**The Molecular Basis of ABA-Mediated Plant Response to Drought**

Agata Daszkowska-Golec and Iwona Szarejko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53128

## **1. Introduction**

'Drought stress is as complicated and difficult to plant biology as cancer is to mammalian biology' said Jian-Kang Zhu, a molecular geneticist at the University of California, River‐ side. The capacity of a plant to turn on or turn off a series of genes that further alter plant physiology and morphology allows a plant to tolerate, escape or avoid drought stress. Many countries around the world experience drought stress in different ways but it always leads to a decreased annual yield of crops. Deciphering the basis of the molecular response to stress and the mechanism for the adaptation and acquisition of tolerance can facilitate the creation of cultivars with increased drought tolerance. Drought response is a complex mech‐ anism that has been investigated using a broad spectrum of 'omics' techniques, such as mo‐ lecular genetics, functional genomics, transcriptomics, proteomics and metabolomics combined with advanced phenotyping techniques. The response of plants to dehydration stress has been extensively studied in a wide range of species with particular emphasis on model plants such as Arabidopsis. Taking advantage of the knowledge already obtained from Arabidopsis and other model species, it is possible to gain insight into the stress re‐ sponse in crops such as barley or wheat.

The best known trigger of the cascade of drought signaling is abscisic acid (ABA). Knowledge about the complexity of ABA signaling in regards to stress response is still full of gaps but the recent identification of ABA receptors and the key factors of the first step of ABA signal transduction in Arabidopsis provided an important insight into this mechanism ([1-4]. The actions of the other ABA signaling components, such as phospha‐ tases, kinases, transcription factors and their roles in abiotic stress response during differ‐ ent developmental stages is also documented in crops [5]. Under drought conditions, ABA induces the expression of many genes whose products are involved in the response

properly cited.

© 2013 Daszkowska-Golec and Szarejko; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 Daszkowska-Golec and Szarejko; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

to drought, among which are positive and negative regulators of ABA signaling, tran‐ scription factors and genes encode enzymes that are involved in the synthesis of osmo‐ protectants. It is important to mention that ABA is not the only phytohormone involved in stress response. There is much evidence of cross-talk between ABA and other phyto‐ hormones, such as jasmonates and ethylene [6].

Recent advances in functional genomics have revealed the importance of posttranscrip‐ tional regulation of gene expression performed by microRNA. Deep sequencing methods have enabled the identification of the miRNA involved in drought response in barley and rice. Further analysis also showed their potential roles in stress signaling by identify‐ ing their targets [7-8].

The molecular basis of drought response and the interaction between genes and proteins involved in this mechanism can be studied using of advanced molecular techniques only when a good drought assay that mimics natural drought conditions can be applied in the laboratory. Many protocols for drought assays have been developed that can be im‐ plemented in the study of different species ranging from Arabidopsis to crops. Another important issue is the method of phenotyping and the spectrum of physiological parame‐ ters that are measured [9]. The techniques used most often are: chlorophyll fluorescence, stomatal conductance and relative water content (RWC) [10-12]. Combining these molec‐ ular techniques with advanced methods of phenotyping would enable drought tolerant forms to be produced. This would contribute to beginning the Blue Revolution advocat‐ ed by Kofi Annan in his April 2000 Millennium Address: "We need a Blue Revolution in agriculture that focuses on increasing productivity per unit of water – more crop per drop". This chapter reviews the newest aspects of the molecular and physiological mech‐ anisms of drought stress response in crops.

#### **2. Abscisic acid – The best known stress messenger**

Since its isolation from cotton in the 1960s [13], the role of abscisic acid (ABA) in plant devel‐ opment and in the response of plants to environmental signals has been extensively studied. Analysis of Arabidopsis under salt and drought stress has revealed the important role ABA plays in response to these stresses [14-16]. Endogenous ABA concentrations increase under drought stress due to induction of ABA biosynthesis genes [14]. The increase in ABA repro‐ grams the gene expression pattern to regulate water relations through adjustment of cellular osmotic pressure, the closure of stomata, a reduced leaf canopy, deeper root growth and changes in root system architecture [17-19].

Biosynthesis of ABA has been relatively well characterized in Arabidopsis and some data is available for other species, such as maize, tomato, potato and barley [20-24]. Knowledge about ABA biosynthesis derived from studies in Arabidopsis is highly applicable to other plant species, because the pathway and the respective genes are conserved in angiosperms. ABA is synthesized through the cleavage of a C40 carotenoid precursor, followed by a twostep conversion of the intermediate xanthoxin to ABA via ABA-aldehyde [25-27]. The path‐ way begins with isopentyl pyrophosphate (IPP) which is the biological isoprene unit and the precursor of all terpenoids, as well as many plant hormones. The next step is the epoxida‐ tion of zeaxanthin and antheraxanthin to violaxanthin which is catalyzed by zeaxanthin ep‐ oxidase (ZEP), which was first identified in tobacco [28]. After a series of violaxanthin modifications which are controlled by the enzyme ABA4, violaxanthin is converted into 9 cis-epoxycarotenoid [29]. Oxidative cleavage of the major epoxycarotenoid 9-cis-neoxanthin by the 9-cis-epoxycarotenoid dioxygenase (NCED) yields a C15 intermediate - xanthoxin [30]. This step is the last one that occurs in the plastid. Xanthoxin is exported to the cyto‐ plasm where two-step reaction via ABA-aldehyde takes place. The first step is catalyzed by a short-chain alcohol dehydrogenase/reductase (SDR) that is encoded by the *AtABA2* (*ABA deficient 2*) gene [31-33] and generates ABA aldehyde. Then the ABA aldehyde oxidase (AAO) with the molybdenum cofactor (MoCo) catalyzes the last step in the biosynthesis pathway - the conversion of ABA-aldehyde into ABA [34].

to drought, among which are positive and negative regulators of ABA signaling, tran‐ scription factors and genes encode enzymes that are involved in the synthesis of osmo‐ protectants. It is important to mention that ABA is not the only phytohormone involved in stress response. There is much evidence of cross-talk between ABA and other phyto‐

Recent advances in functional genomics have revealed the importance of posttranscrip‐ tional regulation of gene expression performed by microRNA. Deep sequencing methods have enabled the identification of the miRNA involved in drought response in barley and rice. Further analysis also showed their potential roles in stress signaling by identify‐

The molecular basis of drought response and the interaction between genes and proteins involved in this mechanism can be studied using of advanced molecular techniques only when a good drought assay that mimics natural drought conditions can be applied in the laboratory. Many protocols for drought assays have been developed that can be im‐ plemented in the study of different species ranging from Arabidopsis to crops. Another important issue is the method of phenotyping and the spectrum of physiological parame‐ ters that are measured [9]. The techniques used most often are: chlorophyll fluorescence, stomatal conductance and relative water content (RWC) [10-12]. Combining these molec‐ ular techniques with advanced methods of phenotyping would enable drought tolerant forms to be produced. This would contribute to beginning the Blue Revolution advocat‐ ed by Kofi Annan in his April 2000 Millennium Address: "We need a Blue Revolution in agriculture that focuses on increasing productivity per unit of water – more crop per drop". This chapter reviews the newest aspects of the molecular and physiological mech‐

Since its isolation from cotton in the 1960s [13], the role of abscisic acid (ABA) in plant devel‐ opment and in the response of plants to environmental signals has been extensively studied. Analysis of Arabidopsis under salt and drought stress has revealed the important role ABA plays in response to these stresses [14-16]. Endogenous ABA concentrations increase under drought stress due to induction of ABA biosynthesis genes [14]. The increase in ABA repro‐ grams the gene expression pattern to regulate water relations through adjustment of cellular osmotic pressure, the closure of stomata, a reduced leaf canopy, deeper root growth and

Biosynthesis of ABA has been relatively well characterized in Arabidopsis and some data is available for other species, such as maize, tomato, potato and barley [20-24]. Knowledge about ABA biosynthesis derived from studies in Arabidopsis is highly applicable to other plant species, because the pathway and the respective genes are conserved in angiosperms. ABA is synthesized through the cleavage of a C40 carotenoid precursor, followed by a twostep conversion of the intermediate xanthoxin to ABA via ABA-aldehyde [25-27]. The path‐

hormones, such as jasmonates and ethylene [6].

104 Abiotic Stress - Plant Responses and Applications in Agriculture

anisms of drought stress response in crops.

changes in root system architecture [17-19].

**2. Abscisic acid – The best known stress messenger**

ing their targets [7-8].

Drought stress has been shown to up-regulate *NCED3* expression in Arabidopsis [14], maize [21], tomato [35], bean [15] and avocado [36]. A significant increase in NCED transcript lev‐ els can be detected within 15 to 30 min after leaf detachment or dehydration treatment [15; 37], indicating activation of NCED genes can be fairly quick. Cheng et al. [32] reported that the *AtNCED3* gene (and *AtZEP* (*Zeaxanthin Epoxidase*) and *AtAAO3* (*ABA aldehyde oxidase*)) could be induced in the Landsberg erecta background by ABA and studies in rice showed that *OsNCED3* expression was induced by dehydration [38]. Immunohistochemical analysis, using antibodies raised against AtNCED3, revealed that the protein is accumulated in the leaf vascular parenchyma cells in response to drought stress. it was not detected under nonstressed conditions. These data indicate that the drought induction of ABA biosynthesis oc‐ curs primarily in vascular tissues and that vascular-derived ABA might trigger stomatal closure via transport to guard cells [39]. *AtNCED3* expression is up-regulated by drought conditions across observed species and decreases after rehydration. At the same time, the expression level of *AtCYP707A1*, *2*, *3* and *4* (*CYTOCHROME P450, FAMILY 707, SUBFAMI‐ LY A, POLYPEPTIDE 1*, *2*, *3*, *4*) were induced by rehydration [40-41]. These genes, which en‐ code the hydroxylases that are responsible mostly for ABA catabolism, were identified in Arabidopsis, rice [42], barley [43], wheat [44] and soybean [45]. *OsABA8ox1* (*ABA-8-hydroxy‐ lase 1*) expression is induced dramatically by rehydration, which can lead to a decrease in the ABA content in rice leaves [42].

The balance between active and inactive ABA is very important for plant stress response and is achieved not only by biosynthesis and catabolism reactions, but also by conjuga‐ tion and deconjugation. ABA can be inactivated at the C-1 hydroxyl group by different chemical compounds that form various conjugates and accumulate in vacuoles or in the apoplastic space [46]. The most widespread conjugate is ABA glucosyl ester (ABA-GE) which is catalyzed by ABA glucosyltransferase [47-48]. Lee et al [49] identified the AtBG1 (BETA-1,3-GLUCANASE 1) protein which is responsible for the release of ABA from ABA-GE. Their findings showed that ABA de-conjugation plays a significant role in providing an ABA pool for plants that allows them to adjust to changing physiological and environmental conditions.

The ability of ABA to move long distances allows it to serve as a critical stress messenger. ABA transport was long assumed to be a diffusive process, mainly due to the ability of ABA to diffuse passively across biological membranes when it is in a protonated state [50]. The last step of ABA biosynthesis occurs in the cytosol where pH is estimated to be 7.2-7.4. In the apoplastic space, where ABA is meant to be transported before reaching the target cell, the pH is estimated to be around 5.0-6.0. Although ABA can be passively transported from a low pH to a higher one with a pH gradient, there is a need for the transporter to allow ABA to get into the target cell and to be exported from the cell to the apoplast. During stress re‐ sponse, the strong alkalization of apoplastic pH would slow ABA diffusive transport from the apoplastic space to the target cells. Because of the predominance of a non-protonated ABA state, there is a need for the existence of ABA transporters. The identification of ABA transporters in target cell membranes, such as the cell membranes of guard cells, has re‐ solved the problem of how ABA gets into the cells when passive transport is decreased un‐ der stress conditions. One of the identified ABA importers is ABCG40 (ARABIDOPSIS THALIANA ATP-BINDING CASSETTE G40) described by Kang et al [51]. The expression of *ABCG40* is not tissue specific and its product localizes in cell membranes [51]. Kuromori et al [52] identified another ABA importer - ABCG22 (ARABIDOPSIS THALIANA ATP-BINDING CASSETTE G22). The gene encoding this transporter is mainly expressed in guard cells. Also, the expulsion of ABA into the intercellular space is mediated by transport‐ ers such as ABCG25 (ARABIDOPSIS THALIANA ATP-BINDING CASSETTE G25). *ABCG25* is expressed mainly in vacuolar tissue, where ABA is synthesized [53].

A breakthrough in understanding ABA signaling occurred recently when several groups identified key ABA receptors. Chemical genetics emerged as the solution for the problem of the identification of receptor. Pyrabactin (4-bromo-N-[pyridine-2-yl methyl]naphthalene-1 sulfonamide) is a synthetic compound that partially mimics the inhibitory effect of ABA during seed germination and seedling development. Using a series of pyrabactin-resistant mutants and the map-based cloning approach, several genes encoding ABA-binding pro‐ teins, among them PYR1 (PYRABACTIN-RESISTANCE 1) have been identified [3]. PYR1 is one of the 14 homologs (PYL – PYRABACTIN RESISTANCE LIKE) present in the Arabidop‐ sis genome [1-4]. After receiving ABA from ABC transporters, the PYR/PYL/RCAR-ABA (PYRABACTIN-RESISTANCE 1/ PYRABACTIN RESISTANCE LIKE/ REGULATORY COM‐ PONENT OF ABA RECEPTOR) complex perceives ABA intracellularly and forms ternary complexes inhibiting clade A of PP2Cs (PROTEIN PHOSPHATASE 2C), the negative regu‐ lators of ABA signaling, such as ABI1 (ABA INSENSITIVE 1), ABI2 (ABA INSENSITIVE 2), HAB1 (HYPERSENSITIVE TO ABA1) [1-2; Table 1].

This allows the activation of down-stream targets of PP2Cs – the Sucrose nonfermenting 1 related subfamily 2 protein kinases (SnRK2), such as SnRK2.2/D, SnRK2.3/E and SnRK2.6/ OST1/E which are the key players in the regulation of ABA signaling [54-57; Figure 1].

The last enzyme, OST1 (OPEN STOMATA1), displays dominant kinase activity during drought stress response when the ABA signal is relayed to the guard cells. Mutants in *OST1* showed a wilty phenotype under water deficit conditions [58]. Mutants for the other two ABA-activated kinases, *SnRK2.2* and *SnRK2.3*, did not show a drought-sensitive phenotype [59]. The triple mutant *snrk2.2/d snrk2.3/I snrk2.6/e* displayed an extremely sensitive pheno‐ type under water deficit conditions. Transcriptomic studies of the triple mutant showed a down-regulation of genes encoding PP2Cs, which suggested a feedback loop in the tran‐ scription regulation of PP2Cs by SnRKs [54].

**Figure 1.** ABA synthesis, catabolism, conjugation and response in a scheme.

The ability of ABA to move long distances allows it to serve as a critical stress messenger. ABA transport was long assumed to be a diffusive process, mainly due to the ability of ABA to diffuse passively across biological membranes when it is in a protonated state [50]. The last step of ABA biosynthesis occurs in the cytosol where pH is estimated to be 7.2-7.4. In the apoplastic space, where ABA is meant to be transported before reaching the target cell, the pH is estimated to be around 5.0-6.0. Although ABA can be passively transported from a low pH to a higher one with a pH gradient, there is a need for the transporter to allow ABA to get into the target cell and to be exported from the cell to the apoplast. During stress re‐ sponse, the strong alkalization of apoplastic pH would slow ABA diffusive transport from the apoplastic space to the target cells. Because of the predominance of a non-protonated ABA state, there is a need for the existence of ABA transporters. The identification of ABA transporters in target cell membranes, such as the cell membranes of guard cells, has re‐ solved the problem of how ABA gets into the cells when passive transport is decreased un‐ der stress conditions. One of the identified ABA importers is ABCG40 (ARABIDOPSIS THALIANA ATP-BINDING CASSETTE G40) described by Kang et al [51]. The expression of *ABCG40* is not tissue specific and its product localizes in cell membranes [51]. Kuromori et al [52] identified another ABA importer - ABCG22 (ARABIDOPSIS THALIANA ATP-BINDING CASSETTE G22). The gene encoding this transporter is mainly expressed in guard cells. Also, the expulsion of ABA into the intercellular space is mediated by transport‐ ers such as ABCG25 (ARABIDOPSIS THALIANA ATP-BINDING CASSETTE G25). *ABCG25*

106 Abiotic Stress - Plant Responses and Applications in Agriculture

is expressed mainly in vacuolar tissue, where ABA is synthesized [53].

HAB1 (HYPERSENSITIVE TO ABA1) [1-2; Table 1].

A breakthrough in understanding ABA signaling occurred recently when several groups identified key ABA receptors. Chemical genetics emerged as the solution for the problem of the identification of receptor. Pyrabactin (4-bromo-N-[pyridine-2-yl methyl]naphthalene-1 sulfonamide) is a synthetic compound that partially mimics the inhibitory effect of ABA during seed germination and seedling development. Using a series of pyrabactin-resistant mutants and the map-based cloning approach, several genes encoding ABA-binding pro‐ teins, among them PYR1 (PYRABACTIN-RESISTANCE 1) have been identified [3]. PYR1 is one of the 14 homologs (PYL – PYRABACTIN RESISTANCE LIKE) present in the Arabidop‐ sis genome [1-4]. After receiving ABA from ABC transporters, the PYR/PYL/RCAR-ABA (PYRABACTIN-RESISTANCE 1/ PYRABACTIN RESISTANCE LIKE/ REGULATORY COM‐ PONENT OF ABA RECEPTOR) complex perceives ABA intracellularly and forms ternary complexes inhibiting clade A of PP2Cs (PROTEIN PHOSPHATASE 2C), the negative regu‐ lators of ABA signaling, such as ABI1 (ABA INSENSITIVE 1), ABI2 (ABA INSENSITIVE 2),

This allows the activation of down-stream targets of PP2Cs – the Sucrose nonfermenting 1 related subfamily 2 protein kinases (SnRK2), such as SnRK2.2/D, SnRK2.3/E and SnRK2.6/ OST1/E which are the key players in the regulation of ABA signaling [54-57; Figure 1].

The last enzyme, OST1 (OPEN STOMATA1), displays dominant kinase activity during drought stress response when the ABA signal is relayed to the guard cells. Mutants in *OST1* showed a wilty phenotype under water deficit conditions [58]. Mutants for the other two ABA-activated kinases, *SnRK2.2* and *SnRK2.3*, did not show a drought-sensitive phenotype One of the earliest plant responses to water deficit condition, and one regulated mainly in an ABA-dependent manner, is the closure of stomata. The closing or opening of the pore is a result of the osmotic shrinking or swelling, of the two surrounding stoma guard cells. ABA acts directly on the guard cells and induces stomata closure via an efflux of potassium and anions from the guard cells [60]. ABA regulation of the membrane ion channels is mediated by increased cytosolic Ca2+ resulting from the release of Ca2+ from intracellular stores and a Ca2+ influx from the extracellular space. It is worth noting that a number of mutations that affect ABA signaling in regards to stomatal action during drought have been characterized. Dominant mutations have been described in genes that encode type-2C phosphatases - ABI1 (ABA INSENSITIVE 1) and ABI2 (ABA INSENSITIVE 2) [61-62], whereas recessive muta‐ tions that lead to supersensitivity to ABA in regards to stomata closure are found in genes that encode farnesyltransferase β-subunit - ERA1 (ENHANCED RESPONSIVE TO ABA1) [63-64], a larger subunit of cap binding complex CBP80 (CAP BINDING PROTEIN 80) [65] and the Sm-like snRNP protein SAD1 (SUPERSENSITIVE TO ABA AND DROUGHT 1) [66].


**Table 1.** The nomenclature of the different soluble receptors and their PP2Cs interactors

#### **3. Abscisic acid is not the only phytohormone in stress response**

The effectiveness of ABA is regulated not only by the length of a drought or the previous stress history of a given plant, but also by other phytohormones such as jasmonates, cytoki‐ nins and ethylene. The role of jasmonic acid (JA) has been well established in regards to plant development and defense responses [67]. Recently, it was also shown that jasmonic acid (JA) and methyl jasmonate (MeJA) are involved in the regulation of drought response. When JA or MeJA are applied exogenously to plants they are converted into a biologically active form (+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile). JA-Ile is then bound by the receptor SCFCOI complex that contains the CORONATINE INSENSITIVE1 (COI1) F-box protein [68-69]. This interaction leads to the degradation of the repressor protein – JAZ (Jasmonate ZIM-domain) by the 26S proteasome, it allows MYC2 (MYC DOMAIN TRANSCRIPTION FACTOR 2) activation of a distinct JA response genes [70-72]. In the absence of JA, JAZ in‐ hibits MYC2 in order to activate the transcription of JA-inducible genes. It was showed that *MYC2* is up-regulated not only by JA, but also by ABA and drought. The described interac‐ tion between the protein specific to jasmonates - JAZ and both jasmonates and also ABA and drought-inducible MYC2 suggest the important regulatory role of JA in an ABA-dependent response to drought. A similar mechanism has been described in rice [73]. It was shown that, in addition to ABA, jasmonates also trigger stomatal closure in response to drought in various species, including Arabidopsis and barley [74-76]. Low endogenous ABA content in the ABA-deficient mutant *aba2* impairs MeJA (methyl-jasmonate)-stimulated Ca2+ elevation, which is, in turn, important metal closure. Furthermore, MeJA stimulates the expression of the ABA biosynthetic gene, *NCED3*. MeJA signaling in guard cells requires the presence of endogenous ABA [77]. Another example of cross talk between ABA and jasmonates during stress response is the up-regulation by JA of *AtPYL4* (*PYRABACTINE LIKE 4*), *AtPYL5* (*PYR‐ ABACTINE LIKE 5*) and *AtPYL6* (*PYRABACTINE LIKE 6*), which are members of the *PYR/PYL/RCAR* ABA receptor family [78]. These studies showed the importance and con‐ servation across the species of the role of JA in ABA-dependent response to drought.

**RCAR PYR/PYL PP2C interactors**

RCAR3 PYL8 HAB1[3], ABI1[4]

RCAR5 PYL11 HAB1[3], ABI1[4] RCAR6 PYL12 PP2CA/AHG3[2]

RCAR8 PYL5 HAB1[3], ABI1[4]

RCAR10 PYL4 HAB1[2], ABI1[4] RCAR11 PYR1 HAB1[2], ABI1[4] RCAR12 PYL1 HAB1[2], ABI1[4]

RCAR13 PYL3 HAB1[2] RCAR14 PYL2 HAB1[2]

**Table 1.** The nomenclature of the different soluble receptors and their PP2Cs interactors

**3. Abscisic acid is not the only phytohormone in stress response**

The effectiveness of ABA is regulated not only by the length of a drought or the previous stress history of a given plant, but also by other phytohormones such as jasmonates, cytoki‐ nins and ethylene. The role of jasmonic acid (JA) has been well established in regards to plant development and defense responses [67]. Recently, it was also shown that jasmonic acid (JA) and methyl jasmonate (MeJA) are involved in the regulation of drought response. When JA or MeJA are applied exogenously to plants they are converted into a biologically active form (+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile). JA-Ile is then bound by the receptor SCFCOI complex that contains the CORONATINE INSENSITIVE1 (COI1) F-box protein [68-69]. This interaction leads to the degradation of the repressor protein – JAZ (Jasmonate ZIM-domain) by the 26S proteasome, it allows MYC2 (MYC DOMAIN TRANSCRIPTION FACTOR 2) activation of a distinct JA response genes [70-72]. In the absence of JA, JAZ in‐ hibits MYC2 in order to activate the transcription of JA-inducible genes. It was showed that *MYC2* is up-regulated not only by JA, but also by ABA and drought. The described interac‐ tion between the protein specific to jasmonates - JAZ and both jasmonates and also ABA and drought-inducible MYC2 suggest the important regulatory role of JA in an ABA-dependent response to drought. A similar mechanism has been described in rice [73]. It was shown that, in addition to ABA, jasmonates also trigger stomatal closure in response to drought in

RCAR9 PYL6 ABI1[1],[4], ABI2[1], HAB1[1]

RCAR2 PYL7 ABI1[4]

RCAR4 PYL10 ABI1[4]

RCAR7 PYL13

108 Abiotic Stress - Plant Responses and Applications in Agriculture

RCAR1 PYL9 ABI1[1],[4], ABI2[1], HAB1[1]

Cytokinins (CKs) are another group of hormones involved in stress responses [79-80]. Cytokinins regulate cell proliferation and differentiation [81]. Abiotic stresses, such as drought, decrease the biosynthesis and transport of CKs from roots to shoots [82]. An in‐ creased concentration of CKs in xylem has been shown to decrease stomatal sensitivity to ABA [83]. The same effect was observed when exogenous CKs were applied [84-85]. When a plant encounters mild drought conditions, it is not necessary to close the stoma‐ ta and further limit its photosynthetic rate. Since the decline in CK content increases the stomatal sensitivity to ABA, avoidance of this phenomenon might help in obtaining a better yield from plants that experience mild drought. CK up-regulation can be achieved by reduced expression of a gene that encodes cytokinin oxidase, an enzyme that de‐ grades CKs. In addition to maintaining a better photosynthetic rate, increased levels of CKs lead to enhanced activity of the cell-cycle genes, and the consequent, increase in cell number may result in improved grain filling [86]. The process of grain filling is actually an increase in cell number and cell filling in the endosperm [87]. There is a generally positive relationship between endosperm cell number and grain weight in wheat [88], barley [89], maize [90] and rice [91]. Thus, endosperm cell number is one important fac‐ tor determining grain weight [87]. Taking into account that endosperm cell number in cereal crops is established during an early phase of development, it is assumed that this step can be regulated by cytokinins [87]. Another manipulation of the CK level in plant tissues was achieved by seed inoculation with CK-producing bacteria, gradually releas‐ ing CKs within the physiological concentration range [92]. Wheat plants in which seeds were treated with such bacteria and grown under mild drought condition gave a 30-60% higher yield than non-treated controls. Since a high level of CKs improves grain quality and photosynthesis rate, and a high level of ABA increases root extension rate, osmopro‐ tectant activity, and solute biosynthesis, another aim of breeders is to obtain a high con‐ tent of both ABA and CKs under mild drought conditions Wilkinson et al. [6].

Ethylene, a gaseous plant hormone that inhibits root growth and development, is involved in stress-induced leaf senescence and can contribute to reducing the rate of photosynthesis [93-95]. ABA can modulate the influence of ethylene on stomatal conductance. Contradicto‐ ry results have been published regarding the role of ethylene in stomatal action. Desikan et al. [96] showed that ethylene induces stomatal closure, whereas Tanaka et al. [97] and Wil‐ kinson and Davies [98] proved that ethylene can antagonize ABA action in the stomata. This is probably due to the fact that the concentration of neither hormone is important for the fi‐ nal effect but rather the ratio of ABA to ethylene [99; 18].

## **4. With a little help from arabidopsis – Transferring knowledge from weeds to crops**

A small genome, short life cycle, small stature, prolific seed production, ease of transforma‐ tion, a completely sequenced genome, a near saturation insertion mutant collection, a ge‐ nome array that contains the entire transcriptome – these are the major advantages of using the model plant Arabidopsis in studies on the molecular basis of responses to environmental stresses including drought. The identification of stress-related genes, their functions and the pathways they are involved in, has been facilitated by an increasing number of molecular tools, genetic resources and the large number of web-based databases available for Arabi‐ dopsis (Table 2).

Genomic resources and results obtained of Arabidopsis provide a resource for exploitation in crops. Using sequence homology, EST (Expressed Sequence Tag) libraries, and the fulllength cDNA repositories available for crop species, there is a possibility of a simple transfer of data revealed in Arabidopsis to identify a gene of interest in a crop species (Figure 2).

**Figure 2.** The pipeline of identification of barley homologous gene based on Arabidopsis and rice information. Gen‐ Bank: http://www.ncbi.nlm.nih.gov/genbank/; TAIR: www.arabidopsis.org; BLAST: http://blast.ncbi.nlm.nih.gov/ Blast.cgi; GeneIndices: http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=barley; GenScan: http:// genes.mit.edu/GENSCAN.html; Splign: http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi; GreenPhyl: http://green‐ phyl.cirad.fr/v2/cgi-bin/index.cgi.


is probably due to the fact that the concentration of neither hormone is important for the fi‐

A small genome, short life cycle, small stature, prolific seed production, ease of transforma‐ tion, a completely sequenced genome, a near saturation insertion mutant collection, a ge‐ nome array that contains the entire transcriptome – these are the major advantages of using the model plant Arabidopsis in studies on the molecular basis of responses to environmental stresses including drought. The identification of stress-related genes, their functions and the pathways they are involved in, has been facilitated by an increasing number of molecular tools, genetic resources and the large number of web-based databases available for Arabi‐

Genomic resources and results obtained of Arabidopsis provide a resource for exploitation in crops. Using sequence homology, EST (Expressed Sequence Tag) libraries, and the fulllength cDNA repositories available for crop species, there is a possibility of a simple transfer of data revealed in Arabidopsis to identify a gene of interest in a crop species (Figure 2).

**Figure 2.** The pipeline of identification of barley homologous gene based on Arabidopsis and rice information. Gen‐ Bank: http://www.ncbi.nlm.nih.gov/genbank/; TAIR: www.arabidopsis.org; BLAST: http://blast.ncbi.nlm.nih.gov/ Blast.cgi; GeneIndices: http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=barley; GenScan: http:// genes.mit.edu/GENSCAN.html; Splign: http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi; GreenPhyl: http://green‐

**4. With a little help from arabidopsis – Transferring knowledge from**

nal effect but rather the ratio of ABA to ethylene [99; 18].

110 Abiotic Stress - Plant Responses and Applications in Agriculture

**weeds to crops**

dopsis (Table 2).

phyl.cirad.fr/v2/cgi-bin/index.cgi.

**Table 2.** Web-based resources for gene expression analysis for Arabidopsis and other species, including crops.

In many cases, not only structural proteins, such as ion channels are conserved between Arabidopsis and other plant species, but also regulatory proteins, such as transcription factors. In addition, it is worth adding that entire transcriptional regulons can also be conserved, as in case of the ABA signalosome PYR/PYL/RCAR-PP2Cs-SNRKs. 'Only after we understand how plants respond to stress — in many cases first in Arabidopsis and then applying the Arabidopsis model to crop plants — will we be able to begin engi‐ neering stress tolerance' [100].

During the last decade, microarrays have become a routine tool for the analysis of tran‐ scripts, not only in model Arabidopsis but also in crops, such as barley and rice. Interesting‐ ly, interspecies comparisons between distantly related species, Arabidopsis and rice or barley revealed conserved patterns of expression in the case of many orthologs genes [101-103]. Comparative analyses showed that orthologous of specific genes in rice or barley are also responsive to stress similar to Arabidoposis [103; 102]. Mochida et al. [104] used publicly available transcriptome data to investigate regulatory networks of the genes in‐ volved in various developmental aspects including drought in barley. On the basis of a com‐ parative analysis between barley and model species, such as Arabidopsis or Brachypodium, modules of genes putatively involved in drought response have been identified. In addition to these computational approaches, Moumeni et al. [105] have undertaken a comparative analysis of the rice root transcriptome under drought stress. They used two pairs each of drought-tolerant and susceptible rice NILs (Near Isogenic Lines). Global gene expression analysis revealed that about 55% of the genes differentially expressed were in rice roots un‐ der drought stress. The drought-tolerant lines showed an up-regulation of the genes in‐ volved in secondary metabolism, amino acid metabolism, response to stimulus, defense response, transcription and signal transduction. Proteomic analysis of drought-sensitive and drought-tolerant barley lines performed by Kausar et al. [106] revealed an increased level of metabolism, photosynthesis and amino acid synthesis-related proteins in tolerant geno‐ types, whereas a decreased level was observed in sensitive forms. The data confirmed the results described previously in other species and should that similar processes play a signifi‐ cant role in barley's adaptation to stress conditions.

## **5. The huge role of tiny molecules (microRNA) in drought response**

Small non-coding RNAs – miRNAs, which were first reported in the nematode *Ceanorhabdi‐ tis elegans* in 1993 [107] and which are responsible for the phenomenon of RNA interference, have become recognized as very important regulatory components of the cell signaling. miRNAs have been shown to be highly conserved gene expression regulators across species [108-109]. The first plant miRNA was isolated from Arabidopsis [110]. To date, approximate‐ ly 5000 plant miRNAs have been identified and deposited in miRbase (19.0 release) includ‐ ing 299 miRNA from Arabidopsis, 135 from Brachypodium, 206 from sorghum, 42 from wheat, 591 from rice, 172 from maize and 67 from barley [111]. miRNAs are small regulatory RNAs of a 20-22 nucleotide length that are encoded by endogenous *MIR* genes. Their pri‐ mary transcripts are partially double-stranded stem-loop structures. Pri-miRNAs in plants are processed by DCL1 (DICER-LIKE 1) HYL1 (HYPONASTIC LEAVES 1), SE (SERRATED) proteins into pre-miRNA hairpin precursors which are finally converted into short duplexes – mature miRNAs. The duplexes are then methylated at the 3' terminus and exported to the cytoplasm. In the cytoplasm, single-stranded miRNAs are incorporated in the AGO (ARGO‐ NAUTE) protein, the catalytic compound of the RISC (RNA-INDUCED SILENCING COM‐ PLEX) complex, and guide the RISC to the target mRNAs by sequence complementarity to negatively regulate their expression [112].

In many cases, not only structural proteins, such as ion channels are conserved between Arabidopsis and other plant species, but also regulatory proteins, such as transcription factors. In addition, it is worth adding that entire transcriptional regulons can also be conserved, as in case of the ABA signalosome PYR/PYL/RCAR-PP2Cs-SNRKs. 'Only after we understand how plants respond to stress — in many cases first in Arabidopsis and then applying the Arabidopsis model to crop plants — will we be able to begin engi‐

During the last decade, microarrays have become a routine tool for the analysis of tran‐ scripts, not only in model Arabidopsis but also in crops, such as barley and rice. Interesting‐ ly, interspecies comparisons between distantly related species, Arabidopsis and rice or barley revealed conserved patterns of expression in the case of many orthologs genes [101-103]. Comparative analyses showed that orthologous of specific genes in rice or barley are also responsive to stress similar to Arabidoposis [103; 102]. Mochida et al. [104] used publicly available transcriptome data to investigate regulatory networks of the genes in‐ volved in various developmental aspects including drought in barley. On the basis of a com‐ parative analysis between barley and model species, such as Arabidopsis or Brachypodium, modules of genes putatively involved in drought response have been identified. In addition to these computational approaches, Moumeni et al. [105] have undertaken a comparative analysis of the rice root transcriptome under drought stress. They used two pairs each of drought-tolerant and susceptible rice NILs (Near Isogenic Lines). Global gene expression analysis revealed that about 55% of the genes differentially expressed were in rice roots un‐ der drought stress. The drought-tolerant lines showed an up-regulation of the genes in‐ volved in secondary metabolism, amino acid metabolism, response to stimulus, defense response, transcription and signal transduction. Proteomic analysis of drought-sensitive and drought-tolerant barley lines performed by Kausar et al. [106] revealed an increased level of metabolism, photosynthesis and amino acid synthesis-related proteins in tolerant geno‐ types, whereas a decreased level was observed in sensitive forms. The data confirmed the results described previously in other species and should that similar processes play a signifi‐

**5. The huge role of tiny molecules (microRNA) in drought response**

Small non-coding RNAs – miRNAs, which were first reported in the nematode *Ceanorhabdi‐ tis elegans* in 1993 [107] and which are responsible for the phenomenon of RNA interference, have become recognized as very important regulatory components of the cell signaling. miRNAs have been shown to be highly conserved gene expression regulators across species [108-109]. The first plant miRNA was isolated from Arabidopsis [110]. To date, approximate‐ ly 5000 plant miRNAs have been identified and deposited in miRbase (19.0 release) includ‐ ing 299 miRNA from Arabidopsis, 135 from Brachypodium, 206 from sorghum, 42 from wheat, 591 from rice, 172 from maize and 67 from barley [111]. miRNAs are small regulatory RNAs of a 20-22 nucleotide length that are encoded by endogenous *MIR* genes. Their pri‐ mary transcripts are partially double-stranded stem-loop structures. Pri-miRNAs in plants

neering stress tolerance' [100].

112 Abiotic Stress - Plant Responses and Applications in Agriculture

cant role in barley's adaptation to stress conditions.

Plant microRNAs are involved in various developmental processes including flowering, and leaf, stem and root development [113-115]. Jones-Rhoades and Bartel [116] drew the atten‐ tion of plant biologists to the miRNA engagement in stress response for the first time. To gain an insight into the role of miRNAs in the regulation of transcripts in response to drought, several projects on the identification of the miRNAs related to stress response in crops were undertaken. Using deep sequencing techniques, Zhou et al [117] identified nine‐ teen new miRNAs that are induced by drought in rice, among them eleven down-regulated and eight up-regulated miRNAs. In addition, they identified nine miRNAs that showed an opposite expression to that observed in drought-stressed Arabidopsis (Table 3). A similar approach was used by Kulcheski et al. [118] in soybean, which revealed 11 miRNAs that are related to drought stress (Table 3). Based on bioinformatic prediction and then verification of the obtained results using RT-qPCR, Xu et al. [119] identified 21 miRNAs differently ex‐ pressed during water stress in maize (Table 3). A similar approach using bioinformatic pre‐ diction of miRNAs on dehydration stress was undertaken by Kantar et al. [7], who found four miRNAs that are related to drought stress in barley (Table 3). Deep sequencing of a small RNA library in the case of barley was performed by Lv et al. [8]. They showed that six miRNAs specific for stress response. hvu-MIRn026a, hvu-MIRn029, hvu-MIR035, hvu-MIR156d exhibited higher expression in response to salt and drought stress, whereas hvu-MIR396d and hvu-MIR399b showed a higher expression only in drought-stressed plants. Additionally, the authors observed that hvu-mir029 was highly expressed after drought treatment and at a very low level under non-stressed conditions, which suggests the impor‐ tant role of this molecule in water deficit response (Table 3).

To understand the function of newly identified miRNAs, the putative target transcripts have to be predicted. In order to identify microRNAs target transcripts, Kantar et al [7] performed computational studies and a modified 5' RLM-RACE (RNA ligase-mediated 5' rapid ampli‐ fication of cDNA ends) in barley. Seven cleaved miRNA transcripts were retrieved from drought-stressed leaf samples as targets for hvu-MIR165, hvu-MIR166, hvu-MIR156, hvu-MIR2055, hvu-MIR171, hvu-MIR172, hvu-MIR397 and hvu-MIR159. The identified targets are mainly transcription factors that play a role in plant development, morphology and de‐ termination of the flowering time. *SCRL6* (*SCARECROW LIKE 6*) encodes a transcription fac‐ tor that is involved in diverse plant developmental processes such as leaf or root growth and is the target of hvu-MIR171, *ARF10* (*AUXIN RESPONSIVE FACTOR 10*) encodes a transcrip‐ tion factor that negatively regulates auxin signaling and is the target of hvu-MIR160, *SBP* (*SQUAMOSA PROMOTER BINDING PROTEIN*) is a transcription factor that is mainly im‐ portant for leaf development and is the target of hvu-MIR156a, and *MYB33* (*MYB DOMAIN*

*PROTEIN 33*) is a transcription factor that is involved in ABA and GA signaling and is the target of hvu-MIR159a [7].


\* red indicates down-regulation by drought, green indicates up-regulation by drought, blue indicates regulation op‐ posite to that observed in Arabidopsis, black indicates no information about regulation by drought

**Table 3.** miRNA related to drought in different crop species.

## **6. From the cell to the organism level – Phenotyping of drought-treated crops**

In order to understand gene-to-phenotype relationships in the plant response to drought stress, it is vital to decipher the physiological and genetic bases of this process. Recent ad‐ vances in crop physiology, genomics and plant phenotyping have provided a broader knowledge and better tools for crop improvement under stress conditions [120]. Maintain‐ ing a high yield under drought conditions has become a priority for breeders. However, the physiological basis of yield maintenance under drought is not yet fully understood, of the complexity of the mechanisms that plants can use to maintain growth in conditions due to water deficit [120]. Quantitative trait loci (QTL) for genes conferring a yield benefit under drought conditions first need to be identified in phenotypic screens and then incorporated into crops using marker-assisted selection [121]. Direct selection for yield in drought-prone environments, however, has proven to be difficult. Drought stress is a dynamic process and can occur at different periods of the crop cycle and with different intensities. Consequently, plants have developed various strategies in response to drought: tolerance, escape and avoidance. Ludlow [122] defined three strategies plants use to cope with drought stress: drought tolerance is the ability of a plant to cope with water deficit through low tissue water potential, drought escape is defined as completion of the life cycle just before a severe drought starts, and drought avoidance is plant maintenance of high tissue water potential by minimizing water loss or maximizing water uptake. The final mechanism conveys the ability to survive and recover rapidly after a severe stress through protective mechanisms, such as cell wall folding, membrane protection, and the accumulation of antioxidants [123-124].

*PROTEIN 33*) is a transcription factor that is involved in ABA and GA signaling and is the

Species Identified miRNA related to drought References

osa-MIR529, osa-MIR896, osa-MIR1030, osa-MIR1035, osa-MIR1050, osa-MIR1088, osa-MIR1126, osa-MIR395, osa-MIR474, osa-MIR845, osa-MIR851, osa-MIR854, osa-MIR901, osa-MIR903 and osa-MIR1125, osa-MIR156, osa-MIR168, osa-MIR170, osa-MIR171, osa-MIR172, osa-

zma-MIR161, zma-MIR397, zma-MIR446, zma-MIR479, zma-MIR530, zma-MIR776, zma-MIR782, zma-MIR815a, zma-MIR818a, zma-MIR820, zma-MIR828, zma-MIR834, zmaMIR1, zma-MIR2, zma-MIR3, zma-MIR4, zma-MIR5, zma-MIR6, zma-MIR7, zma-MIR8, zma-MIR9

[117]

[118]

[119]

osa-MIR170, osa-MIR172, osa-MIR397, osa-MIR408,

MIR319, osa-MIR396, osa-MIR397, osa-MIR408

gma-MIR4415b, gma-MIR-Seq07

**Table 3.** miRNA related to drought in different crop species.

gma-MIR166-5p, gma-MIR169f-3p, gma-MIR1513c, gma-MIR397ab, gma-MIR-Seq13, gma-MIR-Seq11, gma-MIRSeq15, gma-MIR166f, gma-MIR-482bd-3p,

barley hvu-MIR156, hvu-MIR166, hvu-MIR171, hvu-MIR408 [7]

hvu-MIRn026a, hvu-MIRn029, hvu-MIR035, hvu-MIR156d, hvu-

posite to that observed in Arabidopsis, black indicates no information about regulation by drought

MIR396d, hvu-MIR399b [8]

**6. From the cell to the organism level – Phenotyping of drought-treated**

In order to understand gene-to-phenotype relationships in the plant response to drought stress, it is vital to decipher the physiological and genetic bases of this process. Recent ad‐ vances in crop physiology, genomics and plant phenotyping have provided a broader knowledge and better tools for crop improvement under stress conditions [120]. Maintain‐ ing a high yield under drought conditions has become a priority for breeders. However, the physiological basis of yield maintenance under drought is not yet fully understood, of the complexity of the mechanisms that plants can use to maintain growth in conditions due to water deficit [120]. Quantitative trait loci (QTL) for genes conferring a yield benefit under drought conditions first need to be identified in phenotypic screens and then incorporated into crops using marker-assisted selection [121]. Direct selection for yield in drought-prone environments, however, has proven to be difficult. Drought stress is a dynamic process and

\* red indicates down-regulation by drought, green indicates up-regulation by drought, blue indicates regulation op‐

target of hvu-MIR159a [7].

114 Abiotic Stress - Plant Responses and Applications in Agriculture

rice

soybean

maize

**crops**

In order to incorporate traits that confer drought tolerance into molecular breeding pro‐ grams, phenotyping protocols are extremely important [125]. With the wide availability of genetic resources, such as mutant populations (TILLING) or mapping populations, high-throughput phenotyping will become an essential asset in closing the gap between plant physiology and genetics [126- 127]. It is worth noting that a complex set of both abiotic and biotic stresses shapes the natural environment during plant development drought stress is just one of many factors. It is hard to exclude one of the stress path‐ ways and to analyze it in isolation from others because the cascade of stress response is a complicated web of overlapping pathways. When studying drought tolerance in plants, it is very difficult to control and monitor the level and onset of water deficit, since it is a dynamic process and a combination of the available water in the soil and the plant wa‐ ter status. Continuous measurements are needed in order to link the level of drought ex‐ perienced by the plant with the physiological changes occurring in response to it [125]. Under greenhouse conditions, water use can be monitored by weighing the pots or us‐ ing TDR (Time Domain Reflectometry) soil moisture meters [128]. The water supply can be regulated at high-throughput automated screening facilities by using the classical wa‐ ter withdrawal approach [14] and maintaining a constant soil water status [129].

Another difficult issue is how to describe plant response to drought at the physiological lev‐ el using properly chosen physiological, but also morphological, traits. In breeding programs for improved drought tolerance, crop traits associated with the conceptual framework for yield drought adaptation have been proposed by Passioura [130]. This framework has three important drivers: (1) water uptake (WU), (2) water-use efficiency (WUE) and (3) harvest in‐ dex (HI). Several traits are highly associated with these three aspects of Passioura model. With regard to WU, the best method would be direct selection for variation in root architec‐ ture but since this is hard to perform, stomatal conductance, mainly the canopy tempera‐ ture, is measured. This provides indirect indicators of water uptake by roots [131]. To estimate WUE, carbon isotope discrimination is used. A high affinity of Rubisco for the more common 12C isotope over the 13C indicates a lower WUE, whereas a lower discrimina‐ tion value indicates a higher WUE [131]. In the case of HI, the extreme sensitivity of repro‐ ductive processes to drought may result in reproductive failure, which is associated with a low HI value [132].

Water stress reduces photosynthesis in the leaves of higher plants. It is linked with a de‐ creased diffusion of CO2 from the atmosphere to the site of carboxylation [133-134]. Under‐ lying this process is the stomatal closure during short-term drought and photoinhibition damage, and the inactivation of RuBisCO under long-term stress [135].

Stomatal closure is one of the first responses to drought conditions which might result in cell dehydration or runaway xylem cavitation [136]. A good illustration of this process is stomatal behavior in the midday, when either stomatal closure or decreased stomatal conductance can be observed. Both responses are mediated by ABA synthesized in re‐ sponse to dehydration conditions [18]. When decreased stomatal conductance is com‐ bined with sustained high irradiance, leaves are subjected to excess energy relative to the available CO2 and the rate of reducing power can overcome the rate of its use in the Cal‐ vin cycle. These processes lead to the down-regulation of photosynthetic and even photo‐ inhibition. Plants have evolved mechanisms of defense to protect photosynthesis. Such protection can be achieved by the regulated thermal dissipation that occurs in the lightharvesting complexes [137].

Processes associated with the photosynthetic apparatus can be measured using chlorophyll fluorescence. Experiments with chlorophyll fluorescence were first carried out by Kautsky and Hirsch [138]. Since then, this technique has progressed quickly and chlorophyll fluores‐ cence can be easily measured using commercially available chlorophyll fluorimeters which enable the measurements of the photochemical and non-photochemical processes involved in the fluorescence quenching that occurs in the presence of light [139]. The Fv/Fm ratio rep‐ resenting the maximum quantum yield of the primary photochemical reaction of photosys‐ tem II (PSII) is the most often used parameter. Environmental stresses that affect PSII efficiency lead to the characteristic decrease in the value of this parameter [140]. Fluores‐ cence kinetics of chlorophyll a, the 'OJIP/JIP-test' named after the basic steps of the transient by which parameters quantifying PSII behavior are calculated (O is the fluorescence intensi‐ ty F0 (at 50 μs); J is the fluorescence intensities FJ (at 2 ms); I is FI (at 30 ms) and P is the maximal fluorescence intensity, FP = FM) is an informative tool for studying the effects of different environmental stresses on photosynthesis [141-142;10;143]. This analysis offers sim‐ ple equations to express the equilibrium between the inflow and outflow of the entire ener‐ gy flux within PSII; it also provides information about the fate of absorbed energy. Some of the parameters calculated using the JIP-test are related to energy fluxes for light absorption (ABS), the trapping of excitation energy (TR) and electron transport (ETR) per reaction cen‐ ter (RC) or per sample area called cross-section (CS). Their estimates are based on the analy‐ sis of several groups of measured and calculated parameters. Analyses performed using these parameters are quick and the measurements are non-invasive [10].

In addition to the photosynthesis process, it was observed that the alteration of leaf an‐ gle caused by dehydration, towards smaller angles, would diminish intercepted radiation and carbon assimilation, and also have an important protective role against excess solar energy [144]. There is also a correlation between the rate of photosynthesis and the age of the leaf. Younger leaves tend to be more resistant to drought than older ones. When a severe reduction in the size of the leaf canopy occurs, as a result of shedding older leaves, it allows a plant to recover faster following rehydration [145]. Photosynthetic re‐ covery following rehydration plays a pivotal role in drought-tolerance mechanisms and prevents a dramatic decline in crop yields [146]. It was shown that recovery from a se‐ vere stress is a two-step process. The first phase occurs during the first hours or days af‐ ter rewatering and corresponds to an improvement of leaf water status and the reopening of stomata [147]. The second stage lasts a few days and requires the *de novo* synthesis of photosynthetic proteins [148-149].

Water stress reduces photosynthesis in the leaves of higher plants. It is linked with a de‐ creased diffusion of CO2 from the atmosphere to the site of carboxylation [133-134]. Under‐ lying this process is the stomatal closure during short-term drought and photoinhibition

Stomatal closure is one of the first responses to drought conditions which might result in cell dehydration or runaway xylem cavitation [136]. A good illustration of this process is stomatal behavior in the midday, when either stomatal closure or decreased stomatal conductance can be observed. Both responses are mediated by ABA synthesized in re‐ sponse to dehydration conditions [18]. When decreased stomatal conductance is com‐ bined with sustained high irradiance, leaves are subjected to excess energy relative to the available CO2 and the rate of reducing power can overcome the rate of its use in the Cal‐ vin cycle. These processes lead to the down-regulation of photosynthetic and even photo‐ inhibition. Plants have evolved mechanisms of defense to protect photosynthesis. Such protection can be achieved by the regulated thermal dissipation that occurs in the light-

Processes associated with the photosynthetic apparatus can be measured using chlorophyll fluorescence. Experiments with chlorophyll fluorescence were first carried out by Kautsky and Hirsch [138]. Since then, this technique has progressed quickly and chlorophyll fluores‐ cence can be easily measured using commercially available chlorophyll fluorimeters which enable the measurements of the photochemical and non-photochemical processes involved in the fluorescence quenching that occurs in the presence of light [139]. The Fv/Fm ratio rep‐ resenting the maximum quantum yield of the primary photochemical reaction of photosys‐ tem II (PSII) is the most often used parameter. Environmental stresses that affect PSII efficiency lead to the characteristic decrease in the value of this parameter [140]. Fluores‐ cence kinetics of chlorophyll a, the 'OJIP/JIP-test' named after the basic steps of the transient by which parameters quantifying PSII behavior are calculated (O is the fluorescence intensi‐ ty F0 (at 50 μs); J is the fluorescence intensities FJ (at 2 ms); I is FI (at 30 ms) and P is the maximal fluorescence intensity, FP = FM) is an informative tool for studying the effects of different environmental stresses on photosynthesis [141-142;10;143]. This analysis offers sim‐ ple equations to express the equilibrium between the inflow and outflow of the entire ener‐ gy flux within PSII; it also provides information about the fate of absorbed energy. Some of the parameters calculated using the JIP-test are related to energy fluxes for light absorption (ABS), the trapping of excitation energy (TR) and electron transport (ETR) per reaction cen‐ ter (RC) or per sample area called cross-section (CS). Their estimates are based on the analy‐ sis of several groups of measured and calculated parameters. Analyses performed using

damage, and the inactivation of RuBisCO under long-term stress [135].

116 Abiotic Stress - Plant Responses and Applications in Agriculture

these parameters are quick and the measurements are non-invasive [10].

In addition to the photosynthesis process, it was observed that the alteration of leaf an‐ gle caused by dehydration, towards smaller angles, would diminish intercepted radiation and carbon assimilation, and also have an important protective role against excess solar energy [144]. There is also a correlation between the rate of photosynthesis and the age of the leaf. Younger leaves tend to be more resistant to drought than older ones. When a severe reduction in the size of the leaf canopy occurs, as a result of shedding older

harvesting complexes [137].

It is also worth noting that other phenotype analyses should be performed in order to obtain a complete picture of the stress response of a given plant. Relative Water Content (RWC), which was proposed by Sinclair and Ludlow [12], is the most often used assay to assess plant response to a water deficit. This simple test allows the establishment of relative water content in a leaf of control and drought-treated plants. Detached leaves are weighed and sa‐ turated with water for 24 h, then again weighed and dried for 48 h and weighed again. RWC is calculated from the following formula: RWC (%) = [(FM - DM)/(TM - DM)] \* 100, where, FM, DM, and TM are the fresh, dry and turgid masses of the tissue weighted, respectively.

The degree of cell membrane stability (CMS) is considered to be one of the best physiologi‐ cal indicators of drought-stress tolerance. It can be evaluated using measurements of solute leakage from plant tissue [150-151].

In response to drought stress, plants are able to adjust osmotic pressure by synthesizing os‐ moprotectants such as proline, the water soluble carbohydrates that behave like a molecular weapon against dehydration within the cell. There are several methods used in order to esti‐ mate the accumulation of endogenous proline or sugars in drought-treated plants [152].

Several morphological traits that have an impact on drought tolerance have been ob‐ served. Growth inhibition resulting from drought-induced ABA biosynthesis was ob‐ served in plants exposed to stress [153]. A number of studies have shown that wax deposition on the leaf surface increased in response to drought and an associated im‐ provement in drought tolerance was observed in oat, rice, sorghum, wheat and barley plants that had an increased wax layer [154 -157]. Enhanced drought tolerance was also gained by plants having a reduced number of stomata, which was probably dependent on the accumulation of waxes [158]. Yang et al [158] performed analysis on an ox*-win1*/ *shn1* (overexpressor *wax inducer 1*/*shine 1*) mutant. *WIN1*/*SHN1* encodes a transcription factor that regulates the expression of genes that control the accumulation of cuticular wax. Analyses performed by Yang et al [158] showed that induction of *WIN1*/*SHN1* ex‐ pression by drought is correlated with an increased expression of the genes involved in wax accumulation, and on the other hand, a decreased expression of the genes involved in stomatal development. These results suggest that the drought-tolerant phenotype of analyzed by Yang et al [158] forms caused by induction of *WIN1*/*SHN1* may be due to a reduced number of stomata as well as wax accumulation.

There are now several high-throughput phenotyping techniques available for the measure‐ ment of some of the traits described above. One of these is thermal infrared imaging, or in‐ frared thermography (IRT), which is used to measure the leaf or canopy temperature. Evaporation is a main determinant of leaf temperature. There is a direct relationship be‐ tween leaf temperature, transpiration rate and stomatal conductance [159-161]. Drought-tol‐ erant genotypes can maintain a higher stomatal conductance and also a higher rate of photosynthesis, as was mentioned above, thus these genotypes could be identified as having a lower canopy temperature than the sensitive genotypes [162-163].

#### **7. GM crops – are they a solution?**

Genetic modification of crops is a controversial issue. Some aspects of genetic modification that have potential to improve drought tolerance in crops are presented here. Biotechnologi‐ cal approaches may involve the overexpression of genes related to osmotic adjustment, chaperones and antioxidants [reviewed in 164-165]. Also, ectopic expression or suppression of regulatory genes, such as genes that encode transcription factors, is widely used [166]. Re‐ cent studies on rice led to the identification of genes involved in three pathways that can be manipulated in order to improve drought tolerance in crops: the gene that encodes β-caro‐ tene hydroxylase, which confers drought resistance by increasing xanthophylls and ABA synthesis [167], the *DST1* (*DROUGHT AND SALT TOLERANT 1*) gene that regulates stoma‐ tal closure and density under drought stress [168] and the *TLD1*/ *OsGH3.13* (*INCREASED NUMBER OF TILLERS, ENLARGED LEAF ANGLES, AND DWARFISM*) gene whose downregulation enhanced drought tolerance in rice [169]. Although several genes that can im‐ prove the drought tolerance of crops have already been identified, progress in the commercialization of the traits controlled by these genes has been slow [165]. One of the genes that has been successfully introduced into a crop plant and that gave improved drought tolerance in field trials was the gene encoding Cold Shock Protein B (CspB) RNA chaperone from *Bacillus subtilis*. The *CspB* gene is important in the ability of bacteria to adapt to cold, and its overexpression in plants was shown to provide drought tolerance in Arabi‐ dopsis, rice and maize [170]. Results from field experiments showed that a maize line ex‐ pressing the *CspB* gene had a higher yield under water deficit conditions than the control and expressed a yield equivalent to the control under non-stressed conditions. Tests are in progress in 2012 on commercial farms, [171; http://www.monsanto.com/products/Pages/ corn-pipeline.aspx#firstgendroughttolerantcorn]. The value of a biotechnological approach to improving crop yields under drought stress conditions is becoming evident with the first demonstrations of improved drought tolerance in crops in the field (reviewed in [171]).

#### **8. Conclusions and perspectives**

In order to achieve a full understanding of drought-response mechanisms in plants and to make use of this understanding to produce crops with improved drought tolerance, there is a need to combine the data derived from different studies. Detailed analyses of the networks of protein interactions, the co-expression of genes, metabolic factors, etc. should provide in‐ sights into the key regulators of drought response [172-173]. Biotechnological approaches can also be promising in improving drought tolerance in crops based on previously ob‐ tained and integrated knowledge [171].

## **Acknowledgements**

Evaporation is a main determinant of leaf temperature. There is a direct relationship be‐ tween leaf temperature, transpiration rate and stomatal conductance [159-161]. Drought-tol‐ erant genotypes can maintain a higher stomatal conductance and also a higher rate of photosynthesis, as was mentioned above, thus these genotypes could be identified as having

Genetic modification of crops is a controversial issue. Some aspects of genetic modification that have potential to improve drought tolerance in crops are presented here. Biotechnologi‐ cal approaches may involve the overexpression of genes related to osmotic adjustment, chaperones and antioxidants [reviewed in 164-165]. Also, ectopic expression or suppression of regulatory genes, such as genes that encode transcription factors, is widely used [166]. Re‐ cent studies on rice led to the identification of genes involved in three pathways that can be manipulated in order to improve drought tolerance in crops: the gene that encodes β-caro‐ tene hydroxylase, which confers drought resistance by increasing xanthophylls and ABA synthesis [167], the *DST1* (*DROUGHT AND SALT TOLERANT 1*) gene that regulates stoma‐ tal closure and density under drought stress [168] and the *TLD1*/ *OsGH3.13* (*INCREASED NUMBER OF TILLERS, ENLARGED LEAF ANGLES, AND DWARFISM*) gene whose downregulation enhanced drought tolerance in rice [169]. Although several genes that can im‐ prove the drought tolerance of crops have already been identified, progress in the commercialization of the traits controlled by these genes has been slow [165]. One of the genes that has been successfully introduced into a crop plant and that gave improved drought tolerance in field trials was the gene encoding Cold Shock Protein B (CspB) RNA chaperone from *Bacillus subtilis*. The *CspB* gene is important in the ability of bacteria to adapt to cold, and its overexpression in plants was shown to provide drought tolerance in Arabi‐ dopsis, rice and maize [170]. Results from field experiments showed that a maize line ex‐ pressing the *CspB* gene had a higher yield under water deficit conditions than the control and expressed a yield equivalent to the control under non-stressed conditions. Tests are in progress in 2012 on commercial farms, [171; http://www.monsanto.com/products/Pages/ corn-pipeline.aspx#firstgendroughttolerantcorn]. The value of a biotechnological approach to improving crop yields under drought stress conditions is becoming evident with the first demonstrations of improved drought tolerance in crops in the field (reviewed in [171]).

In order to achieve a full understanding of drought-response mechanisms in plants and to make use of this understanding to produce crops with improved drought tolerance, there is a need to combine the data derived from different studies. Detailed analyses of the networks of protein interactions, the co-expression of genes, metabolic factors, etc. should provide in‐ sights into the key regulators of drought response [172-173]. Biotechnological approaches

a lower canopy temperature than the sensitive genotypes [162-163].

**7. GM crops – are they a solution?**

118 Abiotic Stress - Plant Responses and Applications in Agriculture

**8. Conclusions and perspectives**

This work was supported by the European Regional Development Fund through the Inno‐ vative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD "Biotechnological tools for breeding cereals with increased resistance to drought", task 22. The project is realized by POLAPGEN Consortium and is coordinated by the Institute of Plant Genetics, Polish Academy of Sciences in Poznan. Further information about the project can be found at www.polapgen.pl.

## **Author details**

Agata Daszkowska-Golec and Iwona Szarejko

Department of Genetics, Faculty of Biology and Environmental Protection, University of Si‐ lesia, Katowice, Poland

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## **Root Development and Abiotic Stress Adaptation**

L. Sánchez-Calderón, M.E. Ibarra-Cortés and

I. Zepeda-Jazo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55043

## **1. Introduction**

As soon as plants became independent from homogeneous aquatic environments, root-like organsweredeveloped.The interfacebetweenlandandwaterbodieswasprobablythemedium for the earliest land plants. Taking into account that those ancestral root-like organs did not face problems of water and nutrient acquisition, they were probably rather simple. As the earliest plants colonized this medium, the sandy substrate was replaced by heterogeneous soil, promoting more sophisticated vegetation and expanding the limits of land plant colonization. Therefore, to increase the efficiency of exploration of heterogeneous soil, during plant evolu‐ tion the ancestral root-like organ was replaced by a complex root system (RS) as the one we now know [1-3]. Land plants nowadays present a wide diversity of root system architectures (RSA; spatial configuration of the root system) among species, from non-branched to highly com‐ plexbranchingpatterns, achievingthemost effectiveperformance regardinganchorage andthe acquisition of water and nutrients. Each kind of RSA is guided by a genetically controlled postembrionary root developmental program (PERDP). This program is not rigid, and actually permits high phenotypic plasticity in response to stressing environmental conditions. PERDP is essentially driven by two cellular processes, cell division in the apical root meristem and new lateral meristems formed from the pericycle, and cell expansion performed in the root elonga‐ tion area. This particular characteristic permits plants, which are sessile organisms, to change theirrootarchitecturetoadapttoabioticstress[4-6].Soilsprovideplantswithwaterandnutrients; however, nutrients and water are distributed in a heterogeneous or patchy manner. In order to enhance nutrient capture, plant roots have modified their root architecture to explore those nutrient-rich zones. In the last two decades, progress has been made understanding the physiological, molecular and biochemical basis of how the PERDP could be modified by abiotic environmental cues [5, 7]. The aim of this chapter is to provide a review of how abiotic stress modulates post-embryonic plant root development. We will begin with a discussion of origin, anatomy, morphology and kinds of RS. Then, we will review recent advances in the knowl‐

© 2013 Sánchez-Calderón et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Sánchez-Calderón et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

edge of molecular, genetic and cellular processes that modulate post-embryonic root develop‐ ment in the model plant *Arabidopsis thaliana* making emphasis in the cell cycle. We will continue to focus on the modulation of PERDP in response to salinity and water. We will describe the changes in the RS induced by nutrients such as nitrogen, potassium and iron. The modulation of RSA by phosphorous will be discussed taking into account molecular, genetic and cellular responses. Finally, we will discuss how abiotic stress modulates apical root meristem activity.

## **2. Root system**

Raven and Edwards (2001) define: "roots are axial multicelular structures of sporophytes of vascular plants which usually occurs underground, have strictly apical elongation growth, and generally have gravitropic responses which range from positive gravitropism to diagra‐ vitropism, combined with negative phototropism". The apical meristem of one (lower vascular plants) to many (all seed plants) diving cells produces a root cap acropetally and initials of stele, cortex and epidermis basipetally. The branching of roots involves the endogenous origin of new root apical meristems in the pericycle [2]. The most conserved functions of roots present in extant plants are anchorage to substrate, and uptake of water and mineral nutrients. The evolution of multicellular organs such as roots was necessary to successful colonization of land by early plants [1, 4].

#### **2.1. Origin and evolution**

Over 470 million years ago, in the mid-Palaeozoic era, took place one event with far-reaching consequences in the history of the life, the origin and early evolution of embryophytes (land plants).Itappearsthatmarginsofdryingpoolsweretheplacewhereearlyembryophytesevolved from algal ancestors. The earliest land plants probably presented a system of rhizoid-like filaments that performed the rooting functions (anchorage and uptake water and nutrients) helpedbyassociatedfungi.Theygrowinsuperficialsoilproducedforweatheringofrocksurface similarlytobryophytes(mosses).Theirappearancestartedchangesonenergyandnutrientfluxes among terrestrial and freshwater ecosystems and consequently for the evolution of animal, bacteria andfungigroups thatlives inthosehabitats.Roots as theonesweknownowarepresent only in vascular plants (tracheophyta), they evolved in the sporophyte of at least two different lineages of tracheophytes, lycophytes (licopods) and euphyllophytes (ferns and seed plants), duringtheEarlyandmiddleDevonian.RootsofearlyEuphyllophytesstartedtopenetratedeeper into substrate increasing the anchorage and funding the inorganic nutrients produced by rock leaching.InEuphyllophytesafundamentaldifferenceintheanatomyofembryonicrootsamong seed plants and free-sporing monilophytes, suggesting that roots evolved independently. At thistimerootdevelopedmorebranchedaxesandfinerstructuresinvolvedinthenutrientuptake, root hairs. In Carboniferous (300 millions of years ago) gymnosperms appear and their RS is highly branched and depth penetration, they break up rocks letting exposed mayor rock area exposed to weathering. By late Cretaceous (100-65 millions of years) angiosperms are presents showing similar root system as exant angiosperms [1, 2, 8].

During the Devonian period (415–360 million years ago) apparition and radiation of embryo‐ phytes with roots caused large changes to the global level. The early land plants with rhizoidlike filaments that penetrated the top few centimeters of soil, were replaced by plants with deep RS with complex structures.The apparition ofthose organs that activelypenetrate the rock with the capacity of uptake and transport mineral nutrients permitted the development of structur‐ allycomplexabove-groundstructurestophotosynthesis,whichincreasedtheamountsofcarbon fixedonthecontinent.Theincreaseofprimaryproductionofearlylandplantschangedtheglobal carbon cycle and generates new complex soils which increased the border of land inhabited by plants.Ononehand,thehighratesplantproductioninthisperiodalloweddepositionof carbon on continental area from plant-drive organic matter, organic molecules secretedinto the soil; on theotherhand,the increaseweatheringrateofrocksbyrootpenetrationandsecretionoforganic compoundspermittedthemining ofrock-derivedinorganicnutrients.Those changes inhabitat turned up to be a part of a stimuli cycle in plant evolution, as themselves allowed the primary production to rise, which produced changes, and so on. The apparition of RS during Devonian allows that most of land surface was covered by plants, since Carboniferous forest (300 million of years ago), through late Cretaceous where basal angiosperms appeared (100-65 million of years ago) until days [1-3].

#### **2.2. Classification and architecture**

edge of molecular, genetic and cellular processes that modulate post-embryonic root develop‐ ment in the model plant *Arabidopsis thaliana* making emphasis in the cell cycle. We will continue to focus on the modulation of PERDP in response to salinity and water. We will describe the changes in the RS induced by nutrients such as nitrogen, potassium and iron. The modulation of RSA by phosphorous will be discussed taking into account molecular, genetic and cellular responses. Finally, we will discuss how abiotic stress modulates apical root meristem activity.

Raven and Edwards (2001) define: "roots are axial multicelular structures of sporophytes of vascular plants which usually occurs underground, have strictly apical elongation growth, and generally have gravitropic responses which range from positive gravitropism to diagra‐ vitropism, combined with negative phototropism". The apical meristem of one (lower vascular plants) to many (all seed plants) diving cells produces a root cap acropetally and initials of stele, cortex and epidermis basipetally. The branching of roots involves the endogenous origin of new root apical meristems in the pericycle [2]. The most conserved functions of roots present in extant plants are anchorage to substrate, and uptake of water and mineral nutrients. The evolution of multicellular organs such as roots was necessary to successful colonization of land

Over 470 million years ago, in the mid-Palaeozoic era, took place one event with far-reaching consequences in the history of the life, the origin and early evolution of embryophytes (land plants).Itappearsthatmarginsofdryingpoolsweretheplacewhereearlyembryophytesevolved from algal ancestors. The earliest land plants probably presented a system of rhizoid-like filaments that performed the rooting functions (anchorage and uptake water and nutrients) helpedbyassociatedfungi.Theygrowinsuperficialsoilproducedforweatheringofrocksurface similarlytobryophytes(mosses).Theirappearancestartedchangesonenergyandnutrientfluxes among terrestrial and freshwater ecosystems and consequently for the evolution of animal, bacteria andfungigroups thatlives inthosehabitats.Roots as theonesweknownowarepresent only in vascular plants (tracheophyta), they evolved in the sporophyte of at least two different lineages of tracheophytes, lycophytes (licopods) and euphyllophytes (ferns and seed plants), duringtheEarlyandmiddleDevonian.RootsofearlyEuphyllophytesstartedtopenetratedeeper into substrate increasing the anchorage and funding the inorganic nutrients produced by rock leaching.InEuphyllophytesafundamentaldifferenceintheanatomyofembryonicrootsamong seed plants and free-sporing monilophytes, suggesting that roots evolved independently. At thistimerootdevelopedmorebranchedaxesandfinerstructuresinvolvedinthenutrientuptake, root hairs. In Carboniferous (300 millions of years ago) gymnosperms appear and their RS is highly branched and depth penetration, they break up rocks letting exposed mayor rock area exposed to weathering. By late Cretaceous (100-65 millions of years) angiosperms are presents

**2. Root system**

136 Abiotic Stress - Plant Responses and Applications in Agriculture

by early plants [1, 4].

**2.1. Origin and evolution**

showing similar root system as exant angiosperms [1, 2, 8].

The RS consists of all roots that a plant has. It can be classified according to branch structure, root activity or development. The classification based on development is the more typical and useful to analyze the RS growth. This approach ontogenetically classified roots into three categories: primary root (PR), lateral root (LR) and adventitious root (AR; Figure 1 A). This classification reflects the differences between monocotyledonous and dicotyledonous RS. During germination PR is the first root to emerge from seed in both monocotyledonous and dicotyledonous, and is derived from embryonic root. In most of dicotyledonous LR are formed post-embryonically from pericycle cells (Figure 1 B-C) generating a branching system called primary root system. Depending on the length of LR relative to the primary axis (PR), the morphology of the RS will vary between tap rooted (Figure 1 A) and diffuse [3, 6, 9, 10]. Many monocotyledonous form PR and LR in a manner alike to dicotyledonous, in addition form nodal roots (AR) to generate a 'fibrous' adventitious roots system [6, 10, 11]. The morphology of the RS itself is very consistent, depends on the species, however, the spatial configuration of the RS (number, position and growth position of PR, LR and AR) called root system architecture (RSA) is highly variable, even among genetically identical plants. RSA is gener‐ ated during post-embyonic root development and is guided by a plastic genetic program which is modulated by environmental cues [4, 9].

## **3. Root system development**

Root development can be divided in two main stages: a) embryonic development (ED) and b) post-embryonic development (PED). During the ED, through a suite of highly regulated and reproducible stages, the fertilized egg cell rises into an embryo. In the embryo, the primary meristems, body axes and major tissue layers are established [12-15]. Unlike metazoans, almost all the body of the mature plant is generated during the PED. The PE begins during germina‐ tion, when the mitotic activity of meristems commences. Primary root meristems occupy one end of the main body axis and originate the RS [9, 14, 16]. During the post-embrionary root development traits such a i) primary meristems activity, ii) cell elongation, where both determine the anatomy, length and trajectory of roots and iii) de novo formation of secondary meristems and organs increase the branching to explore new soil zones [5, 6].

In Arabidopsis, the root consists of a series of concentric cylinders of different tissues (Figure 1 B), and this pattern is formed by sequential and ordered cell divisions during embryogenesis [17]. The outer epidermal layer covers all root tissues, and by itself contains the trichoblasts, a cell lineage that produces root hairs by tip growth, providing the root with additional anchoring and nutrient uptake surface. Cortex layers give mechanical support and protection while the endodermis forms an ion barrier. Inwards the endodermis, the pericycle cells maintain meristematic properties that can give place to root primordia or diverge into vascular tissues or cambium during secondary root growth. This pattern is established during the embryogenesis by a series of asymmetric and formative divisions [18, 19].

**Figure 1.** Arabidopsis root system. Typical tap root system of dicots (A). Transversal section of primary root (B). Longitudi‐ nal section of primary root meristem (C) and primary root tip. Primary root (PR), lateral roots (LR), adventitious roots (AR), pericycle cell layer (\*), QC cells (arrow), root meristem (RM), elongation area (EA) and differentiation area (DA).

#### **3.1. Cellular proliferation, elongation and differentiation**

Root growth is produced by the biosynthesis of cell wall combined with cell division. In the root meristem (RM) (Figure 1C-D), the cell layers apart from the epidermal and root cap ones are originated around a region that consist of three or four slowly proliferating cells, the quiescent center (QC) (Figure 1 C), which has a role organizing the meristem and is also involved in the stem cell identity maintenance QC removal results in the de novo formation of a new QC with adjacent initial cells and stem cells adjacent to the cortex and endodermal stem cells yield to epidermal initial cells and the lateral root cap [20-22]. Directly upwards from the QC the proximal meristem is located, as the distal meristem is located below, and within the meristems the forward growth is carried on as cells divide and grow there at a steady rate. When reaching certain distance from the meristem, in elongation area (EA)(Figure 1D) division is arrested and the cells start to elongate. Elongated cells are associated with endoreplication, a process of DNA replication without actual cell division which accumulates genome copies in the cell and uses part of the machinery associated with cell cycle, and involves the inacti‐ vation of mitotic CYC-CDK (Cyclin- Cyclin Dependent Kinase) complexes [23-25]. Pericycle and cambium cells, distanced from the root tip, maintain the potential to reenter division, forming LRs or transitional cells at the meristem end, depending on localized auxin responses [26] or oscillating gene expression [27].

#### *3.1.1. Cell cycle*

meristems, body axes and major tissue layers are established [12-15]. Unlike metazoans, almost all the body of the mature plant is generated during the PED. The PE begins during germina‐ tion, when the mitotic activity of meristems commences. Primary root meristems occupy one end of the main body axis and originate the RS [9, 14, 16]. During the post-embrionary root development traits such a i) primary meristems activity, ii) cell elongation, where both determine the anatomy, length and trajectory of roots and iii) de novo formation of secondary

In Arabidopsis, the root consists of a series of concentric cylinders of different tissues (Figure 1 B), and this pattern is formed by sequential and ordered cell divisions during embryogenesis [17]. The outer epidermal layer covers all root tissues, and by itself contains the trichoblasts, a cell lineage that produces root hairs by tip growth, providing the root with additional anchoring and nutrient uptake surface. Cortex layers give mechanical support and protection while the endodermis forms an ion barrier. Inwards the endodermis, the pericycle cells maintain meristematic properties that can give place to root primordia or diverge into vascular tissues or cambium during secondary root growth. This pattern is established during the

**Figure 1.** Arabidopsis root system. Typical tap root system of dicots (A). Transversal section of primary root (B). Longitudi‐ nal section of primary root meristem (C) and primary root tip. Primary root (PR), lateral roots (LR), adventitious roots (AR),

Root growth is produced by the biosynthesis of cell wall combined with cell division. In the root meristem (RM) (Figure 1C-D), the cell layers apart from the epidermal and root cap ones are originated around a region that consist of three or four slowly proliferating cells, the

pericycle cell layer (\*), QC cells (arrow), root meristem (RM), elongation area (EA) and differentiation area (DA).

**3.1. Cellular proliferation, elongation and differentiation**

meristems and organs increase the branching to explore new soil zones [5, 6].

138 Abiotic Stress - Plant Responses and Applications in Agriculture

embryogenesis by a series of asymmetric and formative divisions [18, 19].

The cell cycle is a temporal regulator of proliferative cell division, and it is comprised of mitosis, cytokinesis, post-mitotic interphase (G1), DNA synthetic phase (S) and post-synthetic inter‐ phase (G2)[28]. The conjunction of all these is the key force driving organogenesis and growth in plants and other eukaryotes. The mitotic cycle is driven by the periodic activation of a multicomponent system that relies on CDKs as key regulators. CDKs combine with different CYCs to trigger the transition from the G1 to S phase and the G2 to M phase, and a wide variety of components control the activity of these kinases, thus becoming part of a complex molecular network that is still being studied [29-31]. In plants, a number of core cycle regulators have been revealed to exist [32, 33] and what appears to be distinctive in plants is that they appear to have many more CYCs and CDKs in comparison to animals and yeasts [21, 24]. The reason of this abundance of putative function overlapping components can be the one suggested in [34], postulating that that plants have evolved a combinatorial resource pool consisting of around ninety different CDK-CYC complex variants, thus explaining to an extent the plasticity of plant development regulation, as they provide with a strategy to recognize distinct stimuli and environments, and thus promote different phases of the cell cycle. Cell cycle progression and controlling mechanisms include transcriptional regulation, protein-protein interaction, phosphorylation-dephosphorylation and protein degradation [29, 30, 35, 36]. As recently reviewed [36], the evidence obtained from interaction studies suggests that Arabidopsis CDKA;1 primarily binds to CYCDs to promote the G1/S transition and to CYCA3 to drive the S phase progression while CDKA;1 pairs with CYCD3 to drive the M phase progression. In contrast, CDKBs presumably interact preferentially with CYCA2 and CYCBs to promote the G2/M transition and the M phase progression [37-39]. In Arabidopsis, the accumulation of the CYCB1;1 transcript is correlated with meristematic tissues [40], activated from early S phase in synchronized cells with no significantly increase during G2 phase [41]. Together with environmental and hormonal stimuli, the coordination of the different cell cycle control processes lead to a balance between cell division and expansion that ensures the correct embryonic and post-embryonic development. As part of the extensive toolset that plants possess in order to finely tune the mitotic and endoreplicative cycles, the phase-specific activation of CYC-CDK complexes via temporal transcription is a mechanism that is evidently used but not completely understood in plants. In synchronized Arabidopsis cell cultures many cell cycle genes present highly specific expression windows during the mitotic cell cycle [41, 42]. For example, the expression of several CYCAs is dramatically upregulated at the G1/S transition and S phase, while others are accumulated at G2/M transition, as well as all CYCBs. Most of CYCDs are expressed during G1 and S phases, with the exception of a few ones, like CYCD3:1, expressed during G2-M. In the case of CDKs, CDKA;1 is expressed throughout the cell cycle, with constant transcript levels, the CDKB1s are present from S to M phase, and CDKB2s are detected specifically from late G2 to M phase.

#### *3.1.2. Cell cycle control in root meristem*

The expression windows of cell-cycle control genes can be extrapolated to their expression in the actively dividing cells of the root meristems. In these and all dividing cells, The G1/S transition is generally controlled by the E2F-DP-RBR (E2F-Dimerisation Partner-Retinoblas‐ toma Related) pathway. One of the three Arabidopsis-encoded E2F transcription factors forms dimers with one of the two DP proteins to bind to certain promoter sites in the transcriptional target genes to promote the G1/S transition, including those required for DNA replication and repair. In G1, CYCD-CDKA complexes phosphorylate RBR, releasing the E2F-DP dimers to allow them to bind to the transcriptional activation sites [43-46]. In the other hand, E2Fc-DPb dimers act as transcriptional repressors with yet unknown target genes, although their repressing mechanism appear to be independent from the RBR pathway [47]. Meanwhile, genes expressed during G2 and M phases contain M phase-specific activator (MSA) elements in their promoters, recognized by three Myb repeats (MYB3R) transcription factors, discovered for the first time in tobacco [48]. The Arabidopsis genome encodes five MYB3R proteins (MYB3R1-5), from which MYB3R1 and MYB3R4 are the closest homologs of the G2/M specific transcriptional activators NtMYBA1 and NtMYBA2, with the first having a stable transcript level through the cell cycle, and the latter having an expression peak during G2/M transition, suggesting that MYB3R1 is post-translationally regulated. The expression of many G2 to M specific genes possessing MSA elements in their promoters is visibly down-regulated in the *myb3r1 myb3r4* double mutant, but not completely abolished [49] suggesting an alternative mechanism controlling the transcription of G2 and M phase genes. Additionally to E2F and MYBs, there are other transcription factors that control cell cycle phase-specific gene expres‐ sion, as the DNA-binding with one finger (DOF) transcription factor, OBP1, whose overex‐ pression shortens the cell cycle with elevated expression of many other cell cycle genes, and that normally upregulates the expression of replication-specific transcription factors and CYCD3;3 [50]. Another form of controlling the activity of CYC-CDK complexes is through post-translational mechanisms, and among them, the ubiquitin-mediated degradation of cell cycle proteins is the most determinant for the correct timing in the progression of the cell cycle [51-53]. A number of ubiquitin-dependent degradation pathways have been associated with the mitotic cell cycle, and the E3 ubiquitin ligases participate in all cases, marking target proteins by polyubiquitination and subsequent proteolysis. The Skp-cullin1-F-Box (SCF) E3 ligase regulates primarily the G1/S transition while the Anaphase Promoting Complex/ Cyclosome (APC/C), a Cullin-RING finger E3 ligase, is most active from the M phase to G1 phase. APC/C complex is composed by at least 11 subunits, and in the Arabidopsis genome, all APC/C components except for APC3/CDC27/HOBBIT are encoded by a single gene [54]. All APC/C subunit mutants studied so far accumulate mitotic CYCs in embryo sacs, suggesting that they're substrates of the APC/C [52, 54]. Apart from its core components, APC/C also pairs with co-activators, known as CDC20/FIZZY and CDC20 HOMOLOG1 (CDH1)/FIZZY-RELATED (FZR), which confer substrate specificity and are activated during distinct phases of the cell cycle with equally distinct activities. The Arabidopsis genome contains five CDC20 and three CDH1 genes, also called CELL CYCLE SWITCH 52 (CCS52), and even if their modification of the APC/C activity during the cell cycle is not fully established, CCS52A1 and CCS52A2 participate in meristem maintenance [55]. Notably, they act through different mechanisms and exhibit different expression patterns as well. The expression of CCS52A1 starts at the elongation zone of Arabidopsis roots and stimulates mitotic exit and an entry into the endoreplication cycle, whereas CCS52A2 is expressed at the distal part of the root meristem and is required to maintain the cell identity in the QC. The *ccs52a2* mutation activates the QC cell division, contrasting with the occasional division behavior normally presented, and when its promoter is switched with the one of CCS52A1, the expression of the latter rescues the phenotype in *ccs52a2*, suggesting homologous function. In vertebrates, negative regulators also modify the activity of APC/C. These regulators, called Early mitotic inhibitor1 (Emi1) and Emi2 directly bind to CCS52 and CDC20, inhibiting the APC/C activity. No direct plant orthologs are identified, but recent studies have shown that GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION 1 (OSD) and UV-INSENSITIVE4 (UVI4)/POLYCHROME (PYM) act as their functional homologs in plants [56, 57] by physically interacting with the APC/C activators CCS52 and CDC20. Their overexpression causes an accumulation of CYCB1;2 and CYCA2;3, respectively, by the inactivation of the APC/C, suggesting that it also could have an effect on root meristem maintenance by the inhibition of the APC/C-CCS52 complex activity. Another important way to control and modulate the CYC-CDK complexes activity involves said complexes binding directly to CDK inhibitors, proteins that interfere with the ability of CYC-CDK to phosphorylate their substrates. Plants have two classes of CDK inhibitors – KIP-RELATED PROTEINs (KRPs) and SIAMESE (SIM)/SIAMESE RELATED (SMR). The Arabidopsis genome encodes 7 KRPs, KRP1-7, and at least 13 SIM/SMRs. Recent analyses have shown that all 7 KRPs purify conjoined with CYCDs and CDKA [36] suggesting that they inhibit the activity of the CYCD-CDKA complexes, as it has been proposed previously [58], but not excluding the possibility of them inhibiting the activity of CYCD-CDKB complexes as well [59]. The seven KRPs display overlapping but distinct expression patterns in the Arabi‐ dopsis shoot apex, some of them present strongly in dividing cells, like KRP4 and KRP5, while KRP1 and KRP2 are present in differentiating cells [60]. KRPs have a role driving the endore‐ plication cycle as well, also by inhibiting CDK activities [61, 62]. The SIM/SMR family of CDK inhibitors is found only in plants, and is required to repress the mitotic cell cycle in trichomes via the interaction of SIM with the CYCD-CDKA complex [63]. Another member of the SIM family, SMR1/LGO, is implicated in the control of endoreplication in sepals [64], maintaining the presence of elongated, endoreplication-undergone giant cells in the sepals, which are lost in the *smr1/lgo* because they progressed through additional cell divisions instead of endore‐

possess in order to finely tune the mitotic and endoreplicative cycles, the phase-specific activation of CYC-CDK complexes via temporal transcription is a mechanism that is evidently used but not completely understood in plants. In synchronized Arabidopsis cell cultures many cell cycle genes present highly specific expression windows during the mitotic cell cycle [41, 42]. For example, the expression of several CYCAs is dramatically upregulated at the G1/S transition and S phase, while others are accumulated at G2/M transition, as well as all CYCBs. Most of CYCDs are expressed during G1 and S phases, with the exception of a few ones, like CYCD3:1, expressed during G2-M. In the case of CDKs, CDKA;1 is expressed throughout the cell cycle, with constant transcript levels, the CDKB1s are present from S to M phase, and

The expression windows of cell-cycle control genes can be extrapolated to their expression in the actively dividing cells of the root meristems. In these and all dividing cells, The G1/S transition is generally controlled by the E2F-DP-RBR (E2F-Dimerisation Partner-Retinoblas‐ toma Related) pathway. One of the three Arabidopsis-encoded E2F transcription factors forms dimers with one of the two DP proteins to bind to certain promoter sites in the transcriptional target genes to promote the G1/S transition, including those required for DNA replication and repair. In G1, CYCD-CDKA complexes phosphorylate RBR, releasing the E2F-DP dimers to allow them to bind to the transcriptional activation sites [43-46]. In the other hand, E2Fc-DPb dimers act as transcriptional repressors with yet unknown target genes, although their repressing mechanism appear to be independent from the RBR pathway [47]. Meanwhile, genes expressed during G2 and M phases contain M phase-specific activator (MSA) elements in their promoters, recognized by three Myb repeats (MYB3R) transcription factors, discovered for the first time in tobacco [48]. The Arabidopsis genome encodes five MYB3R proteins (MYB3R1-5), from which MYB3R1 and MYB3R4 are the closest homologs of the G2/M specific transcriptional activators NtMYBA1 and NtMYBA2, with the first having a stable transcript level through the cell cycle, and the latter having an expression peak during G2/M transition, suggesting that MYB3R1 is post-translationally regulated. The expression of many G2 to M specific genes possessing MSA elements in their promoters is visibly down-regulated in the *myb3r1 myb3r4* double mutant, but not completely abolished [49] suggesting an alternative mechanism controlling the transcription of G2 and M phase genes. Additionally to E2F and MYBs, there are other transcription factors that control cell cycle phase-specific gene expres‐ sion, as the DNA-binding with one finger (DOF) transcription factor, OBP1, whose overex‐ pression shortens the cell cycle with elevated expression of many other cell cycle genes, and that normally upregulates the expression of replication-specific transcription factors and CYCD3;3 [50]. Another form of controlling the activity of CYC-CDK complexes is through post-translational mechanisms, and among them, the ubiquitin-mediated degradation of cell cycle proteins is the most determinant for the correct timing in the progression of the cell cycle [51-53]. A number of ubiquitin-dependent degradation pathways have been associated with the mitotic cell cycle, and the E3 ubiquitin ligases participate in all cases, marking target proteins by polyubiquitination and subsequent proteolysis. The Skp-cullin1-F-Box (SCF) E3 ligase regulates primarily the G1/S transition while the Anaphase Promoting Complex/

CDKB2s are detected specifically from late G2 to M phase.

*3.1.2. Cell cycle control in root meristem*

140 Abiotic Stress - Plant Responses and Applications in Agriculture

plication. Recent studies [34] show that both SIM and SMR/LGO are purified jointly with CDKB1;1 while other SMRs interact with CDKA;1, thus suggesting that CDKB1;1 could be directly inhibited by SIM/SMR1 leading to endoreplication onset.

#### *3.1.3. Cell cycle control in post-embryonic root development*

The cell cycle relies not only on its own molecular machinery to determine cellular fate. Postembryonic plant development needs a highly precise coordination of cell cycle-directed signaling to correctly drive cells to form new tissues or cell types, as is evident in root develop‐ ment.Moleculargeneticstudieshaveuncoveredseveralkeyregulatorsinvolvedindevelopmen‐ tal cell cycle control, andmany ofthemhave shownto be transcriptionalregulators, buthow are they linked to cell cycle control has not been well characterized. SHORTROOT (SHR) and SCARECROW (SCR) are members of the GRAS family of transcription factors required for the asymmetric division of cortex/endodermis initial cells (CEI) in the root apical meristem [65, 66]. Thistissue-formativedivisiongeneratestwonewcellularkinds-cortexandendodermis,making the CEI cell division control a key requisite for a proper root development. It has been demon‐ strated that both SHR and SCR directly regulate the expression of CYCD6;1, present at G1 and S phases, by binding to its promoter [67]. CYCD6;1 is expressed specifically in CEI and CEI daughter cells, and the asymmetric division of CEI is significantly decreased in the *cycd6;1* mutants. Additionally, when CYCD6;1 is expressed ectopically in the *shr* mutant background, it partially compensates the division defects presented by the latter, supporting the idea of CYCD6;1beingdownstreamoftheSHR/SCRpathway.Other cell cyclegenes,likeCDKB2;1 and CDKB2;2, have their expression regulated by SHR and SCR, and when these CDKs are overex‐ pressed in endodermal cells, the formative cell division of the CEI is promoted. However, they do not appear to be direct targets of SHR and SCR, implying that the activation of these CDK genes is linked by another control factor. Cell proliferation needs to be restored in the xylempericyclecellsfortheLRinitiationandthisprocesscanbeinducedbyauxininmanyplantspecies, like Arabidopsis. LR development starts by the degradation of INDOLE ACETIC ACID 14(IAA14)/ SOLITARY-ROOT(SLR),dependent onauxin,thatleads to thede-repressionoftwo related AUXIN RESPONSE FACTORs (ARFs), ARF7 and ARF19 [68]. These ARFs are re‐ quired for the subsequent expression of LATERAL ORGAN BOUNDARIES 18 (LBD18] and LBD33 transcription factors, which form a LBD18-LBD33 heterodimer that activates the expression of the E2Fa, one of the E2F genes induced at LR initiation, by binding directly to its promoter [69]. E2Fa expression is increased by auxin treatment at the LR initiation site and this auxin-dependent E2Fa expression is lost in the *iaa14/slr-1* mutant background. Expectedly, the number of LR primordial is decreased in the *e2fa* mutants, evidencing a requirement of E2Fa for LR emerging and establishing a link between auxin signaling and cell cycle progression during LR development. Another unrelated pathway that is also involved in the auxin-induced LR formation has KRP2 downregulated by auxin [70]. Under low auxin conditions, the CYCD2;1- CDKA activity is repressed by the presence of KRP2. Upon auxin treatment, both gene expres‐ sion and protein accumulation of KRP2 is reduced, leading to an increase in the CYCD2;1- CDKAactivityandsubsequentenhancementofLRinduction.Apossiblehyperphosphorylation ofRBRresultingintheactivationofE2FbdirectlycausedbytheCYCD2;1-CDKAcomplexactivity has been suggested [69]. A model on the basis of available information on the density and

orientationofauxintransporters,cellshape,andauxintransportparameterspredictsamaximum auxinconcentrationintheQCanda steepauxingradientintheproximalmeristem,whichdrops according to the cell number from the quiescent center [71, 72]. This agrees with the auxin levels found in protoplasts derived from different apical cell types, as well as with the expression patterns of auxin responsive genes, such as members of the PLETHORA (PLT) family, in the different root tissues [73]. PLT 1 and PLT2 are known to be crucial for interpreting this gradi‐ ent in the terms of root growth and development. They encode for AP2-domain transcription factors, and losing of their function results in the loss of stem cells, arrest of transit-amplifying divisions and reduction of cell expansion [74]. PLT pathway has other effects over cell cycle control. Histone acetyltransferase, a chromatin modifier and required to maintain the divid‐ ing ability in meristem cells, is also requiredto sustainPLTexpression andsupport both transitamplifying divisions and the root stem cell status at the root apex [75]. Moreover, the action of SUMO E3 ligase is vital to repress endoreplication in shoot and root meristems, and in the root, this SUMO E3 ligase acts in the PLT pathway [76]. It can be said then that the root tip is charac‐ terizedbyanauxinmaximum,andauxinisrequiredtosupporttransit-amplifyingdivisions[77].

## **4. Root system development and abiotic stress**

plication. Recent studies [34] show that both SIM and SMR/LGO are purified jointly with CDKB1;1 while other SMRs interact with CDKA;1, thus suggesting that CDKB1;1 could be

The cell cycle relies not only on its own molecular machinery to determine cellular fate. Postembryonic plant development needs a highly precise coordination of cell cycle-directed signaling to correctly drive cells to form new tissues or cell types, as is evident in root develop‐ ment.Moleculargeneticstudieshaveuncoveredseveralkeyregulatorsinvolvedindevelopmen‐ tal cell cycle control, andmany ofthemhave shownto be transcriptionalregulators, buthow are they linked to cell cycle control has not been well characterized. SHORTROOT (SHR) and SCARECROW (SCR) are members of the GRAS family of transcription factors required for the asymmetric division of cortex/endodermis initial cells (CEI) in the root apical meristem [65, 66]. Thistissue-formativedivisiongeneratestwonewcellularkinds-cortexandendodermis,making the CEI cell division control a key requisite for a proper root development. It has been demon‐ strated that both SHR and SCR directly regulate the expression of CYCD6;1, present at G1 and S phases, by binding to its promoter [67]. CYCD6;1 is expressed specifically in CEI and CEI daughter cells, and the asymmetric division of CEI is significantly decreased in the *cycd6;1* mutants. Additionally, when CYCD6;1 is expressed ectopically in the *shr* mutant background, it partially compensates the division defects presented by the latter, supporting the idea of CYCD6;1beingdownstreamoftheSHR/SCRpathway.Other cell cyclegenes,likeCDKB2;1 and CDKB2;2, have their expression regulated by SHR and SCR, and when these CDKs are overex‐ pressed in endodermal cells, the formative cell division of the CEI is promoted. However, they do not appear to be direct targets of SHR and SCR, implying that the activation of these CDK genes is linked by another control factor. Cell proliferation needs to be restored in the xylempericyclecellsfortheLRinitiationandthisprocesscanbeinducedbyauxininmanyplantspecies, like Arabidopsis. LR development starts by the degradation of INDOLE ACETIC ACID 14(IAA14)/ SOLITARY-ROOT(SLR),dependent onauxin,thatleads to thede-repressionoftwo related AUXIN RESPONSE FACTORs (ARFs), ARF7 and ARF19 [68]. These ARFs are re‐ quired for the subsequent expression of LATERAL ORGAN BOUNDARIES 18 (LBD18] and LBD33 transcription factors, which form a LBD18-LBD33 heterodimer that activates the expression of the E2Fa, one of the E2F genes induced at LR initiation, by binding directly to its promoter [69]. E2Fa expression is increased by auxin treatment at the LR initiation site and this auxin-dependent E2Fa expression is lost in the *iaa14/slr-1* mutant background. Expectedly, the number of LR primordial is decreased in the *e2fa* mutants, evidencing a requirement of E2Fa for LR emerging and establishing a link between auxin signaling and cell cycle progression during LR development. Another unrelated pathway that is also involved in the auxin-induced LR formation has KRP2 downregulated by auxin [70]. Under low auxin conditions, the CYCD2;1- CDKA activity is repressed by the presence of KRP2. Upon auxin treatment, both gene expres‐ sion and protein accumulation of KRP2 is reduced, leading to an increase in the CYCD2;1- CDKAactivityandsubsequentenhancementofLRinduction.Apossiblehyperphosphorylation ofRBRresultingintheactivationofE2FbdirectlycausedbytheCYCD2;1-CDKAcomplexactivity has been suggested [69]. A model on the basis of available information on the density and

directly inhibited by SIM/SMR1 leading to endoreplication onset.

*3.1.3. Cell cycle control in post-embryonic root development*

142 Abiotic Stress - Plant Responses and Applications in Agriculture

Abiotic cues as water and nutrient availability limit plant productivity in almost all ecosys‐ tems in the world. Typically, RS has to growth in media where the biotic and abiotic components are distributed heterogeneously. Soils are complex, a broad range of chemical a physical process occurs due to intrinsic soil characteristics and the action of biotic fac‐ tors. Thus, this complexity presents several challenges to survive. As soon as the root makes contact with the soil must sense and integrate biotic and abiotic cues in order to adjust their genetic program of post-embryonic root development (PERD). This capacity to change their PERD allows them change their architecture to find the supplies of water and nutrients that could be limited and localized [3, 4, 12]. Environmental cues such as water, salinity and nutrient can modulate the ARS.

#### **4.1. Regulation of root system architecture by water availability and salinity stress**

Water and salinity can indirectly modulate the RSA because they can produce unfavorable changes in the nutritional composition of the soil, the distribution of said nutrients, the density and compaction of soil, and the type of soil particles [9]. Those interactions complicate the dissection of specific transduction pathways involved in root growth and development [78] The RS is the first to perceive the stress signals for drought and salinity, therefore its devel‐ opment is deeply affected by their availability in soil. In many agriculturally important species, the whole plant growth is inhibited during water starvation, however, RS is more resistant than shoots and continues growing under low water potentials that are completely inhibitors for shoot growth [79]. Notably, while growth of PR is not appreciably affected by water deficit, the number of LRs and its growth are significantly reduced [80]. It has been suggested that the reduction of the LR formation may be caused by the suppression of the activation of the lateral root meristems, not because of the reduction of the initiation in the LR per se, as primordia generation is unaffected [9, 80-82]. Mutants with alterations in the development of LRs respond differently to drought stress [80, 83]. Suppression of the growth of LR by drought has been widely accepted as an adaptive response to ensure the plant survival under unfavorable growing conditions [83]. Another factor that plays an important role in growing and devel‐ opment of plants to tolerate the drought stress is the hydrotropism [84, 85]. A recent study showed that a gradient of moisture generated by water stress causes an immediate degradation of amyloplasts in the columella cells of plant roots, producing a minor response to gravity and an increase of hydrotropism [86]. However, it is unknown how the gravity signals interact with other environmental signals to modulate the direction of root growth. Less known are the adaptations in root morphology and its relevance to salinity tolerance. Many halophytes have developed morphological adaptations, like the formation of specialized organs to expel salt out of their leaves, which allows them to keep the water and take out the salt in an active manner. Glycophytes have not developed permanent changes on its morphology to deal with salt, but they can adjust the root growth and its architecture in response to salinity, like in the case of Arabidopsis [87]. Also it has been observed that Arabidopsis RS exhibit a reduced gravitropism under salt stress, growing against the gravity vector [88]. Arabidopsis RS exposed to a simultaneous salinity and gravity stimuli responded to salinity with a change in growing direction in a way that apparently represents an adaptive arrangement between gravitropic and saline simulation. Control of the relation between gravitropism and hydro‐ tropism allows plants to direct the root growing for a better water uptake, giving an advantage during development of the radical system under stress conditions. It is known that the salt stress inhibits the growth of the PRs in Arabidopsis seedlings, although it has been reported that salt stress also modulates root gravitropism of PR in young seedlings. In vertical position, five day seedlings germinate normally in MS medium (Murashige and Skoog) containing different concentrations of sodium chloride (NaCl), however the direction of root growth changes according to the increase of NaCl concentrations, and the root curves in stressed plants with 150 mM NaCl in the medium [88].These results suggest that the salt stress and the induction of signal translations by stress modulate the direction of the root, despite of the gravity. Some reports suggest that the gravitropic signal and the answers in root apex are controlled, at least partially by Salt Overly Sensitive (SOS) signaling pathway. Therefore, this pathway might interact with the gravity sensor system in the cells of the columella to direct root growth in a coordinated way[88]. Abscisic acid (ABA) and auxins participate in a complex signal system that plays a very important role in the development of the RSA under drought conditions. These hormonal effects (levels) even though are considered as intrinsic [82] can change in response to environmental cues. Cytokinins, gibberellins and abscisic acid are produced in roots to be transported to other tissues, where they play their roles in development and growth. Although auxins are the major determinants of root growth [89], cytokinin and especially abscisic acid [90-92] have been proposed as potential chemical signals in response to water stress to modulate RSA. The decrease in water potential of roots caused by salinity is the factor that triggers the production of ABA in different species [93]. A condition of mild osmotic stress also inhibits the LR formation in a dependent way of ABA [80, 82, 83, 94]. In Arabidopsis, the reduced water availability dramatically inhibits the formation of LR, but not by the suppressing of initiation of LR at the lateral primordia. This inhibition does not occur in lateral root mutant 2 (*lrd2*) nor in two ABA deficient [80, 82]. Abscisic acid and a recently identified gen *LRD2* are linked to repression of LR formation in response to osmotic stress. It is very interesting to note that these regulators are also related to the establishment of RSA without apparent effect of osmotic stress. The mutant *lrd2* presents an altered response to exogenous application of ABA, while ABA-deficient mutants and *lrd2* show an altered response to inhibitors of polar auxin transport [95-97] suggesting a joint interaction of the hormonal signaling pathway in the regulation of LR formation. Some authors propose a model where the promotion or suppression of hormonal signaling pathway and regulators as LRD2 determine the type of LR primordium (LRP) and coordinate the RAS in response to environ‐ mental stimuli [87]. In contrast, under drought stress conditions or osmotic stress, activation of the LR meristem is suppressed by ABA-mediated signals, producing few small LRs [80, 98]. While auxins seem to be the main initialization hormone, pattern and emergence of LRs; ABA is the main hormone that controls the environmental effect (like drought and salt stress) over the RSA [99].

#### *4.1.1. Cellular responses*

#### *4.1.1.1. Epidermis*

generation is unaffected [9, 80-82]. Mutants with alterations in the development of LRs respond differently to drought stress [80, 83]. Suppression of the growth of LR by drought has been widely accepted as an adaptive response to ensure the plant survival under unfavorable growing conditions [83]. Another factor that plays an important role in growing and devel‐ opment of plants to tolerate the drought stress is the hydrotropism [84, 85]. A recent study showed that a gradient of moisture generated by water stress causes an immediate degradation of amyloplasts in the columella cells of plant roots, producing a minor response to gravity and an increase of hydrotropism [86]. However, it is unknown how the gravity signals interact with other environmental signals to modulate the direction of root growth. Less known are the adaptations in root morphology and its relevance to salinity tolerance. Many halophytes have developed morphological adaptations, like the formation of specialized organs to expel salt out of their leaves, which allows them to keep the water and take out the salt in an active manner. Glycophytes have not developed permanent changes on its morphology to deal with salt, but they can adjust the root growth and its architecture in response to salinity, like in the case of Arabidopsis [87]. Also it has been observed that Arabidopsis RS exhibit a reduced gravitropism under salt stress, growing against the gravity vector [88]. Arabidopsis RS exposed to a simultaneous salinity and gravity stimuli responded to salinity with a change in growing direction in a way that apparently represents an adaptive arrangement between gravitropic and saline simulation. Control of the relation between gravitropism and hydro‐ tropism allows plants to direct the root growing for a better water uptake, giving an advantage during development of the radical system under stress conditions. It is known that the salt stress inhibits the growth of the PRs in Arabidopsis seedlings, although it has been reported that salt stress also modulates root gravitropism of PR in young seedlings. In vertical position, five day seedlings germinate normally in MS medium (Murashige and Skoog) containing different concentrations of sodium chloride (NaCl), however the direction of root growth changes according to the increase of NaCl concentrations, and the root curves in stressed plants with 150 mM NaCl in the medium [88].These results suggest that the salt stress and the induction of signal translations by stress modulate the direction of the root, despite of the gravity. Some reports suggest that the gravitropic signal and the answers in root apex are controlled, at least partially by Salt Overly Sensitive (SOS) signaling pathway. Therefore, this pathway might interact with the gravity sensor system in the cells of the columella to direct root growth in a coordinated way[88]. Abscisic acid (ABA) and auxins participate in a complex signal system that plays a very important role in the development of the RSA under drought conditions. These hormonal effects (levels) even though are considered as intrinsic [82] can change in response to environmental cues. Cytokinins, gibberellins and abscisic acid are produced in roots to be transported to other tissues, where they play their roles in development and growth. Although auxins are the major determinants of root growth [89], cytokinin and especially abscisic acid [90-92] have been proposed as potential chemical signals in response to water stress to modulate RSA. The decrease in water potential of roots caused by salinity is the factor that triggers the production of ABA in different species [93]. A condition of mild osmotic stress also inhibits the LR formation in a dependent way of ABA [80, 82, 83, 94]. In Arabidopsis, the reduced water availability dramatically inhibits the formation of LR, but not by the suppressing of initiation of LR at the lateral primordia. This inhibition does not occur

144 Abiotic Stress - Plant Responses and Applications in Agriculture

Root epidermis is the first tissue that makes contact with salt; hence, it is the first to perceive osmotic and ionic changes in cells and the first one that triggers rescue mechanisms. The accumulation of sodium in the cells and the resulting ionic imbalance is the main cause of inhibition of plant growth and yield decrease [100]. Therefore, maintaining low intracellular sodium levels is critical for plant adaptation to water and salinity stress. Plants use different strategies to fight against salinity damage in every organizational level, from cellular, bio‐ chemical, molecular to anatomic, morphological and phenological level. At cellular and molecular level, plants cells keep a low cytosolic sodium (Na+ ) content by means of compart‐ mentalization and ionic transport regulation [100, 101]. During salinity stress, processes of membrane transport play a very special role. Some transport mechanisms implied in the perception of salt stress are: water output of the cell by osmotic gradient, the decrease of the availability of potassium (K+ ) in roots due to the reduced activity of this cation in soil solution, where sodium competes for binding sites for K+ transporters in PM (plasma membrane) including low and high affinity, also the increased efflux of K+ by selective and non-selective channels [102] and finally that these ionic events initially evoked in the PM of epidermal root cells are propagated to intracellular organelles (mainly vacuoles) and other plant tissues such as leaves. Considering the entry of Na+ and K+ loss, preventing worsening of the K+ /Na+ cytosolic relation is a key criterion for resistance to salt stress. Once the stress is perceived, the respective signalization triggers and changes in metabolism and genetic expression take place; all these are related with defense mechanisms [102, 103]. For the response to osmotic changes in metabolic compartments, it occurs an immediate osmotic adjustment by synthesizing compatible osmolytes and inorganic ions capture [104], for the toxic component of stress is performed a compartmentalization of harmful ions and ion transport [105]; and it generally occurs a restriction of unidirectional Na+ entry via non-selective cation channels (NSCC) [105, 106] and high affinity potassium transporters (HKT) [107, 108], the Na+ efflux from the cytosol by the Na+ /H+ exchanger in the PM [100] or its capture by tonoplast [109]; changes of metab‐ olism and signalization by polyamines and Reactive Oxygen Species (ROS) and the antioxidant activity [110, 111].

#### *4.1.1.2. Reactive oxygen species*

ROS fluctuations in time and space can be interpreted as signals to regulate growth, develop‐ ment, cell death and stress responses [112, 113]. Understanding the mechanisms that control ROS signaling in cells in response to water stress and salinity could therefore provide a powerful strategy for increasing crop tolerance to these environmental stress conditions [114]. Among the targets of ROS action at the cellular level, there are ion channels that mediate ion exchange in the PM. In the PM of roots and guard cells H2O2, stimulates the channels activated by hyperpolarization that mediate the influx of Ca2+ and NSCC [112, 115, 116] and inhibit the K+ outward and inward rectifier currents [117]. The stimulation of the influx of Ca2+ in guard cells appears to mediate the induction of stomata closure by ABA [116, 118-120]. At the same time it was reported that the OH• activates a Ca2+ inward and K+ outward currents in epidermal protoplasts derived from mature and growth zone of Arabidopsis roots [115]. A larger stimulation of the inward current of Ca2+ in the growth zone may indicate that ROS are involved in growth regulation via Ca2+ signaling. Moreover, the OH• produced by NADPH oxidase in Arabidopsis root hairs activated a Ca2+ inward rectifier conductance causing an increase in cytosolic Ca2+ allowing the root elongation [112]. Recently it has been reported that under severe water stress autophagy programmed cell death occurs in the region of the root apical meristem [121]. There is evidence that this defense mechanism is promoted by the accumulation of ROS in stressed meristematic cells of root tips. Analysis of the expression of BAX inhibitor-1 (AtBI1, apoptotic inhibitor) and the phenotypic response of the mutant *atbi1-1* under severe water stress indicates that AtBI1 and the pathway of endoplasmic reticulum stress response modulates the induction of PCD by water stress. As a result, thin and short roots induce an increase in their tolerance to stress. These authors also propose that under severe drought stress, plants activate the PCD program in the root apical meristem, removing the apical dominance; so they can remodel the RSA to adapt to stressful environments [122].

A slight drought stress increases the expression of enzymes associated with root morphology (Xyloglucan endotransglucosylase) while other structural proteins (actin and tubulin) are downregulated, these proteins are strongly correlated with root growth since its function is the vesicular carrying in cells with polarized growth (e.g. root hairs) allowing its growth and hence an augmentation in the surface of water uptake. However, when there is a greater stress, these structural proteins increase their expression. It is believed that alterations in the expres‐ sion of these proteins are positively correlated with the of LR development that partially has an indirect effect on whole plant photosynthetic process [123]. While the decrease of lateral root development is a well-known response to water stress, none of the mutants that are resistant to drought stress have a reduced number of LR [124]. Only a few transcription factors have shown to regulate the formation of roots under drought conditions, among them stands the MYB96 transcription factor since it plays an important role in LR growth under drought stress conditions [124], these same authors found that overexpression of MYB96 promotes resistance to drought and reduced lateral root density.

#### **4.2. Regulation of root system architecture by nutrients**

by the Na+

activity [110, 111].

*4.1.1.2. Reactive oxygen species*

146 Abiotic Stress - Plant Responses and Applications in Agriculture

/H+ exchanger in the PM [100] or its capture by tonoplast [109]; changes of metab‐

olism and signalization by polyamines and Reactive Oxygen Species (ROS) and the antioxidant

ROS fluctuations in time and space can be interpreted as signals to regulate growth, develop‐ ment, cell death and stress responses [112, 113]. Understanding the mechanisms that control ROS signaling in cells in response to water stress and salinity could therefore provide a powerful strategy for increasing crop tolerance to these environmental stress conditions [114]. Among the targets of ROS action at the cellular level, there are ion channels that mediate ion exchange in the PM. In the PM of roots and guard cells H2O2, stimulates the channels activated by hyperpolarization that mediate the influx of Ca2+ and NSCC [112, 115, 116] and inhibit the K+ outward and inward rectifier currents [117]. The stimulation of the influx of Ca2+ in guard cells appears to mediate the induction of stomata closure by ABA [116, 118-120]. At the same time it was reported that the OH• activates a Ca2+ inward and K+ outward currents in epidermal protoplasts derived from mature and growth zone of Arabidopsis roots [115]. A larger stimulation of the inward current of Ca2+ in the growth zone may indicate that ROS are involved in growth regulation via Ca2+ signaling. Moreover, the OH• produced by NADPH oxidase in Arabidopsis root hairs activated a Ca2+ inward rectifier conductance causing an increase in cytosolic Ca2+ allowing the root elongation [112]. Recently it has been reported that under severe water stress autophagy programmed cell death occurs in the region of the root apical meristem [121]. There is evidence that this defense mechanism is promoted by the accumulation of ROS in stressed meristematic cells of root tips. Analysis of the expression of BAX inhibitor-1 (AtBI1, apoptotic inhibitor) and the phenotypic response of the mutant *atbi1-1* under severe water stress indicates that AtBI1 and the pathway of endoplasmic reticulum stress response modulates the induction of PCD by water stress. As a result, thin and short roots induce an increase in their tolerance to stress. These authors also propose that under severe drought stress, plants activate the PCD program in the root apical meristem, removing the apical dominance; so they can remodel the RSA to adapt to stressful environments [122].

A slight drought stress increases the expression of enzymes associated with root morphology (Xyloglucan endotransglucosylase) while other structural proteins (actin and tubulin) are downregulated, these proteins are strongly correlated with root growth since its function is the vesicular carrying in cells with polarized growth (e.g. root hairs) allowing its growth and hence an augmentation in the surface of water uptake. However, when there is a greater stress, these structural proteins increase their expression. It is believed that alterations in the expres‐ sion of these proteins are positively correlated with the of LR development that partially has an indirect effect on whole plant photosynthetic process [123]. While the decrease of lateral root development is a well-known response to water stress, none of the mutants that are resistant to drought stress have a reduced number of LR [124]. Only a few transcription factors have shown to regulate the formation of roots under drought conditions, among them stands the MYB96 transcription factor since it plays an important role in LR growth under drought

In soil nutrients such as phosphorus (P), nitrogen (N), potassium (K) and iron (Fe), are distributed in a heterogenous patching pattern. As soon as the PR emerges from the seed, it has to grow. As growth goes on, *de novo* LR are formed to generate the particular RS mor‐ phology and architecture. These nutrients alter root patterning through particular signal transduction pathways. Thus, during their life plants change their PEDP in order to increase exponentially the root-soil interaction area and find the nutrient-rich regions [5, 125-129]. The changes in Arabidopsis root system are specific for each nutrient. P, N and K starvation dramatically alter primary root length (Figure 2).

**Figure 2.** Changes in root system architecture of Arabidopsis seedlings when growth on media depleted of phospho‐ rous (P), nitrogen (N), potassium (P) and iron (Fe).

#### *4.2.1. Phosphate starvation*

Root system in boot monocotyledonous and dicotyledonous plants, present a set of develop‐ mental modifications that tend to increase the exploratory capacity of the plant [130]. When Arabidopsis growth under limiting P conditions their RSA changes dramatically such as reduction in primary root length, increased formation of LRs and greater formation of root hairs [126, 128]. On optimal P conditions the newly formed root cells are added by the mitotic activity of primary meristem. These cells then get away from the meristem and increase their length, and the elongation process ends when the cells start to differentiate. When plants are P starved, cell division in the primary root meristems gradually reduces and the cells start to prematurely differentiate until total inhibition of cell elongation and loss of meristematic activity occur (meristem exhaustion). At the end, root tips change their physiological charac‐ teristics and the exhausted meristem becomes a structure which takes part in P uptake. In this process, root tips locally detect P deficiency, this response being mediated by at least LPR multicopper oxidase genes [12, 131, 132]. Recently, iron (Fe) has been reported to play a role as well in the control of these PED reprogramming [133]. This change of root architecture is due to the fact that, in both meristematic and elongation areas, the content of ROS is reduced as long as the determined PED goes on [134].

In the past decade the changes in RSA evocated by P availability has been widely studied, several genes that regulates the root architectural changes has been idetified, trascription factor such as WRKY75, ZAT6 (ZINC FINGER 6), Pi-responsive R2R3 MYB (MYB62) and BHLH32 (BASIC HELIX\_LOOP\_HELIX 32) [135-138] are key regulators in this response. Mutants affected in the RSA changes induced P availability have been isolated: *pdr2* (*phosphate deficiency response 2*), *lpi* (low phosphorus-insensitive) *siz1* [*S*AP (scaffold attachment factor, acinus, protein inhibitor of activated signal transducer and activator of transcription) and *Miz1* (Msx2 interacting zinc finger), SIZ] [139-141]. It has been reported that ethylene is involved in modulating Pi-starvation-responsive root growth, it may restrict elongation of PR, but promote elongation of LRs [142] HPS4/SABRE (important regulator of cell expansion in Arabidopsis) antagonistically interacts with ethylene signalling to regulate plant responses to Pi starvation. Furthermore, it is shown that Pi-starved *hps4* mutants accumulate more auxin in their root tips than the wild type, which may explain the increased inhibition of their primary root growth when grown under Pi deficiency [143]. Gibberellins and ROS also trigger responses involving DELLAs proteins which control the rate and timing of cell proliferation and they will be dealt with in further sections.

#### *4.2.2. Nitrogen*

Nis fundamentalforbiologicalmolecules, suchasnucleotides,aminoacids,andproteins.Plants needtoacquirenitrogen(N) efficientlyfromthe soilforgrowthanddevelopment.Insoil,nitrate (NO3−) is one of the major N sources for higher plant and their concentrations vary in both time and space. Plants are able to sensing these variations of NO3−, which is one of the most impor‐ tant environmental signals affecting plant physiology and development [144]. The effects of N supplyonplantdevelopmenthavebeenparticularlystudiedinArabidopsis.NO3−-freemedium drastically reduces shoot biomass production and appears to have little effect on PR length (Figure 2). However, NO3− has a dual role on LRs. On one hand, the uniform exposure of RS to high nitrate (>10 mM) inhibits lateral root growth at a specific developmental step correspond‐ ingtotheactivationofthemeristeminLRPaftertheiremergence[145-147].AsahighNO3− supply on only one part of the RS is able to repress lateral root growth on the whole RS, it has been proposed that nitrate accumulation in the aerial tissues is responsible for this LRP arrest, suggestingthatlong-distancesignals totherootareinvolved.Ontheotherhand,whentheentire RS is exposed to low nitrate concentration (10 μM) and only one part of the RS is exposed to a high nitrate, there is local proliferation of LR. NO3− locally promotes LR growth and increased lateralrootgrowthratedue toahigher cellproductioninthe lateralrootmeristem[145,146,148]. The local stimulation of lateral root growth by nitrate-rich patches is a striking example of the

nutrient-induced plasticity of PERDP. This stimulation could be dependent on NRT1.1 (Nitrate Transporter1).This ispartiallydue tothe factthatNRT1.1 repressesLRPemergence andgrowth of young LRs in the absence of nitrate. NRT1.1 transports nitrate and facilitates auxin trans‐ portinaconcentration-dependentmanner.NRT1.1repressesLRgrowthatlownitrateavailabil‐ itybypromotingbasipetalauxintransportoutoftheLRP,towardstheparentalroot[149].MADSbox transcription factor NITRATE REGULATED (ANR1) and Auxin signaling F-box protein 3 (AFB3)arekeyregulatorsofRSAinresponsetonitrateavailability.The*Chlorate-resistant1*mutant (*chl1)* is *ANR1* affected, and is less responsive to the localized NO3−-rich patches similarly to transgenicplants inwhichANR1expressionisdown-regulated.InthetipsofLRandLRP,ANR1 is expressed and is localize with NRT1.1 [150]. The *afb3-1* mutant shows altered root develop‐ ment response to nitrate. *AFB3* is an auxin receptor gene induced by nitrate in the primary root tip and pericycle; its mRNA is the target of miR393 that is induced by the products of NO3− assimilation.

#### *4.2.3. Potasium and iron*

prematurely differentiate until total inhibition of cell elongation and loss of meristematic activity occur (meristem exhaustion). At the end, root tips change their physiological charac‐ teristics and the exhausted meristem becomes a structure which takes part in P uptake. In this process, root tips locally detect P deficiency, this response being mediated by at least LPR multicopper oxidase genes [12, 131, 132]. Recently, iron (Fe) has been reported to play a role as well in the control of these PED reprogramming [133]. This change of root architecture is due to the fact that, in both meristematic and elongation areas, the content of ROS is reduced

In the past decade the changes in RSA evocated by P availability has been widely studied, several genes that regulates the root architectural changes has been idetified, trascription factor such as WRKY75, ZAT6 (ZINC FINGER 6), Pi-responsive R2R3 MYB (MYB62) and BHLH32 (BASIC HELIX\_LOOP\_HELIX 32) [135-138] are key regulators in this response. Mutants affected in the RSA changes induced P availability have been isolated: *pdr2* (*phosphate deficiency response 2*), *lpi* (low phosphorus-insensitive) *siz1* [*S*AP (scaffold attachment factor, acinus, protein inhibitor of activated signal transducer and activator of transcription) and *Miz1* (Msx2 interacting zinc finger), SIZ] [139-141]. It has been reported that ethylene is involved in modulating Pi-starvation-responsive root growth, it may restrict elongation of PR, but promote elongation of LRs [142] HPS4/SABRE (important regulator of cell expansion in Arabidopsis) antagonistically interacts with ethylene signalling to regulate plant responses to Pi starvation. Furthermore, it is shown that Pi-starved *hps4* mutants accumulate more auxin in their root tips than the wild type, which may explain the increased inhibition of their primary root growth when grown under Pi deficiency [143]. Gibberellins and ROS also trigger responses involving DELLAs proteins which control the rate and timing of cell proliferation and they will be dealt

Nis fundamentalforbiologicalmolecules, suchasnucleotides,aminoacids,andproteins.Plants needtoacquirenitrogen(N) efficientlyfromthe soilforgrowthanddevelopment.Insoil,nitrate (NO3−) is one of the major N sources for higher plant and their concentrations vary in both time and space. Plants are able to sensing these variations of NO3−, which is one of the most impor‐ tant environmental signals affecting plant physiology and development [144]. The effects of N supplyonplantdevelopmenthavebeenparticularlystudiedinArabidopsis.NO3−-freemedium drastically reduces shoot biomass production and appears to have little effect on PR length (Figure 2). However, NO3− has a dual role on LRs. On one hand, the uniform exposure of RS to high nitrate (>10 mM) inhibits lateral root growth at a specific developmental step correspond‐ ingtotheactivationofthemeristeminLRPaftertheiremergence[145-147].AsahighNO3− supply on only one part of the RS is able to repress lateral root growth on the whole RS, it has been proposed that nitrate accumulation in the aerial tissues is responsible for this LRP arrest, suggestingthatlong-distancesignals totherootareinvolved.Ontheotherhand,whentheentire RS is exposed to low nitrate concentration (10 μM) and only one part of the RS is exposed to a high nitrate, there is local proliferation of LR. NO3− locally promotes LR growth and increased lateralrootgrowthratedue toahigher cellproductioninthe lateralrootmeristem[145,146,148]. The local stimulation of lateral root growth by nitrate-rich patches is a striking example of the

as long as the determined PED goes on [134].

148 Abiotic Stress - Plant Responses and Applications in Agriculture

with in further sections.

*4.2.2. Nitrogen*

Contrasting with physiological and molecular responses to low K and Fe, changes in RSA have been scarcely described. Potassium deficiencies arrest LR and PR development in Arabidopsis (Figure 2) [129]. K+ transporters play a crucial role in SRA changes in response to K+ availability. Disruption of the root-specific K+ -channel AKT1 in the *akt1-1* Arabidopsis mutant causes reduced ability of plants to grow in low potassium media (100 μM)[151]. In Arabidopsis, changes in the gravitropic behavior of RS were also observed in low potassium media. The genes of the *KUP/HAK/KT* family are homologous to bacterial KUP (TrkD) potassium porters. The *trh1* (tiny root-hair 1) mutant, which is disrupted in *AtKUP4/TRH1* gene shows agravi‐ tropic behavior in its roots independently of K+ concentration in the media when grown on vertical agar plates, and also, *ProTRH1:GUS* expression is limited to the root cap where gravity is sensed. Interestingly, agravitropic responses in *trh1* are complemented by exogenous auxin. This mutation is associated with the loss of auxin pattern in the root apex. Thus, TRH1 is an important part of auxin transport system in Arabidopsis roots [151-153].

Typically, the root architectural changes in response to low availability of Fe include ectopic formation of root hair due to modulation in their position and abundance [154]. Recently, Giehl et al. (2012) analyzed the changes in LR architecture in response to localized Fe supply in wildtype and Fe acquisition and translocation- defective mutant plants. They found that lateral root elongation is highly responsive to local Fe and that the symplastic Fe pool in LR favors local auxin accumulation. They identified the auxin transporter AUX1 as a major Fe-sensitive component in the auxin signaling pathway that mainly directs the rootward auxin stream into LRs that have access to Fe.

#### **4.3. Meristematic activity regulation by abiotic stress**

To cope with environmental changes, plants have to adapt their growth timing and pattern by altering rates of cell proliferation and differentiation. The expression of several cell cycle genes is increased or decreased upon external cues (Figure 3) [155] but it is poorly understood the full molecular basis supporting these transcriptional controls, and if the cell cycle control modifications happen to fall into the post-translational category, the current knowledge is also very limited. However, there have been identified several key players in stress-induced cell cycle modifications that have cast the first light over the understanding the talk between environmental signals and the mitotic or endoreplication cycle. Gibberellins (GAs), plant hormones, promote cell expansion by disrupting growth inhibitory proteins named DELLAs [156] and also promote cell proliferation in Arabidopsis [157]. In the root meristem of GAdeficient mutants, cell division rate is decreased and the phenotype is rescued by GA treat‐ ment. DELLA proteins are also involved in this regulation, as non-degradable forms of DELLA inhibit cell proliferation. Low levels of GAs in GA-deficient mutants enhance the expression of certain CDK inhibitor genes – KRP2, SIM, SMR1 and SMR2- with a DELLA-related mech‐ anism, and cell proliferation defects shown by these mutants can be recovered by overex‐ pressing CYCD3;1. These findings tend to indicate that GA signaling drives cell proliferation by modulating the activity of CYC-CDK complexes, at least partially mediated by the DELLAdependent expression of CDK inhibitors, and thus making DELLA a potential intermediate in the signal transduction channel connecting environmental signals and cell cycle progression. This is proposed to be a consequence of reduced cell expansion and associated division of the endodermis layer in the root apical meristem [158, 159], suggesting a role for the endodermis in controlling the growth rate in the root apical meristem. Another potential link is RICE SALT SENSITIVE 1 (RSS1), controlling the cell cycle progression under various abiotic stress conditions [160]. The *rss1* mutants do not present evident growth defects under normal conditions, but they display hypersensitivity to high salinity, ionic stress and hyperosmotic stress. Under these conditions, in *rss1*, shoot and root meristems are severely affected, showing a reduced population of proliferating cells, leaving RSS1 as a required factor for proliferative cell status in the meristem. RSS1 is expressed during the S phase of the mitotic cycle and its protein is degraded via APC/C during the M/G1 transition. RSS1 interacts with a Type 1 Protein Phosphatase (PP1), known in humans to inactivate Retinoblastoma (Rb) proteins through dephosphorylation, which is inhibitory to the G1/S transition [161]. Sugars can act as signaling molecules in assorted biological processes, and even that sucrose-dependent cyclin expression is known since a decade ago [162], LR formation through sucrose induction is a good example of sugar-dependent reactivation of cell proliferation [163]. This recent study shows that the expression of CYCD4;1 levels in root pericycle cells is dependent on the sucrose availability, and that reduced CYCD4;1 levels in *cyca4;1* mutants or wild-type (wt) roots grown in the absence of sucrose cause LR density to drop. It is not clear how sucrose upregulates CYCD4;1 in specifically in that kind of cells, but these findings suggest that the transcriptional effect has to do with sucrose-dependent regulation of LR density. Notably, auxin does not have an effect over the expression of CYCD4;1 in pericycle cells, and restores the reduced LR density phenotype of cyca4;1 mutants, suggesting that CYCD4;1 has no role in the auxin-mediated LR initiation pathway. CYCD3;1, is also responsive to sucrose availability, but the effects of this over CYCD3;1 activities are not clear [164]. Endoreplication progress is also affected by several environmental signals. E2F3/ DEL1, an atypical E2F present in Arabidopsis, and that functions as a transcriptional repressor, is one of the key regulators that negatively controls the entry into the endoreplicative cycle [165]. It has been suggested that the balance between the transcriptional activator E2Fb and repressor E2Fc controls light-dependent endoreplication through the antagonistic modification of the DEL1 expression [166]. E2Fb and E2Fc compete for the same DNA-binding site of the DEL1 promoter and enhances the DEL1 expression, respectively. Under light conditions, E2Fb is the preferred binding partner, enhancing DEL1 expression and consequently repressing the endoreplicative cycle [167]. In the dark E2Fb is degraded, allowing E2Fc to bind to the DEL1 promoter, repressing DEL1 expression. Ultra‐ violet-B (UVB) radiation damages DNA molecules by forming cyclobutane pyrimidine dimers (CPDs) which prevent DNA transcription and translation. Plants remove CPDs by photolyas‐ es, and these enzymes are encoded by a PHOTOLYASE 1 (PHR1) [168, 169]. It has been shown that in addition to CCS52A2, a known target of DEL1, DEL1 represses the transcription of the PHR1 gene and thereby coordinates DNA repair and endocycle triggering [167]. After UVB treatment, DEL1 expression is strongly downregulated, permitting the upregulation of PHR1 and thus leaving the cell able to repair its DNA.

very limited. However, there have been identified several key players in stress-induced cell cycle modifications that have cast the first light over the understanding the talk between environmental signals and the mitotic or endoreplication cycle. Gibberellins (GAs), plant hormones, promote cell expansion by disrupting growth inhibitory proteins named DELLAs [156] and also promote cell proliferation in Arabidopsis [157]. In the root meristem of GAdeficient mutants, cell division rate is decreased and the phenotype is rescued by GA treat‐ ment. DELLA proteins are also involved in this regulation, as non-degradable forms of DELLA inhibit cell proliferation. Low levels of GAs in GA-deficient mutants enhance the expression of certain CDK inhibitor genes – KRP2, SIM, SMR1 and SMR2- with a DELLA-related mech‐ anism, and cell proliferation defects shown by these mutants can be recovered by overex‐ pressing CYCD3;1. These findings tend to indicate that GA signaling drives cell proliferation by modulating the activity of CYC-CDK complexes, at least partially mediated by the DELLAdependent expression of CDK inhibitors, and thus making DELLA a potential intermediate in the signal transduction channel connecting environmental signals and cell cycle progression. This is proposed to be a consequence of reduced cell expansion and associated division of the endodermis layer in the root apical meristem [158, 159], suggesting a role for the endodermis in controlling the growth rate in the root apical meristem. Another potential link is RICE SALT SENSITIVE 1 (RSS1), controlling the cell cycle progression under various abiotic stress conditions [160]. The *rss1* mutants do not present evident growth defects under normal conditions, but they display hypersensitivity to high salinity, ionic stress and hyperosmotic stress. Under these conditions, in *rss1*, shoot and root meristems are severely affected, showing a reduced population of proliferating cells, leaving RSS1 as a required factor for proliferative cell status in the meristem. RSS1 is expressed during the S phase of the mitotic cycle and its protein is degraded via APC/C during the M/G1 transition. RSS1 interacts with a Type 1 Protein Phosphatase (PP1), known in humans to inactivate Retinoblastoma (Rb) proteins through dephosphorylation, which is inhibitory to the G1/S transition [161]. Sugars can act as signaling molecules in assorted biological processes, and even that sucrose-dependent cyclin expression is known since a decade ago [162], LR formation through sucrose induction is a good example of sugar-dependent reactivation of cell proliferation [163]. This recent study shows that the expression of CYCD4;1 levels in root pericycle cells is dependent on the sucrose availability, and that reduced CYCD4;1 levels in *cyca4;1* mutants or wild-type (wt) roots grown in the absence of sucrose cause LR density to drop. It is not clear how sucrose upregulates CYCD4;1 in specifically in that kind of cells, but these findings suggest that the transcriptional effect has to do with sucrose-dependent regulation of LR density. Notably, auxin does not have an effect over the expression of CYCD4;1 in pericycle cells, and restores the reduced LR density phenotype of cyca4;1 mutants, suggesting that CYCD4;1 has no role in the auxin-mediated LR initiation pathway. CYCD3;1, is also responsive to sucrose availability, but the effects of this over CYCD3;1 activities are not clear [164]. Endoreplication progress is also affected by several environmental signals. E2F3/ DEL1, an atypical E2F present in Arabidopsis, and that functions as a transcriptional repressor, is one of the key regulators that negatively controls the entry into the endoreplicative cycle [165]. It has been suggested that the balance between the transcriptional activator E2Fb and repressor E2Fc controls light-dependent endoreplication through the antagonistic modification of the DEL1 expression [166]. E2Fb and E2Fc compete

150 Abiotic Stress - Plant Responses and Applications in Agriculture

Environmental and nutrient availability condition changes affect root apical meristem organization [170]. ROS and Reactive Nitrogen Species (RNS) have been reported to be rapidly induced by several kinds of environmental stresses in a variety of plant species to regulate the plant response to biotic and abiotic stresses. In particular, oxidative stress caused by drought and salinity, has been proposed that ROS production is an obligatory element of the response to induce an adequate acclimatization process [114]. Therefore, the degree of accumulation of ROS is what determines whether it is a part of the signaling mechanism (low production) or a harmful event (high production) to plants, making the control of production and degradation of ROS the crucial element for plant resistance to stress [114, 171-173]. ROS is never completely eliminated, as it plays an important role in signaling and growth regulation [174]; ROS quenching inhibits the root growth [115], and overexpression in Arabidopsis of a peroxidase localized mainly in the elongation zone stimulates root elongation [175]. This calls for redox control of the cell cycle, which is possibly linked to A-type cyclins, shown to be differentially expressed under oxidative stress in tobacco, resulting in cell cycle arrest [176]. It is also known that low temperatures [177, 178], metals [179] and nutrient deficiency [180] induce the presence of ROS and RNS in specific tissues. These forms of stress affect root morphology by reducing primary root growth and promoting branching, but the mechanisms of the redox generationsensing are not well understood.

The typical response of the Arabidopsis radical system to low phosphorous (P) availability is an example to illustrate how complex these processes are. A recent study showed that ROSs are involved in the developmental adaptation of the RS to low P availability [181]. Rapidly growing roots of plants within a normal P medium synthesize ROS in the elongation zone and QC on the root, whereas seedlings within low P mediums showed a slow growth of the PR, and the ROS normally found in the QC relocate to cortical and epidermal tissues. In a previous study [131], it has been indicated that Arabidopsis plants under low P conditions show a decreased number of cells in the root apical meristem, and it decreases until it is depleted. In these roots, all root apical meristem cells differentiate and the QC is almost indistinguishable. A possible cause of this response to P starvation could be the cell cycle arrest modulated by ROS and CYCAs, but it is more complicated, as the response is also modulated by auxin [170, 182] and gibberellin-DELLA pathways [183]. Interestingly, DELLAs promote survival by reducing the levels of ROS [184], suggesting a link between the gibberellin-DELLA cell cycle

**Figure 3.** Abiotic Stress affects root mitotic cycle. A) Lateral root formation responds to sucrose availability in medium through an unknown link that enhances CycD4;1 expression in pericycle cells, allowing them to proliferate; it also re‐ sponds to low P availability through the activation of the auxin pathway. Auxin controls lateral root initiation through the E2F mechanism, promoting the degradation of IAA14 and thus activating ARF7/18 transcription factors, subse‐ quently activating LBD18/33 factors which in turn bind and activate the promoter of the cell cycle-enabling E2F tran‐ scription factor. B) Meristematic maintenance also responds to diverse environmental changes. Through the gibberellin pathway, DELLA proteins inhibit cell cycle progression by enhancing the accumulation of CDK inhibitors. DELLAs are influenced by various environmental factors including light and temperature. These factors, as well as met‐ als and nutrient deficiency as in low P, promote the accumulation of ROS, known for inhibiting cell cycle in tobacco cells. Interestingly, DELLAs promote survival by lowering the levels of ROS, indicating a novel pathway to maintain cell cycle in the meristems. Salinity affects it by activating RSS1, required to maintain the mitotic cycle in the meristem. The putative mechanism comprises RSS1 interacting with a type 1 protein phosphatase (PP1), regulating its activity at the G1/S transition.

control pathway and ROS pathway in the developmental adaptation to the RS to low P availability It requires further study to precise the way these signals crosstalk and determine the developmental adaptation of the RS to low P availability by means of cell cycle progression control, as well as additional efforts to reveal the manners by which other regulatory pathways responding to abiotic stress interact with and influence the cell cycle control mechanisms.

#### **5. Conclusion**

Sensing and responding to environmental cues by roots enable plants to overcome the challenges posed by their sessile lifestyle [10]. As we mentioned above, RS is important to plants due to a wide variety of processes, including nutrient and water uptake from soil, which is a complex medium with high spatial and temporal environmental variability. Thus, it is not surprising that RSA is highly influenced by environmental cues [9, 148]. The importance of RSA in plant productivity stems from the fact that many soil resources are unevenly distributed or are subject to localized depletion, so that the spatial deployment of the RS will largely determine the ability of a plant to exploit those resources [4].The PERDP which regulates the changes in RSA, can be considered as an evolutionary response to medium with high spatial and temporal variability in resource supplies [148]. The genetic controls regarding root deployment (PERDP) are still largely unknown. A great effort has been made to understand the molecular components that regulate the formation, proliferation and maintenance of meristems, either being embryo or pericycle-originated. Nevertheless, the facts behind their regulation by environmental factors still leave many questions to be solved.

Plants are important to humans, as they provide food, fuel, fibres, medicines and materials. As the global population is projected by the UN to rise to over 9 billion by 2050, the improve‐ ment of crops is becoming an increasingly pressuring issue. The new challenge arisen is to solve the current and future obstacles to the maintenance of food supply security through higher crop yields [10]. Water and nutrient availability limit the productivity in most agricul‐ tural ecosystems. In all environments characterized by low water and nutrient availability, RSA is a fundamental aspect, the acquisition of soil resources by RS systems is therefore a subject of considerable interest in agriculture [4]. RSA and PERDP are important agronomic traits; the right architecture in a given environment allows plants to survive periods of water of nutrient deficit, and compete effectively for resources [9]. Most of drought-resistant rice varieties have a deeper and more highly branched RS than sensitive varieties [9].

Understanding the RSA and the PERDP holds potential for the exploitation and opening of new options for genetic manipulation of the characteristics of the root, in order to both increase food plant yield and optimize agricultural land use. Improved access to deep soil water, inherently reducing the need for irrigation, is one potential benefit that could be achieved by exploitation of RSA. Increase in root branching and root hair in crops may enable plants to make more efficient use of existing soil nutrients and increase stress tolerance, improving yields while decreasing the need for heavy fertilizer application [9, 10]. Understanding which structures and environmental cues that regulate proliferation and elongation of the RS cells will allow us to develop strategies to generate crops that possess greater soil exploration capacities in order of a more efficient usage of nutrients and water present in the soil.

## **Acknowledgements**

control pathway and ROS pathway in the developmental adaptation to the RS to low P availability It requires further study to precise the way these signals crosstalk and determine the developmental adaptation of the RS to low P availability by means of cell cycle progression control, as well as additional efforts to reveal the manners by which other regulatory pathways responding to abiotic stress interact with and influence the cell cycle control mechanisms.

**Figure 3.** Abiotic Stress affects root mitotic cycle. A) Lateral root formation responds to sucrose availability in medium through an unknown link that enhances CycD4;1 expression in pericycle cells, allowing them to proliferate; it also re‐ sponds to low P availability through the activation of the auxin pathway. Auxin controls lateral root initiation through the E2F mechanism, promoting the degradation of IAA14 and thus activating ARF7/18 transcription factors, subse‐ quently activating LBD18/33 factors which in turn bind and activate the promoter of the cell cycle-enabling E2F tran‐ scription factor. B) Meristematic maintenance also responds to diverse environmental changes. Through the gibberellin pathway, DELLA proteins inhibit cell cycle progression by enhancing the accumulation of CDK inhibitors. DELLAs are influenced by various environmental factors including light and temperature. These factors, as well as met‐ als and nutrient deficiency as in low P, promote the accumulation of ROS, known for inhibiting cell cycle in tobacco cells. Interestingly, DELLAs promote survival by lowering the levels of ROS, indicating a novel pathway to maintain cell cycle in the meristems. Salinity affects it by activating RSS1, required to maintain the mitotic cycle in the meristem. The putative mechanism comprises RSS1 interacting with a type 1 protein phosphatase (PP1), regulating its activity at the

Sensing and responding to environmental cues by roots enable plants to overcome the challenges posed by their sessile lifestyle [10]. As we mentioned above, RS is important to

**5. Conclusion**

G1/S transition.

152 Abiotic Stress - Plant Responses and Applications in Agriculture

We thank Biol. V. Limones Briones for their assistance in the literature review. Writing of this paper has been made possible by a financial support from Consejo Nacional de Ciencia y Tecnología (CONACYT) proyecto Ciencia Básica clave CB2010/15685 and Red de Cuerpos Académicos: Biotecnología para el desarrollo de una Agricultura sustentable, UAZ-CA 138.

## **Author details**

L. Sánchez-Calderón1 , M.E. Ibarra-Cortés2 and I. Zepeda-Jazo1,3

1 Unidad de Ciencias Biológicas, Universidad Autónoma de Zacatecas. Zac, México

2 Instituto Tecnológico de Monterrey Campus Querétaro Celaya, Gunajuato, México

3 Current Address: Trayectoria Genómica Alimentaria Universidad de La Ciénega del Esta‐ do de Michoacán de Ocampo. Mich, México

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