protein kinase (MAPK), was found to be highly regulated by salt stress, in terms of its levels and subcellular localization in roots (Baln ka et al., 2000). SIMK is a member of the family of MAPKs(mitogen-activated protein kinases) which are involved in transducing a variety of extracellular signals. It is transiently activated by NaCl, KCl, sorbitol, and in alfalfa reaches maximal activity between 8 and 16 min before a slow inactivation. Unlike other MAPKs in most mammalian and yeast cells, SIMK has a constituive nuclear localization and the activation is not correlated with nucleo-cytoplasmic translocation (Munnik et al., 1999).

The Msapk1 gene, harbouring a unique ankyrin repeat, can be detected in almost every organ of alfalfa. It is found to be induced in the roots of alfalfa upon osmotic stress (Chinchilla et al., 2003). Also MsCPK3, a calmodulin-like domain protein kinase (CPK), was identified in alfalfa. The expression of MsCPK3 is activated by 2,4-Dichlorophenoxyacetic acid (2,4-D), ABA and NaCl but not by kinetin, ABA or salt treatment. Measurement of the recombinant protein activity showed that MsCPK3 involved in auxin and stress related Ca2+ signalling pathways (Davletova et al., 2001).

The AnnMs2 gene, an annexin-like protein from alfalfa, is expressed in various tissues especially in buds, flowers and roots. It is activated in cells or tissues under osmotic stress or by ABA. The recombinant AnnMs2 protein is able to bind to phospholipids in the presence of Ca2+ and immunofluorescence studies showed that it is mainly localized in the nuclear (Kovács et al., 1998).

Another signaling element active oxygen species (AOS), can act as ubiquitous signal molecule in plants. It is a central component in the stress response and it's level determines the type of response (Vranova et al. , 2002).

The gene *Srl*k, from *M. trnucatula*, a leucine-rich repeat RLK (receptor-like protein kinases) is rapidly induced by salt stress in roots. The gene expression study and a *Srl*k promoter-βglucuronidase (GUS) fusion location experiment suggested that Srlk is activated in the root epidermis. Through studies using RNAi and Srlk-TILLING mutants, Srlk would appear to be involved in the regulation of the adaptation of *M.truncatula* roots to salt stress (De Lorenzo et al., 2009).

Heme oxygenase is the rate-limiting enzyme in the breakdown of heme changing into carbon monoxide (CO), iron etc (Shekhawat & Verma, 2010) and it plays a vital role in stress

Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 217

When the AVP1 gene, a vacuolar H+-pyrophosphatase gene from *A. thaliana*, was transformed into alfalfa, over-expression enhanced salt and drought tolerance. Compared to the wild-type, the transgenic plants accumulated more Na+, K+ and Ca2+ in leaves and roots, retained more solutes and water, maintained a higher root activity, had a protected photosynthetic machinery, and maintained a stable cell membrane under abiotic stress conditions (Bao, et al., 2009). Zhao's team reported that co-expressing *Suaeda salsa* SsNHX1 and AVP1 conferred greater salt tolerance to transgenic plants than did SsNHX1 alone (Zhao et al., 2006). These studies provide a promising way for improving salt and drought

Many plants increase freezing tolerance upon exposure to low non-freezing temperatures, a phenomenon known as cold acclimation. Cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury (Thomashow, 1999). In forage grasses, many genes (Mohapatra et al., 1989; Tamura & Yonemaru, 2010; Tominaga et al., 2001; Zhang et al., 2009) and proteins (Kosmala et al.,

In alfalfa, some CAS (cold-acclimation-specific) genes are specifically expressed during coldacclimation and their expression level measured by mRNA abundance is positively correlated with the freezing-tolerance of cultivars (Mohapatra et al., 1989). From alfalfa, MsaCIA, cas15 (cold acclimation-specific gene), cas17, and MsaCIC, induced by low temperature have been isolated (Castonguay et al., 1994; Laberge et al., 1993; Monroy et al., 1993; Wolfraim & Dhindsa 1993), although they were not induced by ABA or other stresses (Mohapatra et al., 1989). Encoding a putative nuclear protein, the cas15 transcript level is increased significantly with cold acclimation but is hardly detectable in the absence of cold acclimation. Thus, the accumulation of cas15 and the prolonged cold acclimation have a close relationship (Monroy et al., 1993). In red clover, the homolog of the alfalfa MsaCIA is induced by cold, but the homolog-like MsaCIB, MsaCIC genes were not induced by cold. The expression level of MsaCIA transcripts was 3 times higher in the cold-acclimated, regenerative, F49R (cold tolerant) genotype, compared to the cold-acclimated, non-regenerative, F49M (cold sensitive) genotype. It was also shown that enhanced expression of MsaCIA and the regenerative trait are either linked (Nelke et al., 1999). MsaCIC is similar to bimodular proteins that are developmentally regulated in other plant species (Castonguay et al., 1994). Calcium, a second messenger, can also play an important role in cold acclimation in alfalfa. The influx of extracellular 45Ca2+ at 4 oC is 15 times higher than at 25 oC. The addition of a calcium ionophore or a calcium channel agonist caused an influx of extracellular 45Ca2+ and induced the expression of *cas* genes (as reporters in low-temperature signal transduction) at 25oC, while the addition of calcium channel blockers inhibited the influx of extracellular 45Ca2+ as well as the expression of cas genes (Monroy & Dhindsa, 1995). Associated with winter survival, the cold acclimation-responsive gene, such as RootCAR1, may act as a molecular marker for identifying winter hardy plants in semi-dormant or non-dormant alfalfa germplasm in winter

Forage crop plants are the foundation of animal husbandry. In general, forage crops are located in harsh environments, especially in China. This implies that forage crop plants contain essential stress-tolerance gene resources. Over previous decades, the study of stress tolerance mechanisms mainly focused on physiology and morphology, the molecular and

tolerance in forage crop plants.

**4.7 Cold-acclimation specific gene** 

2009) are regulated during cold acclimation.

of the seed year (Cunningham et al., 2001).

responses. The expression and protein levels of MsHO1 are higher in alfalfa stems and leaves than in germinating seeds and roots and are induced significantly by some pro-oxidant compounds including hemin and nitric oxide donor sodium nitroprusside (Fu et al., 2011).

### **4.5 Late embryogenesis abundant proteins**

LEA proteins, which are suggested to act as desiccation protectants during seed desiccation and in water-stressed seedlings, can be induced by ABA and various types of water-related stress (Espelund et al., 1992). MtPM5, identified as an atypical hydrophobic LEA protein, during stress, is able to stabilize proteins, but is unable to protect cell membranes. MtPM25 is able to rapidly dissolve aggregates in a non-specific manner and sorption isotherms show that when it is unstructured, it absorbs up to threefold more water than MtEM6 (Boucher et al., 2010). In proteomic analysis of the germination of *M. truncatula* seeds associated to desiccation tolerance (DT), 11 polypeptides were identified as late embryogenesis abundant proteins. The abundance changes of MtEm6 and MtPM25 show the two proteins were related to DT (Boudet et al., 2006).

### **4.6 Transporter genes**

Ion transporters selectively transport ions and maintain them at physiologically relevant concentrations. Sodium transporters in plant cells have been extensively studied. Sodium is compartmentalized into the vacuole, through the operation of the vacuolar Na+/H+ antiporter, down an electrochemical proton gradient generated by the vacuolar H+-ATPase and H+-Ppase (Blumwald et al., 2000). In both prokaryotic and eukaryotic cells, the Na+/H+ exchanger plays a key role in the regulation of cytosolic pH, cell volume and Na+ homeostasis (Padan et al., 2001; Wiebe et al., 2001). Plant Na+/H+ antiporters have been isolated from Arabidopsis (Shi et al., 2000), rice (Fukuda et al., 1999) and forage plants (Li et al., 2009; Tang et al., 2010; Yang et al., 2005). MsNHX1, encoding a vacuolar Na+/H+ antiporter, was isolated from alfalfa. The expression of MsNHX1 was significantly upregulated after treated by NaCl and ABA (Yang et al., 2005). MsNHX1 was located to the vacuolar membrane and can partly complement the NaCl-sensitive phenotypes of a yeast mutant. The expression of MsNHX1 in Arabidopsis enhances the resistance to salt stress (An et al., 2008). To investigate the mechanisms of *Medicago intertexta* and *Melilotus indicus* in salt stress, the expression of four genes coding for NHX-type Na+/H+ antiporters were measured. The result show that three genes are expressed in *M. intertexta* leaves and roots, and one gene in *M. indicus* roots. *NHX* gene expression may trigger *M. intertexta* to cope with tissue Na+ accumulation, while in *M. indicus*, the low Na+ content and the lack of correlation between growth in the presence of NaCl, Na+ content and *NHX* gene expression indicates that different mechanisms are involved in coping with salt stress (Zahran et al., 2007). TrNHX1, isolated from *Trifolium repens* L., can complement the *Δnhx1* and *Δena1- 4Δnhx1* yeast mutants by suppressing their observed phenotypes. A similar result is observed in the presence of LiCl and KCl. Under 150mM NaCl treatment, the expression level of TrNHX1 in roots, shoots and leaves is 1.7, 2.2 and 4.3 times, respectively, that of the controls. The expression levels and Na+ content in organs also have a close relationship (Tang et al., 2010). SsNHX1, isolated from the halophyte *Salsola soda*, could significantly enhance salt- tolerance in transgenic alfalfa under the stress-inducible *rd29A* promoter even at 400 mM NaCl (Li et al., 2011). Also *GoNHX1* was induced by salt, drought and ABA treatment in *Galega orientalis* (Li et al., 2009).

When the AVP1 gene, a vacuolar H+-pyrophosphatase gene from *A. thaliana*, was transformed into alfalfa, over-expression enhanced salt and drought tolerance. Compared to the wild-type, the transgenic plants accumulated more Na+, K+ and Ca2+ in leaves and roots, retained more solutes and water, maintained a higher root activity, had a protected photosynthetic machinery, and maintained a stable cell membrane under abiotic stress conditions (Bao, et al., 2009). Zhao's team reported that co-expressing *Suaeda salsa* SsNHX1 and AVP1 conferred greater salt tolerance to transgenic plants than did SsNHX1 alone (Zhao et al., 2006). These studies provide a promising way for improving salt and drought tolerance in forage crop plants.

### **4.7 Cold-acclimation specific gene**

216 Plants and Environment

responses. The expression and protein levels of MsHO1 are higher in alfalfa stems and leaves than in germinating seeds and roots and are induced significantly by some pro-oxidant compounds including hemin and nitric oxide donor sodium nitroprusside (Fu et al., 2011).

LEA proteins, which are suggested to act as desiccation protectants during seed desiccation and in water-stressed seedlings, can be induced by ABA and various types of water-related stress (Espelund et al., 1992). MtPM5, identified as an atypical hydrophobic LEA protein, during stress, is able to stabilize proteins, but is unable to protect cell membranes. MtPM25 is able to rapidly dissolve aggregates in a non-specific manner and sorption isotherms show that when it is unstructured, it absorbs up to threefold more water than MtEM6 (Boucher et al., 2010). In proteomic analysis of the germination of *M. truncatula* seeds associated to desiccation tolerance (DT), 11 polypeptides were identified as late embryogenesis abundant proteins. The abundance changes of MtEm6 and MtPM25 show the two proteins were

Ion transporters selectively transport ions and maintain them at physiologically relevant concentrations. Sodium transporters in plant cells have been extensively studied. Sodium is compartmentalized into the vacuole, through the operation of the vacuolar Na+/H+ antiporter, down an electrochemical proton gradient generated by the vacuolar H+-ATPase and H+-Ppase (Blumwald et al., 2000). In both prokaryotic and eukaryotic cells, the Na+/H+ exchanger plays a key role in the regulation of cytosolic pH, cell volume and Na+ homeostasis (Padan et al., 2001; Wiebe et al., 2001). Plant Na+/H+ antiporters have been isolated from Arabidopsis (Shi et al., 2000), rice (Fukuda et al., 1999) and forage plants (Li et al., 2009; Tang et al., 2010; Yang et al., 2005). MsNHX1, encoding a vacuolar Na+/H+ antiporter, was isolated from alfalfa. The expression of MsNHX1 was significantly upregulated after treated by NaCl and ABA (Yang et al., 2005). MsNHX1 was located to the vacuolar membrane and can partly complement the NaCl-sensitive phenotypes of a yeast mutant. The expression of MsNHX1 in Arabidopsis enhances the resistance to salt stress (An et al., 2008). To investigate the mechanisms of *Medicago intertexta* and *Melilotus indicus* in salt stress, the expression of four genes coding for NHX-type Na+/H+ antiporters were measured. The result show that three genes are expressed in *M. intertexta* leaves and roots, and one gene in *M. indicus* roots. *NHX* gene expression may trigger *M. intertexta* to cope with tissue Na+ accumulation, while in *M. indicus*, the low Na+ content and the lack of correlation between growth in the presence of NaCl, Na+ content and *NHX* gene expression indicates that different mechanisms are involved in coping with salt stress (Zahran et al., 2007). TrNHX1, isolated from *Trifolium repens* L., can complement the *Δnhx1* and *Δena1- 4Δnhx1* yeast mutants by suppressing their observed phenotypes. A similar result is observed in the presence of LiCl and KCl. Under 150mM NaCl treatment, the expression level of TrNHX1 in roots, shoots and leaves is 1.7, 2.2 and 4.3 times, respectively, that of the controls. The expression levels and Na+ content in organs also have a close relationship (Tang et al., 2010). SsNHX1, isolated from the halophyte *Salsola soda*, could significantly enhance salt- tolerance in transgenic alfalfa under the stress-inducible *rd29A* promoter even at 400 mM NaCl (Li et al., 2011). Also *GoNHX1* was induced by salt, drought and ABA

**4.5 Late embryogenesis abundant proteins** 

related to DT (Boudet et al., 2006).

treatment in *Galega orientalis* (Li et al., 2009).

**4.6 Transporter genes** 

Many plants increase freezing tolerance upon exposure to low non-freezing temperatures, a phenomenon known as cold acclimation. Cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury (Thomashow, 1999). In forage grasses, many genes (Mohapatra et al., 1989; Tamura & Yonemaru, 2010; Tominaga et al., 2001; Zhang et al., 2009) and proteins (Kosmala et al., 2009) are regulated during cold acclimation.

In alfalfa, some CAS (cold-acclimation-specific) genes are specifically expressed during coldacclimation and their expression level measured by mRNA abundance is positively correlated with the freezing-tolerance of cultivars (Mohapatra et al., 1989). From alfalfa, MsaCIA, cas15 (cold acclimation-specific gene), cas17, and MsaCIC, induced by low temperature have been isolated (Castonguay et al., 1994; Laberge et al., 1993; Monroy et al., 1993; Wolfraim & Dhindsa 1993), although they were not induced by ABA or other stresses (Mohapatra et al., 1989). Encoding a putative nuclear protein, the cas15 transcript level is increased significantly with cold acclimation but is hardly detectable in the absence of cold acclimation. Thus, the accumulation of cas15 and the prolonged cold acclimation have a close relationship (Monroy et al., 1993). In red clover, the homolog of the alfalfa MsaCIA is induced by cold, but the homolog-like MsaCIB, MsaCIC genes were not induced by cold. The expression level of MsaCIA transcripts was 3 times higher in the cold-acclimated, regenerative, F49R (cold tolerant) genotype, compared to the cold-acclimated, non-regenerative, F49M (cold sensitive) genotype. It was also shown that enhanced expression of MsaCIA and the regenerative trait are either linked (Nelke et al., 1999). MsaCIC is similar to bimodular proteins that are developmentally regulated in other plant species (Castonguay et al., 1994). Calcium, a second messenger, can also play an important role in cold acclimation in alfalfa. The influx of extracellular 45Ca2+ at 4 oC is 15 times higher than at 25 oC. The addition of a calcium ionophore or a calcium channel agonist caused an influx of extracellular 45Ca2+ and induced the expression of *cas* genes (as reporters in low-temperature signal transduction) at 25oC, while the addition of calcium channel blockers inhibited the influx of extracellular 45Ca2+ as well as the expression of cas genes (Monroy & Dhindsa, 1995). Associated with winter survival, the cold acclimation-responsive gene, such as RootCAR1, may act as a molecular marker for identifying winter hardy plants in semi-dormant or non-dormant alfalfa germplasm in winter of the seed year (Cunningham et al., 2001).

Forage crop plants are the foundation of animal husbandry. In general, forage crops are located in harsh environments, especially in China. This implies that forage crop plants contain essential stress-tolerance gene resources. Over previous decades, the study of stress tolerance mechanisms mainly focused on physiology and morphology, the molecular and

Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 219

Blumwald, E., G. S. Aharon and M. P. Apse. (2000). Sodium transport in plant cells.

Boucher, V., Buitink, J., Lin, X., Boudet, J., Hoekstra, F. A., Hunder Tmark, M., Renard, D. &

Boudet, J., Buitink, J., Hoekstra, F. A., Rogniaux, H., Larré, C., Satour, P. & Leprince, O.

proteins associated with desiccation tolerance. *Plant Physiol*. 140: 1418-1436. Bryan, D.M., Chen, Y.R., Mitchel, D.B., Stephen, R.B., Chris, B.,.Dirk, I., Kathleen, D.H. &

Bryan, D.M., Julia, M., & Kim, S.J.(2000) Iron-superoxide dismutase expression in transgenic

Cao, Y. J., Wei, Q., Liao, Y., Song, H. L., Li, X., Xiang, C. B. & Kuai, B. K. (2009). Ectopic

Castonguay, Y., Laberge, S., Nadeau, P. & Vézina, L. P. (1994). A cold-induced gene from

Chao, Y., Kang, J., Sun, Y., Yang, Q., Wang, P., Wu, M., Li, Y., Long, R. & Qin. Z. (2009).

Chaves, M.M. (1991). Effects of water deficits on carbon assimilation. *Journal of Experimental* 

Chaves, M.M., Maroco, J.P. & Pereira, J.S. (2003). Understanding plant response to drought-

Chen, W.J. & Zhu. T. (2004). Networks of transcription factors with roles in environmental

Chen, J. R., Lü, J. J., Wang, T. X., Chen, S. Y. & Wang, H. F.(2009). Activation of a DRE-

Crowe, J.H., F.A. Hoekstra, and L.M. Crowe. 1992.Anhydroviosis. *Annu.Rev.Physiol.* 54:579-

Chen, T. H. H. & Murata. N. (2011). Glycinebetaine protects plants against abiotic stress:

Chen, X. F., Wang, Z., Wang, X. M., Dong, J., Ren, J. & Gao,H.W. (2009). Isolation and

Chinchilla, D., Merchan, F., Megias, M., Kondorosi, A., Sousa, C. & Crespi, M. (2003).

induced by osmotic stress in alfalfa. *Plant molecular biology.* 51: 555-566. Cirousse, C., Bournoville, R. & Bonnemain, J.L. (1996). Water deficit-induced changes in

transgenic *alfalfa* (*Medicago sativa* L.) *Plant Physiol.* , *103*, 1155–1163.

oxidative stress tolerance. *Plant Physiol.* 2000, *122*, 1427–1437.

from Medicago sativa L. *Molecular biology reports*. 36: 2315-2321.

from genes to the whole plant. *Functional Plant Biol*. 30:239-264.

region. *In Vitro Cellular & Developmental Biology-Plant.* 45: 1-11.

drought and salt stress. *Plant cell reports*. 28: 579-588.

proteins. *Plant molecular biology*. 24: 799-804.

stress response. *Trends Plant Sci*. 9:591–596

orientalis. *Genes & genetic systems.* 84(2): 101-109.

*Plant Physiol.* 111:109-113.

*Botany* 42: 1–16.

599.

Leprince,O. (2010). MtPM25 is an atypical hydrophobic late embryogenesis‐abundant protein that dissociates cold and desiccation‐aggregated

(2006). Comparative analysis of the heat stable proteome of radicles of Medicago truncatula seeds during germination identifies late embryogenesis abundant

Johan, B. (1993) . Superoxide dismutase enhances tolerance of freezing stress in

*alfalfa* increases winter survival without a detectable increase in photosynthetic

overexpression of AtHDG11 in tall fescue resulted in enhanced tolerance to

Medicago sativa encodes a bimodular protein similar to developmentally regulated

Molecular cloning and characterization of a novel gene encoding zinc finger protein

binding transcription factor from Medicago truncatula by deleting a Ser/Thr-rich

mechanisms and biotechnological applications. Plant, Cell & Environment. 34: 1-20.

characterization of GoRAV, a novel gene encoding a RAV-type protein in Galegae

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is

concentrations in proline and some other amino acids in the phloem sap of Alfalfa.

*Biochimica et Biophysica Acta (BBA)-Biomembranes*. 1465: 140-151.

proteins. *Plant, Cell & Environment.* 33: 418-430.

genetic mechanism being less understood. The discovery and use of stress-toleranceassociated genes to confer forage plant stress tolerance, is clearly a promising approach. New studies aimed at revealing the signaling transduction, transcriptional regulation and gene responses in forage plants, will contribute to this end.

### **5. References**


genetic mechanism being less understood. The discovery and use of stress-toleranceassociated genes to confer forage plant stress tolerance, is clearly a promising approach. New studies aimed at revealing the signaling transduction, transcriptional regulation and

Abogadallah, G. M., Nada, R. M., Malinowski R. & Quick. P. (2011). Overexpression of

Adams, P., Nelson, D.E., Amada, S.Y., Chmara, W., Jensen, .R.G., Bohnert, H.J. & Griffiths,

An, B. Y.; Luo, Y.; Li, J. R., Qiao, W. H., Zhang X. S., & Gao. X. Q. (2008). Expression of a

Ariel, F., Diet, A., Verdenaud, M., Gruber, V., Frugier, F., Chan R., & Crespi, M. (2010).

Ashraf, M. & Foolad, M. (2007). Roles of glycine betaine and proline in improving plant abiotic stress resistance. *Environmental and Experimental Botany*. 59: 206-216. Aubert, S., Hennion, F., Bouchereau, A., Gout, E., Bligny R., & DORNE, A. J. (1999).

Bagga, S., Rochford, J., Klaene, Z., Kuehn G.D., & Phillips, G.C. (1997). Putrescine

Baln ka, F., Ovecka M., & Hirt, H. (2000). Salt stress induces changes in amounts and

Bao, A. K., Wang, S. M., Wu, G. Q., Xi, J. J., Zhang J. L. & Wang. C.M. (2009).

Bartels, D. (2001). Targeting detoxification pathways: an efficient approach to obtain plants

Bastola, D. R., Pethe V. V. & Winicov, I. (1998). Alfin1, a novel zinc-finger protein in alfalfa

Bhatnagar-Mathur, P., Vadez V. & Sharma, K. K. (2008). Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. *Plant cell reports*. 27: 411-424.

with multiple stress tolerance. *Trends Plant Sci.* 6:284–286.

transgenic Arabidopsis. *Acta Agronomica Sinica*. 34(4): 557-564.

HARDY, an AP2/ERF gene from Arabidopsis, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium

H. (1998). Growth and development of *Mesembryanthemum crystallinum*

vacuolar Na+/H+ antiporter gene of alfalfa enhances salinity tolerance in

Environmental Regulation of Lateral Root Emergence in Medicago truncatula Requires the HD-Zip I Transcription Factor HB1. *The Plant Cell*. 22: 2171-2183. Armengaud, P., Thiery, L., Buhot, N., Grenier‐de March G., & Savouré, A. (2004).

Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. *Physiologia Plantarum*. 120: 442-

Subcellular compartmentation of proline in the leaves of the subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br. In vivo13C‐NMR study. *Plant, Cell &* 

aminopropyltransferase is responsible for biosynthesis of spermidine, spermine, and multiple uncommon polyamines in osmotic stress-tolerant alfalfa. *Plant* 

localization of the mitogen-activated protein kinase SIMK in alfalfa roots.

Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). *Plant Science*. 176: 232-240.

roots that binds to promoter elements in the salt-inducible MsPRP2 gene. *Plant* 

gene responses in forage plants, will contribute to this end.

alexandrinum L. *Planta*: 1-12.

*Environment.* 22: 255-259.

*Physiology*. 114: 445-454.

*Protoplasma.* 212: 262-267.

*molecular biology.* 38: 1123-1135.

(Aizoaceae). *New Phytol.* 138: 171–190.

**5. References** 

450.


Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 221

Jin, H., Sun, Y., Yang, Q., Chao, Y., Kang, J. & Li, Y. (2010). Screening of genes induced by

Jin, T., Chang, Q., Li, W., Yin, D., Li, Z., Wang, D., Liu, B. & Liu, L. (2010). Stress-inducible

Jonak, C., Kiegerl, S., Ligterink, W., Barker, P. J., Huskisson, N. S. & Hirt, H. (1996). Stress

Kishor,P. B. K., Hong, Z., Miao, G. H., Hu, C. A. A. & Verma, D. P. S. (1995). Overexpression

confers osmotolerance in transgenic plants. *Plant Physiology*. 108: 1387-1394. Kosmala, A., Bocian, A., Rapacz, M., Jurczyk, B. & Zwierzykowski, Z. (2009). Identification

Kovács, I., Ayaydin, F., Oberschall, A., Ipacs, I., Bottka, S., Pongor, S., Dudits, D. & Tóth, E.

and abscisic acid responsive gene in alfalfa. *The Plant Journal.* 15(2): 185-197. Laberge, S., Castonguay,Y. &Vézina, L. P. (1993). New cold-and drought-regulated gene

Laude, H.M. (1953). The nature of summer dormancy in perennial grasses. *Botanical Gazette.* 

Li, W., Wang, D., Jin, T., Chang, Q., Yin, D., Xu, S., Liu, B. & Liu, L. (2011). The Vacuolar

Li, X., Cheng, X., Liu, J., Zeng, H., Han, L. & Tang, W. (2011). Heterologous expression of the

Liu, L., White, M. J. & MacRae, T. H. (1999). Transcription factors and their genes in higher

Liu, Z. H., Zhang, H. M., Li, G. L., Guo, X. L., Chen, S. Y., Liu, G. B. & Zhang, Y. M. (2011).

Norton, M., Lelievre, F. & Volaire, F. (2006). Summer dormancy in *Dactylis glomerata* L., the

contrasting cultivars. *Australian Journal of Agricultural Research.* 57:565–575. Mansour, M. M. F. (1998). Protection of plasma membrane of onion epidermal cells by

Marcum, K. B. (1994). Salinity Tolerance Mechanisms of Six C4 Turfgrasses. *J. Amer. Soc.* 

transgenic Lolium perenne plants. *Plant Biotechnology Reports.* 5: 61-69. Li, X., Wang, Z., Wang, X. M., Gao, H.W., Chen, X. F., Dong, J. & Xu, B. (2009). Cloning and

from Medicago sativa. *Plant Physiology.* 101: 1411-1412.

plants. *European Journal of Biochemistry.* 262: 247-257.

betaine aldehyde dehydrogenase. *Euphytica.* 178: 363-372.

expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. *Plant cell,* 

signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought. *Proceedings of the National Academy of Sciences of the United States of* 

of [delta]-pyrroline-5-carboxylate synthetase increases proline production and

of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis plants with distinct levels of frost tolerance. *Journal of experimental botany.* 

C. (1998). Immunolocalization of a novel annexin‐like protein encoded by a stress

Na+/H+ Antiporter Gene SsNHX1 from the Halophyte Salsola soda Confers Salt Tolerance in Transgenic Alfalfa (Medicago sativa L.). *Plant Molecular Biology* 

Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in

Analysis of a Vacuolar Na+/H+ Antiporter Gene in *Galega orientalis. Plant physiol.* 

Enhancement of salt tolerance in alfalfa transformed with the gene encoding for

inuence of season of sowing and a simulated mid-summer storm on two

glycinebetaine and proline against NaCl stress. *Plant Physiology and Biochemistry.* 

salt stress from Alfalfa. *Molecular biology reports.* 37: 745-753.

*tissue and organ culture.* 100: 219-227.

*America.* 93: 11274-11279.

60(12): 3595-3609.

114:282–292.

*Reporter.* 29: 278-290.

*Commun.* 45(5):1-5.

36(10): 767-772.

*Hort. Sci.* 119(4):779–784.


Cunningham, S., Volenec, J. & Teuber, J. (2001). Winter hardiness, root physiology, and

DaCosta, M., & Huang, B.R. (2006). Osmotic adjustment associated with variation in bentgrass tolerance to drought sress. *J.Amer.Soc.Hort.Sci.* 131(3): 338-344. Davletova, S., Mészáros, T., Miskolczi, P., Oberschall, A., T r k, K., Magyar, Z., Dudits, D. &

De Lorenzo, Merchan, L., F., Laporte, P., Thompson, R., Clarke, J., Sousa, C. & Crespi, M.

Espelund, M., S b e‐Larssen, S., Hughes, D. W., Galau, G. A., Larsen, F. & Jakobsen, K. S.

Feng, C. J., Luo, X. Y., Sha, W. & Wang, F. G. (2005). Effect of low temperature stress on SOD, POD activity and proline content of *alfalfa*. *Pratacultural Sci. 22*:29–32. Fougere, F., Rudulier, D.L. & Streeter,J.G. (1991). Effects of Salt Stress on Amino Acid,

Fu, G. Q., Xu, S., Xie, Y. J., Han, B., Nie, L. , Shen, W. B. & Wang, R. (2011). Molecular

Fukuda, A., Nakamura, A. & Tanaka. Y. (1999). Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. *Biochim Biophys Acta.* 1446: 149–155. Geerts, P., Buldgen, A., Diallo, T. & Dieng, A. (1998). Drought resistance by six Senegalese

Gimeno-Gilles, C., Gervais, M. L., Planchet, E., Satour, P., Limami, A.M. & Lelievre, E.

Halliwell, B. & Gutteridge, J.M.C. (1999). Free Radicals in Biology and Medicine. New York,

Hare,P.D., Gress, W. A. & VanStaden, J. (1998). Dissecting the roles od osmolyte

Hoeketra, F. A., Golovina, E. A. & Buitink, J. (2001). Mechanisms of plant desiccation

Ingram, J. & Bartels, D. (1996). Molecular basis of dehydration tolerance in plants. *Annu.Rev.* 

James, V. A., Neibaur, I. & Altpeter, F. (2008). Stress inducible expression of the DREB1A

transcription factor from xeric, Hordeum spontaneum L. in turf and forage grass (Paspalum notatum Flugge) enhances abiotic stress tolerance. *Transgenic research.*

accumulation during stress. *Plant Cell Environ.* 21:535-553.

Medicago truncatula roots to salt stress. *The Plant Cell.* 21: 668-690.

101'alfalfa. *Crop science.* 41: 1091-1098.

*of experimental botany*. 52(355): 215-221.

osmotic stress. *The Plant Journal.* 2(2): 241-252.

Alfalfa (*Medicago sativa* L.) .*Plant Physiol.*, 96:1228-1236.

Biochemistry;doi:10.1016/j.plaphy.2011.01.018 .

*Trop. Grasslands.* 32:235-242.

Oxford University Press.

tolerance. *Trends Plant Sci.* 6:431-438.

*Plant Physiol.Plant. Mol. Biol.* 47:377-403.

310.

17(1): 93-104.

gene expression in successive fall dormancy selections from 'Mesilla'and 'CUF

Deák, M. (2001). Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells. *Journal* 

(2009). A novel plant leucine-rich repeat receptor kinase regulates the response of

(1992). Late embryogenesis‐abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and

Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of

cloning, characterization, and expression of an alfalfa (Medicago sativa L.) heme oxygenase-1 gene, MsHO1, which is pro-oxidants-regulated. Plant Physiology and

local strains of *Andropogen gayanus* var. *bisquamulatus* through osmoregulation.

(2011). A stress-associated protein containing A20/AN1 zing-finger domains expressed in Medicago truncatula seeds. *Plant Physiology and Biochemistry.* 49: 303-


Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 223

Qian, Y. & Fry, J. D. (1997). Water relation and drought tolerance of four turfgrasses. *J. Amer.* 

Qian, Y. L., Fry, J. D. & Upham,W.S. (1997). Rooting anddrought avoidance of warm-Season

Qu, C. P., Xu, Z. R., Liu, G. J., Liu, C., Li, Y., Wei, Z. G. & Liu, G. F. (2010). Differential

Rubio, M. C., Gonzalez, E. M., Minchin, F. R., Webb, K. J., Arrese‐Igor, C., Ramos, J. &

Sakuma, Y., Liu, Q., Dubouzet, J. G., Abe, H., Shinozaki, K. & Yamaguchi- Shinozaki,K.

Samis, K., Bowley, S. & McKersie,B. (2002). Pyramiding Mn‐superoxide dismutase

Scandalios, J. G. (1997). *Oxidative Stress and the Molecular Biology of Antioxidant Defenses*; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 1997, 2–11. Schat, H., Sharma, S. S. & Vooijs, R. (1997). Heavy metal‐induced accumulation of free

Shekhawat, G. & Verma, K. (2010). Haem oxygenase (HO): an overlooked enzyme of plant metabolism and defence. *Journal of experimental botany*. 61(9): 2255-2270. Sheng, L. (2010). The study on stress tolerance of forage. *Chinese Qinghai journal of animal and* 

Shi, H., Ishitani, M., Kim, C. & Zhu, J. K. (2000). The Arabidopsis thaliana salt tolerance gene

Shinozaki, K. & Yamaguchi-Shinozaki, K. (2000). Molecular responses to dehydration and

Shinozaki, K., Yamaguchi-Shinozaki, K. & Seki, M. (2003). Regulatory network of gene

Singh, K. B., Foley, R. C. & O ate-Sánchez, L. (2002). Transcription factors in plant defense

Song, F. N., Yang, C. P., Liu, X. M. & Li, G. B. (2006). Effect of salt stress on activity of

Suárez, R. C. & Iturriaga, C. (2009). Enhanced tolerance to multiple abiotic stresses in transgenic alfalfa accumulating trehalose. *Crop science*. 49: 1791-1799. Sun, S. J., Guo, S. Q., Yang, X., Bao, Y. M., Tang, H. J., Sun, H., Huang, J. & Zhang, H. S.

in salt tolerance in rice. *Journal of Experimental Botany.* 61(10): 2807–2818.

and stress responses. *Current Opinion in Plant Biology*. 5: 430-436.

superoxide dismutase(SOD) in *Ulmus pumila* L*. J. For. Res. 17*:13–16.

*Biochemical and biophysical research communications*. 290: 998-1009.

Expression of Copper-Zinc Superoxide Dismutase Gene of Polygonum sibiricum Leaves, Stems and Underground Stems, Subjected to High-Salt Stress. *International* 

Becana, M. (2002). Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases. *Physiologia* 

(2002). DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression.

transgenes to improve persistence and biomass production in alfalfa. *Journal of* 

proline in a metal‐tolerant and a nontolerant ecotype of Silene vulgaris. *Physiologia* 

SOS1 encodes a putative Na+/H+ antiporter. *Proc Natl Acad Sci USA*. 97:6896–6901.

low temperature: differences and cross-talk between two stress signaling pathways.

expression in the drought and cold stress responses. *Current Opinion in Plant* 

(2010). Functional analysis of a novel Cys2/His2-type zinc finger protein involved

*Soc. Hort. Sci.* 122:129-133.

*Plantarum.* 115: 531-540.

*Plantarum*. 101(3): 477-482.

*veterinary sciences*. 4(4):43-44.

*Biology.* 6: 410-417.

*Current Opinion in Plant Biology*. 3: 217-223.

turfgrasses and tall fescue. *Crop Sci.* 37:905–910.

*Journal of Molecular Sciences*. 11: 5234-5245.

*experimental botany*. 53(372): 1343-1350.


McKersie, B. D., Bowley, S. R., Harjanto, E. & Leprince, O. (1996). Water-deficit tolerance

McKersie, B. D., Bowley, S. R. & Jones,K. S. (1999). Winter survival of transgenic alfalfa overexpressing superoxide dismutase. *Plant Physiology.* 119: 839-847. McKersie, B. D., Chen, Y., de Beus, M., Bowley, S. R., Bowler, C., Inze, D., D'Halluin, K. &

transgenic alfalfa (Medicago sativa L.). *Plant Physiology.* 103: 1155-1163. McKersie, B. D., Murnaghan, J., Jones, K. S. & Bowley, S. R. (2000). Iron-superoxide

McWilliam, J. R. (1968). The nature of the perennial response in Mediterranean grasses. II.

Mo, Y., Liang, G., Shi, W. & Xie, J. (2011). Metabolic responses of alfalfa (Medicago Sativa L.)

Mohapatra, S. S., Wolfraim, L., Poole, R. J. & Dhindsa, R. S. (1989). Molecular cloning and

Monroy, A. F., Castonguay, Y., Laberge, S., Sarhan, F., Vezina, L. P. & Dhindsa, R. S. (1993).

Monroy, A. F. & Dhindsa, R. S. (1995). Low-temperature signal transduction: Induction of

Morgan, J.M. (1983). Osmoregulation as a selection criterion for drought tolerance in wheat.

Munnik, T., Ligterink, W., Meskiene, I., Calderini, O., Beyerly, J., Musgrave, A. & Hirt, H.

moderate and severe hyper-osmotic stress. *The Plant Journal*. 20(4): 381-388. Munns, R., James, R. A. & La¨uchli, A. (2006). Approaches to increasing the salt tolerance of wheat and other cereals. *Journal of Experimental Botany.* 57:1025–1043. Nelke, M., Nowak, J., Wright, J. M., McLean, N. L., Laberge, S., Castonguay, Y. & Vezina, L.

Niu, Y., Hu, T., Zhou, Y. & Hasi, A. (2010). Isolation and characterization of two Medicago

Nobel, P. S. (1980). Adaption of plants to water and high temperature. NewYork:John

Padan, E., Venturi, M., Gerchman, Y. & Dover, N. (2001). Na+/H+ antiporters. Biochimica et

*Plant Physiology*. 111: 1177-1181.

*Biotechnology.* 10(7): 1117-1124.

*Austral.J.Agr.Res*. 34:607-614.

*Euphytica.* 105: 211-217.

Wiley Sons. 43-45.

temperature. *Plant Physiology*. 102: 873-893.

*Plant Physiology and Biochemistry*. 48: 971-976.

Biophysica Acta (BBA)-Bioenergetics. 1505: 144-157.

*Physiology*. 89: 375-380.

122: 1427-1437.

409.

331.

and field performance of transgenic alfalfa overexpressing superoxide dismutase.

Botterman, J. (1993). Superoxide dismutase enhances tolerance of freezing stress in

dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance. *Plant Physiology*.

Senescence, summer dormancy and survival in Phalaris. *Aust. J. Agr. Res*. 19:397–

leaves to low and high temperature induced stresses. *African Journal of* 

relationship to freezing tolerance of cold-acclimation-specific genes of alfalfa. *Plant* 

A new cold-induced alfalfa gene is associated with enhanced hardening at subzero

cold acclimation-specific genes of Alfalfa by calcium at 25oC. *The Plant Cell*. 7: 321-

(1999). Distinct osmo‐sensing protein kinase pathways are involved in signalling

P. (1999). Enhanced expression of a cold-induced gene coding for a glycine-rich protein in regenerative somaclonal variants of red clover (Trifolium pratense L.).

falcate AP2/EREBP family transcription factor cDNA, MfDREB1 and MfDREB1s.


Molecular and Genetic Analysis of Abiotic Stress Resistance of Forage Crops 225

Wiebe, C. A., DiBattista, E. R. & Fliegel, L. (2001). Functional role of polar amino acid

Wilson, J.R. & Ludlow, M. M. (1983). Time trends for change in osmotic adjustment and

Winicov, I. (1993). cDNA encoding putative zinc finger motifs from salt-tolerant alfalfa

Winicov, I. (2000). Alfin1 transcription factor overexpression enhances plant root growth

Winicov, I. & Bastola, D.R. (1999). Transgenic Overexpression of the Transcription

Xiong, Y. & Fei, S. Z. (2006). Functional and phylogenetic analysis of a DREB/CBF-like gene

Yamaguchi-Shinozaki, K. & Shinozaki, K. (2006). Transcriptional regulatory networks in

Yang, Q. C., Wu, M., Wang, P.Q. & Zhou, X.L. (2005). Cloning and expression analysis of a vacuolar Na+/H+ antiporter gene from alfalfa. *DNA Seq*. 16(5):352-357. Yang, S.Q., Ren, G. X., Yang, G. H., Feng, Y. Z., & Zhang, Q. (2007). Effects of Water stress on

Yang, W. L., Liu, J. M., Chen, F., Liu, Q., Gong, Y. D. & Zhao. N. M. (2006). Identification of

Zahran, H. H., Marín‐Manzano, M. C., Sánchez‐Raya, A. J., Bedmar, E. J., Venema, K. &

Zhang, C., Fei, S., Warnke, S., Li, L. & Hannapel, D. (2009). Identification of genes

Zhang, J. Y., Broeckling, C. D., Blancaflor, E. B., Sledge, M. K., Sumner, L. W. & Wang, Z. Y.

Under Abiotic Stresses. *Journal of Integrative Plant Biology*. 48(3): 334-340. Yan, Q., Ma, Y. S., & Shi, J. J. (2007). Cold-stress tolerance study of forage cultivals in

cellular responses and tolerance to dehydration and cold stresses. *Annu. Rev. Plant* 

osmoregulation substances and chlorophyll fluorescent parameter for forage grass.

Festuca arundinacea Schreb Cat1 Catalase Gene and Analysis of its Expression

Sanjiangyuan region. Heilongjiang animal science and veterinary medicine. 12: 64-

Rodríguez‐Rosales, M. P. (2007). Effect of salt stress on the expression of NHX‐type ion transporters in Medicago intertexta and Melilotus indicus plants. *Physiologia* 

associated with cold acclimation in perennial ryegrass. *Journal of Plant Physiology*.

(2005).Overexpression of WXP1, a putative Medicago truncatula AP2 domaincontaining transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (Medicago sativa). *The Plant* 

Improves Salinity Tolerance of the Plants. *Plant Physiology*. 120: 473-480. Wolfraim, L. A. & Dhindsa, R. S. (1993). Cloning and sequencing of the cDNA for cas17, a cold acclimation-specific gene of alfalfa. *Plant Physiology*. 103: 667-668. Xiong, L. & Zhu, J. K. (2001). Abiotic stress signal transduction in plants: molecular and

water relations of leaves of Cenchrus ciliaris during and after water stress. *Aust. J.* 

under normal and saline conditions and improves salt tolerance in alfalfa. *Planta.*

FactorAlfin1 Enhances Expression of the Endogenous MsPRP2Gene in Alfalfa and

residues in Na+/H+ exchangers. *Biochemical Journal*. 357: 1-10.

(Medicago sativa L.) cells. *Plant Physiology*. 102: 681-682.

genetic perspectives. *Physiologia Plantarum*. 112: 152-166.

*Acta Bot . Boreal-occident Sin*. 27(9): 1826-1832.

in perennial ryegrass (Lolium perenne L.). *Planta*. 224: 878-888.

*Plant Physiol*. 10:15-24.

210: 416-422.

*Biol.* 57: 781-803.

*Plantarum*. 131: 122-130.

166: 1436-1445.

*Journal*. 42: 689-707.

66.


Tamura, K. & Yonemaru, J. (2010). Next‐generation sequencing for comparative

Takatsuji, H. (1998). Zinc-finger transcription factors in plants. *Cell Mol Life Sci*. 54(6): 582–

Sheng, L. (2010). The study of forage stress tolerance. *Chinese Qinghai journal of animal and* 

Thomashow, M.F. (1999). Plant cold acclimation: freezing tolerance genes and regulatory

Thomason, W. W., Berry, L., Liu, L. L. (1969). Localization and secretion of salt gland of *Tam* 

Tominaga, Y., Kanazawa, A. & Shimamoto, Y. (2001). Identification of cold-responsive genes

Turner, N. C. (1979). Drought resistance and adaptation to water deficits in crop

Volaire, F., Conejero, G., Lelievre.,F. (2001). Drought survival and dehydration

Vranova, E., Inzé, D. & Van Breusegem, F. (2002). Signal transduction during oxidative

Wang, J. P. & Bughrara, S. S. (2008). Morpho-physiological responses of several fescue

Wang, R. G., Chen, S. L., Liu, L. Y., Hao, Z. Y., Weng, H. J., Li, H., Yang, S. & Duan. S. (2005).

Wang, W.X., Vinocur, B. & Altman, A. (2003). Plant responses to drought, salinity and

Wang, W. X., Vinocur, B., Shoseyov, O. & Altman, A. (2001). Biotechnology of plant osmotic

Wang, X.M., Chen, X.F., Liu, Y., Gao, H.W., Wang, Z. & Sun, G.Z. (2011). CkDREB gene in

Wang, X.M., Dong, J., Liu, Y. & Gao, H.W. (2010). A Novel Dehydration-Responsive

White, R. H., Engelke, M. C., Morton, S. J. & Ruemmele, B. A. (1992). Competitive turgor

in perennial ryegrass (Lolium perenne L.) by a modified differential display

tolerance in *Dactylis glomerata* and *Poa bulbosa*. *Australian Journal of Plant* 

Genotypic differences in antioxidative ability and salt tolerance of three poplars

extreme temperatures: towards genetic engineering for stress tolerance. *Planta*. 218:

stress tolerance: physiological and molecular considerations. *Acta Hort.* 560:285–

Caragana korshinskii is involved in the regulation of stress response to multiple abiotic stresses as an AP2/EREBP transcription factor. *Molecular biology reports*. 38:

Element-Binding Protein from Caragana korshinskii Is Involved in the Response to Multiple Abiotic Stresses and Enhances Stress Tolerance in Transgenic Tobacco.

Trifolium repens L. *Plant Molecular Biology Reporter*. 28: 102-111.

mechanisms. *Annual Review of Plant Biology*. 50: 571-599.

*arix aphy lla*. *Proc Nat Acad Sci USA*, 63:310-317.

plant.New York:Johns Wiley and Sons. 343-372.

stress. *Journal of experimental botany*. 53(372): 1227.

grasses to drought stress. *Hortscience*. 43(3):776–783.

under salt stress. *J. Beijing For. Univ. 27*:46–52.

*Plant Molecular Biology Reporter*. 28: 664-675.

maintenance in tall fescue. *Crop sci*. 31:251-256.

method. *Grassland Science*. 47(5): 516-519.

596.

1–14.

292.

2801-2811.

*veterinary sciences*. 40(4):43-44.

*Physiology*. 28:743–754.

transcriptomics of perennial ryegrass (Lolium perenne L.) and meadow fescue (Festuca pratensis Huds.) during cold acclimation. *Grassland Science*. 56: 230-239. Tang, R., Li, C., Xu, K., Du, Y. & Xia, T. (2010). Isolation, Functional Characterization, and

Expression Pattern of a Vacuolar Na+/H+ Antiporter Gene TrNHX1 from


**10** 

*Spain* 

**Salt Stress in Vascular Plants and Its** 

Among abiotic stresses, high salinity is the most severe environmental stress, impairing crop production on at least 20% of irrigated land worldwide. In addition, the increased salinity of arable land is expected to have devastating global effects, resulting in up to 50% land loss by the middle of the 21st century. Furthermore, there is a deterioration of about 2 million ha (1%

Critically, the problem of salinization is increasing due to the accumulation of tons of salts into the soil as a consequence of bad agricultural practices (e.g., use of fertilizers on a massive scale), draining of aquifers, and a limited regional rainfall. Irrigated land is particularly at risk with approximately one-third being significantly affected by salinity. Despite its relatively small area, irrigated land is estimated to produce one-third of world's

Water scarcity in arid and semi-arid regions has forced the increased use of recycled wastewater and desalination of salty groundwater resources for agriculture use. Current technologies to desalinate or purify recycled waste-water for agricultural use can effectively reduce the concentrations of most toxic elements with the significant exception of boron (B). Boron in recycled water is often concentrated substantially as a result of the recycling process and as such can significantly impact agricultural soils. Therefore irrigation with saline groundwater containing high B concentration occurs in parts of the world where

The relations between salinity and mineral nutrition of horticultural crops are extremely complex and a complete understanding of the intricate interactions involved would require the input from multidisciplinary team of scientists. Thus, although information about their independent effects is abundant, information about the combined effects of salinity and B is

Plants, due to their sessile nature, have developed several mechanisms to tolerate the various stresses to which that may be encountered during their life cycles. Most plants are

of world agricultural lands) because of salinity each year (Mahajan & Tuteja, 2005).

food (Munns, 2002), so salinization of this resource is particularly critical.

**1. Introduction** 

there is a notable scarcity of water.

**2. Resistance to salinity - range of tolerance** 

very limited.

 **Interaction with Boron Toxicity** 

Ildefonso Bonilla1 and Agustín González-Fontes2

*2Departamento de Fisiología, Anatomía y Biología Celular,* 

*1Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid* 

*Universidad Pablo de Olavide, Sevilla* 


### **Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity**

Ildefonso Bonilla1 and Agustín González-Fontes2

*1Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 2Departamento de Fisiología, Anatomía y Biología Celular, Universidad Pablo de Olavide, Sevilla Spain* 

### **1. Introduction**

226 Plants and Environment

Zhao, H. & Bughrara, S. S. (2008). Isolation and characterization of cold-regulated

Zhu, B., Su, J., Chang, M., Verma, D. P. S., Fan, Y. L. & Wu, R. (1998). Overexpression of a

*Molecular Genetics and Genomics*. 279: 585-594.

salt-stress in transgenic rice. *Plant Science*. 139: 41-48.

transcriptional activator LpCBF3 gene from perennial ryegrass (Lolium perenne L.).

△1-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water-and

Among abiotic stresses, high salinity is the most severe environmental stress, impairing crop production on at least 20% of irrigated land worldwide. In addition, the increased salinity of arable land is expected to have devastating global effects, resulting in up to 50% land loss by the middle of the 21st century. Furthermore, there is a deterioration of about 2 million ha (1% of world agricultural lands) because of salinity each year (Mahajan & Tuteja, 2005).

Critically, the problem of salinization is increasing due to the accumulation of tons of salts into the soil as a consequence of bad agricultural practices (e.g., use of fertilizers on a massive scale), draining of aquifers, and a limited regional rainfall. Irrigated land is particularly at risk with approximately one-third being significantly affected by salinity. Despite its relatively small area, irrigated land is estimated to produce one-third of world's food (Munns, 2002), so salinization of this resource is particularly critical.

Water scarcity in arid and semi-arid regions has forced the increased use of recycled wastewater and desalination of salty groundwater resources for agriculture use. Current technologies to desalinate or purify recycled waste-water for agricultural use can effectively reduce the concentrations of most toxic elements with the significant exception of boron (B). Boron in recycled water is often concentrated substantially as a result of the recycling process and as such can significantly impact agricultural soils. Therefore irrigation with saline groundwater containing high B concentration occurs in parts of the world where there is a notable scarcity of water.

The relations between salinity and mineral nutrition of horticultural crops are extremely complex and a complete understanding of the intricate interactions involved would require the input from multidisciplinary team of scientists. Thus, although information about their independent effects is abundant, information about the combined effects of salinity and B is very limited.

### **2. Resistance to salinity - range of tolerance**

Plants, due to their sessile nature, have developed several mechanisms to tolerate the various stresses to which that may be encountered during their life cycles. Most plants are

Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity 229

These receptors trigger different signal transduction cascades, among which are found

 These signaling cascades are at the core of the induction of the expression of certain genes that are directly or indirectly involved in protection against this stress. According to the response time we distinguish between early and late response genes. The first, induced in minutes, are transcription factors that determine the expression of late genes, which themselves are involved in stress tolerance and detoxifying enzymes. They comprise ion channels, enzymes, metabolites synthesizing protective chaperone,

 Some of the induced genes are involved in the synthesis of diffusible signals (e.g., ABA, ethylene, salicylic acid) acting in a second wave of signaling, which now affects to the

When we analyze the response of plants to stresses of different nature, there is a redundancy in the mechanisms deployed. Thus, plants show cross-tolerance, which means that a plant resistant to a particular condition can develop tolerance to other forms of stress. Although the mechanisms by which develops cross-tolerance occurs remain unknown, it is suspected that cross-tolerance between salinity, drought and cold stress are due to the common consequences (osmotic and oxidative stresses) (Mahajan & Tuteja, 2005; Tester &

Salt stress is a complex problem, in which experiments show induction of 194 genes in *Arabidopsis*. It requires a perfect orchestration between genetic, epigenetic modifications, pre- and post-transcriptional regulation and also post-translational control. Below we expose the main signaling pathways and safeguard mechanisms induced by salinity (Tuteja,

Signaling is an area of greatest potential for plant research, either in relation to how to detect nutrients, abiotic factors or other organisms (symbionts, harmless or pathogenic). It also is of major interest to optimize the production in agricultural systems. In the case of salt stress we know that the Ca2+-mediated signaling plays a crucial role, followed by ABA-mediated signaling, and with the participation of other routes, such as the MAP kinases (Mahajan &

Calcium levels in the cytoplasm are maintained below 1 µM by a delicate balance held by carriers that are present in the endoplasmic reticulum (ER), chloroplast and in the vacuole.

In the presence of high salt concentration, at first place there is an increase in cytosolic Ca2+ level coming from the apoplast. Then, the entry of Ca2+ derived from cellular organelles takes place, which is determined by the action of inositol triphosphate (IP3) formed by the enzyme phospholipase C. This will generate several waves of Ca2+ to form a signaling pathway (so called "Ca signature"), which is decoded by several calcium-binding proteins. In *Arabidopsis*, three genes involved in this decoding activity have been described, known as

This homeostasis is a prerequisite for the action of this cation as a second messenger.

as secondary signals Ca2+, inositol phosphate (IP), ABA and ROS.

whole organism and determines a global adaptation.

among others.

Davenport, 2003).

2007).

Tuteja, 2005).

**5.1.1 Ca2+ signaling**

**5. The case of salt stress** 

**5.1 Signaling of salt stress** 

very sensitive to soil salinity and are known as *glycophytes,* whereas salt-tolerant plants are known as *halophytes.* In general, *glycophytes* cannot grow at 100 mM NaCl, whereas *halophytes* can grow at salinities over 250 mM NaCl.

These adaptations include the synthesis of compatible osmolytes, proper maintenance of ionic balance, the synthesis of detoxifying enzymes of reactive oxygen species (ROS), and other responses. Playing an important role in the proper induction of these responses are: at first, the Ca2+ signaling; then, the signaling mediated by abscisic acid (ABA) and, finally, the MAP (Mitogen-Activated Protein) kinases signaling cascade (Tuteja, 2007).

### **3. Problems caused by salinity**

Salinity alters the smooth operation of the plant due to factors ranging from the cellular level to physiological level (Hasegawa et al., 2000; Mahajan & Tuteja, 2005; Munns, 2002), which are outlined as follows:


There is therefore a link between the immediate cellular alterations to salt stress and physiological changes taking place in the plant, and which focalize the problems in ionic imbalance and an insurmountable decrease in water potential values for the plant.

### **4. The response to stress and cross-tolerance**

When stress affects any plant a series of responses are triggered at both cellular and systemic level (Tuteja, 2007; Zhu, 2002). According to these authors, synthetically the stress response goes as follows:

 Different receptors (ion channels, receptors serine/threonine kinase or histidine kinase, or G-protein-coupled receptors) located in the plasma membrane perceived stress at that localization.


When we analyze the response of plants to stresses of different nature, there is a redundancy in the mechanisms deployed. Thus, plants show cross-tolerance, which means that a plant resistant to a particular condition can develop tolerance to other forms of stress. Although the mechanisms by which develops cross-tolerance occurs remain unknown, it is suspected that cross-tolerance between salinity, drought and cold stress are due to the common consequences (osmotic and oxidative stresses) (Mahajan & Tuteja, 2005; Tester & Davenport, 2003).

### **5. The case of salt stress**

228 Plants and Environment

very sensitive to soil salinity and are known as *glycophytes,* whereas salt-tolerant plants are known as *halophytes.* In general, *glycophytes* cannot grow at 100 mM NaCl, whereas

These adaptations include the synthesis of compatible osmolytes, proper maintenance of ionic balance, the synthesis of detoxifying enzymes of reactive oxygen species (ROS), and other responses. Playing an important role in the proper induction of these responses are: at first, the Ca2+ signaling; then, the signaling mediated by abscisic acid (ABA) and, finally, the

Salinity alters the smooth operation of the plant due to factors ranging from the cellular level to physiological level (Hasegawa et al., 2000; Mahajan & Tuteja, 2005; Munns, 2002),

Salinity affects the physiology and metabolism of the plant, as it causes both

 Due to the high salt concentration, soil water potential (Ψ) becomes more negative, which hinders water uptake by the plant. These salt levels also affect the absorption of nutrients such as K+, Ca2+, or NO3- owing to competition of these ions with Na+

 A physiological damage is observed in leaf transpiration and also a growth inhibition, which increases the toxic effects of the salt within the plant. These alterations might respond, according to recent findings in *Arabidopsis*, to a failure in cortical microtubule organization and helical growth of this plant. A salt buildup also occurs in the old leaves, and the death of them could be a key strategy for plant survival. It has also been

 At cellular level, the presence of Na+ alters the K+/Na+ cytosolic ratio, even though the Na+ remained extracellular. The alteration in the levels of K+, due to its importance for plant growth, causes a disruption of osmotic balance, the malfunctioning of the stomata

 Also at cellular level, the entry of Na+ alters the cell membrane potential, thereby allowing the entry of Cl- ions. Thus, the Na+ in concentrations around 100 mM is toxic to cell metabolism and may inhibit some essential enzymes, expansion and cell division, and membrane organization. Associated with this stress, many ROS are produced, with

There is therefore a link between the immediate cellular alterations to salt stress and physiological changes taking place in the plant, and which focalize the problems in ionic

When stress affects any plant a series of responses are triggered at both cellular and systemic level (Tuteja, 2007; Zhu, 2002). According to these authors, synthetically the stress

 Different receptors (ion channels, receptors serine/threonine kinase or histidine kinase, or G-protein-coupled receptors) located in the plasma membrane perceived stress at

imbalance and an insurmountable decrease in water potential values for the plant.

MAP (Mitogen-Activated Protein) kinases signaling cascade (Tuteja, 2007).

*halophytes* can grow at salinities over 250 mM NaCl.

**3. Problems caused by salinity** 

hyperosmotic and hyperionic stresses.

observed a decline in photosynthetic activity.

and the inhibition of some enzyme activities.

**4. The response to stress and cross-tolerance** 

response goes as follows:

that localization.

the oxidative damage that this entails (Grattan & Grieve, 1999).

which are outlined as follows:

and Cl-.

Salt stress is a complex problem, in which experiments show induction of 194 genes in *Arabidopsis*. It requires a perfect orchestration between genetic, epigenetic modifications, pre- and post-transcriptional regulation and also post-translational control. Below we expose the main signaling pathways and safeguard mechanisms induced by salinity (Tuteja, 2007).

### **5.1 Signaling of salt stress**

Signaling is an area of greatest potential for plant research, either in relation to how to detect nutrients, abiotic factors or other organisms (symbionts, harmless or pathogenic). It also is of major interest to optimize the production in agricultural systems. In the case of salt stress we know that the Ca2+-mediated signaling plays a crucial role, followed by ABA-mediated signaling, and with the participation of other routes, such as the MAP kinases (Mahajan & Tuteja, 2005).

### **5.1.1 Ca2+ signaling**

Calcium levels in the cytoplasm are maintained below 1 µM by a delicate balance held by carriers that are present in the endoplasmic reticulum (ER), chloroplast and in the vacuole. This homeostasis is a prerequisite for the action of this cation as a second messenger.

In the presence of high salt concentration, at first place there is an increase in cytosolic Ca2+ level coming from the apoplast. Then, the entry of Ca2+ derived from cellular organelles takes place, which is determined by the action of inositol triphosphate (IP3) formed by the enzyme phospholipase C. This will generate several waves of Ca2+ to form a signaling pathway (so called "Ca signature"), which is decoded by several calcium-binding proteins. In *Arabidopsis*, three genes involved in this decoding activity have been described, known as

Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity 231

inhibition, among others. The main mechanisms involved in resistance to high salt concentrations, namely, restoration of ionic balance, synthesis of compatible osmolytes (also called osmoprotectants, i.e., compatible solutes) such as proline or glycine betaine,

Excess of Na+ ions produce an imbalance in the ionic equilibrium, which is maintained through the joint action of pumps, together with other ions, and mediated largely by Ca2+ signaling. Thus, during salt stress, the expression of numerous channels is modified by the

There are channels of many different types of channels, which perform different functions for the maintenance of ionic and osmotic homeostasis. On one hand, we have those with greater selectivity for the ion K+ than for Na+, such as the KIRC channel (K+ Inward-Rectifying Channel) that mediates the entry of K+ after hyperpolarization of the membrane and accumulates this ion over ion Na+; another example of this type of channel is the HKT channel (Histidine Kinase Transporter), a low affinity transporter for Na+, which prevents the entry of Na+ into the cytosol. On the other hand, the entry of Na+ in plant cells is determined by the NSCC channel (NonSpecific Cation Channel). Another type of channel is provided by the KORC channel (K+ Outward-Rectifying Channel), which is activated after depolarization of the membrane, mediates the efflux of K+ and Na+ entry, which thus is accumulated in the cytosol. To place this movement requires a number of carriers that generate the H+ gradient needed for channel maintenance. In that sense, transporters as NHX (Na+/H+ exchanger) that allows the accumulation of Na+ in vacuoles are necessary, alleviating the effects of stress; or as channel CAX1 (H+/Ca2+ antiporter) responsible for the

Both are osmoprotectants synthesized by many plants in response to various stresses, including salt, and whose main function is to relieve the effects of this stress (Chen & Murata, 2008; Delauney & Verma, 1993). They are not the only ones; there are other osmolytes as polyols and alcohol sugars, whose functions are centered in the maintenance of

Glycine betaine (GB) is synthesized naturally from the choline by the action of the choline monooxygenase and betaine aldehyde dehydrogenase enzymes. In plants where GB is not produced, the overexpression of GB synthesizing genes in transgenic plants resulted in the production of enough amount of GB. With the inclusion of these genes in various plants and other organisms, it has been shown greater tolerance to salinity. Likewise, direct foliar application of the compound also improves the plant response to a saline environment

The synthesis of proline is a frequent response in salt stress. This osmolyte is accumulated in the cytosol and allows proper osmotic adjustment. Furthermore, this amino acid also stabilizes subcellular structures, buffers the redox potential and blocks free radicals. It is synthesized from glutamic acid by the action of enzymes pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR). Again, the inclusion of these genes in various plants has improved the tolerance of these transgenic plants to high salt concentrations. The induction of P5CS gene seems be mediated by ABA, as its mRNA

expression of detoxifying enzymes of ROS, and helicases are shown in Figure 1.

calcium-dependent partner SOS2-SOS3, as already discussed.

maintenance of Ca2+ homeostasis (Tuteja, 2007).

the osmotic balance, the cell pressure and the protein folding.

**6.2 Proline and glycine betaine** 

(Tuteja, 2007).

**6.1 Ionic balance** 

*SOS1*, *SOS2* and *SOS3* (Salt Sensitive Overlay), although only *SOS3* encodes a calcineurin Blike protein (CBL), that is, a calcium sensor. The pathway follows with the interaction of SOS3 with SOS2, a serine/threonine kinase that catalyzes the phosphorylation of SOS1 (an antiporter Na+/H+) and this determines its activation state. This antiporter is involved in maintaining ionic balance, as we will detail later. In addition of SOS1, other channels and transporters as HKT, NHX and CAX1 are regulated by the SOS2-SOS3 pathway. This pathway is the most important in salinity tolerance, as it seeks to restore the ion balance (Mahajan et al., 2008; Zhu, 2002).

Calcium levels also induce genes responsible for enzymes of ABA synthesis pathway, such as zeaxanthin oxidase, 9-*cis*-epoxycaotenoid dioxygenase, ABA-aldehyde oxidase and molybdenum cofactor sulfurase (Tuteja, 2007). The induction of these genes leads to increased levels of ABA, which acts as a second messenger, as explained later.

Maintenance and restoration of calcium homeostasis is determined by the channel CAX1, an H+/Ca2+ antiporter present in the membrane of the vacuole. The main role of this network is the restoration of basal levels of Ca2+ in the cytoplasm, but is also important in order to maintain the osmotic balance during stress.

### **5.1.2 ABA signaling**

The genes responsible for ABA synthesis are induced by salt stress. Once accumulated, this hormone mediates the induction of genes involved in both its own synthesis and its degradation. But when it comes from salt stress responses, genes such as *RD29A* or *RD22* (Responsive to Dehydration), *COR15A* or *COR47* (COld Responsive), the pea DNA helicase 45 (*PDH45*), or pyrroline-5 carboxylate synthetase (*P5CS*, involved in the synthesis of proline), among others, are regulated by ABA (Tuteja, 2007). However, there are activation pathways of those genes independently of ABA.

At the molecular level, there have been described several transcription factors regulated by ABA as well the *cis*-regulatory elements that they recognize. Genes involved in tolerance to salinity are under the control of these promoters and their transcription factors. There are several examples, such as the AREB transcription factor (a leucine zipper transcription factor) that recognizes the ABRE region; or as the DREB2A and DREB2B transcription factors that recognize the region known as DRE/CRT. Other regulatory elements are MYCRS and MYBRS regions recognized by RD22 and MYC/MYB factors that help in the activation of the response, and could be the bridge between different stresses, and an explanation for cross-tolerance (Mahajan & Tuteja, 2005).

### **5.1.3 Signalling by MAP kinases**

The pathway of MAP kinases is well known as a signal transduction system, both intracellular and extracellular. Increases have been detected in the expression of different MAPK, MAPKK and MAPKKK in *Arabidopsis*, alfalfa, tobacco and others, suggesting that this signaling pathway is working on an appropriate response to salt stress (Zhang et al., 2006).

### **6. Response to salt stress**

As already stated, the major damages of salinity resulting from the alterations of Na+ occur at the ion balance and osmotic levels, which then lead to problems of toxicity and enzyme inhibition, among others. The main mechanisms involved in resistance to high salt concentrations, namely, restoration of ionic balance, synthesis of compatible osmolytes (also called osmoprotectants, i.e., compatible solutes) such as proline or glycine betaine, expression of detoxifying enzymes of ROS, and helicases are shown in Figure 1.

### **6.1 Ionic balance**

230 Plants and Environment

*SOS1*, *SOS2* and *SOS3* (Salt Sensitive Overlay), although only *SOS3* encodes a calcineurin Blike protein (CBL), that is, a calcium sensor. The pathway follows with the interaction of SOS3 with SOS2, a serine/threonine kinase that catalyzes the phosphorylation of SOS1 (an antiporter Na+/H+) and this determines its activation state. This antiporter is involved in maintaining ionic balance, as we will detail later. In addition of SOS1, other channels and transporters as HKT, NHX and CAX1 are regulated by the SOS2-SOS3 pathway. This pathway is the most important in salinity tolerance, as it seeks to restore the ion balance

Calcium levels also induce genes responsible for enzymes of ABA synthesis pathway, such as zeaxanthin oxidase, 9-*cis*-epoxycaotenoid dioxygenase, ABA-aldehyde oxidase and molybdenum cofactor sulfurase (Tuteja, 2007). The induction of these genes leads to

Maintenance and restoration of calcium homeostasis is determined by the channel CAX1, an H+/Ca2+ antiporter present in the membrane of the vacuole. The main role of this network is the restoration of basal levels of Ca2+ in the cytoplasm, but is also important in order to

The genes responsible for ABA synthesis are induced by salt stress. Once accumulated, this hormone mediates the induction of genes involved in both its own synthesis and its degradation. But when it comes from salt stress responses, genes such as *RD29A* or *RD22* (Responsive to Dehydration), *COR15A* or *COR47* (COld Responsive), the pea DNA helicase 45 (*PDH45*), or pyrroline-5 carboxylate synthetase (*P5CS*, involved in the synthesis of proline), among others, are regulated by ABA (Tuteja, 2007). However, there are activation

At the molecular level, there have been described several transcription factors regulated by ABA as well the *cis*-regulatory elements that they recognize. Genes involved in tolerance to salinity are under the control of these promoters and their transcription factors. There are several examples, such as the AREB transcription factor (a leucine zipper transcription factor) that recognizes the ABRE region; or as the DREB2A and DREB2B transcription factors that recognize the region known as DRE/CRT. Other regulatory elements are MYCRS and MYBRS regions recognized by RD22 and MYC/MYB factors that help in the activation of the response, and could be the bridge between different stresses, and an

The pathway of MAP kinases is well known as a signal transduction system, both intracellular and extracellular. Increases have been detected in the expression of different MAPK, MAPKK and MAPKKK in *Arabidopsis*, alfalfa, tobacco and others, suggesting that this signaling pathway is working on an appropriate response to salt stress (Zhang et al.,

As already stated, the major damages of salinity resulting from the alterations of Na+ occur at the ion balance and osmotic levels, which then lead to problems of toxicity and enzyme

increased levels of ABA, which acts as a second messenger, as explained later.

(Mahajan et al., 2008; Zhu, 2002).

**5.1.2 ABA signaling** 

maintain the osmotic balance during stress.

pathways of those genes independently of ABA.

explanation for cross-tolerance (Mahajan & Tuteja, 2005).

**5.1.3 Signalling by MAP kinases**

**6. Response to salt stress** 

2006).

Excess of Na+ ions produce an imbalance in the ionic equilibrium, which is maintained through the joint action of pumps, together with other ions, and mediated largely by Ca2+ signaling. Thus, during salt stress, the expression of numerous channels is modified by the calcium-dependent partner SOS2-SOS3, as already discussed.

There are channels of many different types of channels, which perform different functions for the maintenance of ionic and osmotic homeostasis. On one hand, we have those with greater selectivity for the ion K+ than for Na+, such as the KIRC channel (K+ Inward-Rectifying Channel) that mediates the entry of K+ after hyperpolarization of the membrane and accumulates this ion over ion Na+; another example of this type of channel is the HKT channel (Histidine Kinase Transporter), a low affinity transporter for Na+, which prevents the entry of Na+ into the cytosol. On the other hand, the entry of Na+ in plant cells is determined by the NSCC channel (NonSpecific Cation Channel). Another type of channel is provided by the KORC channel (K+ Outward-Rectifying Channel), which is activated after depolarization of the membrane, mediates the efflux of K+ and Na+ entry, which thus is accumulated in the cytosol. To place this movement requires a number of carriers that generate the H+ gradient needed for channel maintenance. In that sense, transporters as NHX (Na+/H+ exchanger) that allows the accumulation of Na+ in vacuoles are necessary, alleviating the effects of stress; or as channel CAX1 (H+/Ca2+ antiporter) responsible for the maintenance of Ca2+ homeostasis (Tuteja, 2007).

### **6.2 Proline and glycine betaine**

Both are osmoprotectants synthesized by many plants in response to various stresses, including salt, and whose main function is to relieve the effects of this stress (Chen & Murata, 2008; Delauney & Verma, 1993). They are not the only ones; there are other osmolytes as polyols and alcohol sugars, whose functions are centered in the maintenance of the osmotic balance, the cell pressure and the protein folding.

Glycine betaine (GB) is synthesized naturally from the choline by the action of the choline monooxygenase and betaine aldehyde dehydrogenase enzymes. In plants where GB is not produced, the overexpression of GB synthesizing genes in transgenic plants resulted in the production of enough amount of GB. With the inclusion of these genes in various plants and other organisms, it has been shown greater tolerance to salinity. Likewise, direct foliar application of the compound also improves the plant response to a saline environment (Tuteja, 2007).

The synthesis of proline is a frequent response in salt stress. This osmolyte is accumulated in the cytosol and allows proper osmotic adjustment. Furthermore, this amino acid also stabilizes subcellular structures, buffers the redox potential and blocks free radicals. It is synthesized from glutamic acid by the action of enzymes pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR). Again, the inclusion of these genes in various plants has improved the tolerance of these transgenic plants to high salt concentrations. The induction of P5CS gene seems be mediated by ABA, as its mRNA

Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity 233

Chaperones are involved in the folding of protein, under both normal and abnormal situations. Although it is depending of the kind of chaperones, the ability to isolate the nascent protein or unfolded protein from the high saline cytoplasm can help to achieve the

Sugar metabolism is the source of carbon backbones for the whole cell metabolism. Under salt stress a series of cell changes occurs, which include synthesis of osmoprotectants and induction of gene transcription and protein synthesis involved in stress tolerance. Interestingly, genes encoding gliceraldehyde-3-phosphate dehydrogenase, sucrosephosphate synthase and sucrose synthase are upregulated in response to drought (Ingram & Bartells, 1996), and increased levels of these enzymes are also associated with other environmental stresses in plants. Therefore, given the cross-tolerance between drought and salt stress (Munns, 2002), a similar gene response is very likely that also occurs against salt

Finally, careful genetic and biochemical analyses have demonstrated that maturation of *N*glycans is necessary for plants stress tolerance, and that *N-*glycans are essential not only for protein folding but also for in vivo functions of plant glycoproteins (Kang et al., 2008).

stress, which would reflect increased energy and carbon backbone requirements.

Fig. 1. Regulation of ion homeostasis by SOS pathway and other related pathways in

relation to mechanisms of stress tolerance.

folded state of essential proteins (Choi et al., 2004).

accumulates quickly in response to the treatment with this plant hormone (Chinnusamy et al., 2005; Vinocur & Altman, 2005).

### **6.3 ROS detoxifying enzymes**

Salt stress, like many other stresses, involves the development of ROS such as singlet oxygen (O21), the superoxide radical (O2-•), hydrogen peroxide (H2O2) and hydroxyl radical (HO•). These species are capable of producing lipid peroxidation, as well as DNA, RNA, and protein oxidative damages that compromise cell and plant viability. For detoxification of these ROS, plants have a battery of enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, and GSH reductase, all induced under salt stress. Furthermore, enzymes such as aldehyde dehydrogenase are also induced, allowing more tolerance to salt stress by eliminating aldehydes produced in reactions between ROS and lipids or proteins (Vinocur & Altman, 2005).

### **6.4 Helicases**

Various stresses, including salinity, induce the expression of genes involved in gene expression machinery, such as several helicases with DEAD box. The answer is given in the presence of Na+ ions and mediated by ABA. Furthermore, phosphorylation sites in these proteins have been described, which may be a contact point for Ca2+ signaling. These helicases have the function of the opening of duplex DNA or RNA, and in this second case we speak of an RNA chaperone function, to avoid unfavorable folds (Owttrim, 2006).

Although the actual mechanism by which helicases increase tolerance to salinity remains unknown, there are two prevailing hypotheses:


### **6.5 LEA proteins**

LEA proteins were first discovered in seed embryogenesis and germination, because they are accumulated in the first phase, and constitute over the 4% of cytoplasmic proteins in some seeds. They constitute a diverse family, but their sequences are enriched in polar amino acid, charged or uncharged, as glicines, glutamic acid or lysines. This hydrophilic character can explain their diverse functions, which include water retention as the protein D-19 from cotton; stabilization of other proteins by hydrophilic and hydrophobic interactions as RAB proteins; formation of a solvation surface around proteins, similar to the sugar solvation surface induced by salinity too; and ion sequestering as HVA1 protein in barley. ABA is inducing LEA protein synthesis in embryogenesis, as has been revealed in some mutants in the ABA signaling, and the same procedure will be controlling the expression of these proteins under salinity (Chourey et al., 2003).

### **6.6 Other responses**

As other stresses, salt stress induces more responses that mentioned above. A brief comment about chaperones and sugar metabolism follows.

accumulates quickly in response to the treatment with this plant hormone (Chinnusamy et

Salt stress, like many other stresses, involves the development of ROS such as singlet oxygen (O21), the superoxide radical (O2-•), hydrogen peroxide (H2O2) and hydroxyl radical (HO•). These species are capable of producing lipid peroxidation, as well as DNA, RNA, and protein oxidative damages that compromise cell and plant viability. For detoxification of these ROS, plants have a battery of enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, and GSH reductase, all induced under salt stress. Furthermore, enzymes such as aldehyde dehydrogenase are also induced, allowing more tolerance to salt stress by eliminating aldehydes produced in reactions between ROS and lipids or proteins

Various stresses, including salinity, induce the expression of genes involved in gene expression machinery, such as several helicases with DEAD box. The answer is given in the presence of Na+ ions and mediated by ABA. Furthermore, phosphorylation sites in these proteins have been described, which may be a contact point for Ca2+ signaling. These helicases have the function of the opening of duplex DNA or RNA, and in this second case

Although the actual mechanism by which helicases increase tolerance to salinity remains

 They would stabilize the mRNA transcriptional or translational level. In response to various stresses, secondary structures in the 5' end of mRNA can be performed, and this

They would alter gene expression in association with protein complexes of DNA

LEA proteins were first discovered in seed embryogenesis and germination, because they are accumulated in the first phase, and constitute over the 4% of cytoplasmic proteins in some seeds. They constitute a diverse family, but their sequences are enriched in polar amino acid, charged or uncharged, as glicines, glutamic acid or lysines. This hydrophilic character can explain their diverse functions, which include water retention as the protein D-19 from cotton; stabilization of other proteins by hydrophilic and hydrophobic interactions as RAB proteins; formation of a solvation surface around proteins, similar to the sugar solvation surface induced by salinity too; and ion sequestering as HVA1 protein in barley. ABA is inducing LEA protein synthesis in embryogenesis, as has been revealed in some mutants in the ABA signaling, and the same procedure will be controlling the

As other stresses, salt stress induces more responses that mentioned above. A brief comment

we speak of an RNA chaperone function, to avoid unfavorable folds (Owttrim, 2006).

which could be preventing the proper processing of the RNA.

expression of these proteins under salinity (Chourey et al., 2003).

about chaperones and sugar metabolism follows.

al., 2005; Vinocur & Altman, 2005).

**6.3 ROS detoxifying enzymes** 

(Vinocur & Altman, 2005).

unknown, there are two prevailing hypotheses:

**6.4 Helicases** 

processing.

**6.5 LEA proteins** 

**6.6 Other responses** 

Chaperones are involved in the folding of protein, under both normal and abnormal situations. Although it is depending of the kind of chaperones, the ability to isolate the nascent protein or unfolded protein from the high saline cytoplasm can help to achieve the folded state of essential proteins (Choi et al., 2004).

Sugar metabolism is the source of carbon backbones for the whole cell metabolism. Under salt stress a series of cell changes occurs, which include synthesis of osmoprotectants and induction of gene transcription and protein synthesis involved in stress tolerance. Interestingly, genes encoding gliceraldehyde-3-phosphate dehydrogenase, sucrosephosphate synthase and sucrose synthase are upregulated in response to drought (Ingram & Bartells, 1996), and increased levels of these enzymes are also associated with other environmental stresses in plants. Therefore, given the cross-tolerance between drought and salt stress (Munns, 2002), a similar gene response is very likely that also occurs against salt stress, which would reflect increased energy and carbon backbone requirements.

Finally, careful genetic and biochemical analyses have demonstrated that maturation of *N*glycans is necessary for plants stress tolerance, and that *N-*glycans are essential not only for protein folding but also for in vivo functions of plant glycoproteins (Kang et al., 2008).

Fig. 1. Regulation of ion homeostasis by SOS pathway and other related pathways in relation to mechanisms of stress tolerance.

Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity 235

As above mentioned, plant growth and yield are severely affected by salt stress in many regions of the world as a result of osmotic effects, ion toxicities, and mineral disorders (Hasegawa et al., 2000). The response of plants to salinity depends not only on the total ion concentration in the external medium, but also on the chemical nature of the involved ions (Curtin et al., 1993). Nevertheless, most experimental works for salinity studies in plants

NaCl toxicity is apparent by ion toxicity and osmotic stress. Ion toxicity is due to specific damages by the high levels of Na+ and Cl-, reduction of K+ uptake being a main consequence, among others. Moreover NaCl treatment can increase the electrolyte leakage in tomato roots, indicating that the integrity of their plasma membranes is altered with salt

Boron-rich soils with high contents of naturally occurring salinity, and irrigation with groundwater containing high concentrations of salts and B are two common ways by which plants can be subjected to a double stress: salinity and excess B (Nable et al., 1997). For instance, soils with salt and B accumulation occur in South Australia (Marcar et al., 1999) and Jordan River Valley in Israel and Jordan (Yermiyahu et al., 2008); irrigation water with high levels of salts and B has been well documented in San Joaquin Valley in California (Grieve al., 2010), and the Lluta Valley in Chile (Bastías et al., 2004b), among other places. At first glance, one might think that between salt stress and B toxicity there would be an additive or synergistic relationship. In other words, either the outcome of the two stresses, when occur simultaneously, is equivalent to the sum of the effects of both stresses when applied separately (additive response), or the outcome of the both combined stresses is greater than the sum of them acting separately (synergistic response). However, interactions between salinity and B toxicity are rather complex and it has been reported that an antagonistic response can also exist when both stresses appear simultaneously (Bastías et al., 2004a; Yermiyahu et al., 2008). As summarized by Yermiyahu et al. (2008), there is no agreement regarding mutual relations between salt stress and B toxicity (Grieve et al., 2010). Apparently, antagonism between salt stress and B toxicity can be a consequence of lower toxicity of NaCl in the presence of high B, lower toxicity of B in the presence of high NaCl,

the salt toxicity, since the increased addition of B to the soil did not affect leaf Na+ content in pepper plants (Yermiyahu et al., 2008), or even decreased it in wheat (Holloway & Alston,

in the leaves as a consequence of the reduced Cl- uptake. Nevertheless, this amelioration is

In turn, reduced leaf B contents with the increase of NaCl concentration in the irrigation water have been widely reported. As an explanation it has been proposed that reduced rates of transpiration would limit the leaf accumulation of B that is transported through the xylem

Another alternative proposal is that the interaction between salinity and B toxicity could be related to aquaporin functionality (Bastías et al., 2004a; Martínez-Ballesta et al., 2008a, 2008b). Under excess external B, significant B transport takes place through the plasma membrane aquaporins (Dordas et al., 2000). The lower aquaporin functionality found in NaCl-treated plants could be related to the reduction of B contents in plants subjected to combined B and NaCl, in comparison with plants treated only with B, which could explain the beneficial effect of salinity against B toxicity (Bastías et al., 2004a). In turn, under salt

1992). Therefore, B would positively affect to salt stress through decreased Cl-

uptake owing to high B could reduce

accumulation

**9. Salinity and excess boron** 

stress (Bastías et al., 2010).

have been performed with NaCl as a salinizing chemical.

or both. Several reports support that diminution of Cl-

through an as-yet-unknown mechanism.

(Yermiyahu et al., 2008).

### **7. Boron toxicity**

Boron (B) is likely the micronutrient whose concentration inside vascular plants must be kept within the narrowest range for achievement optimal growth and, for this purpose, excess B in soils is more difficult to manage than its deficiency, which can be prevented by fertilization (Herrera-Rodríguez et al., 2010). Although B is found in all cellular compartments (Dannel et al., 2002), its presence is particularly notable in the cell wall where most B is located forming complexes with pectic and galacturonic derivatives with a specific *cis*-diol configuration (Bonilla et al., 2010; Camacho-Cristóbal et al., 2008; O'Neill et al., 1996). Once B is inside the roots, it moves passively with the transpiration stream and it is accumulated in mature leaves, where it can be translocated depending on the presence or not of sugar alcohols capable of forming mobile B-polyol complexes through phloem (Brown et al., 1999). Overall increased B content in mature leaves indicates B immobility, whereas higher B content in young meristematic tissues suggests B mobility. In most plants, B mobility is restricted to the xylem, as they do not biosynthesize significant amounts of polyols (Brown et al., 1999; Brown & Shelp, 1997).

B toxicity is an agricultural problem that reduces crop yield worldwide. Toxicity takes place in vascular plants as B accumulates in shoots, generally following a pattern from leaf base to tip, that leads to chlorosis and necrosis (Marschner, 1995; Reid et al., 2004). As B toxicity alters metabolism and cell division, its symptoms are also manifested as a slowing-down and inhibition of growth, especially in roots, and reductions in yield, along with loss of fruit quality (Grieve et al., 2010; Nable et al., 1997).

Furthermore, high irradiance appears to increase the harmful effects of B toxicity, probably because elevated B contents may impair plant mechanisms to cope with photooxidation stress (Reid et al., 2004).

According to Reid et al. (2004), excess B would overbind compounds with hydroxyl groups in the *cis*-configuration that could explain how this mineral stress exerts its harmful effect. Thus, extra B may bind with pectic polysaccharides and thereby altering cell wall structure; also excess B could alter the structure of primary metabolic compounds through binding to the ribose moieties of ATP (adenosine triphosphate), NADH (nicotinamide adenine dinucleotide, reduced form), NADPH (nicotinamide adenine dinucleotide phosphate, reduced form); finally, high internal B concentrations may negatively affect plant development by binding to ribose of RNA.

Recent reports show changes in gene expressions as a consequence of high internal B concentration (Kasajima & Fujiwara, 2007; Öz et al., 2009; Pang et al., 2010). Although the mechanism by which excess B promotes these changes remains unknown despite many efforts are being made to elucidate it (Kasai et al., 2011), the involvement of a signaling cascade is likely.

### **8. Boron toxicity tolerance**

Boron tolerance appears to be associated with the ability to limit B accumulation in both roots and shoots. Thus, *Bot1* expression, a gene providing tolerance to excess B, was localized in barley roots and youngest leaf blades (Sutton et al., 2007). Other genes encoding B efflux transporters and conferring tolerance to B toxicity in cultivars of wheat (cv. India) and barley (cv. Sahara), namely *TaBOR2* and *HvBOR2*, respectively, have also been reported (Reid, 2007). Therefore, boric acid/borate efflux transporters appear to be key determinants of plant B tolerance, and provide a molecular basis for the generation of highly B-tolerant crops (Takano et al., 2008).

### **9. Salinity and excess boron**

234 Plants and Environment

Boron (B) is likely the micronutrient whose concentration inside vascular plants must be kept within the narrowest range for achievement optimal growth and, for this purpose, excess B in soils is more difficult to manage than its deficiency, which can be prevented by fertilization (Herrera-Rodríguez et al., 2010). Although B is found in all cellular compartments (Dannel et al., 2002), its presence is particularly notable in the cell wall where most B is located forming complexes with pectic and galacturonic derivatives with a specific *cis*-diol configuration (Bonilla et al., 2010; Camacho-Cristóbal et al., 2008; O'Neill et al., 1996). Once B is inside the roots, it moves passively with the transpiration stream and it is accumulated in mature leaves, where it can be translocated depending on the presence or not of sugar alcohols capable of forming mobile B-polyol complexes through phloem (Brown et al., 1999). Overall increased B content in mature leaves indicates B immobility, whereas higher B content in young meristematic tissues suggests B mobility. In most plants, B mobility is restricted to the xylem, as they do not biosynthesize significant amounts of

B toxicity is an agricultural problem that reduces crop yield worldwide. Toxicity takes place in vascular plants as B accumulates in shoots, generally following a pattern from leaf base to tip, that leads to chlorosis and necrosis (Marschner, 1995; Reid et al., 2004). As B toxicity alters metabolism and cell division, its symptoms are also manifested as a slowing-down and inhibition of growth, especially in roots, and reductions in yield, along with loss of fruit

Furthermore, high irradiance appears to increase the harmful effects of B toxicity, probably because elevated B contents may impair plant mechanisms to cope with photooxidation

According to Reid et al. (2004), excess B would overbind compounds with hydroxyl groups in the *cis*-configuration that could explain how this mineral stress exerts its harmful effect. Thus, extra B may bind with pectic polysaccharides and thereby altering cell wall structure; also excess B could alter the structure of primary metabolic compounds through binding to the ribose moieties of ATP (adenosine triphosphate), NADH (nicotinamide adenine dinucleotide, reduced form), NADPH (nicotinamide adenine dinucleotide phosphate, reduced form); finally, high internal B concentrations may negatively affect plant

Recent reports show changes in gene expressions as a consequence of high internal B concentration (Kasajima & Fujiwara, 2007; Öz et al., 2009; Pang et al., 2010). Although the mechanism by which excess B promotes these changes remains unknown despite many efforts are being made to elucidate it (Kasai et al., 2011), the involvement of a signaling

Boron tolerance appears to be associated with the ability to limit B accumulation in both roots and shoots. Thus, *Bot1* expression, a gene providing tolerance to excess B, was localized in barley roots and youngest leaf blades (Sutton et al., 2007). Other genes encoding B efflux transporters and conferring tolerance to B toxicity in cultivars of wheat (cv. India) and barley (cv. Sahara), namely *TaBOR2* and *HvBOR2*, respectively, have also been reported (Reid, 2007). Therefore, boric acid/borate efflux transporters appear to be key determinants of plant B tolerance, and provide a molecular basis for the generation of highly B-tolerant

**7. Boron toxicity** 

polyols (Brown et al., 1999; Brown & Shelp, 1997).

quality (Grieve et al., 2010; Nable et al., 1997).

development by binding to ribose of RNA.

stress (Reid et al., 2004).

cascade is likely.

**8. Boron toxicity tolerance** 

crops (Takano et al., 2008).

As above mentioned, plant growth and yield are severely affected by salt stress in many regions of the world as a result of osmotic effects, ion toxicities, and mineral disorders (Hasegawa et al., 2000). The response of plants to salinity depends not only on the total ion concentration in the external medium, but also on the chemical nature of the involved ions (Curtin et al., 1993). Nevertheless, most experimental works for salinity studies in plants have been performed with NaCl as a salinizing chemical.

NaCl toxicity is apparent by ion toxicity and osmotic stress. Ion toxicity is due to specific damages by the high levels of Na+ and Cl-, reduction of K+ uptake being a main consequence, among others. Moreover NaCl treatment can increase the electrolyte leakage in tomato roots, indicating that the integrity of their plasma membranes is altered with salt stress (Bastías et al., 2010).

Boron-rich soils with high contents of naturally occurring salinity, and irrigation with groundwater containing high concentrations of salts and B are two common ways by which plants can be subjected to a double stress: salinity and excess B (Nable et al., 1997). For instance, soils with salt and B accumulation occur in South Australia (Marcar et al., 1999) and Jordan River Valley in Israel and Jordan (Yermiyahu et al., 2008); irrigation water with high levels of salts and B has been well documented in San Joaquin Valley in California (Grieve al., 2010), and the Lluta Valley in Chile (Bastías et al., 2004b), among other places.

At first glance, one might think that between salt stress and B toxicity there would be an additive or synergistic relationship. In other words, either the outcome of the two stresses, when occur simultaneously, is equivalent to the sum of the effects of both stresses when applied separately (additive response), or the outcome of the both combined stresses is greater than the sum of them acting separately (synergistic response). However, interactions between salinity and B toxicity are rather complex and it has been reported that an antagonistic response can also exist when both stresses appear simultaneously (Bastías et al., 2004a; Yermiyahu et al., 2008). As summarized by Yermiyahu et al. (2008), there is no agreement regarding mutual relations between salt stress and B toxicity (Grieve et al., 2010).

Apparently, antagonism between salt stress and B toxicity can be a consequence of lower toxicity of NaCl in the presence of high B, lower toxicity of B in the presence of high NaCl, or both. Several reports support that diminution of Cl uptake owing to high B could reduce the salt toxicity, since the increased addition of B to the soil did not affect leaf Na+ content in pepper plants (Yermiyahu et al., 2008), or even decreased it in wheat (Holloway & Alston, 1992). Therefore, B would positively affect to salt stress through decreased Cl accumulation in the leaves as a consequence of the reduced Cl uptake. Nevertheless, this amelioration is through an as-yet-unknown mechanism.

In turn, reduced leaf B contents with the increase of NaCl concentration in the irrigation water have been widely reported. As an explanation it has been proposed that reduced rates of transpiration would limit the leaf accumulation of B that is transported through the xylem (Yermiyahu et al., 2008).

Another alternative proposal is that the interaction between salinity and B toxicity could be related to aquaporin functionality (Bastías et al., 2004a; Martínez-Ballesta et al., 2008a, 2008b). Under excess external B, significant B transport takes place through the plasma membrane aquaporins (Dordas et al., 2000). The lower aquaporin functionality found in NaCl-treated plants could be related to the reduction of B contents in plants subjected to combined B and NaCl, in comparison with plants treated only with B, which could explain the beneficial effect of salinity against B toxicity (Bastías et al., 2004a). In turn, under salt

Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity 237

acid region (Kobayashi et al., 1999), and B is also essential to the structure and function of the cell forming borate ester cross-linked rhamnogalacturonan II dimer (O'Neill et al., 1996). Besides this structural feature, it has been highlighted that both nutrients share other characteristics, namely, preferential distribution to apoplast, scarce mobility, very low cytosolic concentration, and signaling functions (Bonilla et al., 2004; Camacho-Cristóbal et al., 2008; González-Fontes et al., 2008). Thus, it would not be surprising that Ca2+ and B within the cell could contribute co-ordinately to some physiological role, and that the combined addition of both nutrients can palliate the harmful effects caused by salinity.

Tolerance to salinity involves processes in many different parts of the plant and can occur at a wide range of organizational levels, from the molecular level to the whole plant. Consequently improvements in salinity tolerance result from close interactions among molecular biologists, geneticists, biotechnologists, and physiologists and benefit from feedback from plant breeders and agronomists. The mechanism of high salt tolerance is just beginning to be understood. However, much effort is still required to understand in detail each product of genes induced by salinity stress and their interacting partners to elucidate

On the other hand, the mechanism of the relationship between B and salinity is not yet elucidated. Nevertheless, the palliative effect of B under saline conditions may be due to an improvement of the functionality of aquaporins, to prevention of salt-induced nutrient imbalance, interactions between B and Ca with respect to cell wall stability, or to a lower Cl-

We are grateful to Mr. Isidro Abreu for reading the manuscript and for his help in figure 1. This work was supported by Ministerio de Educación y Ciencia (grant BIO2008-05736-CO2- 01 to I.B.), Ministerio de Ciencia e Innovación (grant BFU2009-08397 to A.G.-F.), MICROAMBIENTE CM Program from Comunidad de Madrid (grant to I.B.), and by Junta

Alpaslan, M. & Gunes, A. (2001). Interactive effects of boron and salinity stress on the

Bastías, E.; Alcaraz-López, C.; Bonilla, I.; Martínez-Ballesta, M.C.; Bolaños, L. & Carvajal, M.

Bastías, E.; Fernández-García, N. & Carvajal, M. (2004a). Aquaporin functionality in roots of

Bastías, E.I.; González-Moro, M.B. & González-Murua, C. (2004b). *Zea mays* L. amylacea

apoplastic calcium. *Journal of Plant Physiology*, Vol.167, pp. 54-60

boron are available. *Plant and Soil*, Vol.267, pp. 73-84

growth, membrane permeability and mineral composition of tomato and cucumber

(2010). Interactions between salinity and boron toxicity in tomato plants involve

*Zea mays* in relation to the interactive effects of boron and salinity. *Plant Biology*,

from the Lluta Valley (Arica-Chile) tolerates salinity stress when high levels of

de Andalucía (grants BIO-266 and CVI-4721 to A.G.-F.), Spain.

plants. *Plant and Soil*, Vol.236, pp. 123-128

the complexity of the signal transduction pathways involved in high salinity stress.

**11. Conclusion** 

uptake.

**12. Acknowledgment** 

**13. References** 

Vol.5, pp. 415-421

stress, the activity of specific membrane components can be influenced by B regulating the functions of certain aquaporin isoforms as possible components of the salinity tolerance mechanism (Martínez-Ballesta et al., 2008b).

In addition, it has been proposed that salt-tolerant plants may be more resistant to toxic B levels because their salt-exclusion mechanisms also contribute to reduce internal B concentrations (Alpaslan & Gunes, 2001).

### **10. Calcium and boron ameliorate salt tolerance**

It is well known that salt stress leads to a Ca2+ and K+ deficiency and to other nutrient disorders (Cramer et al., 1987; Marschner, 1995). The external supply of Ca2+ to the soil ameliorates the damage caused by salinity (La Haye & Epstein, 1971), likely because the integrity of the membrane and its selective capacity is maintained by an adequate supply of Ca2+ (Cramer & Läuchli, 1996).

Interestingly, a balanced addition of B and Ca2+ together increased tolerance to salt stress in nodulated nitrogen-fixing pea plants, as extra Ca2+ can recover nodulation inhibited by salinity and extra B also contributes to nodule development and functionality (El-Hamdaoui et al., 2003a, 2003b). Furthermore, a proper B and Ca2+ supplement restores iron content in salt-stressed pea nodules (Bolaños et al., 2006), as well as the germination in salt-stressed pea seeds (Figure 2).

In addition, it has been proposed that interactions among NaCl, B and Ca2+ appear to be involved in the stability of the cell wall in plants (Cassab, 1998) and nodules (Bolaños et al., 2003).

It is widely known that both Ca2+ and B are needed for cell wall structure. Ca2+ stabilizes pectic polysaccharides in cell walls by ionic and coordinate bonding in the polygalacturonic

Fig. 2. Effects of supplement with combined boron (**A,** control: 9.3 µM*;* **B,** +6B: 55.8 µM) and calcium (+Ca: 0.68 mM; +2Ca: 1.36 mM; +4Ca: 2.72 mM; +8Ca: 5.44 mM) on germination of *Pisum sativum* cv. Argona seeds after 6 days under salt stress (75 mM NaCl). Boron was added as H3BO3 and Ca as CaCl2. Seeds treated with combined +6B and +4Ca had a germination similar to that of those treated without NaCl (control seeds).

acid region (Kobayashi et al., 1999), and B is also essential to the structure and function of the cell forming borate ester cross-linked rhamnogalacturonan II dimer (O'Neill et al., 1996). Besides this structural feature, it has been highlighted that both nutrients share other characteristics, namely, preferential distribution to apoplast, scarce mobility, very low cytosolic concentration, and signaling functions (Bonilla et al., 2004; Camacho-Cristóbal et al., 2008; González-Fontes et al., 2008). Thus, it would not be surprising that Ca2+ and B within the cell could contribute co-ordinately to some physiological role, and that the combined addition of both nutrients can palliate the harmful effects caused by salinity.

### **11. Conclusion**

236 Plants and Environment

stress, the activity of specific membrane components can be influenced by B regulating the functions of certain aquaporin isoforms as possible components of the salinity tolerance

In addition, it has been proposed that salt-tolerant plants may be more resistant to toxic B levels because their salt-exclusion mechanisms also contribute to reduce internal B

It is well known that salt stress leads to a Ca2+ and K+ deficiency and to other nutrient disorders (Cramer et al., 1987; Marschner, 1995). The external supply of Ca2+ to the soil ameliorates the damage caused by salinity (La Haye & Epstein, 1971), likely because the integrity of the membrane and its selective capacity is maintained by an adequate supply of

Interestingly, a balanced addition of B and Ca2+ together increased tolerance to salt stress in nodulated nitrogen-fixing pea plants, as extra Ca2+ can recover nodulation inhibited by salinity and extra B also contributes to nodule development and functionality (El-Hamdaoui et al., 2003a, 2003b). Furthermore, a proper B and Ca2+ supplement restores iron content in salt-stressed pea nodules (Bolaños et al., 2006), as well as the germination in salt-stressed

In addition, it has been proposed that interactions among NaCl, B and Ca2+ appear to be involved in the stability of the cell wall in plants (Cassab, 1998) and nodules (Bolaños et al.,

It is widely known that both Ca2+ and B are needed for cell wall structure. Ca2+ stabilizes pectic polysaccharides in cell walls by ionic and coordinate bonding in the polygalacturonic

Fig. 2. Effects of supplement with combined boron (**A,** control: 9.3 µM*;* **B,** +6B: 55.8 µM) and calcium (+Ca: 0.68 mM; +2Ca: 1.36 mM; +4Ca: 2.72 mM; +8Ca: 5.44 mM) on germination of *Pisum sativum* cv. Argona seeds after 6 days under salt stress (75 mM NaCl). Boron was added as H3BO3 and Ca as CaCl2. Seeds treated with combined +6B and +4Ca had a

germination similar to that of those treated without NaCl (control seeds).

mechanism (Martínez-Ballesta et al., 2008b).

concentrations (Alpaslan & Gunes, 2001).

Ca2+ (Cramer & Läuchli, 1996).

pea seeds (Figure 2).

2003).

**10. Calcium and boron ameliorate salt tolerance** 

Tolerance to salinity involves processes in many different parts of the plant and can occur at a wide range of organizational levels, from the molecular level to the whole plant. Consequently improvements in salinity tolerance result from close interactions among molecular biologists, geneticists, biotechnologists, and physiologists and benefit from feedback from plant breeders and agronomists. The mechanism of high salt tolerance is just beginning to be understood. However, much effort is still required to understand in detail each product of genes induced by salinity stress and their interacting partners to elucidate the complexity of the signal transduction pathways involved in high salinity stress.

On the other hand, the mechanism of the relationship between B and salinity is not yet elucidated. Nevertheless, the palliative effect of B under saline conditions may be due to an improvement of the functionality of aquaporins, to prevention of salt-induced nutrient imbalance, interactions between B and Ca with respect to cell wall stability, or to a lower Cluptake.

### **12. Acknowledgment**

We are grateful to Mr. Isidro Abreu for reading the manuscript and for his help in figure 1. This work was supported by Ministerio de Educación y Ciencia (grant BIO2008-05736-CO2- 01 to I.B.), Ministerio de Ciencia e Innovación (grant BFU2009-08397 to A.G.-F.), MICROAMBIENTE CM Program from Comunidad de Madrid (grant to I.B.), and by Junta de Andalucía (grants BIO-266 and CVI-4721 to A.G.-F.), Spain.

### **13. References**


Salt Stress in Vascular Plants and Its Interaction with Boron Toxicity 239

El-Hamdaoui, A.; Redondo-Nieto, M.; Torralba, B.; Rivilla, R.; Bonilla, I. & Bolaños, L.

González-Fontes, A.; Rexach, J.; Navarro-Gochicoa, M.T.; Herrera-Rodríguez, M.B.; Beato,

Grieve, C.M.; Poss, J.A.; Grattan, S.R.; Suarez, D.L. & Smith, T.E. (2010). The combined

Hasegawa, P.; Bressan, R.A.; Zhu, J.-K. & Bohnert, H. (2000). Plant cellular and molecular

Herrera-Rodríguez, M.B.; González-Fontes, A.; Rexach, J.; Camacho-Cristóbal, J.J.;

Ingram, J. & Bartels, D. (1996). The molecular basis of dehydration tolerance in plants. *Annual Review of Plant Physiology and Plant Molecular Biology*, Vol.47, pp. 377-403 Kang, J.S.; Frank, J.; Kang, C.H.; Kajiura, H.; Vikram, M.; Ueda, A.; Kim, S.; Bahk, J.D.;

Kasai, K.; Takano, J.; Miwa, K.; Toyoda, A. & Fujiwara, T. (2011). High boron-induced

*Arabidopsis thaliana*. *The Journal of Biological Chemistry*, Vol.286, pp. 6175-6183 Kasajima, I. & Fujiwara, T. (2007). Identification of novel *Arabidopsis thaliana* genes which are induced by high levels of boron. *Plant Biotechnology*, Vol.24, pp. 355-360 Kobayashi, M.; Nakagawa, H.; Asaka, T. & Matoh, T. (1999). Borate-rhamnogalacturonan II

La Haye, P.A. & Epstein, E. (1971). Calcium and salt tolerant by bean plants. *Physiologia* 

Mahajan, S.; Pandey, G.K. & Tuteja, N. (2008). Calcium- and salt stress signaling in plants:

Mahajan, S. & Tuteja, N. (2005). Cold, salinity and drought stresses: An overview. *Archives of* 

Marcar, N.E.; Guo, J. & Crawford, D.F. (1999). Response of *Eucalyptus camaldulensis* Dehnh.,

Marschner, H. (1995). *Mineral Nutrition of Higher Plants* (2nd edition), Academic Press, ISBN

*Australian Journal of Agricultural Research*, Vol.43, pp. 987-1001

*& Environment*, Vol.26, pp. 1003-1011

*Scientia Horticulturae*, Vol. 78, pp. 127-157

*Horticultarae*, Vol.125, pp. 179-187

*Plant Physiology*, Vol.119, 199-203

*Biochemistry and Biophysics*, Vol.444, pp. 139-158

chloride. *Plant and Soil*, Vol.208, pp. 251-257

*Plantarum*, Vol.83, pp. 497-499

0-12-473543-6, London

*Biology*, Vol.51, pp. 464-497

pp. 5933-5938

146-158

fixing pea plants. *Plant and Soil*, Vol.251, pp. 93-103

pea (*Pisum sativum*) symbiosis and nodule development under salt stress. *Plant, Cell* 

(2003b). Influence of boron and calcium on the tolerance to salinity of nitrogen-

V.M.; Maldonado, J.M. & Camacho-Cristóbal, J.J. (2008). Is boron involved solely in structural roles in vascular plants? *Plant Signaling & Behavior*, Vol.3, pp. 24-26 Grattan, S.R. & Grieve, C.M. (1999). Salinity-mineral nutrient relations in horticultural crops.

effects of salinity and excess boron on mineral ion relations in broccoli. *Scientia* 

responses to high salinity. *Annual Review of Plant Physiology and Plant Molecular* 

Maldonado, J.M. & Navarro-Gochicoa, M. T. (2010). Role of boron in vascular plants and response mechanisms to boron stresses. *Plant Stress*, Vol.4, pp. 115-122 Holloway, R.E. & Alston, M. (1992). The effects of salt and boron on growth of wheat.

Triplett, B.; Fujiyama, K.; Lee, S.Y.; Schaewen, A. von & Koiwa, H. (2008). Salt tolerance of *Arabidopsis thaliana* requires maturation of *N*-glycosylated proteins in the Golgi apparatus. *Proceedings of the National Academy of Sciences of USA*, Vol.105,

ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in

bonding reinforced by Ca2+ retains pectic polysaccharides in higher-plant cell walls.

Shedding light on SOS pathway. *Archives of Biochemistry and Biophysics*, Vol.471, pp.

*E. globulus* Labill. ssp. *globulus* and *E. grandis* W. Hill to excess boron and sodium


Bolaños, L.; El-Hamdaoui, A. & Bonilla, I. (2003). Recovery of development and

Bolaños, L.; Martín, M.; El-Hamdaoui, A.; Rivilla, R. & Bonilla, I. (2006). Nitrogenase inhibition in

Bonilla, I.; Abadía, J.; Bolaños, L. (2010). Introduction to mineral nutrition of plants, In:

Brown, P.H.; Hu, H. & Roberts, W.G. (1999). Occurrence of sugar alcohols determines boron

Brown, P.H. & Shelp, B.J. (1997). Boron mobility in plants. *Plant and Soil*, Vol. 193, pp. 85-101 Camacho-Cristóbal, J.J.; Rexach, J. & González-Fontes, A. (2008). Boron in plants: deficiency and toxicity. *Journal of Integrative Plant Biology*, Vol.50, pp. 1247-1255 Cassab, G.I. (1998). Plant cell wall proteins. *Annual Review of Plant Physiology and Plant* 

Chen, T.H.H. & Murata, N. (2008). Glycinebetaine: an effective protectant against abiotic

Chinnusamy, V.; Jangerdof, A. & Zhu, J.-K. (2005). Understanding and improving salt

Choi, S.K. ; Adachi, M. ; Yoshikawa M. ; Maruyama, N. & Utsumi, S. (2004). Soybean

Chourey, K.; Ramani, S. & Apte, S.K. (2003). Accumulation of LEA proteins in salt (NaCl)

Cramer, G.R. & Läuchli, A. (1996). Ion activities in solution in relation to Na+–Ca2+

Curtin, D.; Steppuhn, H. & Selles, F. (1993). Plant responses to sulfate and chloride salinity: growth and ionic relations. *Soil Science Society of America Journal*, Vol.57, pp. 1304-1310 Dannel, F.; Pfeffer, H. & Romheld, V. (2002). Update on boron in higher plants -uptake, primary translocation and compartmentation. *Plant Biology*, Vol.4, pp. 193-204 Delauney, A.J. & Verma, D.P.S. (1993). Proline biosynthesis and osmoregulation. *The Plant* 

Dordas, C.; Chrispeels, M.J. & Brown, P.H. (2000). Permeability and channel-mediated

El-Hamdaoui, A.; Redondo-Nieto, M.; Rivilla, R.; Bonilla, I. & Bolaños, L. (2003a). Effects of

glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability. *Bioscience, Biotechnology, and* 

stressed young seedlings of rice (*Oryza sativa* L.) cultivar Bura Rata and their degradation during recovery from salinity stress. *Journal of Plant Physiology*,

interactions at the plasmalemma. *Journal of Experimental Botany*, Vol.37, pp. 241-250 Cramer, G.R.; Lynch, J.; Lauchli, A. & Epstein, E. (1987). Influx of Na+, K+, and Ca2+ into

roots of salt-stressed cotton seedlings. Effects of supplemental Ca2+. *Plant* 

transport of boric acid across membrane vesicles isolated from squash roots. *Plant* 

boron and calcium nutrition on the establishment of the *Rhizobium leguminosarum-*

stress in plants. *Trends in Plant Science*, Vol.13, pp. 499-505

tolerance in plants. *Crop Science*, Vol.45, pp. 437-448

can be alleviated by B and Ca. *Plant and Soil*, Vol.280, pp. 135-142

pp. 1493-1497

Vol.267, pp. 97-107

*Horticultural Science*, Vol.124, pp. 347-352

*Molecular Biology*, Vol. 49, pp. 281-309.

*Biochemistry*, Vol.68, pp. 1991-1994

*Physiology*, Vol. 83, pp. 510–516

*Journal*, Vol.4, pp. 215-223

*Physiology*, Vol.124, pp. 1349-1361

Vol.160, pp. 1165-1174

functionality of nodules and plant growth in salt-stressed *Pisum sativum-Rhizobium leguminosarun* symbiosis by boron and calcium. *Journal of Plant Physiology*, Vol.160,

nodules from pea plants grown under salt stress occurs at the physiological level and

*Agricultural Sciences: Topics in Modern Agriculture*, A. González-Fontes, A. Gárate & I. Bonilla (Eds.), pp. 145-171, Studium Press, ISBN 1-933699-48-5, Houston, USA Bonilla, I.; El-Hamdaoui, A.; Bolaños, L. (2004). Boron and calcium increase *Pisum sativum*

seed germination and seedling development under salt stress. *Plant and Soil*,

toxicity symptoms of ornamental species. *Journal of the American Society for* 

pea (*Pisum sativum*) symbiosis and nodule development under salt stress. *Plant, Cell & Environment*, Vol.26, pp. 1003-1011


**11** 

**An Efficient Method to Screen for Salt** 

*Arabidopsis thaliana* is the most widely used model organism in plant molecular biology (Bressan et al., 2001) and it is an ideal model system for many reasons. Arabidopsis can be cultured in solid and liquid media and in soil. Greenhouses and growth chambers are suitable for Arabidopsis growth, meaning that different environmental conditions can be selected. Compared with crop plants, such as rice, wheat and tomato, Arabidopsis is more attractive due to its small size, high fecundity and short life cycle. Arabidopsis can be stably transformed using *Agrobacterium tumefaciens*-mediated transfer of T-DNA. Using the vacuum-infiltration procedure, transformants can be obtained at high efficiency. The small genome size of Arabidopsis meant that it was the first model plant to have its whole genome sequenced. Mutant lines, especially T-DNA insertion lines, of most Arabidopsis genes are obtained easily from several large seed stock libraries around the world. In conclusion, as a plant genetic model, Arabidopsis has played a significant role in characterizing the biological functions of plant genes, including salt

Nevertheless, Arabidopsis is a typical glycophyte in that does not display tolerance to intense salt stress. Thus, to solve this problem, a halophytic model system needs be developed. Any new model plant must provide experimental expediency similar to that of Arabidopsis. Salt cress (*Thellungiella salsuginea*), which is closely related to Arabidopsis, has emerged as a candidate (Amtmann, 2009). Like Arabidopsis, salt cress meets certain criteria that are important for any model plant, such as small size, short life cycle, self-pollination, high seed number, small genome and efficient transformation. Salt cress can withstand dramatic salinity shocks up to 500 mM NaCl and it can grow in high salt environments that are lethal to Arabidopsis (Bressan et al., 2001). Salt cress does not produce salt glands or other complex morphological alterations either before or after salt adaptation. Expressedsequence tag (EST) analyses of several hundred salt cress clones have shown that there is approximately 90 to 95% identity between salt cress and Arabidopsis cDNA sequences

**1. Introduction** 

stress-related genes.

 **Tolerance Genes in Salt Cress** 

Huawei Zhang, Gang Li, Yiyue Zhang,

*Institute of Genetics and Developmental Biology,* 

Ran Xia, Jing Wang and Qi Xie *State Key Laboratory of Plant Genomics, National Center for Plant Gene Research,* 

*Chinese Academy of Sciences, No.1 West* 

*Beichen Road, Beijing* 

*China* 


## **An Efficient Method to Screen for Salt Tolerance Genes in Salt Cress**

Huawei Zhang, Gang Li, Yiyue Zhang, Ran Xia, Jing Wang and Qi Xie *State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No.1 West Beichen Road, Beijing China* 

### **1. Introduction**

240 Plants and Environment

Martínez-Ballesta, M.C.; Bastías, E. & Carvajal, M. (2008a). Combined effect of boron and

Martínez-Ballesta, M.C.; Bastías, E.; Zhu, C.; Schäffner, A.R.; González-Moro, B.; González-

Munns, R. (2002). Comparative physiology of salt and water stress. *Plant, Cell &* 

Nable, R.O.; Bañuelos, G.S. & Paull, J.G. (1997). Boron toxicity. *Plant and Soil*, Vol.193, pp.

O'Neill, M.A.; Warrenfeltz, D.; Kates, K.; Pellerin, P.; Doco, T.; Darvill, A.G. & Albersheim,

Owttrim, G.W. (2006). RNA helicase and abiotic stress. *Nucleic Acids Research*, Vol.34, pp.

Öz, M.T.; Yilmaz, R.; Eyidogan, F.; Graaff, L.D.; Yücel, M. & Öktem, H.A. (2009). Microarray

Pang, Y.; Li, L.; Ren, F.; Lu, P.; Wei, P.; Cai, J.; Xin, L.; Zhang, J.; Chen, J. & Wang, X. (2010).

toxicity in *Arabidopsis*. *Journal of Genetics and Genomics*, Vol.37, pp. 389-397 Reid, R. (2007). Identification of boron transporter genes likely to be responsible for

Reid, R.J.; Hayes, J.E.; Posti, A.; Stangoulis, J.C.R. & Graham, R.D. (2004). A critical analysis of the causes of boron toxicity in plants. *Plant, Cell & Environment*, Vol.27, pp. 1405-1414 Sutton, T.; Baumann, U.; Hayes J.; Collins, N.C.; Shi, B.J.; Schnurbusch, T.; Hay, A.; Mayo,

arising from efflux transporter amplification. *Science*, Vol.318, pp. 1446-1449 Takano, J.; Miwa, K. & Fujiwara, T. (2008) Boron transport mechanisms: collaboration of channels and transporters. *Trends in Plant Science*, Vol.13, pp. 451-457 Tester, M. & Davenport, R. (2003). Na+ tolerance and Na+ transport in higher plants. *Annals* 

Tuteja, N. (2007). Mechanisms of high salinity tolerance in plants. *Methods in Enzymology*,

Vinocur, B. & Altman, A. (2005). Recent advances in engineering plant tolerance to abiotic stress: Achievements and limitations. *Current Opinion in Biotechnology*, Vol.16, pp. 123-132 Yermiyahu, U.; Ben-Gal, A.; Keren, R. & Reid, R.J. (2008). Combined effect of salinity and excess boron on plant growth and yield. *Plant and Soil*, Vol.304, pp. 73-87 Zhang, T.; Liu,Y.; Yang, T.; Zhang, L.; Xu, S.; Xue, L. & An, L. (2006). Diverse signals converge at MAPK cascades in plant. *Plant Physiology and Biochemistry,* Vol.44, pp. 274-283 Zhu, J.-K. (2002). Salt and drought stress signal transduction in plants. *Annual Review of* 

*Turkish Journal of Agriculture and Forestry*, Vol.33, pp. 191-202

Vol.3, pp. 844-845

181-198

3220-3230

1673-1678

*Environment*, Vol.25, pp. 239-250

*Chemistry*, Vol.271, pp. 22923-22930

*of Botany*, Vol.91, pp. 503-527

*Plant Biology*, Vol.53, pp. 247-273

Vol.428, pp. 419-438

salinity on water transport. The role of aquaporins. *Plant Signaling & Behavior*,

Murua, C. & Carvajal, M. (2008b). Boric acid and salinity effects on maize roots. Response of aquaporins ZmPIP1 and ZmPIP2, and plasma membrane H+-ATPase, in relation to water and nutrient uptake. *Physiologia Plantarum*, Vol.132, pp. 479-490

P. (1996). Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester: In vitro conditions for the formation and hydrolysis of the dimer. *Journal of Biological* 

analysis of late response to boron toxicity in barley (*Hordeum vulgare* L.) leaves.

Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron

tolerance to boron toxicity in wheat and barley. *Plant and Cell Physiology*, Vol. 48,

G.; Pallotta, M.; Tester, M. & Langridge, P. (2007). Boron-toxicity tolerance in barley

*Arabidopsis thaliana* is the most widely used model organism in plant molecular biology (Bressan et al., 2001) and it is an ideal model system for many reasons. Arabidopsis can be cultured in solid and liquid media and in soil. Greenhouses and growth chambers are suitable for Arabidopsis growth, meaning that different environmental conditions can be selected. Compared with crop plants, such as rice, wheat and tomato, Arabidopsis is more attractive due to its small size, high fecundity and short life cycle. Arabidopsis can be stably transformed using *Agrobacterium tumefaciens*-mediated transfer of T-DNA. Using the vacuum-infiltration procedure, transformants can be obtained at high efficiency. The small genome size of Arabidopsis meant that it was the first model plant to have its whole genome sequenced. Mutant lines, especially T-DNA insertion lines, of most Arabidopsis genes are obtained easily from several large seed stock libraries around the world. In conclusion, as a plant genetic model, Arabidopsis has played a significant role in characterizing the biological functions of plant genes, including salt stress-related genes.

Nevertheless, Arabidopsis is a typical glycophyte in that does not display tolerance to intense salt stress. Thus, to solve this problem, a halophytic model system needs be developed. Any new model plant must provide experimental expediency similar to that of Arabidopsis. Salt cress (*Thellungiella salsuginea*), which is closely related to Arabidopsis, has emerged as a candidate (Amtmann, 2009). Like Arabidopsis, salt cress meets certain criteria that are important for any model plant, such as small size, short life cycle, self-pollination, high seed number, small genome and efficient transformation. Salt cress can withstand dramatic salinity shocks up to 500 mM NaCl and it can grow in high salt environments that are lethal to Arabidopsis (Bressan et al., 2001). Salt cress does not produce salt glands or other complex morphological alterations either before or after salt adaptation. Expressedsequence tag (EST) analyses of several hundred salt cress clones have shown that there is approximately 90 to 95% identity between salt cress and Arabidopsis cDNA sequences

An Efficient Method to Screen for Salt Tolerance Genes in Salt Cress 243

**3.2 Generation of a library of Arabidopsis transgenic lines overexpressing salt cress** 

Salt cress cDNAs were constructed into pGreen and driven by the double CaMV 35S promoters. A selectable marker gene (*NPTII*) was chosen to identify positively transformed plants. As *RepA* and *Mob* were removed from pGreen, it was unable to replicate in the *Agrobacterium*. To remedy this problem, a co-resident, pSoup, was used to provide the

The plasmids isolated from the cDNA library were mixed with pSoup and introduced into *Agrobacterium* strain EHA105. Wild type Arabidopsis plants were transformed and T0 seeds were sown. With efficient kan resistance selection, a total of 2,000 individual transgenic lines

High salt stress has multiple adverse effects on plant growth and development, such as inhibition of seed germination, retardation of plant growth and acceleration of senescence. To evaluate whether a plant is tolerant to salt, the following parameters can be monitored:

Seed germination rate has been used for isolating abiotic stress tolerant or abscisic acid insensitive genes in many previous reports. However, seed quality and storage time can influence germination rate, in spite of their genetic background. In addition, fast germination is not necessary or sufficient for salt tolerance, as some lines that germinate faster than the wild type plants under high salinity conditions ultimately were found to be more sensitive to salt stress. Thus, germination rate is not sufficient for identifying salt tolerant plants. Another method for screening for salt tolerance plants is to germinate seeds on media containing salt and then monitor growth or survival rates. This method is better than the seed germination method. However it needs a consistent treatment and high quantities of seeds. Root elongation is a parameter used widely for the identification of salt stress tolerant plants. Seeds are germinated on agar plates without salt. Then, 4-days-old seedlings are transplanted onto new agar plates containing the desired concentrations of salt. Finally, root length is measured at several time points to calculate the growth rate. However, while this method is efficient for identifying salt sensitive mutants, it is not able to identify salt tolerant mutants. If using the rate of fresh weight increase as the salt tolerance parameter, the plants should be grown in liquid medium and be kept alive after being weighed. This method is time consuming and unsuitable for high-throughput screening. Therefore, a new strategy had to be applied to avoid these disadvantages. To reduce the influence of seed quality on plant growth, plants were grown in soil before salt treatment. Among all the parameters used to evaluate salt tolerance ability, the most important one was selected in this present study, specifically survival rate. If most lines contain a single insertion, 75% of transgenic plants would contain one copy of an expressed salt cress gene. When this gene is related to salt tolerance, 75% of plants should have the phenotype. To save screening time. T1 plants were analyzed because salt cress cDNA would be expressed

A high-throughput screen system was set up to isolate transgenic lines with the ability to tolerate high salinity. Using wild type plants as the control, seeds of transgenic lines were germinated on MS plates and then transplanted into soil. Approximately three weeks after

**3.3 Screening for salt tolerant lines from the transgenic Arabidopsis library** 

seed germination, root elongation, fresh weight increase and survival rate.

**genes** 

replication function *in trans* for pGreen.

in approximately 75% of T1 plants.

were obtained and T1 seeds of these lines were harvested.

(Zhang et al., 2008). As a result, more than 10 years ago, salt cress started to be used for studies examining the mechanisms underlying salt tolerance.

### **2. Importance**

Forward and reverse genetic studies of salt cress were encumbered by a lack of genomic information and poor mutant line storage. So, a new method is required that combines the advantages of Arabidopsis and salt cress, while avoiding any important disadvantages. Here, an overexpression system is presented, which has been used previously in Arabidopsis (LeClere and Bartel, 2001)*.* A similar system has been development by another group independently to mine stress tolerance genes from salt cress (Du et al., 2008). In our lab, a cDNA library of salt cress was generated from salt-treated seedlings and that was driven by double cauliflower mosaic virus (CaMV) 35S promoters. Using *A. tumefaciens*mediated transformation*,* the salt cress cDNA library was randomly overexpressed in Arabidopsis. T1 transgenic plants were grown in soil, treated with NaCl and the survival rates of transgenic lines were monitored. The salt cress cDNAs expressed in these lines were identified by PCR amplification and sequencing. To confirm the initial screening results, both salt cress genes and their homologs in Arabidopsis were re-overexpressed in Arabidopsis Salt tolerance ability of the off-spring of these re-transformed lines and mutants of the homologous genes in Arabidopsis was examined. After this screening, candidate genes were chosen for further investigation of their biological functions.

### **3. Results**

### **3.1 Generation of salt cress cDNA library**

Binary Ti vectors are the plasmid vectors used for the *Agrobacterium*-mediated plant transformation because they are able to replicate in both the *E. coli* and *Agrobacterium* species. High level efficiency of *in vivo* recombination is needed for the construction of cDNA libraries, but the large size of the common binary Ti vectors limit the *in vivo* recombination efficiency. Moreover, the size of binary Ti vectors needs to be limited to allow for large pieces of DNA to be transferred into plants. Plasmid manipulations and cDNA library construction are also easier if the vectors replicate in *E. coli* to high copy number. A binary Ti vector with double cauliflower mosaic virus (CaMV) 35S promoter and NOS terminal, named pGreen, fulfilled these demands and was used in the present study (Hellens RP, 2000). To keep the size of pGreen to a minimum, *RepA* and *Mob*, which were necessary for plasmid replication in *Agrobacterium*, were removed. The cloning site of pGreen was based on the well known plasmid, pBluescript, meaning that it was relatively simple to rearrange selective marker and reporter genes.

For wide coverage of salt cress transcripts in relation to salt stress, whole plants were collected at different time points after NaCl treatment. Total mRNA was purified from a mixture of plant tissues and at different time points the cDNA library was constructed in pGreen. Approximately 1 x106 colonies were collected from LB agar for the construction of the primary library. The titer of the cDNA library in the host bacteria, *E. coli* XL1-blue MRF ' (Stratagene), was ca. 1 x107 colonies/μL. To analyze the insert size and recombination rate in the primary library, 100 colonies were randomly selected and the plasmids were digested by *Eco*RI/*Xho*I. Insert sizes ranged from 500 bp to 2 kb (mean size of ca. 1 kb), while efficiency of recombination was ca. 89%.

(Zhang et al., 2008). As a result, more than 10 years ago, salt cress started to be used for

Forward and reverse genetic studies of salt cress were encumbered by a lack of genomic information and poor mutant line storage. So, a new method is required that combines the advantages of Arabidopsis and salt cress, while avoiding any important disadvantages. Here, an overexpression system is presented, which has been used previously in Arabidopsis (LeClere and Bartel, 2001)*.* A similar system has been development by another group independently to mine stress tolerance genes from salt cress (Du et al., 2008). In our lab, a cDNA library of salt cress was generated from salt-treated seedlings and that was driven by double cauliflower mosaic virus (CaMV) 35S promoters. Using *A. tumefaciens*mediated transformation*,* the salt cress cDNA library was randomly overexpressed in Arabidopsis. T1 transgenic plants were grown in soil, treated with NaCl and the survival rates of transgenic lines were monitored. The salt cress cDNAs expressed in these lines were identified by PCR amplification and sequencing. To confirm the initial screening results, both salt cress genes and their homologs in Arabidopsis were re-overexpressed in Arabidopsis Salt tolerance ability of the off-spring of these re-transformed lines and mutants of the homologous genes in Arabidopsis was examined. After this screening, candidate

Binary Ti vectors are the plasmid vectors used for the *Agrobacterium*-mediated plant transformation because they are able to replicate in both the *E. coli* and *Agrobacterium* species. High level efficiency of *in vivo* recombination is needed for the construction of cDNA libraries, but the large size of the common binary Ti vectors limit the *in vivo* recombination efficiency. Moreover, the size of binary Ti vectors needs to be limited to allow for large pieces of DNA to be transferred into plants. Plasmid manipulations and cDNA library construction are also easier if the vectors replicate in *E. coli* to high copy number. A binary Ti vector with double cauliflower mosaic virus (CaMV) 35S promoter and NOS terminal, named pGreen, fulfilled these demands and was used in the present study (Hellens RP, 2000). To keep the size of pGreen to a minimum, *RepA* and *Mob*, which were necessary for plasmid replication in *Agrobacterium*, were removed. The cloning site of pGreen was based on the well known plasmid, pBluescript, meaning that it was relatively

For wide coverage of salt cress transcripts in relation to salt stress, whole plants were collected at different time points after NaCl treatment. Total mRNA was purified from a mixture of plant tissues and at different time points the cDNA library was constructed in pGreen. Approximately 1 x106 colonies were collected from LB agar for the construction of the primary library. The titer of the cDNA library in the host bacteria, *E. coli* XL1-blue MRF ' (Stratagene), was ca. 1 x107 colonies/μL. To analyze the insert size and recombination rate in the primary library, 100 colonies were randomly selected and the plasmids were digested by *Eco*RI/*Xho*I. Insert sizes ranged from 500 bp to 2 kb (mean size of ca. 1 kb), while efficiency

studies examining the mechanisms underlying salt tolerance.

genes were chosen for further investigation of their biological functions.

**3.1 Generation of salt cress cDNA library** 

simple to rearrange selective marker and reporter genes.

of recombination was ca. 89%.

**2. Importance** 

**3. Results** 

### **3.2 Generation of a library of Arabidopsis transgenic lines overexpressing salt cress genes**

Salt cress cDNAs were constructed into pGreen and driven by the double CaMV 35S promoters. A selectable marker gene (*NPTII*) was chosen to identify positively transformed plants. As *RepA* and *Mob* were removed from pGreen, it was unable to replicate in the *Agrobacterium*. To remedy this problem, a co-resident, pSoup, was used to provide the replication function *in trans* for pGreen.

The plasmids isolated from the cDNA library were mixed with pSoup and introduced into *Agrobacterium* strain EHA105. Wild type Arabidopsis plants were transformed and T0 seeds were sown. With efficient kan resistance selection, a total of 2,000 individual transgenic lines were obtained and T1 seeds of these lines were harvested.

### **3.3 Screening for salt tolerant lines from the transgenic Arabidopsis library**

High salt stress has multiple adverse effects on plant growth and development, such as inhibition of seed germination, retardation of plant growth and acceleration of senescence. To evaluate whether a plant is tolerant to salt, the following parameters can be monitored: seed germination, root elongation, fresh weight increase and survival rate.

Seed germination rate has been used for isolating abiotic stress tolerant or abscisic acid insensitive genes in many previous reports. However, seed quality and storage time can influence germination rate, in spite of their genetic background. In addition, fast germination is not necessary or sufficient for salt tolerance, as some lines that germinate faster than the wild type plants under high salinity conditions ultimately were found to be more sensitive to salt stress. Thus, germination rate is not sufficient for identifying salt tolerant plants. Another method for screening for salt tolerance plants is to germinate seeds on media containing salt and then monitor growth or survival rates. This method is better than the seed germination method. However it needs a consistent treatment and high quantities of seeds. Root elongation is a parameter used widely for the identification of salt stress tolerant plants. Seeds are germinated on agar plates without salt. Then, 4-days-old seedlings are transplanted onto new agar plates containing the desired concentrations of salt. Finally, root length is measured at several time points to calculate the growth rate. However, while this method is efficient for identifying salt sensitive mutants, it is not able to identify salt tolerant mutants. If using the rate of fresh weight increase as the salt tolerance parameter, the plants should be grown in liquid medium and be kept alive after being weighed. This method is time consuming and unsuitable for high-throughput screening.

Therefore, a new strategy had to be applied to avoid these disadvantages. To reduce the influence of seed quality on plant growth, plants were grown in soil before salt treatment. Among all the parameters used to evaluate salt tolerance ability, the most important one was selected in this present study, specifically survival rate. If most lines contain a single insertion, 75% of transgenic plants would contain one copy of an expressed salt cress gene. When this gene is related to salt tolerance, 75% of plants should have the phenotype. To save screening time. T1 plants were analyzed because salt cress cDNA would be expressed in approximately 75% of T1 plants.

A high-throughput screen system was set up to isolate transgenic lines with the ability to tolerate high salinity. Using wild type plants as the control, seeds of transgenic lines were germinated on MS plates and then transplanted into soil. Approximately three weeks after

An Efficient Method to Screen for Salt Tolerance Genes in Salt Cress 245

The salt tolerant phenotype of the plants must be confirmed by other methods for the following reasons. First, environment factors, such as illumination and irrigation, are not identical for every plant, and can lead to variations among plants, even when the salt treatment is not added. Second, there is often more than one insert identified in a single transgenic line. By using the screening result alone, it is not clear which gene is responsible for the salt tolerance. Third, co-suppression can often occur. Thus, it is possible that the homologous gene in the transgenic plants is expressed at lower levels than in wild type plants, and the salt tolerant phenotype is due to reductions in the

Two different strategies were adopted to solve these problems. The first approach was reoverexpression. The candidate salt cress genes and their homologs in Arabidopsis were overexpressed in the Arabidopsis wild type plants. Then the salt tolerability of these transgenic lines was checked as described above. If transgenic plants gained salt tolerant ability, the genes were considered to be related to salt stress tolerance. The second approach was to check the phenotype of homologous gene mutant lines of Arabidopsis, and RNAi lines of salt cress. If these genes were indispensable for salt tolerance, elimination or reduction of their expression level should result in increased salt sensitivity of the plants.

In this present study, a simple high-throughput method to mine salt tolerance genes from salt cress has been developed by ectopically expressing salt cress cDNA in Arabidopsis. This method does not require any genomic or cDNA sequence information for salt cress. It is convenient for gene cloning by a single PCR instead of mapping. Moreover, based on the information on homologous genes in Arabidopsis, the functional analyses of candidate salt cress genes is much simpler. Finally, gain-of-function mutants can uncover gene functions that would never be revealed by conventional loss-of-function approaches. This approach could be applied to mine genes related to interesting phenotypes in other stress or

*A. thaliana* Columbia and salt cress (*T. salsuginea*) were used in this study. Seeds of Arabidopsis and salt cress were surface-sterilized with 10% bleach and then washed three times with sterile water. Sterile seeds were plated on MS medium plus 1.5% sucrose. Arabidopsis seeds were stratified in darkness for 2 to 4 d at 4 ºC, while salt cress seeds were stratified for three weeks under the same conditions. Then, the seeds were transferred to a tissue culture room at 22 ºC operating a 16-h light/8-h dark photoperiod. After two weeks, seedlings were potted in soil and placed in a growth chamber at 22 ºC and 50% humidity

For a wide coverage of transcripts related to salt stress, when salt cress plants in soil had generated four to six true leaves, they were treated with 400 mM NaCl. Leaf and root

**3.5 Validation of the salt-tolerance functions of the isolated candidate genes** 

expression of these genes.

**4. Conclusion** 

developmental conditions.

**5.1 Plant material** 

**5. Materials and methods** 

operating a 16-h light/8-h dark photoperiod.

**5.2 Generation of the salt cress cDNA library** 

germination, plants were treated with 200 mM NaCl. It was a robust screening, as most wild type plants died. The lines with a survival rate of greater than 70% were identified to be putative salt tolerance lines because about 25% of the T1 plants were wild type. We routinely screened more than 1,000 lines in soil and yielded a total of 20 candidate lines.

For confirming the salt tolerant ability of the candidate lines, the T2 seeds of each line were subjected to a secondary screen. Plasmolysis often occurs when plants are treated with high salinity without pre-conditioning. These plants tend to die of plasmolysis, rather than ionic or osmotic toxicity. This situation was avoided by adding salt in steps of 50 mM to allow the plants to adjust to the increasing salinity. Ten high salt stress tolerance lines of the 20 candidate lines were confirmed at this stage.

### **3.4 Isolation of the inserted salt cress cDNA expressed in the salt tolerant lines and identification of homologs in Arabidopsis**

After screening, the next step was to identify the salt cress genes in these salt tolerant lines. Insert sequences were amplified by PCR and sequenced. Homologous genes in Arabidopsis were identified using BLAST.

Fig. 1. Isolation of insertion sequences by PCR. ST1 and ST2 are two salt tolerant lines. The plant overexpressing pGreen-GFP was used as a positive control. Arrows indicate the amplified sequences from transgenic lines to be sequenced.

### **3.5 Validation of the salt-tolerance functions of the isolated candidate genes**

The salt tolerant phenotype of the plants must be confirmed by other methods for the following reasons. First, environment factors, such as illumination and irrigation, are not identical for every plant, and can lead to variations among plants, even when the salt treatment is not added. Second, there is often more than one insert identified in a single transgenic line. By using the screening result alone, it is not clear which gene is responsible for the salt tolerance. Third, co-suppression can often occur. Thus, it is possible that the homologous gene in the transgenic plants is expressed at lower levels than in wild type plants, and the salt tolerant phenotype is due to reductions in the expression of these genes.

Two different strategies were adopted to solve these problems. The first approach was reoverexpression. The candidate salt cress genes and their homologs in Arabidopsis were overexpressed in the Arabidopsis wild type plants. Then the salt tolerability of these transgenic lines was checked as described above. If transgenic plants gained salt tolerant ability, the genes were considered to be related to salt stress tolerance. The second approach was to check the phenotype of homologous gene mutant lines of Arabidopsis, and RNAi lines of salt cress. If these genes were indispensable for salt tolerance, elimination or reduction of their expression level should result in increased salt sensitivity of the plants.

### **4. Conclusion**

244 Plants and Environment

germination, plants were treated with 200 mM NaCl. It was a robust screening, as most wild type plants died. The lines with a survival rate of greater than 70% were identified to be putative salt tolerance lines because about 25% of the T1 plants were wild type. We routinely screened more than 1,000 lines in soil and yielded a total of 20 candidate lines. For confirming the salt tolerant ability of the candidate lines, the T2 seeds of each line were subjected to a secondary screen. Plasmolysis often occurs when plants are treated with high salinity without pre-conditioning. These plants tend to die of plasmolysis, rather than ionic or osmotic toxicity. This situation was avoided by adding salt in steps of 50 mM to allow the plants to adjust to the increasing salinity. Ten high salt stress tolerance lines of the 20

**3.4 Isolation of the inserted salt cress cDNA expressed in the salt tolerant lines and** 

After screening, the next step was to identify the salt cress genes in these salt tolerant lines. Insert sequences were amplified by PCR and sequenced. Homologous genes in Arabidopsis

Fig. 1. Isolation of insertion sequences by PCR. ST1 and ST2 are two salt tolerant lines. The plant overexpressing pGreen-GFP was used as a positive control. Arrows indicate the

amplified sequences from transgenic lines to be sequenced.

candidate lines were confirmed at this stage.

**identification of homologs in Arabidopsis** 

were identified using BLAST.

In this present study, a simple high-throughput method to mine salt tolerance genes from salt cress has been developed by ectopically expressing salt cress cDNA in Arabidopsis. This method does not require any genomic or cDNA sequence information for salt cress. It is convenient for gene cloning by a single PCR instead of mapping. Moreover, based on the information on homologous genes in Arabidopsis, the functional analyses of candidate salt cress genes is much simpler. Finally, gain-of-function mutants can uncover gene functions that would never be revealed by conventional loss-of-function approaches. This approach could be applied to mine genes related to interesting phenotypes in other stress or developmental conditions.

### **5. Materials and methods**

### **5.1 Plant material**

*A. thaliana* Columbia and salt cress (*T. salsuginea*) were used in this study. Seeds of Arabidopsis and salt cress were surface-sterilized with 10% bleach and then washed three times with sterile water. Sterile seeds were plated on MS medium plus 1.5% sucrose. Arabidopsis seeds were stratified in darkness for 2 to 4 d at 4 ºC, while salt cress seeds were stratified for three weeks under the same conditions. Then, the seeds were transferred to a tissue culture room at 22 ºC operating a 16-h light/8-h dark photoperiod. After two weeks, seedlings were potted in soil and placed in a growth chamber at 22 ºC and 50% humidity operating a 16-h light/8-h dark photoperiod.

### **5.2 Generation of the salt cress cDNA library**

For a wide coverage of transcripts related to salt stress, when salt cress plants in soil had generated four to six true leaves, they were treated with 400 mM NaCl. Leaf and root

An Efficient Method to Screen for Salt Tolerance Genes in Salt Cress 247

type plants were used as the control. About three weeks after germination, plants had generated four to six true leaves. Then, the plants were treated with 50 mM NaCl. Salt concentration was increased by 50 mM every four days until a final concentration of 200 mM. Plants were allowed to keep on growing under these conditions. The final survival rate of salt tolerant lines would be expected to be much greater than that of wild type

**5.6 Isolation of the inserted salt cress cDNA and identification of homologous genes** 

Since salt cress cDNAs were introduced to Arabidopsis by T-DNA insertion, the flanking sequences of cDNAs were conserved in the genome of transgenic plants. Using appropriate primers (pGreen-sense: 5'-GGAACTACTCACACATTATTATGGAG-3'; and pGreenantisense: 5'-CATTTGGAGAGGACACGCTG-3'), cDNA inserts were amplified by PCR. PCR products were cloned into T-vectors and sequenced. If more than one band emerged from the PCR as a result of multiple insertions, each band should be sequenced. According to previous studies, most salt cress genes had homologous genes in Arabidopsis.

This work was supported by grants from the Ministry of Science and Technology (MST, No 863-2007AA021402) and National Natural Science Foundation of China (NSFC, No

Amtmann, A. (2009). Learning from evolution: Thellungiella generates new knowledge on

Bechtold, N., and Pelletier, G. (1998). In planta Agrobacterium-mediated transformation of

Bressan, R.A., Zhang, C., Zhang, H., Hasegawa, P.M., Bohnert, H.J., and Zhu, J.K. (2001).

Du, J., Huang, Y.P., Xi, J., Cao, M.J., Ni, W.S., Chen, X., Zhu, J.K., Oliver, D.J., and Xiang,

Hellens RP, E.E., Leyland NR, Bean S, Mullineaux PM. (2000). pGreen: a versatile and

LeClere, S., and Bartel, B. (2001). A library of Arabidopsis 35S-cDNA lines for identifying

essential and critical components of abiotic stress tolerance in plants. Mol Plant 2, 3-

adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82, 259-

Learning from the Arabidopsis experience. The next gene search paradigm. Plant

C.B. (2008). Functional gene-mining for salt-tolerance genes with the power of

flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant

plants.

**in Arabidopsis** 

**6. Acknowledgments** 

30700050).

**7. References** 

12.

266.

Physiol 127, 1354-1360.

Mol Biol. 42, 819-832.

Arabidopsis. Plant J 56, 653-664.

novel mutants. Plant Mol Biol 46, 695-703.

Arabidopsis homologs were identified using BLAST.

tissues of plants were collected at 0, 15 min, 30 min, 1 h, 3 h, 6 h and 12 h. Rosettes were cut off from the plants directly and the roots were washed with water before collection. The collected tissues were mixed together, frozen in liquid nitrogen and ground to a powder. The powder was resuspended in extraction buffer (50 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid [EDTA], 2% sodium dodecyl sulfate [SDS], 10 mM LiCl; pH 6.0) and mixed with an equal volume phenol/chloroform (1:1, pH 6.0) at 65 ºC. This mixture was vortexed and centrifuged at 4 ºC for 15 min at 10,000 *g*. The supernatant was phenolized twice and precipitated with an equal volume of 4 M LiCl. After centrifugation, the RNA pellet was resuspended in Tris-EDTA (TE) buffer. The supernatant was phenolized again and then precipitated by adding 0.1 volumes of 3 M NaAc (pH 5.3) and three volumes of pure ethanol. The RNA pellet was washed and resuspended in TE buffer.

Total mRNA was purified, and double strand cDNA was synthesized from 5 μg of mRNA using a cDNA synthesis kit (Stratagene, CA, USA). The double-stranded cDNA, containing *Eco*RI and *Xho*I ends, was ligated into the *Eco*RI/*Xho*I-digested pGreen vector (Hellens RP, 2000). After ligation, the library was dialyzed with distilled water and electroporated into *Escherichia coli* XL1-Blue MRF' (Stratagene) .The transformants were plated on Luria Bertani (LB) agar containing 50 μg/mL kanamycin sulfate (kan).

### **5.3 Transformation of the cDNA library into Arabidopsis**

Plasmids of the salt cress cDNA library were collected from *E. coli* XL1-Blue MRF' and electroporated into *Agrobacterium* strain EHA105 with the co-resident plasmid pSoup. The transformed agrobacteria were selected on LB agar containing 50 μg/mL kan and 100 μg/L rifampicin. All agrobacterial colonies were washed off the agar plates, resuspended and then diluted in agrobacterial infiltration medium to an OD600 of 0.7 for Arabidopsis transformation. When plants were at their peak of flowering, the Arabidopsis flowers were dipped using a vacuum infiltration method as described previously (Bechtold and Pelletier, 1998). For maintaining a high level of humidity, plants were covered for 24 h after dipping. Then, the plants were transferred to a greenhouse and allowed to grow to maturity. T0 seeds were harvested in bulk and germinated on half-strength MS medium containing 50 μg/mL kan. The positive transgenic seedlings were transplanted into soil for the production of T1 seeds.

### **5.4 Screening for salt tolerance lines from the transgenic Arabidopsis library**

T1 seeds were surface-sterilized, germinated and cultured as detailed in the 'Plant material' Section above. For each line, 18 plants were chosen at random and transplanted into two pots. Wild type plants were used as the control. By approximately three weeks after germination, each plant had generated four to six true leaves. Then, the plants were treated with 200 mM NaCl. Normally, *A. thaliana* Columbia wild type plants are unable to withstand such a high concentration of NaCl. T1 lines with survival rates greater than 70% were chosen as salt tolerant candidates.

#### **5.5 Confirmation of salt tolerance in the candidate lines by re-screening**

To confirm the salt tolerant phenotype, T2 seeds were used. T2 seeds were surfacesterilized, germinated and cultured as detailed in the 'Plant material' Section above. Wild type plants were used as the control. About three weeks after germination, plants had generated four to six true leaves. Then, the plants were treated with 50 mM NaCl. Salt concentration was increased by 50 mM every four days until a final concentration of 200 mM. Plants were allowed to keep on growing under these conditions. The final survival rate of salt tolerant lines would be expected to be much greater than that of wild type plants.

### **5.6 Isolation of the inserted salt cress cDNA and identification of homologous genes in Arabidopsis**

Since salt cress cDNAs were introduced to Arabidopsis by T-DNA insertion, the flanking sequences of cDNAs were conserved in the genome of transgenic plants. Using appropriate primers (pGreen-sense: 5'-GGAACTACTCACACATTATTATGGAG-3'; and pGreenantisense: 5'-CATTTGGAGAGGACACGCTG-3'), cDNA inserts were amplified by PCR. PCR products were cloned into T-vectors and sequenced. If more than one band emerged from the PCR as a result of multiple insertions, each band should be sequenced. According to previous studies, most salt cress genes had homologous genes in Arabidopsis. Arabidopsis homologs were identified using BLAST.

### **6. Acknowledgments**

This work was supported by grants from the Ministry of Science and Technology (MST, No 863-2007AA021402) and National Natural Science Foundation of China (NSFC, No 30700050).

### **7. References**

246 Plants and Environment

tissues of plants were collected at 0, 15 min, 30 min, 1 h, 3 h, 6 h and 12 h. Rosettes were cut off from the plants directly and the roots were washed with water before collection. The collected tissues were mixed together, frozen in liquid nitrogen and ground to a powder. The powder was resuspended in extraction buffer (50 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid [EDTA], 2% sodium dodecyl sulfate [SDS], 10 mM LiCl; pH 6.0) and mixed with an equal volume phenol/chloroform (1:1, pH 6.0) at 65 ºC. This mixture was vortexed and centrifuged at 4 ºC for 15 min at 10,000 *g*. The supernatant was phenolized twice and precipitated with an equal volume of 4 M LiCl. After centrifugation, the RNA pellet was resuspended in Tris-EDTA (TE) buffer. The supernatant was phenolized again and then precipitated by adding 0.1 volumes of 3 M NaAc (pH 5.3) and three volumes of pure ethanol. The RNA pellet was washed and resuspended in TE

Total mRNA was purified, and double strand cDNA was synthesized from 5 μg of mRNA using a cDNA synthesis kit (Stratagene, CA, USA). The double-stranded cDNA, containing *Eco*RI and *Xho*I ends, was ligated into the *Eco*RI/*Xho*I-digested pGreen vector (Hellens RP, 2000). After ligation, the library was dialyzed with distilled water and electroporated into *Escherichia coli* XL1-Blue MRF' (Stratagene) .The transformants were plated on Luria Bertani

Plasmids of the salt cress cDNA library were collected from *E. coli* XL1-Blue MRF' and electroporated into *Agrobacterium* strain EHA105 with the co-resident plasmid pSoup. The transformed agrobacteria were selected on LB agar containing 50 μg/mL kan and 100 μg/L rifampicin. All agrobacterial colonies were washed off the agar plates, resuspended and then diluted in agrobacterial infiltration medium to an OD600 of 0.7 for Arabidopsis transformation. When plants were at their peak of flowering, the Arabidopsis flowers were dipped using a vacuum infiltration method as described previously (Bechtold and Pelletier, 1998). For maintaining a high level of humidity, plants were covered for 24 h after dipping. Then, the plants were transferred to a greenhouse and allowed to grow to maturity. T0 seeds were harvested in bulk and germinated on half-strength MS medium containing 50 μg/mL kan. The positive transgenic seedlings were transplanted into soil for the production of T1

**5.4 Screening for salt tolerance lines from the transgenic Arabidopsis library** 

**5.5 Confirmation of salt tolerance in the candidate lines by re-screening** 

T1 seeds were surface-sterilized, germinated and cultured as detailed in the 'Plant material' Section above. For each line, 18 plants were chosen at random and transplanted into two pots. Wild type plants were used as the control. By approximately three weeks after germination, each plant had generated four to six true leaves. Then, the plants were treated with 200 mM NaCl. Normally, *A. thaliana* Columbia wild type plants are unable to withstand such a high concentration of NaCl. T1 lines with survival rates greater than 70%

To confirm the salt tolerant phenotype, T2 seeds were used. T2 seeds were surfacesterilized, germinated and cultured as detailed in the 'Plant material' Section above. Wild

(LB) agar containing 50 μg/mL kanamycin sulfate (kan).

**5.3 Transformation of the cDNA library into Arabidopsis** 

buffer.

seeds.

were chosen as salt tolerant candidates.


**12** 

 **and Food Safety** 

*Tallahassee, Florida 32317* 

 *Tallahassee, Florida 32317* 

*1,2USA 3India* 

and Karamthotsivasankar Naik3

**Impact of Drought Stress on Peanut** 

**(***Arachis hypogaea* **L.) Productivity** 

Devaiah M. Kambiranda1, Hemanth KN. Vasanthaiah1, Ramesh Katam1, Athony Ananga2, Sheikh M. Basha1

Peanut (*Arachis hypogaea* L.) is one of the world's most important legumes. It is grown primarily for its high quality edible oil and protein. Peanut is grown on 35.5 million ha across 82 countries in the world. More than half of the production area, which accounts for 70% of the peanut growing area fall under arid and semi-arid regions, where peanuts are frequently subjected to drought stresses for different duration and intensities (Reddy et al., 2003). An annual estimated loss in peanut production equivalent to over US\$520 million is caused by drought. Further, drought is also known to predispose peanut to aflatoxin contamination (Blankenship et al., 1984; Cole et al., 1989) making them unfit for human consumption. Yield losses due to drought are highly variable in nature depending on timing, intensity, and duration, coupled with other location-specific environmental stress factors such as high irradiance and temperature. In the United States peanuts contribute to more than \$4 billion to the country's economy each year. In USA majority of the peanut are grown under rain-fed conditions and only limited acreage is irrigated. Frequent failure of rains late in the season has resulted in decreased yield, poor quality peanuts and aflatoxin contamination. Furthermore, increased worldwide demand for water due to rapid population growth and irrigation practices have resulted in declines in aquifers limiting availability of water for irrigation. To meet future food-supply demands, crop production will have to increase, but it must do so under the constraints of less water and, most likely, less farm land. Agricultural Research Service (ARS) scientists with the Plant Stress and

**1. Introduction** 

*1Plant Biotechnology Laboratory, College of Agriculture* 

*Florida A & M University, 6505 Mahan Drive,* 

*2Center for Viticulture, College of Agriculture Florida A & M University, 6505 Mahan Drive* 

*University, Kadiri, Andhra Pradesh 515591,* 

*3Agriculture Research Station, ANGR Agricultural* 

Zhang, Y., Lai, J., Sun, S., Li, Y., Liu, Y., Liang, L., Chen, M., and Xie, Q. (2008). Comparison analysis of transcripts from the halophyte Thellungiella halophila. J Integr Plant Biol 50, 1327-1335.

### **Impact of Drought Stress on Peanut (***Arachis hypogaea* **L.) Productivity and Food Safety**

Devaiah M. Kambiranda1, Hemanth KN. Vasanthaiah1, Ramesh Katam1, Athony Ananga2, Sheikh M. Basha1 and Karamthotsivasankar Naik3 *1Plant Biotechnology Laboratory, College of Agriculture Florida A & M University, 6505 Mahan Drive, Tallahassee, Florida 32317 2Center for Viticulture, College of Agriculture Florida A & M University, 6505 Mahan Drive Tallahassee, Florida 32317 3Agriculture Research Station, ANGR Agricultural University, Kadiri, Andhra Pradesh 515591, 1,2USA 3India* 

### **1. Introduction**

248 Plants and Environment

Zhang, Y., Lai, J., Sun, S., Li, Y., Liu, Y., Liang, L., Chen, M., and Xie, Q. (2008). Comparison

Biol 50, 1327-1335.

analysis of transcripts from the halophyte Thellungiella halophila. J Integr Plant

Peanut (*Arachis hypogaea* L.) is one of the world's most important legumes. It is grown primarily for its high quality edible oil and protein. Peanut is grown on 35.5 million ha across 82 countries in the world. More than half of the production area, which accounts for 70% of the peanut growing area fall under arid and semi-arid regions, where peanuts are frequently subjected to drought stresses for different duration and intensities (Reddy et al., 2003). An annual estimated loss in peanut production equivalent to over US\$520 million is caused by drought. Further, drought is also known to predispose peanut to aflatoxin contamination (Blankenship et al., 1984; Cole et al., 1989) making them unfit for human consumption. Yield losses due to drought are highly variable in nature depending on timing, intensity, and duration, coupled with other location-specific environmental stress factors such as high irradiance and temperature. In the United States peanuts contribute to more than \$4 billion to the country's economy each year. In USA majority of the peanut are grown under rain-fed conditions and only limited acreage is irrigated. Frequent failure of rains late in the season has resulted in decreased yield, poor quality peanuts and aflatoxin contamination. Furthermore, increased worldwide demand for water due to rapid population growth and irrigation practices have resulted in declines in aquifers limiting availability of water for irrigation. To meet future food-supply demands, crop production will have to increase, but it must do so under the constraints of less water and, most likely, less farm land. Agricultural Research Service (ARS) scientists with the Plant Stress and

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 251

show large diurnal variation with high values in the morning when solar radiation and vapor pressure deficits are low, followed by low values around midday and gradual increase after midday (Erickson & Ketring, 1985). Osmotic potential follows the same pattern but ranges less widely than leaf water potential. Transpiration rate generally correlates to the incident solar radiation under sufficient water availability. However, drought stressed plants transpire less than unstressed plants. Subramaniam & Maheswari, (1990) reported that leaf water potential, transpiration rate and photosynthetic rate decreased progressively with increasing duration of water stress indicating that plants under mild stress were postponing tissue dehydration. Stomatal conductance decreased almost steadily during the stress period indicating that stomatal conductance was more sensitive than transpiration during the initial stress period. Stirling et al., (1989) found that under water deficit conditions the leaves exhibited marked diurnal variation in leaf turgor, while pegs showed less variation and maintained much higher turgor levels largely because of their lower solute potentials. Marked osmotic adjustment occurred in growing leaves but not in mature ones, allowing them to maintain higher turgor during periods of severe stress. This adjustment was rapidly lost when stress was released (Ali Ahmad & Basha, 1998). Bhagsari et al., (1976) reported that water potential of leaves and immature fruits were similar under drought stress conditions. It is a general observation that under severe moisture stress conditions, young pods lose their turgor and shrivel. Azam Ali (1984) reported that stomatal resistance of older leaves was greater than that of younger leaves. Leaves also become thicker under moderate drought stress (Reddy & Rao, 1968). The developing leaves of groundnut have an unusual thick layer of cells devoid of chloroplasts with lower epidermis below the sponge parenchyma. Cells of this layer are considered to be water storage cells (Reddy & Rao, 1968). During moisture stress, the opposing leaflets of tri-foliate leaf come together and orient themselves parallel to incident solar radiation, in an effort to reduce solar radiation load on the leaf. Leaf expansion is more sensitive to soil water deficit than stomatal closure (Black et al., 1985). Drought reduces leaf area by slowing leaf expansion and reducing the supply of carbohydrates. Reddy and Rao (1968) reported that severe drought stress decreased the levels of chlorophyll *a*, *b* and total chlorophyll. The decrease in chlorophyll was attributed to the inhibition of chlorophyll synthesis as well as to accelerated turnover of chlorophyll

Periodic water stress leads to anatomical changes such as a decrease in size of cells and intercellular spaces, thicker cell walls and greater development of epidermal tissue. Nitrogen fixation by leguminous plants is reduced by moisture stress due to a reduction in leg haemoglobin in nodules, specific nodule activity and number of arid regions. In addition, dry weight of nodules is significantly reduced in moisture stressed plants. Moisture stress also delays nodule formation in leguminous crops (Reddi & Reddy, 1995). There is considerable evidence that N, P and K uptake of peanut is reduced by drought

Leakage of solutes as a consequence of membrane damage is a common response of groundnut tissue to drought stress. Metabolic process is affected by water deficits. Severe water deficits cause decreases in enzymatic activity. Complex carbohydrates and proteins are broken down by enzymes into simpler sugars and amino acids, respectively (Pandey et al., 1984). Accumulation of soluble compounds in cells increases osmotic potential and reduces water loss from cells. Proline, an amino acid, accumulates whenever there is moisture stress. Accumulation of proline is greater in the later stages of drought stress and

already present.

stress (Kulkarni et al., 1988).

Germplasm Development Research Unit, Lubbock, Texas, the National Peanut Research Laboratory (NPRL) in Dawson, Georgia, and ICRISAT, India are working with cooperators to help peanut farmers maintain and improve their production in a changing environment.

Drought-stressed plants lose moisture from pods which leads to the reduction in the seeds physiological activity, thereby increasing the susceptibility to fungal invasion. Besides affecting food quality, drought stress is also known to alter nutritional quality of peanut seed proteins. Since peanut lack desirable genetic variation in drought and aflatoxin tolerance several conventional as well as molecular breeding techniques were adopted to improve drought and aflatoxin tolerance (Mehan et al., 1986; Dorner et al., 1989; Holbrook et al., 2000). Recently several advanced molecular tools have been developed to screen drought tolerance in peanut genotypes. Effect of drought stress on peanut is being studied at the molecular and cellular level, which has generated enormous amount of genomic and proteomic data that displays the mechanism by which peanut plants respond to drought stress. Engineering peanuts to withstand drought stress has been achieved *via* different strategies, while few of them have succeeded in developing improved peanut genotypes that withstand drought stress others are in the process of developing advanced genotypes. This chapter will highlight selected as well as most significant achievements made to understand and overcome drought stress in peanuts.

### **2. Effect of drought on plant performance**

### **2.1 Drought stresses reduce plant productivity**

Drought stress has been the major environmental factor contributing to the reduced agricultural productivity and food safety worldwide. Drought stress perceived by the plant from its surrounding environment varies spatially and temporally at several different scales. Drought affects membrane lipids and photosynthetic responses (Lauriano et al., 2000) and yield in peanuts (Suther & Patel, 1992). Water deficit affects thylakoid electron transport, phosphorylation, carboxylation and photosynthesis. Changes in the lipid content and composition are common in water-stressed plants and this increases membrane permeability. This causes damage and membrane disruption as well as reduction in photosynthesis. Maintaining membrane integrity under drought conditions will determine the plants resistance towards stress. Plants have several mechanisms for adaptation to water and heat stress including stomatal conductance, paraheliotropism, and osmotic adjustments. Arid and semi-arid environments typically have hot days and cool nights. Since there is a lack of water vapor in the air, the temperature at night drops making the night cooler but the day hotter. This can be stressful to the plant.

#### **2.2 Plant responses to drought**

Drought stress has adverse influence on water relations (Babu & Rao, 1983), photosynthesis (Bhagsari et al., 1976), mineral nutrition, metabolism, growth and yield of groundnut (Suther & Patel, 1992). In addition, drought conditions influence the growth of weeds, agronomic management and, nature and intensity of insects, pests and diseases (Wightman et al., 1989). Parameters like relative water content (RWC), leaf water potential, stomatal resistance, rate of transpiration, leaf temperature and canopy temperature influences water relations in peanut during drought. Stressed plants have lower RWC than non-stressed plants. For example, relative water content of non-stressed plants range from 85 to 90%, while in drought stressed plants, it may be as low as 30% (Babu & Rao, 1983). Peanut leaves

Germplasm Development Research Unit, Lubbock, Texas, the National Peanut Research Laboratory (NPRL) in Dawson, Georgia, and ICRISAT, India are working with cooperators to help peanut farmers maintain and improve their production in a changing environment. Drought-stressed plants lose moisture from pods which leads to the reduction in the seeds physiological activity, thereby increasing the susceptibility to fungal invasion. Besides affecting food quality, drought stress is also known to alter nutritional quality of peanut seed proteins. Since peanut lack desirable genetic variation in drought and aflatoxin tolerance several conventional as well as molecular breeding techniques were adopted to improve drought and aflatoxin tolerance (Mehan et al., 1986; Dorner et al., 1989; Holbrook et al., 2000). Recently several advanced molecular tools have been developed to screen drought tolerance in peanut genotypes. Effect of drought stress on peanut is being studied at the molecular and cellular level, which has generated enormous amount of genomic and proteomic data that displays the mechanism by which peanut plants respond to drought stress. Engineering peanuts to withstand drought stress has been achieved *via* different strategies, while few of them have succeeded in developing improved peanut genotypes that withstand drought stress others are in the process of developing advanced genotypes. This chapter will highlight selected as well as most significant achievements made to

Drought stress has been the major environmental factor contributing to the reduced agricultural productivity and food safety worldwide. Drought stress perceived by the plant from its surrounding environment varies spatially and temporally at several different scales. Drought affects membrane lipids and photosynthetic responses (Lauriano et al., 2000) and yield in peanuts (Suther & Patel, 1992). Water deficit affects thylakoid electron transport, phosphorylation, carboxylation and photosynthesis. Changes in the lipid content and composition are common in water-stressed plants and this increases membrane permeability. This causes damage and membrane disruption as well as reduction in photosynthesis. Maintaining membrane integrity under drought conditions will determine the plants resistance towards stress. Plants have several mechanisms for adaptation to water and heat stress including stomatal conductance, paraheliotropism, and osmotic adjustments. Arid and semi-arid environments typically have hot days and cool nights. Since there is a lack of water vapor in the air, the temperature at night drops making the night cooler but

Drought stress has adverse influence on water relations (Babu & Rao, 1983), photosynthesis (Bhagsari et al., 1976), mineral nutrition, metabolism, growth and yield of groundnut (Suther & Patel, 1992). In addition, drought conditions influence the growth of weeds, agronomic management and, nature and intensity of insects, pests and diseases (Wightman et al., 1989). Parameters like relative water content (RWC), leaf water potential, stomatal resistance, rate of transpiration, leaf temperature and canopy temperature influences water relations in peanut during drought. Stressed plants have lower RWC than non-stressed plants. For example, relative water content of non-stressed plants range from 85 to 90%, while in drought stressed plants, it may be as low as 30% (Babu & Rao, 1983). Peanut leaves

understand and overcome drought stress in peanuts.

**2. Effect of drought on plant performance 2.1 Drought stresses reduce plant productivity** 

the day hotter. This can be stressful to the plant.

**2.2 Plant responses to drought** 

show large diurnal variation with high values in the morning when solar radiation and vapor pressure deficits are low, followed by low values around midday and gradual increase after midday (Erickson & Ketring, 1985). Osmotic potential follows the same pattern but ranges less widely than leaf water potential. Transpiration rate generally correlates to the incident solar radiation under sufficient water availability. However, drought stressed plants transpire less than unstressed plants. Subramaniam & Maheswari, (1990) reported that leaf water potential, transpiration rate and photosynthetic rate decreased progressively with increasing duration of water stress indicating that plants under mild stress were postponing tissue dehydration. Stomatal conductance decreased almost steadily during the stress period indicating that stomatal conductance was more sensitive than transpiration during the initial stress period. Stirling et al., (1989) found that under water deficit conditions the leaves exhibited marked diurnal variation in leaf turgor, while pegs showed less variation and maintained much higher turgor levels largely because of their lower solute potentials. Marked osmotic adjustment occurred in growing leaves but not in mature ones, allowing them to maintain higher turgor during periods of severe stress. This adjustment was rapidly lost when stress was released (Ali Ahmad & Basha, 1998). Bhagsari et al., (1976) reported that water potential of leaves and immature fruits were similar under drought stress conditions. It is a general observation that under severe moisture stress conditions, young pods lose their turgor and shrivel. Azam Ali (1984) reported that stomatal resistance of older leaves was greater than that of younger leaves. Leaves also become thicker under moderate drought stress (Reddy & Rao, 1968). The developing leaves of groundnut have an unusual thick layer of cells devoid of chloroplasts with lower epidermis below the sponge parenchyma. Cells of this layer are considered to be water storage cells (Reddy & Rao, 1968). During moisture stress, the opposing leaflets of tri-foliate leaf come together and orient themselves parallel to incident solar radiation, in an effort to reduce solar radiation load on the leaf. Leaf expansion is more sensitive to soil water deficit than stomatal closure (Black et al., 1985). Drought reduces leaf area by slowing leaf expansion and reducing the supply of carbohydrates. Reddy and Rao (1968) reported that severe drought stress decreased the levels of chlorophyll *a*, *b* and total chlorophyll. The decrease in chlorophyll was attributed to the inhibition of chlorophyll synthesis as well as to accelerated turnover of chlorophyll already present.

Periodic water stress leads to anatomical changes such as a decrease in size of cells and intercellular spaces, thicker cell walls and greater development of epidermal tissue. Nitrogen fixation by leguminous plants is reduced by moisture stress due to a reduction in leg haemoglobin in nodules, specific nodule activity and number of arid regions. In addition, dry weight of nodules is significantly reduced in moisture stressed plants. Moisture stress also delays nodule formation in leguminous crops (Reddi & Reddy, 1995). There is considerable evidence that N, P and K uptake of peanut is reduced by drought stress (Kulkarni et al., 1988).

Leakage of solutes as a consequence of membrane damage is a common response of groundnut tissue to drought stress. Metabolic process is affected by water deficits. Severe water deficits cause decreases in enzymatic activity. Complex carbohydrates and proteins are broken down by enzymes into simpler sugars and amino acids, respectively (Pandey et al., 1984). Accumulation of soluble compounds in cells increases osmotic potential and reduces water loss from cells. Proline, an amino acid, accumulates whenever there is moisture stress. Accumulation of proline is greater in the later stages of drought stress and

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 253

adversely affected during drought stress through cell membrane-mediated mechanisms. Drought stress and drought stress mediated-fungal infection compromise peanut defense and exacerbate aflatoxin formation in the seeds (Guo et al., 2005). Thus, breeding for drought tolerance has been accepted as one of the strategies for developing aflatoxintolerant peanut cultivars, which would not only minimize water usage but also help expand peanut production in marginal and sub-marginal soils. Success in this effort has been slow due to lack of genetic resources and lack of information on the relationship or interaction between the pathways affected due to drought and or pathogen invasion. However, to date, few peanut cultivars with natural pre-harvest resistance to aflatoxin production have been

Efforts to improve peanuts that focus on yield as the only environmental method for screening of tolerance are seen to have a high variability in yield as well as differences in exactly reproducing stress conditions. A more-integrated approach for peanut breeding is needed to offer success in developing stress-tolerant varieties. Understanding physiological and molecular genetics may lead to the understanding of stress response and aid in development of new varieties with stress tolerance. So, a high-yielding cultivar that continues to produce well under drought conditions is a priority to enable stability of production. That is why much research for the last decade has attempted to improve performance by selecting plants with good pod yield under adverse conditions. As well as spending time testing plants in large-scale trials under different conditions, a study of plant physiology has revealed the features of the plant that correlate best with drought

Research in the previous decade had developed low-cost, rapid and easily measured indicators for three significant physiological features of drought-tolerance *viz.* amount of water transpired (T), water-use efficiency (W) and harvest index (HI), thus allowing their potential quantification in large numbers of breeding populations. The application of this physiological model in peanut-breeding programs has not been possible because of practical difficulties associated with measurement of the traits under field conditions. The USDA germplasm collection numbers over 9000 accessions of *A. hypogaea* (Holbrook, 2001) and about 800 accessions of *Arachis* species. Large *Arachis* species collections are also maintained at Texas A&M University and N. C. State University. The US breeding program is focused more on yield, grade, seed size and developing disease tolerant germplasm, and less on drought tolerance. Identifying drought tolerant genotypes with emphasis to reduce preharvest aflatoxin contamination is being conducted at the USDA-ARS, Tifton, GA., The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India and The National Center of Genetic Resources (CENARGEN), Brazil. The largest collection of domesticated peanut germplasm is located at ICRISAT, where there are 14,310 accessions from 92 countries while CENARGEN has 413 accessions of *Arachis* species (Upadhyaya et al., 2001a). A new drought tolerant groundnut variety, ICGV 91114, is becoming very popular in Anantapur district in Andhra Pradesh, India, where it is now replacing a 7 decade old variety TMV 2. ICGV 91114 has also been released in Orissa, India and is doing

identified through field screening.

tolerance.

very well in Karnataka, India.

**3. Breeding for crop improvement 3.1 Breeding towards drought tolerance** 

therefore its concentration is considered a good indicator of moisture stress (Reddi & Reddy, 1995).

### **2.3 Effect of drought during flowering and pod formation**

### **2.3.1 Flowering**

The start of flowering is not delayed by drought stress (Boote & Ketring, 1990). The rate of flower production is reduced by drought stress during flowering but the total number of flowers per plant is not affected due to an increase in the duration of flowering (Gowda & Hegde, 1986; Janamatti et al., 1986; Meisner & Karnok, 1992). A significant burst in flowering on alleviation of stress is a unique feature in the pattern of flowering under moisture stress, particularly when drought is imposed just prior to re-productive development (Janamatti et al., 1986). When stress is imposed during 30–45 days after sowing the first flush of flowers produced up to 45 days do not form pegs during that time, however, flowers produced after re-watering compensated for this loss (Gowda & Hegde, 1986).

### **2.3.2 Pod formation**

Peanut plants may experience water stress during pegging and pod development and then may have adequate amount of water (Jogloy et al., 1996). This would result in a drastic reduction of crop yield, and the magnitude of reduction would depend on peanut cultivars. Not only the yield of peanut but also the quality of products decreases under drought stress (Rucker et al., 1995). Peg elongation, which is turgor dependent, is delayed due to drought stress (Boote & Ketring, 1990). Pegs fail to penetrate effectively into air-dry soil, especially in crusted soils. Often, within 4 days of withholding water, the soil surface becomes too dry for peg penetration. Skelton & Shear (1971) reported that adequate root zone moisture could keep pegs alive until pegging zone moisture content is sufficient to allow penetration and initiation of pod development. Once pegs are in the soil, adequate moisture and darkness are needed for pod development. Adequate pod zone moisture is critical for development of pegs into pods and adequate soil water in the root zone cannot compensate for lack of pod zone water for the first 30 days of peg development. Dry pegging zone soil delayed pod and seed development. Soil water deficits in the pegging and root zone decreased pod and seed growth rates by approximately 30% and decreased weight per seed from 563 to 428 mg. Peg initiation growth during drought stress demonstrated ability to suspend development during the period of soil water deficit and to re-initiate pod development after the drought stress was relieved (Sexton et al., 1988). It has frequently been reported that under water stress, pegging and seed set responses of various peanut cultivars varied substantially, this leads to a large reduction in pod yield, and the reduction percentage also varies among peanut cultivars (Haris et al., 1988, Nageswara Rao et al., 1989).

### **2.4 Relationship of drought tolerance and aflatoxin contamination**

Drought stress has a strong effect on biocompetitive (phytoalexins, antifungal proteins) or protective compounds (phenols), which influence the growth of *Aspergillus* fungus and aflatoxin synthesis, as well as the proper maturation of peanut seeds. Aflatoxin contamination threat increases with increasing seed maturity. As the seed moisture content decreases during drought, the capacity of seed to produce phytoalexins decreases resulting in *Aspergillus* invasion and aflatoxin production. Some of the enzymes that are induced in response to fungal attack such as chitinases, osmotins, peroxidases, and proteases are also adversely affected during drought stress through cell membrane-mediated mechanisms. Drought stress and drought stress mediated-fungal infection compromise peanut defense and exacerbate aflatoxin formation in the seeds (Guo et al., 2005). Thus, breeding for drought tolerance has been accepted as one of the strategies for developing aflatoxintolerant peanut cultivars, which would not only minimize water usage but also help expand peanut production in marginal and sub-marginal soils. Success in this effort has been slow due to lack of genetic resources and lack of information on the relationship or interaction between the pathways affected due to drought and or pathogen invasion. However, to date, few peanut cultivars with natural pre-harvest resistance to aflatoxin production have been identified through field screening.

### **3. Breeding for crop improvement**

252 Plants and Environment

therefore its concentration is considered a good indicator of moisture stress (Reddi & Reddy,

The start of flowering is not delayed by drought stress (Boote & Ketring, 1990). The rate of flower production is reduced by drought stress during flowering but the total number of flowers per plant is not affected due to an increase in the duration of flowering (Gowda & Hegde, 1986; Janamatti et al., 1986; Meisner & Karnok, 1992). A significant burst in flowering on alleviation of stress is a unique feature in the pattern of flowering under moisture stress, particularly when drought is imposed just prior to re-productive development (Janamatti et al., 1986). When stress is imposed during 30–45 days after sowing the first flush of flowers produced up to 45 days do not form pegs during that time, however, flowers produced after

Peanut plants may experience water stress during pegging and pod development and then may have adequate amount of water (Jogloy et al., 1996). This would result in a drastic reduction of crop yield, and the magnitude of reduction would depend on peanut cultivars. Not only the yield of peanut but also the quality of products decreases under drought stress (Rucker et al., 1995). Peg elongation, which is turgor dependent, is delayed due to drought stress (Boote & Ketring, 1990). Pegs fail to penetrate effectively into air-dry soil, especially in crusted soils. Often, within 4 days of withholding water, the soil surface becomes too dry for peg penetration. Skelton & Shear (1971) reported that adequate root zone moisture could keep pegs alive until pegging zone moisture content is sufficient to allow penetration and initiation of pod development. Once pegs are in the soil, adequate moisture and darkness are needed for pod development. Adequate pod zone moisture is critical for development of pegs into pods and adequate soil water in the root zone cannot compensate for lack of pod zone water for the first 30 days of peg development. Dry pegging zone soil delayed pod and seed development. Soil water deficits in the pegging and root zone decreased pod and seed growth rates by approximately 30% and decreased weight per seed from 563 to 428 mg. Peg initiation growth during drought stress demonstrated ability to suspend development during the period of soil water deficit and to re-initiate pod development after the drought stress was relieved (Sexton et al., 1988). It has frequently been reported that under water stress, pegging and seed set responses of various peanut cultivars varied substantially, this leads to a large reduction in pod yield, and the reduction percentage also varies among

**2.3 Effect of drought during flowering and pod formation** 

re-watering compensated for this loss (Gowda & Hegde, 1986).

peanut cultivars (Haris et al., 1988, Nageswara Rao et al., 1989).

**2.4 Relationship of drought tolerance and aflatoxin contamination** 

Drought stress has a strong effect on biocompetitive (phytoalexins, antifungal proteins) or protective compounds (phenols), which influence the growth of *Aspergillus* fungus and aflatoxin synthesis, as well as the proper maturation of peanut seeds. Aflatoxin contamination threat increases with increasing seed maturity. As the seed moisture content decreases during drought, the capacity of seed to produce phytoalexins decreases resulting in *Aspergillus* invasion and aflatoxin production. Some of the enzymes that are induced in response to fungal attack such as chitinases, osmotins, peroxidases, and proteases are also

1995).

**2.3.1 Flowering** 

**2.3.2 Pod formation** 

### **3.1 Breeding towards drought tolerance**

Efforts to improve peanuts that focus on yield as the only environmental method for screening of tolerance are seen to have a high variability in yield as well as differences in exactly reproducing stress conditions. A more-integrated approach for peanut breeding is needed to offer success in developing stress-tolerant varieties. Understanding physiological and molecular genetics may lead to the understanding of stress response and aid in development of new varieties with stress tolerance. So, a high-yielding cultivar that continues to produce well under drought conditions is a priority to enable stability of production. That is why much research for the last decade has attempted to improve performance by selecting plants with good pod yield under adverse conditions. As well as spending time testing plants in large-scale trials under different conditions, a study of plant physiology has revealed the features of the plant that correlate best with drought tolerance.

Research in the previous decade had developed low-cost, rapid and easily measured indicators for three significant physiological features of drought-tolerance *viz.* amount of water transpired (T), water-use efficiency (W) and harvest index (HI), thus allowing their potential quantification in large numbers of breeding populations. The application of this physiological model in peanut-breeding programs has not been possible because of practical difficulties associated with measurement of the traits under field conditions. The USDA germplasm collection numbers over 9000 accessions of *A. hypogaea* (Holbrook, 2001) and about 800 accessions of *Arachis* species. Large *Arachis* species collections are also maintained at Texas A&M University and N. C. State University. The US breeding program is focused more on yield, grade, seed size and developing disease tolerant germplasm, and less on drought tolerance. Identifying drought tolerant genotypes with emphasis to reduce preharvest aflatoxin contamination is being conducted at the USDA-ARS, Tifton, GA., The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India and The National Center of Genetic Resources (CENARGEN), Brazil. The largest collection of domesticated peanut germplasm is located at ICRISAT, where there are 14,310 accessions from 92 countries while CENARGEN has 413 accessions of *Arachis* species (Upadhyaya et al., 2001a). A new drought tolerant groundnut variety, ICGV 91114, is becoming very popular in Anantapur district in Andhra Pradesh, India, where it is now replacing a 7 decade old variety TMV 2. ICGV 91114 has also been released in Orissa, India and is doing very well in Karnataka, India.

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 255

Improvement of drought tolerance is an important area of research for groundnut breeding programs. Recent advances in the area of crop genomics offer tools to assist in breeding (Varshney et al., 2005, 2006). The identification of genomic regions associated with drought tolerance would enable breeders to develop improved cultivars with increased drought tolerance using marker-assisted selection (Ribaut et al., 1996). To make selection on large populations of progeny for breeding work, the accessions must be grown out and tested for traits. This is time consuming and subject to environmental variability. The scarcity of DNA polymorphism in cultivated peanut posses a considerable obstacle in genetic mapping of peanut. The Texas Peanut Breeding and Genetics Program is working on a long-term program to integrate modern physiological and molecular methods with plant breeding, to develop peanut varieties that can be grown efficiently under reduced water inputs and high heat stress. There are RFLP (Restricted Fragment Length Polymorphism) maps of wild type x cultivar crosses but the polymorphisms are too low for a cultivated x cultivated species cross; therefore, new markers are needed (Burow et al., 2001). Restricted Fragment Length Polymorphism markers also have disadvantages of using radioisotope, and results take longer to obtain than the use of PCR-based methods. Burow et al., (2001) study focused on finding traits useful in selecting genotypes for drought and heat tolerance. Heat stress was determined by fluorescence from cultivars grown in a high thermal stress greenhouse environment. Selections were made for drought and heat tolerance and crosses were made for further progeny evaluation. Further, they suggested that the research would entail sequencing cDNA in mapped RFLP clones to start the development of molecular markers in

A considerable number of SSR sequences have been identified from peanut genome by several research groups (Hopkins et al., 1999; He et al., 2003; Ferguson et al., 2004; Moretzsohn et al., 2005; Proite et al., 2007; Cuc et al., 2008). SSR markers developed from these repeat sequences offer promising genetic and genomic tools in peanut research. Genetic diversity of peanut germplasm has been studied in Valencia (Krishna et al., 2004), mini-core collection (Barkley et al., 2007), and in Chinese (Tang et al., 2007) and Japanese peanut germplasm collections (Naito et al., 2008) using SSR markers. Genetic linkage maps with SSR markers have been constructed for diploid AA genome (Moretzsohn et al., 2005), BB genome (Moretzsohn et al., 2009), tetraploid AABB genome derived from a cross of cultivated with amphidiploids (Fonceka et al., 2009), and tetraploid AABB genome in the cultivated peanut (Hong et al., 2008, Varshney et al., 2009; Hong et al., 2010). Although an exceedingly large number of SSRs have been identified, the polymorphic SSR markers may not be sufficient for the construction of a saturated linkage map in the cultivated peanut, provide enough meaningful markers for marker-assisted selection in peanut breeding programs, or sufficient coverage of important domains of the peanut genome for functional

To identify the genomic regions suitable for marker-assisted breeding strategies, it is important to establish accurate phenotyping methods, develop highly saturated molecular marker-based genetic linkage maps, and then identify QTLs (quantitative trait loci) associated with traits of interest. Several studies were conducted in the past that reported identification of QTLs for drought tolerance or related traits. A RIL mapping population comprising of 318 F8/F9/F10 lines derived from a cross of TAG 24 x ICGV 86031 was phenotyped for transpiration (T, g plant-1), transpiration efficiency (TE, g biomass kg-1 water transpired), SLA (cm2 g-1), SCMR, leaf area (LA, cm2 plant-1), shoot plus pod dry

**4.1.1 Molecular markers** 

peanut.

genomics research.

In another study at Main Oilseeds Research Station, Junagadh Agricultural University, Junagadh, around 130 genotypes/crosses from different breeding trials (these were identified as potential drought tolerant with the help of visual observations such as retention of greenness at harvest, thickness of foliage, dwarfness combined with greenness, etc.) were screened for higher yield than local check varieties under simulated drought conditions in the summer season of 1999, 2000 and 1997. In the second phase of investigation, yield performance of these selected crosses/entries was assessed in comparison with three varieties GG-2, GG-5 (local checks) and J-11 (national check) at three naturally drought prone locations viz., at Targhadia (Main Dry Farming Research Station), Manavadar, Nanakandhasar and Jamkhambhalia, Gujarat, India in terms of pod yield. The basic advantage in selecting yield as the selection criteria is that it integrates all the additive effects of many underlying mechanisms of drought tolerance. Seven crosses and two genotypes with three controls (check varieties) were grown in a randomized complete block design with four replications for three consecutive Kharif seasons – 1999, 2000 and 2001. The results clearly indicated that the selected crosses/genotypes are at par with the local cultivated varieties of groundnut with respect to pod yields. In fact, they could even be termed superior because under extreme conditions of water deficit during 1999 and 2000 they recorded significantly higher pod yield than the local checks. Hence, the crosses GG-2 x NCAC 17135, GG-2 x PI 259747, J 11 x PI 259747, S 206 x FESR-8, Kisan x FESR-S-PI-B1-B, and the genotypes JB 223 and 224 could be termed as drought tolerant genotypes. Hence, it is suggested that these lines/genotypes could be grown under regions of limited rainfall. These lines may be also used as parents in breeding programs for developing drought tolerant groundnut cultivars.

### **3.2 Limitations of traditional breeding**

Crop improvement in terms of production, desirable traits and resistance to drought stress is a pre-requisite in modern day agriculture. Conventional breeding for developing drought-tolerant crop varieties is time-consuming and labor intensive due to the quantitative nature of drought tolerance and difficulties in selection for drought tolerance (Ribaut et al., 1997). Combining high levels of resistance into higher yielding cultivars with acceptable market traits continues to be difficult (Holbrook & Stalker, 2003). Breeding programs, aimed at incorporating resistance genes from wild *Arachis* relatives have proved largely unsuccessful due to genetic incompatibility. Due to limitations of conventional peanut breeding either because of limited gene pool or the restricted range of organisms between which genes can be transferred, new *omics* techniques in addition to conventional methods are needed to develop peanut cultivars with resistance to drought and pre-harvest aflatoxin contamination.

### **4. Applications of molecular breeding tools for crop improvement**

### **4.1 Genomic approach**

Peanut is a polyploid with a large genome size, complete sequencing will be too expensive and labor intensive to perform with current resources. Research with molecular aspects of the peanut genome began in the 1980s when protein and isozyme variation in *A. hypogaea* was determined to be of little use for characterizing variation within the cultivated peanut. Although large numbers of polymorphisms were detected among other species in the genus (Lu & Pickersgill, 1993; Stalker et al., 1994), the number of markers was too small to be routinely used in breeding programs.

### **4.1.1 Molecular markers**

254 Plants and Environment

In another study at Main Oilseeds Research Station, Junagadh Agricultural University, Junagadh, around 130 genotypes/crosses from different breeding trials (these were identified as potential drought tolerant with the help of visual observations such as retention of greenness at harvest, thickness of foliage, dwarfness combined with greenness, etc.) were screened for higher yield than local check varieties under simulated drought conditions in the summer season of 1999, 2000 and 1997. In the second phase of investigation, yield performance of these selected crosses/entries was assessed in comparison with three varieties GG-2, GG-5 (local checks) and J-11 (national check) at three naturally drought prone locations viz., at Targhadia (Main Dry Farming Research Station), Manavadar, Nanakandhasar and Jamkhambhalia, Gujarat, India in terms of pod yield. The basic advantage in selecting yield as the selection criteria is that it integrates all the additive effects of many underlying mechanisms of drought tolerance. Seven crosses and two genotypes with three controls (check varieties) were grown in a randomized complete block design with four replications for three consecutive Kharif seasons – 1999, 2000 and 2001. The results clearly indicated that the selected crosses/genotypes are at par with the local cultivated varieties of groundnut with respect to pod yields. In fact, they could even be termed superior because under extreme conditions of water deficit during 1999 and 2000 they recorded significantly higher pod yield than the local checks. Hence, the crosses GG-2 x NCAC 17135, GG-2 x PI 259747, J 11 x PI 259747, S 206 x FESR-8, Kisan x FESR-S-PI-B1-B, and the genotypes JB 223 and 224 could be termed as drought tolerant genotypes. Hence, it is suggested that these lines/genotypes could be grown under regions of limited rainfall. These lines may be also used as parents in breeding programs for developing drought

Crop improvement in terms of production, desirable traits and resistance to drought stress is a pre-requisite in modern day agriculture. Conventional breeding for developing drought-tolerant crop varieties is time-consuming and labor intensive due to the quantitative nature of drought tolerance and difficulties in selection for drought tolerance (Ribaut et al., 1997). Combining high levels of resistance into higher yielding cultivars with acceptable market traits continues to be difficult (Holbrook & Stalker, 2003). Breeding programs, aimed at incorporating resistance genes from wild *Arachis* relatives have proved largely unsuccessful due to genetic incompatibility. Due to limitations of conventional peanut breeding either because of limited gene pool or the restricted range of organisms between which genes can be transferred, new *omics* techniques in addition to conventional methods are needed to develop peanut cultivars with resistance to drought and pre-harvest

**4. Applications of molecular breeding tools for crop improvement**

Peanut is a polyploid with a large genome size, complete sequencing will be too expensive and labor intensive to perform with current resources. Research with molecular aspects of the peanut genome began in the 1980s when protein and isozyme variation in *A. hypogaea* was determined to be of little use for characterizing variation within the cultivated peanut. Although large numbers of polymorphisms were detected among other species in the genus (Lu & Pickersgill, 1993; Stalker et al., 1994), the number of markers was too small to be

tolerant groundnut cultivars.

aflatoxin contamination.

**4.1 Genomic approach** 

routinely used in breeding programs.

**3.2 Limitations of traditional breeding** 

Improvement of drought tolerance is an important area of research for groundnut breeding programs. Recent advances in the area of crop genomics offer tools to assist in breeding (Varshney et al., 2005, 2006). The identification of genomic regions associated with drought tolerance would enable breeders to develop improved cultivars with increased drought tolerance using marker-assisted selection (Ribaut et al., 1996). To make selection on large populations of progeny for breeding work, the accessions must be grown out and tested for traits. This is time consuming and subject to environmental variability. The scarcity of DNA polymorphism in cultivated peanut posses a considerable obstacle in genetic mapping of peanut. The Texas Peanut Breeding and Genetics Program is working on a long-term program to integrate modern physiological and molecular methods with plant breeding, to develop peanut varieties that can be grown efficiently under reduced water inputs and high heat stress. There are RFLP (Restricted Fragment Length Polymorphism) maps of wild type x cultivar crosses but the polymorphisms are too low for a cultivated x cultivated species cross; therefore, new markers are needed (Burow et al., 2001). Restricted Fragment Length Polymorphism markers also have disadvantages of using radioisotope, and results take longer to obtain than the use of PCR-based methods. Burow et al., (2001) study focused on finding traits useful in selecting genotypes for drought and heat tolerance. Heat stress was determined by fluorescence from cultivars grown in a high thermal stress greenhouse environment. Selections were made for drought and heat tolerance and crosses were made for further progeny evaluation. Further, they suggested that the research would entail sequencing cDNA in mapped RFLP clones to start the development of molecular markers in peanut.

A considerable number of SSR sequences have been identified from peanut genome by several research groups (Hopkins et al., 1999; He et al., 2003; Ferguson et al., 2004; Moretzsohn et al., 2005; Proite et al., 2007; Cuc et al., 2008). SSR markers developed from these repeat sequences offer promising genetic and genomic tools in peanut research. Genetic diversity of peanut germplasm has been studied in Valencia (Krishna et al., 2004), mini-core collection (Barkley et al., 2007), and in Chinese (Tang et al., 2007) and Japanese peanut germplasm collections (Naito et al., 2008) using SSR markers. Genetic linkage maps with SSR markers have been constructed for diploid AA genome (Moretzsohn et al., 2005), BB genome (Moretzsohn et al., 2009), tetraploid AABB genome derived from a cross of cultivated with amphidiploids (Fonceka et al., 2009), and tetraploid AABB genome in the cultivated peanut (Hong et al., 2008, Varshney et al., 2009; Hong et al., 2010). Although an exceedingly large number of SSRs have been identified, the polymorphic SSR markers may not be sufficient for the construction of a saturated linkage map in the cultivated peanut, provide enough meaningful markers for marker-assisted selection in peanut breeding programs, or sufficient coverage of important domains of the peanut genome for functional genomics research.

To identify the genomic regions suitable for marker-assisted breeding strategies, it is important to establish accurate phenotyping methods, develop highly saturated molecular marker-based genetic linkage maps, and then identify QTLs (quantitative trait loci) associated with traits of interest. Several studies were conducted in the past that reported identification of QTLs for drought tolerance or related traits. A RIL mapping population comprising of 318 F8/F9/F10 lines derived from a cross of TAG 24 x ICGV 86031 was phenotyped for transpiration (T, g plant-1), transpiration efficiency (TE, g biomass kg-1 water transpired), SLA (cm2 g-1), SCMR, leaf area (LA, cm2 plant-1), shoot plus pod dry

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 257

Fig. 1. DDRT PCR cDNA amplification from drought susceptible (JL-24) and drought tolerant peanut genotype (K1375). Arrows pointed upwards show peanut transcripts drought induced (PTDI) and arrows pointing downwards show peanut transcripts drought

reduced water loss and protection of cellular components.

AhWSI 308) and several zinc finger protein transcripts are preferentially induced under drought treatments in peanut plants. Also among the upstream signaling components they observed induction of transcripts of calmodulins (AhWSI 227, AhWSI 228), G protein (AhWSI 551), MAPKK (AhWSI 28) and several receptor kinases during drought treatments. In addition, specific upregulation of hormone responsive genes such as auxin-repressed proteins (AhWSI 306, AhWSI 468, AhWSI 467), brassinosteroid responsive BRH1 (AhWSI 36), cytokinin-repressed protein CR9 (AhWSI 465), GA like proteins (AhWSI 291, AhWSI 464) was observed during drought treatments. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. Guo et al., (2006) identified a novel PLD gene in peanut, encoding a putative phospholipase D (a main enzyme responsible for the drought-induced degradation of membrane phospholipids in plants). PLD expression was induced faster by drought stress in the drought-sensitive lines than in the drought-tolerant lines, suggesting that peanut PLD may be involved in drought sensitivity responses, which could be useful as a tool in germplasm screening for drought tolerance. Gene expression in leaves of peanut plants submitted to progressive drought stress was studied by Drame et al., (2007). This study revealed that a good correlation exists with the agronomical and physiological responses during drought in peanuts. This study demonstrated that phospolipase Dα and LEA transcripts accumulation could contribute to

**4.1.3 Microarray based screening for monitoring gene expression during drought**  Microarray technology employing cDNAs or oligonucleotides is a powerful tool for analyzing gene expression profiles of plants exposed to abiotic stresses such as drought,

suppressed (PTDS).

weight (DW, g plant-1), and total dry matter (TDM, g plant-1, which includes root dry weight) and carbon discrimination ratio (d13C) during post-rainy season in 2004 and 2005 by Ravi et al., (2011). A genetic map containing 191 SSR loci based on a single mapping population (TAG 24 9 ICGV 86031), segregating for drought and surrogate traits was developed. This study suggests deployment of modern approaches like marker-assisted recurrent selection or genomic selection instead of marker-assisted backcrossing approach for breeding drought tolerance in peanut.

### **4.1.2 Gene expression during drought stress in peanuts**

Abiotic stress is a growing concern for peanut cultivation. Many production areas are in semiarid environments or have unreliable rainfall, and global climate changes and growing demand for fresh water pose major challenges. Physiological adaptation and selection for drought tolerance have been studied by many researchers (Reddy et al., 2003). Study of peanut genomics has been limited by biological constraints, and many basic tools of genomics have yet to be developed (Gepts et al., 2005). The peanut genome is large, making insertional mutagenesis and whole-genome sequencing expensive using current technology, and requiring large genomic libraries for physical mapping and positional cloning. To date, 136,901 peanut sequences, including 87,688 ESTs from cultivated peanuts and 39,866 nucleotide sequences have been deposited in the NCBI EST database. Out of which 52 nucleotide sequences and 25,914 EST sequences are available in response to drought treatments.

One of the major molecular responses that plants exhibit to drought stress is altered expression of genes, related to different pathways associated with stress perception, signal transduction, regulators and synthesis of a number of compounds (Ramanjulu & Bartels, 2002; Sreenivasulu et al., 2007). Several hundred genes that respond to drought stress at the transcriptional level have been identified in model crop *Arabidopsis* by microarray technology and other means (Seki et al., 2002; Shinozaki and Yamaguchi- Shinozaki., 2007). The adaptive mechanisms under stress are a net effect of altered cell metabolism resulting from regulated expression of stress responsive genes. The resurrection plants have better capabilities to cope with severe drought conditions; hence, several studies have been conducted to discover what key genes are involved in enabling these plants to survive desiccation.

Differential display reverse transcriptase PCR was used to identify genes induced and suppressed in peanut seed during drought. A total of 1235 differential display products were observed in irrigated samples, compared to 950 differential display products in stressed leaf samples (Jain et al., 2001). In another experiment, seven transcripts were found induced following stress of which two transcripts were suppressed in drought stressed immature pods of tolerant variety K1375 (Devaiah et al., 2007) (Fig. 1). These products demonstrated qualitative and quantitative differences in the gene expression during drought stress in peanuts.

Subtractive hybridization was used to identify about 700 genes from cDNA library prepared from peanut plants that were subjected to gradual process of drought stress adaptation (Govind et al., 2009). Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Many families of transcription factors including AP2/EREBP (AhWSI 279), bHLH (AhWSI 111, AhWSI 40), bZIP (AhWSI 20), CCAAT box (AhWSI 117), Homeobox (AhWSI6 11), Jumonji (AhWSI 72, AhWSI 116), NAC (AhWSI 153,

weight (DW, g plant-1), and total dry matter (TDM, g plant-1, which includes root dry weight) and carbon discrimination ratio (d13C) during post-rainy season in 2004 and 2005 by Ravi et al., (2011). A genetic map containing 191 SSR loci based on a single mapping population (TAG 24 9 ICGV 86031), segregating for drought and surrogate traits was developed. This study suggests deployment of modern approaches like marker-assisted recurrent selection or genomic selection instead of marker-assisted backcrossing approach

Abiotic stress is a growing concern for peanut cultivation. Many production areas are in semiarid environments or have unreliable rainfall, and global climate changes and growing demand for fresh water pose major challenges. Physiological adaptation and selection for drought tolerance have been studied by many researchers (Reddy et al., 2003). Study of peanut genomics has been limited by biological constraints, and many basic tools of genomics have yet to be developed (Gepts et al., 2005). The peanut genome is large, making insertional mutagenesis and whole-genome sequencing expensive using current technology, and requiring large genomic libraries for physical mapping and positional cloning. To date, 136,901 peanut sequences, including 87,688 ESTs from cultivated peanuts and 39,866 nucleotide sequences have been deposited in the NCBI EST database. Out of which 52 nucleotide sequences and 25,914 EST sequences are available in response to drought

One of the major molecular responses that plants exhibit to drought stress is altered expression of genes, related to different pathways associated with stress perception, signal transduction, regulators and synthesis of a number of compounds (Ramanjulu & Bartels, 2002; Sreenivasulu et al., 2007). Several hundred genes that respond to drought stress at the transcriptional level have been identified in model crop *Arabidopsis* by microarray technology and other means (Seki et al., 2002; Shinozaki and Yamaguchi- Shinozaki., 2007). The adaptive mechanisms under stress are a net effect of altered cell metabolism resulting from regulated expression of stress responsive genes. The resurrection plants have better capabilities to cope with severe drought conditions; hence, several studies have been conducted to discover what key genes are involved in enabling these plants to survive

Differential display reverse transcriptase PCR was used to identify genes induced and suppressed in peanut seed during drought. A total of 1235 differential display products were observed in irrigated samples, compared to 950 differential display products in stressed leaf samples (Jain et al., 2001). In another experiment, seven transcripts were found induced following stress of which two transcripts were suppressed in drought stressed immature pods of tolerant variety K1375 (Devaiah et al., 2007) (Fig. 1). These products demonstrated qualitative and quantitative differences in the gene expression during

Subtractive hybridization was used to identify about 700 genes from cDNA library prepared from peanut plants that were subjected to gradual process of drought stress adaptation (Govind et al., 2009). Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Many families of transcription factors including AP2/EREBP (AhWSI 279), bHLH (AhWSI 111, AhWSI 40), bZIP (AhWSI 20), CCAAT box (AhWSI 117), Homeobox (AhWSI6 11), Jumonji (AhWSI 72, AhWSI 116), NAC (AhWSI 153,

for breeding drought tolerance in peanut.

treatments.

desiccation.

drought stress in peanuts.

**4.1.2 Gene expression during drought stress in peanuts** 

Fig. 1. DDRT PCR cDNA amplification from drought susceptible (JL-24) and drought tolerant peanut genotype (K1375). Arrows pointed upwards show peanut transcripts drought induced (PTDI) and arrows pointing downwards show peanut transcripts drought suppressed (PTDS).

AhWSI 308) and several zinc finger protein transcripts are preferentially induced under drought treatments in peanut plants. Also among the upstream signaling components they observed induction of transcripts of calmodulins (AhWSI 227, AhWSI 228), G protein (AhWSI 551), MAPKK (AhWSI 28) and several receptor kinases during drought treatments. In addition, specific upregulation of hormone responsive genes such as auxin-repressed proteins (AhWSI 306, AhWSI 468, AhWSI 467), brassinosteroid responsive BRH1 (AhWSI 36), cytokinin-repressed protein CR9 (AhWSI 465), GA like proteins (AhWSI 291, AhWSI 464) was observed during drought treatments. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. Guo et al., (2006) identified a novel PLD gene in peanut, encoding a putative phospholipase D (a main enzyme responsible for the drought-induced degradation of membrane phospholipids in plants). PLD expression was induced faster by drought stress in the drought-sensitive lines than in the drought-tolerant lines, suggesting that peanut PLD may be involved in drought sensitivity responses, which could be useful as a tool in germplasm screening for drought tolerance. Gene expression in leaves of peanut plants submitted to progressive drought stress was studied by Drame et al., (2007). This study revealed that a good correlation exists with the agronomical and physiological responses during drought in peanuts. This study demonstrated that phospolipase Dα and LEA transcripts accumulation could contribute to reduced water loss and protection of cellular components.

### **4.1.3 Microarray based screening for monitoring gene expression during drought**

Microarray technology employing cDNAs or oligonucleotides is a powerful tool for analyzing gene expression profiles of plants exposed to abiotic stresses such as drought,

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 259

important peanuts and may play an important role in peanut growth, development, and

Proteomics studies have been carried out in leaf and immature peanut pods in response to drought stress. Identification and development of drought-tolerant genotype/s is the potential means to reduce aflatoxin contamination. Difference in biochemical response of peanut genotypes with varying degree of drought tolerance was monitored by withholding irrigation for various intervals. Changes in seed protein composition in response to drought stress were measured using two-dimensional electrophoresis followed by Mass spectroscopy. Mass spectroscopy analysis revealed down-regulation of methionine rich proteins (MRPs) and arachin proteins in drought-susceptible (DS) genotypes, while these proteins continue to express in drought-tolerant (DT) genotypes. Up-regulation of mRNA transcripts in DT genotypes indicated their association with stress tolerance. Continued expression of these proteins seems to enhance drought tolerance, reduce aflatoxin level and enhance nutritional value of peanut. These studies have revealed that drought stress suppresses expression of several seed storage proteins such as arachin, methionine-rich

Changes in the seed protein content and composition during 14 days of desiccation was determined by Mazhar and Basha (2002) using a combination of electrophoretic and immunochemical techniques. Following desiccation, the protein content of 'white' (most immature) and 'orange' (Intermediate maturity stage: Drexler and Williams, 1979) seed increased, while that of the 'brown' (more mature) seed were not affected. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed no major qualitative differences in protein composition during desiccation. However, immunoblotting with antidehydrin antisera revealed presence of several new proteins in the desiccated samples compared with the controls. One of the dehydrin-like proteins, was found to be related to water-stress, while the other proteins appeared to be the storage proteins accumulated as the seed matured *in vitro*. Capillary electrophoresis (CE) showed major changes in protein quantity and quality of 'white' seed (Immature) during the 0–14 days of desiccation. In contrast, in the 'orange' and 'brown' seeds (more mature) changes in protein composition were less significant. Their results indicated that several dehydrin-like proteins expressed in

In 2007, Basha and his co-workers carried out a study to determine changes in seed polypeptide composition among drought-tolerant (Vemana and K-1375) and droughtsusceptible peanut genotypes (M-13 and JL-220) following water stress (WS) for 7, 14 and 28 d. They found that water stress had variable effect on peanut seed polypeptide composition (Fig. 2A) among the DT and DS genotypes. WS affected polypeptides with apparent molecular weight (Mr) around 70, 35, 25, 20, 18 and 14 kDa, and isoelectric points between 4.0 and 6.0 pH. The maximum response to WS occurred between 0 to 7 d, and additional periods (14 and 28 d) of stress caused only limited changes in seed polypeptide composition. These responses included over-expression, suppression, and appearance of new proteins in water-stressed seed compared to irrigated control. These data revealed that seed polypeptide composition of drought-tolerant peanut genotypes (Vemana and K-1375) was least affected while that of drought-susceptible genotypes (M-13 and JL-220) significantly

peanuts during desiccation but not all of them are related to drought stress.

response to environmental stress.

**4.2.1 Protein expression during drought stress** 

proteins, conarachin, etc (Basha et al., 2007).

**4.2 Proteomic approach** 

altered due to WS (Fig. 2).

high salinity, or cold, or to ABA treatment (Seki et al., 2001, 2002a, 2002b; Kreps et al., 2002). There are two predominant varieties of microarray technology available; the cDNA microarray (Seki et al., 2001, 2002a, 2002b) and the oligonucleotide microarray. cDNA Microarray was used to screen peanut genotypes by Luo et al., (2005). In this study, resistance genes in response to *Aspergillus parasiticus* infection under drought stress were identified using microarray and real-time PCR. A peanut genotype (A13) which is believed to be tolerant to drought and pre-harvest aflatoxin contamination was used to study gene expression. A total of 52 up-regulated genes were detected in response to drought apart from genes that were expressed due to biotic stress. Reactive oxygen scavengers glutathione S-transferase GST, superoxide dismutase (Cu–Zn), lactoylglutathione lyase, ascorbate peroxidase, lipoxygenase 1, Lipoxygenase 1, lactoylglutathione lyase, superoxide dismutase (Cu–Zn), stress proteins like drought-induced protein RPR-10, cytochrome P450, NOI protein, cold-regulated LTCOR12, low temperature and salt responsive protein, LTI6B, auxin-induced protein, ultraviolet-B-repressible protein, embryonic abundant protein, salt tolerance-like protein, proline-rich protein APG isolog, 10 kDa protein precursor, salt tolerance-like protein, NOI protein, embryonic abundant protein, ultraviolet-B-repressible protein, auxin-induced protein, osmotin-like protein, cell-autonomous heat shock cognate protein 7 and heat shock protein 81-2 were observed to be induced during drought.

High-density oligonucleotide microarray was developed for peanut using 49,205 publicly available ESTs and the utility of this array were tested for expression profiling in a variety of peanut tissues (Payton et al., 2009) to identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, transcript levels in pod, peg, leaf, stem, and root tissues. A set of 108 putatively podspecific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root was detected. The transcripts significantly overrepresented in pod including genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense were also observed. This Microarray chip represents sequences available from various drought stress treatments and hence, can be used as tool to monitor gene expression profile in genotype screening for drought tolerance.

### **4.1.4 Micro RNA could modify regulator gene expression during drought in peanuts**

Micro RNAs are a new class of small, endogenous RNAs that play a regulatory role in the cell by negatively affecting gene expression at the post-transcriptional level. MicroRNAs have been shown to control numerous genes involved in various biological and metabolic processes. Recently MicroRNAs (miRNAs) were isolated in peanuts by Zhao et al., (2010). In this study, they used next generation high through-put Solexa sequencing technology to clone and identify both conserved and species-specific miRNAs in peanut. Next generation high through-put Solexa sequencing showed that peanuts have a complex small RNA population and the length of small RNAs varied, 24-nt being the predominant length for a majority of the small RNAs. Combining the deep sequencing and bioinformatics, they discovered 14 novel miRNA families as well as 75 conserved miRNAs in peanuts. All 14 novel peanut miRNAs were considered to be species-specific because no homologs have been found in other plant species except ahy-miRn1, which has a homolog in soybean. qRT-PCR analysis demonstrated that both conserved and peanut-specific miRNAs were expressed in peanuts. This study led to the discovery of 14 novel and 22 conserved miRNA families from peanut. These results show that regulatory miRNAs exist in agronomicallyimportant peanuts and may play an important role in peanut growth, development, and response to environmental stress.

### **4.2 Proteomic approach**

258 Plants and Environment

high salinity, or cold, or to ABA treatment (Seki et al., 2001, 2002a, 2002b; Kreps et al., 2002). There are two predominant varieties of microarray technology available; the cDNA microarray (Seki et al., 2001, 2002a, 2002b) and the oligonucleotide microarray. cDNA Microarray was used to screen peanut genotypes by Luo et al., (2005). In this study, resistance genes in response to *Aspergillus parasiticus* infection under drought stress were identified using microarray and real-time PCR. A peanut genotype (A13) which is believed to be tolerant to drought and pre-harvest aflatoxin contamination was used to study gene expression. A total of 52 up-regulated genes were detected in response to drought apart from genes that were expressed due to biotic stress. Reactive oxygen scavengers glutathione S-transferase GST, superoxide dismutase (Cu–Zn), lactoylglutathione lyase, ascorbate peroxidase, lipoxygenase 1, Lipoxygenase 1, lactoylglutathione lyase, superoxide dismutase (Cu–Zn), stress proteins like drought-induced protein RPR-10, cytochrome P450, NOI protein, cold-regulated LTCOR12, low temperature and salt responsive protein, LTI6B, auxin-induced protein, ultraviolet-B-repressible protein, embryonic abundant protein, salt tolerance-like protein, proline-rich protein APG isolog, 10 kDa protein precursor, salt tolerance-like protein, NOI protein, embryonic abundant protein, ultraviolet-B-repressible protein, auxin-induced protein, osmotin-like protein, cell-autonomous heat shock cognate

protein 7 and heat shock protein 81-2 were observed to be induced during drought.

expression profile in genotype screening for drought tolerance.

High-density oligonucleotide microarray was developed for peanut using 49,205 publicly available ESTs and the utility of this array were tested for expression profiling in a variety of peanut tissues (Payton et al., 2009) to identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, transcript levels in pod, peg, leaf, stem, and root tissues. A set of 108 putatively podspecific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root was detected. The transcripts significantly overrepresented in pod including genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense were also observed. This Microarray chip represents sequences available from various drought stress treatments and hence, can be used as tool to monitor gene

**4.1.4 Micro RNA could modify regulator gene expression during drought in peanuts**  Micro RNAs are a new class of small, endogenous RNAs that play a regulatory role in the cell by negatively affecting gene expression at the post-transcriptional level. MicroRNAs have been shown to control numerous genes involved in various biological and metabolic processes. Recently MicroRNAs (miRNAs) were isolated in peanuts by Zhao et al., (2010). In this study, they used next generation high through-put Solexa sequencing technology to clone and identify both conserved and species-specific miRNAs in peanut. Next generation high through-put Solexa sequencing showed that peanuts have a complex small RNA population and the length of small RNAs varied, 24-nt being the predominant length for a majority of the small RNAs. Combining the deep sequencing and bioinformatics, they discovered 14 novel miRNA families as well as 75 conserved miRNAs in peanuts. All 14 novel peanut miRNAs were considered to be species-specific because no homologs have been found in other plant species except ahy-miRn1, which has a homolog in soybean. qRT-PCR analysis demonstrated that both conserved and peanut-specific miRNAs were expressed in peanuts. This study led to the discovery of 14 novel and 22 conserved miRNA families from peanut. These results show that regulatory miRNAs exist in agronomically-

### **4.2.1 Protein expression during drought stress**

Proteomics studies have been carried out in leaf and immature peanut pods in response to drought stress. Identification and development of drought-tolerant genotype/s is the potential means to reduce aflatoxin contamination. Difference in biochemical response of peanut genotypes with varying degree of drought tolerance was monitored by withholding irrigation for various intervals. Changes in seed protein composition in response to drought stress were measured using two-dimensional electrophoresis followed by Mass spectroscopy. Mass spectroscopy analysis revealed down-regulation of methionine rich proteins (MRPs) and arachin proteins in drought-susceptible (DS) genotypes, while these proteins continue to express in drought-tolerant (DT) genotypes. Up-regulation of mRNA transcripts in DT genotypes indicated their association with stress tolerance. Continued expression of these proteins seems to enhance drought tolerance, reduce aflatoxin level and enhance nutritional value of peanut. These studies have revealed that drought stress suppresses expression of several seed storage proteins such as arachin, methionine-rich proteins, conarachin, etc (Basha et al., 2007).

Changes in the seed protein content and composition during 14 days of desiccation was determined by Mazhar and Basha (2002) using a combination of electrophoretic and immunochemical techniques. Following desiccation, the protein content of 'white' (most immature) and 'orange' (Intermediate maturity stage: Drexler and Williams, 1979) seed increased, while that of the 'brown' (more mature) seed were not affected. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed no major qualitative differences in protein composition during desiccation. However, immunoblotting with antidehydrin antisera revealed presence of several new proteins in the desiccated samples compared with the controls. One of the dehydrin-like proteins, was found to be related to water-stress, while the other proteins appeared to be the storage proteins accumulated as the seed matured *in vitro*. Capillary electrophoresis (CE) showed major changes in protein quantity and quality of 'white' seed (Immature) during the 0–14 days of desiccation. In contrast, in the 'orange' and 'brown' seeds (more mature) changes in protein composition were less significant. Their results indicated that several dehydrin-like proteins expressed in peanuts during desiccation but not all of them are related to drought stress.

In 2007, Basha and his co-workers carried out a study to determine changes in seed polypeptide composition among drought-tolerant (Vemana and K-1375) and droughtsusceptible peanut genotypes (M-13 and JL-220) following water stress (WS) for 7, 14 and 28 d. They found that water stress had variable effect on peanut seed polypeptide composition (Fig. 2A) among the DT and DS genotypes. WS affected polypeptides with apparent molecular weight (Mr) around 70, 35, 25, 20, 18 and 14 kDa, and isoelectric points between 4.0 and 6.0 pH. The maximum response to WS occurred between 0 to 7 d, and additional periods (14 and 28 d) of stress caused only limited changes in seed polypeptide composition. These responses included over-expression, suppression, and appearance of new proteins in water-stressed seed compared to irrigated control. These data revealed that seed polypeptide composition of drought-tolerant peanut genotypes (Vemana and K-1375) was least affected while that of drought-susceptible genotypes (M-13 and JL-220) significantly altered due to WS (Fig. 2).

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 261

The mechanisms of drought response have been investigated extensively in *Arabidopsis* (Bray et al., 1997; Shinozaki et al., 2003). However, the response of peanut to drought stress has not been extensively studied using genetic engineering. Classical breeding for drought tolerance in peanut is difficult because of variability in time, intensity, and duration of stress. In certain breeding programs, plants with genetic variability to drought have been identified and used to introduce this trait in genotypes with desirable agronomic characteristics. Thus in peanut classical breeding has and continues to have some success but the process is slow and limited by the availability of suitable genes for breeding. Beyond this there has been limited progress in breeding for drought tolerance because of limited characterization of associated traits and the fact that potential component traits of drought tolerance such as Transpiration, Transpiration Efficiency, or Harvest Index (Passioura, 1977) do not have simply additive effects (Bhatnagar-Mathur et al., 2007) in peanut. Molecular markers have been used to aid in the breeding process, but the low level of polymorphism in cultivated peanut has interfered with this approach. Although peanut germplasm with reduced drought tolerance have been identified and screened in breeding populations (Holbrook et al., 2000), peanut growers currently cannot rely fully on the available drought tolerant cultivars, as they are location specific. Therefore, the use of genetic engineering technology to over-express drought tolerant genes in peanut is an

**5. Transgenic peanut tolerant to drought** 

attractive prospective way to improve tolerance.

peanut.

**5.1 Developing drought tolerant peanut through genetic engineering** 

**5.2 ABA-independent gene regulation to drought stress** 

improvement for drought tolerance through genetic engineering.

Development of drought tolerant peanut by genetic engineering requires the identification of key genetic determinants underlying stress tolerance in peanut plants, and introducing these genes into peanut crops. The effect of drought can trigger a wide array of physiological responses in plants, and this can affect a large number of genes. For example, Sahi et al., (2006) through their gene expression experiments have identified several hundred genes which are either induced or repressed during drought. *Arabidopsis* has played an important role in the elucidation of the basic processes underlying stress tolerance, and the knowledge achieved has been transferred to several food crops (Zhang et al., 2004). Most of the genes that are known to be involved in stress tolerance were initially isolated from *Arabidopsis*. Several stress induced genes that have been introduced in other plants by genetic engineering have resulted in increased tolerance of transgenic plants to drought. Therefore the same techniques that have been used in other crops can be used in

There are two transcription factors *DREB1* and *DREB2*, which have been identified to be important in the ABA-independent drought tolerant pathways that induce the expression of drought tolerant genes. When the native form of *DREB1* and the constitutively active form of *DREB2* are over-expressed, tolerance of transgenic *Arabidopsis* plants to drought is increased. Even though these genes were initially identified in *Arabidopsis* plants, their existence and function in stress tolerance have been reported in many other important plants, such as tomato, barley, rice, canola, maize, rye, wheat, maize and soybean. This is an indication that these genes are conserved, and they perform a universal stress defense mechanism in plants. This is why the *DREB* genes can be used as suitable targets for peanut

Fig. 2. Differential response of seed proteins of Drought Tolerant and Drought Susceptible Peanut Genotypes to Water Stress

Recently, Kottapalli and co-workers (2009) analyzed peanut genotypes from the US minicore collection for changes in leaf proteins during reproductive growth under water-deficit stress. One and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected drought-tolerant and drought-susceptible genotypes. A total of 102 protein bands/spots were analyzed by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine nonredundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1L-myo-inositol-1-phosphate synthase, which aid in inter and intracellular stress signaling were found to be more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. They also found a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. In contrast, Katam et al. (2007) found up-regulation of leaf proteins following drought stress in DT genotypes and down-regulation in DS genotypes. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.

### **5. Transgenic peanut tolerant to drought**

260 Plants and Environment

Fig. 2. Differential response of seed proteins of Drought Tolerant and Drought Susceptible

Recently, Kottapalli and co-workers (2009) analyzed peanut genotypes from the US minicore collection for changes in leaf proteins during reproductive growth under water-deficit stress. One and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected drought-tolerant and drought-susceptible genotypes. A total of 102 protein bands/spots were analyzed by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine nonredundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1L-myo-inositol-1-phosphate synthase, which aid in inter and intracellular stress signaling were found to be more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. They also found a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. In contrast, Katam et al. (2007) found up-regulation of leaf proteins following drought stress in DT genotypes and down-regulation in DS genotypes. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of

Peanut Genotypes to Water Stress

water-deficit stress tolerance in peanut.

The mechanisms of drought response have been investigated extensively in *Arabidopsis* (Bray et al., 1997; Shinozaki et al., 2003). However, the response of peanut to drought stress has not been extensively studied using genetic engineering. Classical breeding for drought tolerance in peanut is difficult because of variability in time, intensity, and duration of stress. In certain breeding programs, plants with genetic variability to drought have been identified and used to introduce this trait in genotypes with desirable agronomic characteristics. Thus in peanut classical breeding has and continues to have some success but the process is slow and limited by the availability of suitable genes for breeding. Beyond this there has been limited progress in breeding for drought tolerance because of limited characterization of associated traits and the fact that potential component traits of drought tolerance such as Transpiration, Transpiration Efficiency, or Harvest Index (Passioura, 1977) do not have simply additive effects (Bhatnagar-Mathur et al., 2007) in peanut. Molecular markers have been used to aid in the breeding process, but the low level of polymorphism in cultivated peanut has interfered with this approach. Although peanut germplasm with reduced drought tolerance have been identified and screened in breeding populations (Holbrook et al., 2000), peanut growers currently cannot rely fully on the available drought tolerant cultivars, as they are location specific. Therefore, the use of genetic engineering technology to over-express drought tolerant genes in peanut is an attractive prospective way to improve tolerance.

### **5.1 Developing drought tolerant peanut through genetic engineering**

Development of drought tolerant peanut by genetic engineering requires the identification of key genetic determinants underlying stress tolerance in peanut plants, and introducing these genes into peanut crops. The effect of drought can trigger a wide array of physiological responses in plants, and this can affect a large number of genes. For example, Sahi et al., (2006) through their gene expression experiments have identified several hundred genes which are either induced or repressed during drought. *Arabidopsis* has played an important role in the elucidation of the basic processes underlying stress tolerance, and the knowledge achieved has been transferred to several food crops (Zhang et al., 2004). Most of the genes that are known to be involved in stress tolerance were initially isolated from *Arabidopsis*. Several stress induced genes that have been introduced in other plants by genetic engineering have resulted in increased tolerance of transgenic plants to drought. Therefore the same techniques that have been used in other crops can be used in peanut.

### **5.2 ABA-independent gene regulation to drought stress**

There are two transcription factors *DREB1* and *DREB2*, which have been identified to be important in the ABA-independent drought tolerant pathways that induce the expression of drought tolerant genes. When the native form of *DREB1* and the constitutively active form of *DREB2* are over-expressed, tolerance of transgenic *Arabidopsis* plants to drought is increased. Even though these genes were initially identified in *Arabidopsis* plants, their existence and function in stress tolerance have been reported in many other important plants, such as tomato, barley, rice, canola, maize, rye, wheat, maize and soybean. This is an indication that these genes are conserved, and they perform a universal stress defense mechanism in plants. This is why the *DREB* genes can be used as suitable targets for peanut improvement for drought tolerance through genetic engineering.

Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 263

Fig. 3. Schematic representation of Cloning and Agrobacterium mediated genetic

Although significant progress is being made to elucidate the genetic mechanisms underlying drought tolerance in peanut, considerable challenges still remain. In field conditions, peanut plants are subjected to variable levels of multiple stresses, and hence, the response of peanut to a combination of stresses deserves much more attention. In other words, the response of plants to multiple stresses cannot be inferred from the response to individual stress. Therefore, it is very important to test newly developed varieties to multiple stresses, and to perform extensive field studies under diverse environments to

Ali Ahmad, M.; & Basha, S.M. (1998). Effect of Water Stress on Composition of Peanut

Athmaram, T.N.; Bali G. & Devaiah, K.M. (2006). Integration and expression of Bluetongue

VP2 gene in somatic embryos of peanut through particle bombardment method,

Leaves, *Peanut Science*, Vol. 25, No.1, pp. 31–34.

*Vaccine,* Vol. 24, No.15, pp. 2994-3000.

transformation in peanut

**7. Conclusion** 

assess their tolerance.

**8. References** 

### **5.3 Peanut transformation systems**

Peanut transformation has been accomplished by several different methods. Ozias-Akins et al., (1993) reported the first successful transformation of peanut with accompanying plant regeneration by utilizing the microbombardment technique. Micro-bombardment has since been completed in peanut with a number of genes conferring disease resistance (Ozias-Akins & Gill, 2001; Magbanua et al., 2000; Yang et al., 1998; Higgins et al., 2004; Athmaram et al., 2006). However, its efficiency levels remain low and the process takes several months from when the initial transformation event is induced until plant maturity (Egnin et al., 1998). A highly-efficient and faster technique is needed to transform peanut, and *Agrobacterium*-mediated transformation appears to offer the possibility to achieve this goal. Cheng et al., (1996) used this method on a Valencia-type peanut, but other investigators have been unable to expand the methodology to other genotypes thus restricting its usefulness. To date, biolistic methodologies are more reliable in peanut than other transformation methodologies and single constructs can be inserted into the peanut genome. Individual genes that confer agronomic traits have been integrated into the peanut genome such as bialophos resistance (*bar*) for herbicide tolerance (Brar et al., 1994), *Bacillus thuringiensis* (*Bt*) toxin *cryIA(c)* for insect resistance (Singsit et al., 1997), viral nucleocapsid or coat protein genes for virus resistance (Higgings et al., 2004), chitinase, glucanase, and oxalate oxidase to control fungal diseases (Chenault et al., 2005; Livngstone et al., 2005; Rohini and Rao, 2001). But, in studying drought tolerance in transgenic peanut plants, Bhatnagar-Mathur et al., (2007), introduced a transcription factor *DREB1A* from *Arabidopsis thaliana*, in a drought-sensitive peanut cultivar JL-24 through *Agrobacterium tumefaciens*mediated gene transfer (Fig.3). The stress inducible expression of *DREB1A* in these transgenic plants did not result in growth retardation or visible phenotypic alterations. They were successful in developing transgenic events of peanut with the *DREB1A* transcription factor that is specifically expressed under a stress responsive promoter such as *A. thaliana rd29A*. Thus, their study opens ways to other scientist to dwell more on producing transgenic peanut with drought tolerance.

### **6. Future research**

Classical plant breeding programs, which are relatively inexpensive, are not well adapted for utilizing advanced technologies associated with genomics. Hence, a large percentage of scientists who perform genomic research are mainly interested in the molecular function of specific genes or processes and are usually less interested in marker development for phenotypic selection applications. On the other hand, plant breeders need markers to facilitate selection and are generally not interested in developing large data sets for sequencing specific genes. Although the gap between the producer of genomic information (molecular biologist) and the user (plant breeders) is very wide, there is enormous potential for interactions among disciplines for plant improvement. Indeed, increasing research efforts in engineering for production of drought-tolerant peanut crops should be employed. There are certain genes that are expressed at elevated levels when a plant encounters stress, and it is important to understand that tolerance to drought is a complex process, and it is unlikely to be under the control of a single gene. Therefore, it is wise to combine conventional screening efforts, marker assisted selection and genetic engineering to switch on a transcription factor regulating the expression of several genes related to drought tolerance.

Fig. 3. Schematic representation of Cloning and Agrobacterium mediated genetic transformation in peanut

### **7. Conclusion**

262 Plants and Environment

Peanut transformation has been accomplished by several different methods. Ozias-Akins et al., (1993) reported the first successful transformation of peanut with accompanying plant regeneration by utilizing the microbombardment technique. Micro-bombardment has since been completed in peanut with a number of genes conferring disease resistance (Ozias-Akins & Gill, 2001; Magbanua et al., 2000; Yang et al., 1998; Higgins et al., 2004; Athmaram et al., 2006). However, its efficiency levels remain low and the process takes several months from when the initial transformation event is induced until plant maturity (Egnin et al., 1998). A highly-efficient and faster technique is needed to transform peanut, and *Agrobacterium*-mediated transformation appears to offer the possibility to achieve this goal. Cheng et al., (1996) used this method on a Valencia-type peanut, but other investigators have been unable to expand the methodology to other genotypes thus restricting its usefulness. To date, biolistic methodologies are more reliable in peanut than other transformation methodologies and single constructs can be inserted into the peanut genome. Individual genes that confer agronomic traits have been integrated into the peanut genome such as bialophos resistance (*bar*) for herbicide tolerance (Brar et al., 1994), *Bacillus thuringiensis* (*Bt*) toxin *cryIA(c)* for insect resistance (Singsit et al., 1997), viral nucleocapsid or coat protein genes for virus resistance (Higgings et al., 2004), chitinase, glucanase, and oxalate oxidase to control fungal diseases (Chenault et al., 2005; Livngstone et al., 2005; Rohini and Rao, 2001). But, in studying drought tolerance in transgenic peanut plants, Bhatnagar-Mathur et al., (2007), introduced a transcription factor *DREB1A* from *Arabidopsis thaliana*, in a drought-sensitive peanut cultivar JL-24 through *Agrobacterium tumefaciens*mediated gene transfer (Fig.3). The stress inducible expression of *DREB1A* in these transgenic plants did not result in growth retardation or visible phenotypic alterations. They were successful in developing transgenic events of peanut with the *DREB1A* transcription factor that is specifically expressed under a stress responsive promoter such as *A. thaliana rd29A*. Thus, their study opens ways to other scientist to dwell more on producing

Classical plant breeding programs, which are relatively inexpensive, are not well adapted for utilizing advanced technologies associated with genomics. Hence, a large percentage of scientists who perform genomic research are mainly interested in the molecular function of specific genes or processes and are usually less interested in marker development for phenotypic selection applications. On the other hand, plant breeders need markers to facilitate selection and are generally not interested in developing large data sets for sequencing specific genes. Although the gap between the producer of genomic information (molecular biologist) and the user (plant breeders) is very wide, there is enormous potential for interactions among disciplines for plant improvement. Indeed, increasing research efforts in engineering for production of drought-tolerant peanut crops should be employed. There are certain genes that are expressed at elevated levels when a plant encounters stress, and it is important to understand that tolerance to drought is a complex process, and it is unlikely to be under the control of a single gene. Therefore, it is wise to combine conventional screening efforts, marker assisted selection and genetic engineering to switch on a transcription factor regulating the expression of several genes related to drought

**5.3 Peanut transformation systems** 

transgenic peanut with drought tolerance.

**6. Future research** 

tolerance.

Although significant progress is being made to elucidate the genetic mechanisms underlying drought tolerance in peanut, considerable challenges still remain. In field conditions, peanut plants are subjected to variable levels of multiple stresses, and hence, the response of peanut to a combination of stresses deserves much more attention. In other words, the response of plants to multiple stresses cannot be inferred from the response to individual stress. Therefore, it is very important to test newly developed varieties to multiple stresses, and to perform extensive field studies under diverse environments to assess their tolerance.

### **8. References**


Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 265

Chenault, K.D.; Melouk, H.A. & Payton, M.E. (2005). Field reaction to *Sclerotinia* blight

Cheng, M.; Jarret, R., Li, Z., Xing, A. & Demski, J. (1996). Production of Fertile Transgenic

Cole, R.J.; Sanders, T.H., Dorner, J.W. & Blankenship, P.D. Environmental conditions

Cuc, L.M.; Mace, E.S., Crouch, J.H., Quang, V.D., Long, T.D. & Varshney, R.K. (2008).

Devaiah, K.M.; Bali, G., Athmaram, T.N. & Basha, S.M. (2007). Identification of Two New

Dorner, J. W.; Cole, R.J., Sanders, T.H. & Blankenship, P.D. (1989). Interrelationship of kernel

Drexler, S. & Williams, E. J. (1979). A nondestructive method of peanut pod maturity

Drame, K.N.; Clavel, D., Repellin, A., Passaquet, C. & Fodil, Y.Z. (2007). Water Deficit

Egnin, M.; Mora, A. & Prakash, C.S. (1998). Factors enhancing Agrobacterium tumefaciens-

Erickson, P.I.& Ketring, D.L. (1985). Evaluation of Genotypes for Resistance to Water Stress

Ferguson, M.E.; Burow, M.D., Schulze, S.R., Bramel, P.J., Paterson, A.H., Kresovich, S. &

*hypogaea* L.), *Theoretical And Applied Genetics*, Vol.108, No.6,pp. 1064-1070. Foncéka, D.; Hodo-Abalo, T., Rivallan, R., Faye, I., Sall, M.N., Ndoye, O., Fávero, A.P.,

Gepts, P.; Beavis W.D., Brummer, E.C., Shoemaker, R.C., Stalker, H.T., Weeden, N.F. &

Basis of A Recent Allotetraploid. *BMC Plant Biology,* Vol.9,pp.103.

511–515.

*Reports*, Vol.15,pp. 653-657.

India; 1989, pp.279-287.

Vol.105, pp.117-128.

*BMC Plant Biology*, Vol.8, pp.55.

*Growth Regulation*, Vol.52,pp. 249–258.

classlficaiton. *Proc. APRES,* Vol.11, pp.57.

*Developmental Biology-Plant*, Vol.34, pp.310-318.

*In Situ*, *Crop Science*, Vol.25,pp. 870–876.

*Physiology*, Vol.137,pp. 1228–1235.

*and Biochemistry*, Vol.45,pp. 236-243.

among transgenic peanut lines containing antifungal genes, *Crop Science, Vol.*45,pp.

Peanut (*Arachis hypogaea* L.) Plants Using *Agrobacterium tumefaciens*, *Plant Cell* 

required to induce preharvest aflatoxin contamination of groundnuts: summary of six years' research. In *Aflatoxin contamination of groundnuts*. ICRISAT, Patancheru,

Isolation And Characterization of Novel Microsatellite Markers and Their Application for Diversity Assessment in Cultivated Groundnut (*Arachis hypogaea*).

Genes from Drought Tolerant Peanut Up-Regulated In Response to Drought, *Plant* 

water activity, soil-temperature, maturity, and phytoalexin production in preharvest aflatoxin contamination of drought-stressed peanuts. *Mycopathologia*

Induces Variation In Expression Of Stress-Responsive Genes In Two Peanut (*Arachis hypogaea* L.) Cultivars with Different Tolerance to Drought, *Plant Physiology* 

mediated gene transfer in peanut (*Arachis hypogaea* L.), *In Vitro Cellular &* 

Mitchell, S. (2004). Microsatellite Identification and Characterization in Peanut (*A.* 

Bertioli, D.J., Glaszmann, J.C., Courtois, B. & Rami, J.F. (2009). Genetic Mapping of Wild Introgressions into Cultivated Peanut: A Way toward Enlarging the Genetic

Young, N.D. (2005). Legumes as a Model Plant Family. Genomics for Food and Feed Report of the Cross-Legume Advances through Genomics Conference, *Plant* 


Azam Ali, S.N. (1984). Environmental and Physiological Control of Transpiration by Groundnut Crops, *Agricultural and Forest Meteorology*, Vol. 33, pp.129-140. Babu, V.R. & Rao, D.V.M. (1983). Water Stress Adaptations in The Ground Nut (*Arachis* 

Basha, S.M.; Katam, R. & Naik, K.S.S. (2007). Differential Response of Peanut Genotypes to

Barkley, N.A.; Dean, R., Pittman, R.N., Wang, M., Holbrook, C.C. & Pederson, G.A. (2007).

SSR Markers and Sequencing, *Genetical Research*, Vol. 89, No.2, pp. 93-106. Bartels, D. & Sunkar, R. (2005). Drought and salt tolerance in plants, *Critical Reviews in Plant* 

Bhagsari, A.S.; Brown, R.H.& Schepers, J.S. (1976). Effect of Moisture Stress on

Bhatnagar-Mathur, P.; Devi, M.J., Serraj, R., Yamaguchi-Shinozaki, K., Vadez, V. & Sharma,

Bhatnagar-Mathur, P.; Devi, M.J., Reddy, D., Lavanya, M., Vadez, V., Serrej, R., Yamaguchi-

Black, C.R.; Tang, D.Y., Ong, C.K., Solon, A. & Simmonds, L.P. (1985). Effects of Soil

Blankenship, P.D.; Cole, R.J., Sanders, T.H. & Hill, R.A. (1984). Effect of geocarposphere

Brar, G.S.; Cohen, B.A., Vick, C.L.& Johnson, G.W. (1994). Recovery of transgenic peanut

Chen, H.; Holbrook, C.C. & Guo, B.Z. (2006). Peanut Seed Transcriptome: Construction Of

Bray, E. (1997)*.* Plant responses to water deficit, *Trends in Plant Science*, Vol. 2,pp. 48–54. Burow, M.D.; Simpson, C.E., Starr, J.L. & Paterson A.H. (2001). Transmission Genetics of

conditions, *International Arachis Newsletter*, Vol.24, pp. 33–34.

water limiting conditions, *Plant Cell Reports*, Vol.26, pp. 2071-2082.

*Physiology & Biochemistry*, Vol. 10,pp. 64–80.

*Sciences*, Vol.24, pp. 23-58.

*Science*, Vol.16, pp. 712–715.

Vol.100, pp. 313–328.

Research CSSA-SSSA, Madison.

*Research and Education Society*, Vol.38. pp. 73.

*Journal,* Vol.5,pp. 745–753.

Vol.159, pp. 823-837.

Water Stress. *Peanut Science* Vol.34, pp. 96-104.

*hypogaea* L.) – Foliar Characteristics and Adaptations to Moisture Stress, *Plant* 

Genetic Diversity of Cultivated and Wild-Type Peanuts Evaluated With M13-Tailed

Photosynthesis and Some Related Physiological Characteristics in Peanuts, *Crop* 

K.K. (2004). Evaluation of transgenic groundnut lines under water limited

Shinozaki K. & Sharma, K. (2007). Stress-inducible expression of at DREB1A in transgenic peanut (*Arachis hypogaea* L.) increases transpiration efficiency under

Moisture on Water Relations And Water Use of Groundnut Stands, *New Phytologist*,

temperature on pre-harvest colonization of drought-stressed peanuts by *Aspergillus flavus* and subsequent aflatoxin contamination. *Mycopathologia*, Vol.85, pp.69-74. Boote, K.J. & Ketring, D.L. (1990). Peanut. In: Stewart B.A. And Nielson D.R. (Eds), Irrigation

Of Agricultural Crops. Asa- Groundnut - A Global Perspective. International Crops

(*Arachis hypogaea* L.) plants from elite cultivars utilizing ACCELL technology, *Plant* 

Chromatin from A Synthetic Amphidiploid to Cultivated Peanut (*Arachis hypogaea L.*): Broadening the Gene Pool of a Monophyletic Polyploid Species, *Genetics*,

Six Peanut Seed cDNA Libraries from Two Peanut Cultivars, *American Peanut* 


Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 267

Holbrook, C.C.& Stalker, H.T. (2003). Peanut Breeding and Genetic Resources. *Plant Breeding* 

Hong, Y.; Chen, X., Liang, X., Liu, H., Zhou, G., Li, S., Wen, S., Holbrook, C.C.& Guo, B.

Hong, Y.B.; Liang, X.Q., Chen, X.P., Liu, H.Y., Zhou, G.Y., Li, S.X. & Wen, S.J. (2008).

Hopkins, M.S.; Casa, A.M., Wang, T., Michell, S.E., Dean, R.E., Kochert, G.D.& Kresovich, S.

Hopkins, M.S.; Casa, A.M., Wang, T., Mitchell, S.E., Dean, R.E., Kochert, G.D.& Kresovich, S.

Iuchi, S.; Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T., Tabata, S., Kakubari, Y.,

abscisic acid biosynthesis in Arabidopsis, *Plant Journal*, Vol.27,pp. 325–333. Jain, A.K.; Basha, S.M. & Holbrook, C.C. (2001). Identification of Drought-Responsive

Janamatti, V.S.; Sashidharv, R., Prasad, I.G., Sastry, K.S.K. (1986). Effect Of Cycles Of

Jogloy, S.; Patanothai, A., Toomsan, S. & Isleib, T.G. Breeding peanut to fit into Thai

Kasuga, M.; Setsuko, M., Kazuo, S. & Kazuko, Y.S. (2004). A combination of the *Arabidopsis*

Kawasaki, S.; Miyake, C., Kohci, T., Fujii, S., Uchida, M. & Yokota, A. (2000). Response Of

Kottapalli, K.R.; Rakwal, R., Shibato, J., Burow, G., Tissue, D., Burke, J., Puppala, N., Burow,

Kreps, J.A.; Wu, Y., Chang, H.S., Zhu, T., Wang, X. & Harper, J. (2002). Transcriptome

(*Arachis hypogaea* L.) Genome. *BMC Plant Biology*, Vol.10,pp. 17.

*hypogaea* L.). *Agricultural Sciences in China*, Vol.7, No.8,pp. 915-921.

(SSSR) in Peanuts, *Crop Science*, Vol.39,pp.1243-1247.

(SSR) In Peanut, *Crop Science*, Vol.39, 1243-1247.

*Agricultural Research*, Vol.1, No.2,pp. 136–142.

Arlington, Virginia, USA, 25-31 March, 1996: pp 353-362.

(2010). A SSR-Based Composite Genetic Linkage Map for the Cultivated Peanut

Construction of Genetic Linkage Map Based On Ssr Markers In Peanut (*Arachis* 

(1999). Discovery and Characterization of Polymorphic Simple Sequence Repeats

(1999). Discovery And Characterization Of Polymorphic Simple Sequence Repeats

Yamaguchi-Shinozaki, K. & Shinozaki, K. (2001). Regulation of drought tolerance by gene manipulation of 9-cisepoxycarotenoid dioxygenase, a key enzyme in

Transcripts in Peanut (*Arachis hypogaea* L.), *Electronic Journal of Biotechnology*, Vol.4,

Moisture Stress On Flowering Pattern, Flower Production, Gynophore Length and Their Relationship To Pod Yield In Bunch Types Of Groundnut, *Journal of* 

cropping systems. Proc. of the Peanut Collaborative Research Support Program-International Research Symposium and Workshop, Two Jima Quality Inn,

DREB1A gene and stress-inducible *rd29A* promoter improved drought and lowtemperature stress tolerance in tobacco by gene transfer. *Plant and Cell Physiology*,

Wild Watermelon To Drought Stress: Accumulation Of an ARGE Homologue and Citrulline In Leaves During Water Deficits, *Plant Cell Physiology*, Vol.102,pp. 1353-

M. & Payton P. (2009). Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes, *Plant Cell & Environment*, Vol.32,

changes for Arabidopsis in response to salt, osmotic, and cold stress, *Plant* 

*Review*, Vol.22,pp. 297-356.

No.2,pp. 59-67.

Vol.45, No.3,pp. 346-350.

1354.

No.4,pp. 380-407.

*Physiology*, Vol.130,pp. 2129-2141.


Govind, G.; Harshavardhan, V.T., Patricia, J.K., Dhanalakshmi, R., Senthil, K.M.,

Gowda, A. & Hegde, B.R. (1986). Moisture Stress and Hormonal Influence on The Flowering Behavior And Yield Of Groundnut, *Plant Physiology*, Vol.66,pp. 835–837. Guo, B.; Luo, M., Dang, P., He, G.& Holbrook, C.C. (2004). Peanut Expressed Sequence Tag

Guo, B.Z.; Xu, G., Cao, Y.G., Holbrook, C.C. & Lynch, R.E. (2006). Identification and

Hajhaeidri, M.; Abdollahian-Noghabi, M., Askari, H., Hedari, M., Sadeghian, S.Y., Ober, E.S.

Harris, D.; Matthews, R.B., Nageswara Rao, R.C.& Williams, J.H. (1988). The Physiological

L.) In Response To Drought, *Experimental Agriculture*, Vol.24,pp.215-226. He, G.& Prakash, C.S. (1997). Identification of Polymorphic DNA Markers in Cultivated

He, G.; Meng, R., Newman, M., Gao, G., Pittman, R.N.& Prakash C.S. (2003). Microsatellites

He, G.H.; Meng, R., Gao, H., Guo, B., Gao, G., Newman, M., Pittman, R.N.& Prakash, C.S.

He, G.H.; Meng, R., Newman, M., Gao, G., Pittman, R.N. & Prakash, C.S. (2003).

Higgins, C.; Hall, R., Mitter, N., Cruickshank, A.& Dietzgen, R. (2004). Peanut Stripe

Holbrook, C.C. (2001). Status of the *Arachis* Germplasm Collection in The United States,

Holbrook, C.C.; Kvien, C.K., Rucker, K.S., Wilson, D.M., Hook, J.E.& Matheron, M.E. (2000).

Peanut (*Arachis hypogaea* L.), *Euphytica*, Vol.97,pp. 143-149.

Peanut (*Arachis hypogaea* L.), *Euphytica*, Vol.142,pp. 131-136.

Protein Gene Sequences, *Transgenic Research*, Vol.13,pp. 59-67.

peanut genotypes, *Peanut Science*, Vol.27,pp. 45–48.

Susceptibilities In Peanut (*Arachis hypogaea*), *Planta*, Vol.223,pp. 512-520. Guo, B.Z.; Chen, X., Dang, P., Scully, B.T., Liang, X., Holbrook, C.C., Yu, J. & Culbreath, A.K.

*Http://Www.Cropscience.Org.Au/Icsc2004/Poster/ 3/1/424\_Guob.Htm*.

Vol.281, No.6, pp.591-605

*Biology*, Vol.8,pp.12.

Vol.3,pp. 3.

*Plant Biology*, Vol.3,pp.3.

*Peanut Science*, Vol.28,pp. 84-89.

Drought Stress, *Proteomics*, Vol.5, 950-960.

Sreenivasulu, N. & Udayakumar, M. (2009). Identification and Functional Validation Of A Unique Set of Drought Induced Genes Preferentially Expressed In Response To Gradual Water Stress In Peanut, *Molecular Genetics & Genomics,* 

(EST) Project and The Marker Development For Cultivated Peanut (*Arachis hypogaea*), *Proceedings of the International Crop Science Congress*,

Characterization of Phospholipase D and Its Association With Drought

(2008). Peanut Gene Expression Profiling In Developing Seeds At Different Reproduction Stages During *Aspergillus parasiticus* Infection, *BMC Developmental* 

& Hosseini Salekdeh G.N. (2005). Proteome Analysis of Sugar Beet Leaves Under

Basis for yield Differences between Four Genotypes of Groundnut (*Arachis hypogaea*

as DNA Markers In Cultivated Peanut (*Arachis hypogaea* L.). *BMC Plant Biology*,

(2005). Simple Sequence Repeat Markers for Botanical Varieties of Cultivated

Microsatellites as DNA Markers in Cultivated Peanut (*Arachis hypogaea* L.), *BMC* 

Potyvirus Resistance in Peanut (*Arachis hypogaea* L.) Plants Carrying Viral Coat

Preharvest aflatoxin contamination in drought-tolerant and drought-intolerant


Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 269

Moretzsohn, M.C.; Barbosa, A.V.G., Alves-Freitas, D.M.T., Teixeira, C., Leal-Bertioli, S.C.M.,

Ozias-Akins, P. & Gill, R. (2001). Progress In the Development of Tissue Culture and

Ozias-Akins P.; Schnall, J.A., Anderson, W.F., Singsit, C., Clemente, T.E., Adang, M.J. &

Pandey, R.K.; Herrera, W.A.T., Villepas, A.N. & Pendelton, J.W. (1984). Drought Response Grain Legumes Under Irrigation Gradient. *Agronomy Journal*, Vol.76,pp. 557–560. Passioura, J. B. (1977). Physiology of grain yield in wheat growing on stored water.

Payton, P.; Kottapalli, K.R., Rowland, D., Faircloth, W., Guo, B., Burow, M., Puppala, N. &

Pellegrineschi, A.; Matthew, R., Mario, P., Rosa Maria, R., Rosaura, A., Kazuko, Y.S. &

Proite, K.; Leal-Bertioli, S.C.M., Bertioli, D.J., Moretzsohn, M.C., Silva, F.R., Martins, N.F.,

Ramanjulu, S. & Bartels, D. (2002). Drought- And Desiccation-Induced Modulation of Gene

Ravi, K.; Vadez, V., Isobe, S., Mir, R.R., Guo, Y., Nigam, S.N., Gowda, M.V.C.,

Reddi, G.H.S. & Reddy, T.Y. (1995). Efficient Use of Irrigation Water. Kalyani Publishers,

Reddy, A.J. & Rao, I.M. (1968). Influence of Induced Water Stress On Chlorophyll

Reddy, T.Y.; Reddy, V.R., Anbumozhi, V. (2003). Physiological Responses of Groundnut

Ribaut, J.M.; Jiang, C., Gonzalez-De-Leon, D., Edmeades, G.O. & Hoisington, D.A. (1997).

Expression in Plants. *Plant Cell & Environment*, Vol.25,pp. 141–151.

during Pre-Flowering Phase, *Agronomy Journal*, Vol.80,pp.431-438.

Transformed Embryogenic Callus, *Plant Science*, Vol.93,pp. 185-194.

oligonucleotide microarrays. *BMC Genomics*, Vol.12, No.10,pp.265.

*Australian Journal Plant Physiology*, Vol.3,pp.560–565.

Marker Development. *BMC Plant Biology*, Vol.7,pp. 7.

*Theoretical & Applied Genetics*, Vol.122, 1119–1132.

*Plant Growth Regulation*, Vol.41,pp.75–88.

*Peanut Science*, Vol.28,pp. 123-131.

Vol.47, No.3,pp. 493-500.

New Delhi.

Vol.5, No.3,pp. 118–121.

Guimarães, P.M., Pereira, R.W., Lopes, C.R., Cavallari, M.M., Valls, J.F.M., Bertioli, D.J. & Gimenes, M.A. (2009). A Linkage Map for The B-Genome Of *Arachis*  (Fabaceae) and Its Synteny To The A-Genome, *BMC Plant Biology*, Vol.9,pp. 40. Nageswara Rao, R.C.; Williams, J.H., Sivakumar, M.V.K. & Wadia, K.R.D. (1998). Effect of

Water Deficit At Different Growth Phases Of Peanut. Ii. Response to Drought

Transformation Methods Applicable To The Production Of Transgenic Peanut,

Weissinger, A.K. (1993). Regeneration of Transgenic Peanut Plants from Stably

Gallo, M. (2009). Gene expression profiling in peanut using high density

David, H. (2004). Stress-induced expression in wheat of the *Arabidopsis thaliana* DREB1A gene delays water stress symptoms under greenhouse conditions. *Genome*,

Guimarães, P.M. (2007). ESTs from a Wild *Arachis* Species for Gene Discovery and

Radhakrishnan, T., Bertioli, D.J., Knapp, S.J. & Varshney, R.K. (2011). Identification Of Several Small Main-Effect QTLs and a Large Number of epistatic QTLs For Drought Tolerance Related Traits In Groundnut (*Arachis hypogaea* L.).

Components Of Proximal And Distal Leaflets of Groundnut Plants, *Current Science*,

(*Arachis hypogea* L.) To Drought Stress and Its Amelioration: A Critical Review.

Identification of Quantitative Trait Loci under Drought Conditions In Tropical


Krishna, G.K.; Zhang, J., Burow, M., Pittman, R.N., Delikostadinov, S.G., Lu, Y. & Puppala,

Kulkarni, J.H.; Ravindra, V., Sojitra, V.K. & Bhatt, D.M. (1988). Growth, nodulation and N

Lauriano, J.A.; Lidon, F.C., Carvalho, C.A., Campos, P.S. & Matos, M.D.C. (2000). Drought

Liu, Q.; Kasuga, M., Yoh, S., Hiroshi, A.B.E., Setsuko, M., Kazuko, Y.S. & Kazuo, S. (1998).

Livingstone, D. M.; Hampton, J. L., Phipps, P. M. & Grabau, E. A. (2005). Enhancing

Lu, J. & Pickersgill, B. (1993). Isozyme Variation and Species Relationships In Peanut And Its

Luo, M.; Dang, P., Guo, B.Z., He, G., Holbrook, C.C., Bausher, M.G. & Lee, R.D. (2005a).

Magbanua, Z.; Wilde, H., Roberts, J., Chowdhury, K., Abad, J., Moyer, J., Wetzstein, H. &

Mazhar, H.& Basha, S.M. (2002). Effect of Desiccation of Peanut (*Arachis hypogaea* L.) Seed Protein Composition. *Environmental & Experimental Botany*, Vol.47,pp.67-75. Meisner, C.A. (1991). Peanut Roots, Shoot and Yield and Water Stress. *Dissertation Abstracts International. Biological Sciences and Engineering*. Vol.52, No.1,pp. 38–48. Mehan, V.K.; McDonald, D., Ramakrishna, N. & Williams, J.H. (1986). Effect of cultivar and

Moretzsohn, M.C.; Leoi, L., Proite, K., Guimara, P.M., Leal-Bertioli, S.C.M., Gimenes, M.A.,

contamination with aflatoxin. *Peanut Science*, Vol.13,pp.46-50.

(Fabaceae). *Theoretical & Applied Genetics*, Vol.111,pp. 1060-1071.

Development in Cultivated Peanut, *Crop Science*, Vol.45,pp. 346-353. Luo, M.; Liang, X.Q., Dang, D., Holbrook, C.C., Bausher, M.G., Lee, R.D. & Guo, B.Z.

different phenophase, *Oleagineus,*Vol*.* 43,pp. 415–419.

Cultivars, *Photosynthetica (Prague)*, Vol.38,pp. 7-12.

Vol.10, No.8,pp. 1391-1406.

*Plant Physiology,* Vol.137,pp.:1354–1362.

*Molecular Breeding*, Vol.6,pp.227-236.

697.

550-560.

Vol.169,pp. 695-703.

N. (2004). Genetic Diversity Analysis in Valencia Peanut (*Arachis hypogaea* L.) Using Microsatellite Markers, *Cellular and Molecular Biology Letters*, Vol.9, No.4a,pp. 685-

uptake of groundnut (*Arachis hypogaea* L.) as influenced by water deficits stress at

Effects On Membrane Lipids And Photosynthetic Activity In Different Peanut

Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and lowtemperature-responsive gene expression, respectively, in *Arabidopsis, The Plant Cell*,

resistance to *Sclerotinia minor* in peanut by expressing a barley oxalate oxidase gene.

Wild Relatives (*Arachis* L. —Leguminosae), *Theoretical & Applied Genetics*, Vol.85,pp.

Generation of Expressed Sequence Tags (ESTs) For Gene Discovery and Marker

(2005b). Microarray-Based Screening of differentially Expressed Genes In Peanut In Response To *Aspergillus parasiticus* Infection And Drought Stress. *Plant Science*,

Parrott, W. (2000). Field Resistance to Tomato Spotted Wilt Virus in Transgenic Peanut (*Arachis hypogaea* L.) Expressing an Antisense Nucleocapsid Gene Sequence.

date of harvest on infection of peanut seed by *Aspergillus flavus* and subsequent

Martins, W.S., Valls J.F.M., Grattapaglia, D. & Bertioli, D.J. (2005). A Micro Satellite–Based, Gene–Rich Linkage Map For The AA Genome Of *Arachis*


Impact of Drought Stress on Peanut (Arachis hypogaea L.) Productivity and Food Safety 271

Singsit, C.; Adang, M.J., Lynch R.E., Anderson, W.F., Wang, A., Cardineau, G. & Ozias-

Skelton, B.J. & Shear, G.M. (1971). Calcium Translocation in the Peanut (*Arachis hypogae* L.).

Sreenivasulu, N.; Radchuk, V., Strickert, M., Miersch, O., Weschke, W. & Wobus, U. (2006).

Stalker, H.T.; Phillips, T.G., Murphy, J.P. & Jones, T.M. (1994). Diversity of Isozyme Patterns

Stirling, C.M.; Black, C.R. & Ong, C.K. (1989). The response of groundnut (*Arachis hypogae* 

Stockinger, E.J.; Gilmour, S.J. & Thomashow, M.F. (1997). *Arabidopsis thaliana CBF1* encodes

Subramaniam, V.B. & Maheswari, M. (1990). Physiological Responses of Groundnut to

Suther, D.M. & Patel, M.S. (1992). Yield and Nutrient Absorption by Groundnut and Iron

Tang, R.; Gao, G., He, L., Han, Z., Shan, S., Zhong, R., Zhou, C., Jiang, J., Li, Y. & Zhuang, W.

Upadhyaya, H.D.; Nigam, S.N., Mehan, V.K., Reddy, A.G.S. & Yellaiah, N. (2001).

Varshney, R.K.; Hoisington, D. & Tyagi, A.K. (2006). Advances in Cereals Genomics and Applications in Crop Breeding, *Trends Biotechnology*, Vol.24,pp. 490–499. Varshney, R.K.; Bertioli, D.J., Moretzsohn, M.C., Vadez, V., Krishnamurthy, L., Aruna, R.,

Wheatley, A.R.D.; Whiteman, J.A., Williams, J.H. & Wheatly, S.J. (1989). The Influence of

91278, ICGV 91283, and ICGV 91284. *Crop Science*, Vol.41,pp. 559–600. Varshney, R.K.; Graner, A. & Sorrells, M.E. (2005). Genomics assisted Breeding for Crop

Improvement. *Trends In Plant Science*, Vol.10,pp. 621–630.

*hypogaea* L.). *Theoritical Applied Genetics*, Vol.118,pp.729-739.

Water Stress. *Indian Journal of Plant Physiology*, Vol.2, 130–135.

in Arachis Species, *Theoretical & Applied Genetics*, Vol.87, 746-755.

Vol.6,pp. 169–176.

Vol.94,pp. 1035–1040.

*Soil Science*, Vol.40,pp. 594–596.

*Agronomy Journal*, Vol.63,pp. 409–412.

Seed. *Plant Journal*, Vol.47, No.2,pp. 310–327.

*Experimental Botany*, Vol.40, No. 221,pp. 1363–1373.

*Genetics and Genomics*, Vol.34, No.5,pp. 449-459.

Akins, P. (1997). Expression of a *Bacillus thuringiensis* cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer. *Transgenic Research*,

Gene Expression Patterns Reveal Tissue-Specific Signaling Networks Controlling Programmed Cell Death and ABA-Regulated Maturation In Developing Barley

L.) to timing of irrigation. II. 14C partitioning and plant water status. *Journal* 

an AP2 domain-containing transcription activator that binds to the C-repeat/DRE, a *cis* acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. *Proceedings of National Academy of Sciences*,

Availability in Soil as Influenced By Lime and Soil Water, *Journal of Indian Society of* 

(2007). Genetic Diversity in Cultivated Groundnut Based on SSR Markers. *Journal of* 

Registration of *Aspergillus flavus* Seed Infection Resistant Peanut Germplasm ICGV

Nigam, S.N., Moss, B.J., Seetha, K., Ravi, K., He, G., Knapp, S.J. & Hoisington, D.A. (2009). The first SSR-based genetic linkage map for cultivated groundnut (*Arachis* 

Drought Stress on The Distribution Of Insects On Four Groundnut Genotypes Grown Near Hyderabad. *India Bulletin of Entomology Research*, Vol.79,pp. 567–577.

Maize. 2. Yield Components and Marker Assisted Selection Strategies. *Theoretical & Applied Genetics*, Vol.94, 887*-*896.


Ribaut, J.M.; Hoisington, D.A., Deutsch, J.A., Jian, C. & Gonzalez, De Leon. (1996).

Rohini, V. K. & Rao, K. S. (2001). Transformation of peanut (*Arachis hypogaea* L.) with

Rucker, K.S.; Kevin, C.K., Holbrook, C.C. & Hook, J.E. (1995). Identification of Peanut

Sahi C.; Singh, A., Kumar, K., Bumwald, E. & Grover, A. (2006). Salt stress response in rice:

Salekeh, G.H.; Siopongco, H.J., Wade, I.J., Ghareyazie, B. & Bennett, J. (2002a). A Proteomics

Salekeh, G.H.; Siopongco, H.J., Wade, I.J., Ghareyazie, B. & Bennett, J. (2002b). A Proteomics

Seki, M.; Narusaka, M. & Ishida, J. (2002a). Monitoring the expression profiles of ca. 7000

Seki, M.; Narusaka, M. & Kamiya, A. (2002). Functional Annotation of a Full-Length

Seki, M.; Ishida, J. & Narusaka, M. (2002b). Monitoring the expression pattern of ca. 7000

Seki, M.; Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P.,

Sexton, P.J.; Benett, J.M. & Boote, K.J. (1997). The Effect of Dry Use Efficiency and Carbon

Shinozaki, K. & Yamaguchi-Shinozaki K. (2007). Gene Networks Involved In Drought Stress Response And Tolerance. *Journal of Experimental Botany*, Vol.58, No.2,pp. 221–227. Shinozaki K.; Yamaguchi-Shinozaki, K. & Seki, M*.* (2003). Regulatory network of gene

length cDNA microarray. *The Plant Journal*, Vol.31, 279-292.

Arabidopsis cDNA Collection. *Science*, 296: 141–145.

*Functional and Integrative Genomics*, Vol.2,pp. 282-291.

microarray. *The Plant Cell*, Vol.13,pp. 61-72.

*Peanut Science*, Vol.24,pp. 19–24.

*Biology*, Vol.6,pp. 410-417.

*Applied Genetics*, Vol.94, 887*-*896.

*Plant Physiology*, Vol.117,pp. 1263-1253.

*Genomic*, DOI 10.1007/s10142-006-0032-5

*Science,* Vol.160,pp.889–898.

*Research*, Vol.76,pp. 199-219.

No.1,pp. 14–18.

1131-1145.

Maize. 2. Yield Components and Marker Assisted Selection Strategies. *Theoretical &* 

Identification of QTL under Drought Conditions In Tropical Maize. 1. Flowering Parameters and the ASI, *Theoretical & Applied Genetics*, Vol.92, No.7,pp. 905-914. Riccardi, F.; Gazeau, P., De Vienne, D. & Zivy, M. (1998). Protein Changes in Response to

Progressive Water Deficit In Maize: Quantitative Variations And Identification,

tobacco chitinase gene: variable response of transformants to leaf spot disease. *Plant* 

Genotypes with Improved Drought Avoid- Ance Traits, *Peanut Science*, Vol.22,

genetics, molecular biology, and comparative genomics. *Functional & Integrative* 

Approach to Analyzing Drought and Salt-Responsiveness in Rice. *Field Crops* 

Analysis of Rice Leaves during Drought Stress and Recovery. *Proteomics*, Vol.2,pp.

Arabidopsis genes under drought, cold, and high-salinity stresses using a full-

Arabidopsis genes under ABA treatments using a full-length cDNA microarray.

Hayashizaki, Y. & Shinozaki, K. (2001). Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA

Isotope Discrimination in Peanut Under Pegging Zone Soil On Pod Formation.

expression in the drought and cold stress responses. *Current Opinion in Plant* 


Yamaguchi-Shinozaki, K. & Shinozaki, K. (1994). A Novel *cis*-acting element in an

Yang, H.; Singsit, C., Wang, A., Gonsalves, D. & Ozias-Akins P. (1998). Transgenic Peanut

Zhang, H.; Sreenivasulu, N., Weschke, W., Stein, N., Rudd, S. & Radchuk, V. (2004). Large-

Zhao, C.Z.; Xia, H., Frazier, T.P., Yao, Y.Y., Bi, Y.P., Li, A.Q., Li, M.J., Li, C.S., Zhang, B.H. &

high-salt stress. *Plant Cell*, Vol.6,pp. 251-264.

*Journal*, Vol.40,pp. 276-290.

Strategies, Agricultural Research Magazine, January 2010 (*Http://Www.Worldandi.Com/Subscribers /Feature\_Detail.Asp?Num=27275*)

peanuts (*Arachis hypogaea* L.). *BMC Plant Biology*, Vol.10,pp. 3.

Arabidopsis gene is involved in responsiveness to drought, low-temperature, or

Plants Containing A Nucleocapsid Protein Gene Of Tomato Spotted Wilt Virus Show Divergent Levels of Gene Expression. *Plant Cell Reports*, Vol.17,pp. 693-699. Yao, S. & Durham, S. Preparing Peanuts For The Future: New Cultivars And Farming

scale analysis of the barley transcriptome based on expressed sequence tags. *Plant* 

Wang X.J. (2010). Deep sequencing identifies novel and conserved microRNAs in

### *Edited by Hemanth KN. Vasanthaiah and Devaiah Kambiranda*

Changing environmental condition and global population demands understanding the plant responses to hostile environment. Significant progress has been made over the past few decades through amalgamation of molecular breeding with non-conventional breeding. Understanding the cellular and molecular mechanisms to stress tolerance has received considerable scientific scrutiny because of the uniqueness of such processes to plant biology, and also its importance in the campaign "Freedom From Hunger". The main intention of this publication is to provide a state-of-the-art and up-to-date knowledge of recent developments in understanding of plant responses to major abiotic stresses, limitations and the current status of crop improvement. A better insight will help in taking a multidisciplinary approach to address the issues affecting plant development and performance under adverse conditions. I trust this book will act as a platform to excel in the field of stress biology.

Plants and Environment

Plants and Environment

*Edited by Hemanth KN. Vasanthaiah* 

*and Devaiah Kambiranda*

Photo by noLimit46 / iStock