**7. CDF/MTP**

The cation diffusion facilitator (CDF) family of proteins plays an important role in the maintenance of cation homeostasis in all forms of life from bacteria, yeast, plants to mammals [82]. Generally, CDF proteins are involved in binding and efflux of cations such as Zn, Fe, Co, Cd, and Mn from the cytoplasm either by sequestrating into internal organelles like vacuole or effluxing from the cell [82, 83]. CDF transporters also influence the cation accumulation, metal ion tolerance, signal transduction cascades, oxidative stress resistance, and protein turnover in cells [84]. In plants, CDF members are called Metal Tolerance Proteins (MTPs) and as Solute carrier family 30 (SLC30) in vertebrates [84].

The first plant CDF protein identified was ZAT (zinc transporter of *Arabidopsis thaliana*), because of its role in heavy metal tolerance in Arabidopsis. Later it was renamed as AtMTP1 (Metal Tolerance Protein 1) [85]. MTPs are a group of proteins that play an important role in heavy metal homeostasis in plants [82, 83]. MTP members are present in all the three kingdoms (Archaea, Eubacteria, and Eukaryotes). The plant CDF family can be classified into three subgroups phylogenetically: Zn-CDF, Fe/Zn-CDF, and Mn-CDF [83] based on their main substrate transported: Zn, Zn, and Fe, or Mn [82, 84].

Rice genome has 10 MTP genes [84]. Studies have shown that Rice Metal Tolerance Protein1 (OsMTP1) gene expression is induced by Cd and OsMTP1 belongs to the Zn-CDF subgroup. In rice, there are five Mn-CDFs, three Zn-CDFs, one Fe/Zn- CDF, and one unclassified CDF.

*OsMTP1* was characterized recently [86, 87]. In mature leaves and stem, it is highly expressed [86]. Generally, OsMTP1 transports Zn but can also transport Co, Fe, and Cd. Earlier OsMTP1 has been shown to transport Ni [86]. The vacuolar localization of OsMTP1 in the tonoplast, compartmentalizes primarily Zn, but also Co, Fe, and Cd, and serves as a detoxification system when these metals are available in excess. OsMTP1 is expressed constitutively and upregulated by Cd [86]. OsMTP1, OsMTP5, and OsMTP12 belong to the Zn-CDF subgroup [82, 84]. Expression of OsMTP1 in leaves, stems, roots, and flowers is relatively low and spatially and temporally regulated during development of rice. Also, it shows differential response to Cd stress. Transgenic assays in rice have shown that OsMTP1 expression levels can change plant cation absorption and in turn has affect on Zn, Ni, and Cd contents [86].

plant throughout its growth and developmental stages [74]. Enhanced activity of OsHMA3 is related to increased storage of Cd in roots and its decreased transport to the shoot and the final accumulation in rice grains [74]. OsHMA2 is localized at the root pericycle and plays a

*OsHMA3* gene selectively sequesters Cd into the vacuoles thus limits the root-to-shoot translocation of Cd [75, 76]. In rice plant, *OsHMA2* gene has also been shown to be involved in the

In root cells, OsHMA4 is a vacuolar membrane-localized transporter and is involved in sequestering Cu into the vacuoles. OsHMA4 specifically transports Cu. Increased Cu accumulation in rice grain due to increased root-to-shoot translocation of Cu has been observed when OsHMA4 function is lost. In rice OsHMA4–OsHMA9 are members of the Cu/Ag subgroup of HMAs. OsHMA5 is a Cu transporter, localized to the plasma membrane [81]. In rice, OsHMA5 is involved in transferring Cu into the xylem for its root-to-shoot translocation and/ or Cu detoxification in roots [81]. OsHMA4 is induced under long-term exposure of excess Cu and its expression is suppressed by Cu deficiency. In mature root zone, OsHMA4 is localized at the pericycle [81]. OsHMA4 regulates the cellular Cu concentration before loading to the xylem depending on its environmental concentration. OsHMA3 is localized in all root cells [75]. In future, the mechanism responsible for the transporter substrate specificity of the

The cation diffusion facilitator (CDF) family of proteins plays an important role in the maintenance of cation homeostasis in all forms of life from bacteria, yeast, plants to mammals [82]. Generally, CDF proteins are involved in binding and efflux of cations such as Zn, Fe, Co, Cd, and Mn from the cytoplasm either by sequestrating into internal organelles like vacuole or effluxing from the cell [82, 83]. CDF transporters also influence the cation accumulation, metal ion tolerance, signal transduction cascades, oxidative stress resistance, and protein turnover in cells [84]. In plants, CDF members are called Metal Tolerance Proteins (MTPs) and as Solute

The first plant CDF protein identified was ZAT (zinc transporter of *Arabidopsis thaliana*), because of its role in heavy metal tolerance in Arabidopsis. Later it was renamed as AtMTP1 (Metal Tolerance Protein 1) [85]. MTPs are a group of proteins that play an important role in heavy metal homeostasis in plants [82, 83]. MTP members are present in all the three kingdoms (Archaea, Eubacteria, and Eukaryotes). The plant CDF family can be classified into three subgroups phylogenetically: Zn-CDF, Fe/Zn-CDF, and Mn-CDF [83] based on their

Rice genome has 10 MTP genes [84]. Studies have shown that Rice Metal Tolerance Protein1 (OsMTP1) gene expression is induced by Cd and OsMTP1 belongs to the Zn-CDF subgroup. In rice, there are five Mn-CDFs, three Zn-CDFs, one Fe/Zn- CDF, and one unclassified CDF.

major role in of transport of Zn and Cd during xylem loading [74, 75].

translocation of Cd through xylem from root to shoot [79, 80].

HMAs needs to be studied.

160 Rice Crop - Current Developments

carrier family 30 (SLC30) in vertebrates [84].

main substrate transported: Zn, Zn, and Fe, or Mn [82, 84].

**7. CDF/MTP**

In rice, OsMTP8.1 is the first Mn-CDF member to be identified. It is localized on to the tonoplast, and its over expression in rice enhances Mn accumulation and tolerance [88]. OsMTP9 is also a Mn-CDF member, which is involved in the uptake and translocation of Mn in rice plant [89]. Mn and other heavy metals induce the expression of *OsMTP11* in rice. In rice plant, *OsMTP1* is involved in Zn and Cd homeostasis/stress and mediates their translocation from roots to the aerial parts [86]. In most rice plant tissues *OsMTP11* is constitutively expressed.

OsMTP8.1 is localized to the tonoplast and involved in the detoxification of manganese by sequestering excess manganese to the vacuoles [90]. In rice root, OsMTP9 is polarly localized at the proximal side of both exodermis and endodermis opposite to Nramp5. The cooperative transport by Nramp5 and MTP9 efficiently transport Mn leading to its high accumulation in rice [91].

The Mn-CDF group in plants is further clustered into two subgroups, Groups 8 and 9 [82]. In the rice genome, there are three members (MTP9/11/11.1) of Group 9. MTP9 shows much higher expression in the roots than in the basal region and shoots. The expression is unaffected by the deficiency of iron, zinc, copper, and manganese. The expression of MTP9 in roots is eightfold higher in the basal parts than that in apical parts. At the reproductive growth stage, MTP9 is also expressed in other organs such as nodes and leaf sheath in addition to the roots. MTP9 is polarly located at the proximal side of the exodermis and the endodermis, which is in opposition to Nramp5 [71]. Therefore, MTP9 at the proximal side of the exodermis releases manganese taken up by Nramp5 to the apoplast of a spoke-like structure in the aerenchyma, whereas MTP9 at the proximal side of endodermis further releases manganese toward the apoplast of stele including xylem vessels. Thus polar localization of transporters plays an important role in the directional transport of minerals. Recently, a number of transporters have been found to show polar localization. However, our understanding of the molecular mechanism underlying polar localization is still very poor. Also, MTP9 is different from other members of the Mn-CDF group as it shows a distinct expression pattern in tissue and subcellular localization. MTP9 is mainly expressed in the roots, but MTP8.1 from rice [90] in the same group is mainly expressed in shoots rather than roots. Different from other members, rice MTP9 is localized to the plasma membrane. These differences are associated with the role of MTP9 in manganese uptake in rice roots. In conclusion, MTP9 is a plasma membrane-localized efflux transporter for manganese uptake and translocation in rice roots. The polar localization of MTP9 and Nramp5 at both the exodermis and the endodermis leads to efficient and unidirectional flux of manganese from the soil solution to the stele.

automatically adjusted to the varying availability of metal ions in the environment, due to the cooperation between tonoplast-localized transporters and ion chelators. Hence vacuoles work

Heavy Metal and Mineral Element-Induced Abiotic Stress in Rice Plant

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163

Plants have developed several defense mechanisms like chelation, excretion, and subcellular compartmentalization to combat heavy metal toxicity as it severely affects its overall growth

A large lytic vacuole (LV) is present in most of the plant cells which occupies about 80% of the cell volume. In plants, LVs undergo less metabolism and acts as a store house which can accumulate a huge amount of minerals and water thus play a major role in turgor generation. LVs also function as a store house for other xenobiotic and toxic compounds and also reduce their impact in the cytoplasm where several sensitive processes take place. LV also store plant secondary metabolites and proteins involved in plant defense against pathogens and herbivores and release them when subjected to cellular damage. LV has acidic pH around 4–5, and this acidic environment helps in the degradation of both exogenous and endogenous

Plant LVs are equivalent to lysosomes in animal and vacuoles in yeast and acts as degradation and waste storage compartments. The vacuolar tonoplast of higher plants and fungi as

from these, inner organellar membranes also contain multidrug resistance-associated protein (MRP) type ATP binding cassette (ABC) transporters, chloride channels (CLC) type ion chan-

Storage of nutrient minerals in the LV is important in buffering any variations in the supply of nutrients like when plants are grown in nutrient-rich conditions, they will deposit large quantities of such nutrients in vacuoles of vegetative tissues. This helps the plant to survive during subsequent periods of nutrient deficiency by remobilizing from the vacuolar store. Thus, the solute composition of vacuoles is highly dynamic and reflects changes in the environment

The vacuole is a major organelle in higher plants functioning as a store for metabolites, mineral nutrients, and toxicants. Studies have shown that in addition to its storage role, the vacuole also contributes to long-distance transport of metals, through the modulation of vacuolar sequestration capacity (VSC) which is basically controlled by cytosolic metal chelators and

VSC regulates the long-distance transport of mineral nutrients in plants. Zhang et al. [103] isolated the two vacuolar membrane-localized metal transporters OsVIT1 and OsVIT2 in rice. Both *OsVIT1* and *OsVIT2* primarily function to sequester Fe/Zn into vacuoles across the vacuolar membrane. In rice plant, flag leaves show a high expression of *OsVIT1* and *OsVIT2*. *OsVIT1* and *OsVIT2* along with VSC play an important role in Fe and Zn long-distance trans-

tonoplast-localized transporters, or the interaction between them [102].


**8.1. Metal sequestration in vacuoles by tonoplast transporters**

well as lysosomes of animal cells share very similar H<sup>+</sup>

nels, cation channels, and aquaporins [99].

and the plant developmental stage [101].

location between flag leaves and seeds.

as a buffering zone.

and development [99].

compounds [100].

### **8. Root-to-shoot translocation and metal chelation in cytoplasm**

Plants that prevent or limit the entry of metals from roots to shoots are categorized as excluders. On the contrary, plants that can transfer metals from root–to-shoot via the xylem along the transpiration stream by increasing the uptake of metals in roots, thus increasing the sequestration of metals in the aerial parts are considered as accumulators/hyperaccumulators. The transfer of metals from the roots to the aerial parts in plants helps to reduce the damage caused by the heavy metals on their root. The translocation of metals from root to the aerial parts is an essential process for the overall growth and development of the plant.

Studies of long-distance root-to-shoot metal transport within plants mainly emphasizes on transporters that are localized to either xylem parenchyma cells or phloem companion cells, as they are directly associated with xylem and phloem loading or unloading thus majorly contribute to the metal redistribution process within the plant. The movement of heavy metals from roots to shoots is facilitated when metals are chelated with ligands such as organic acids, amino acids, and thiols. The movement of metal cations across the xylem cell wall is restricted when the metals are not chelated by ligands due to high cation exchange capability of the xylem wall.

The chelation of metals with NA provides improved tolerance against the restriction by the xylem cell wall. NA facilitates the chelation and transport of divalent ions of metal Ni, Cu, and Zn [92]. Synthesis of NA by trimerization of S-adenosylmethionine is facilitated by nicotianamine synthase (NAS) [93]. In rice plant, increased accumulation of Fe, Zn, and Cu is associated with over expression of the gene *NAS3* [94].

During the process of long-distance transport of metals, some chelators, like nicotianamine [41], glutathione (GSH), and phytochelatins [95], also play a vital role.

Metals pass through xylem unloading process before their distribution and detoxification in the shoot and followed by their redistribution via the phloem. After unloading, the metals either enter into the nearby cells or are symplastically transported or they are apoplastically distributed throughout the leaf tissue [96]. For the symplastic transport of metals across the leaf via the YLS transporter proteins, chelation of metals to NA is required [97].

Excess metals in the plant are sequestered in various aerial plant parts, such as trichomes, leaf epidermal cell vacuole, and mesophyll vacuole. Not much study has been done on the transport of metals through the phloem sap. Nicotianamine is the only molecule to be identified as a phloem metal transporter which is associated with the transport of Fe, Cu, Zn, and Mn [98].

In plants, the vacuole sequestration capacity (VSC) also plays an important role in the longdistance transport and sequestration of metals. Vacuolar metal sequestration capacities are automatically adjusted to the varying availability of metal ions in the environment, due to the cooperation between tonoplast-localized transporters and ion chelators. Hence vacuoles work as a buffering zone.

#### **8.1. Metal sequestration in vacuoles by tonoplast transporters**

The polar localization of MTP9 and Nramp5 at both the exodermis and the endodermis leads

Plants that prevent or limit the entry of metals from roots to shoots are categorized as excluders. On the contrary, plants that can transfer metals from root–to-shoot via the xylem along the transpiration stream by increasing the uptake of metals in roots, thus increasing the sequestration of metals in the aerial parts are considered as accumulators/hyperaccumulators. The transfer of metals from the roots to the aerial parts in plants helps to reduce the damage caused by the heavy metals on their root. The translocation of metals from root to the aerial parts is an essential process for the overall growth and development of the

Studies of long-distance root-to-shoot metal transport within plants mainly emphasizes on transporters that are localized to either xylem parenchyma cells or phloem companion cells, as they are directly associated with xylem and phloem loading or unloading thus majorly contribute to the metal redistribution process within the plant. The movement of heavy metals from roots to shoots is facilitated when metals are chelated with ligands such as organic acids, amino acids, and thiols. The movement of metal cations across the xylem cell wall is restricted when the metals are not chelated by ligands due to high cation exchange capability

The chelation of metals with NA provides improved tolerance against the restriction by the xylem cell wall. NA facilitates the chelation and transport of divalent ions of metal Ni, Cu, and Zn [92]. Synthesis of NA by trimerization of S-adenosylmethionine is facilitated by nicotianamine synthase (NAS) [93]. In rice plant, increased accumulation of Fe, Zn, and Cu is

During the process of long-distance transport of metals, some chelators, like nicotianamine

Metals pass through xylem unloading process before their distribution and detoxification in the shoot and followed by their redistribution via the phloem. After unloading, the metals either enter into the nearby cells or are symplastically transported or they are apoplastically distributed throughout the leaf tissue [96]. For the symplastic transport of metals across the

Excess metals in the plant are sequestered in various aerial plant parts, such as trichomes, leaf epidermal cell vacuole, and mesophyll vacuole. Not much study has been done on the transport of metals through the phloem sap. Nicotianamine is the only molecule to be identified as a phloem metal transporter which is associated with the transport of Fe, Cu, Zn, and Mn [98]. In plants, the vacuole sequestration capacity (VSC) also plays an important role in the longdistance transport and sequestration of metals. Vacuolar metal sequestration capacities are

to efficient and unidirectional flux of manganese from the soil solution to the stele.

**8. Root-to-shoot translocation and metal chelation in cytoplasm**

plant.

of the xylem wall.

162 Rice Crop - Current Developments

associated with over expression of the gene *NAS3* [94].

[41], glutathione (GSH), and phytochelatins [95], also play a vital role.

leaf via the YLS transporter proteins, chelation of metals to NA is required [97].

Plants have developed several defense mechanisms like chelation, excretion, and subcellular compartmentalization to combat heavy metal toxicity as it severely affects its overall growth and development [99].

A large lytic vacuole (LV) is present in most of the plant cells which occupies about 80% of the cell volume. In plants, LVs undergo less metabolism and acts as a store house which can accumulate a huge amount of minerals and water thus play a major role in turgor generation. LVs also function as a store house for other xenobiotic and toxic compounds and also reduce their impact in the cytoplasm where several sensitive processes take place. LV also store plant secondary metabolites and proteins involved in plant defense against pathogens and herbivores and release them when subjected to cellular damage. LV has acidic pH around 4–5, and this acidic environment helps in the degradation of both exogenous and endogenous compounds [100].

Plant LVs are equivalent to lysosomes in animal and vacuoles in yeast and acts as degradation and waste storage compartments. The vacuolar tonoplast of higher plants and fungi as well as lysosomes of animal cells share very similar H<sup>+</sup> -ATPases that acidify the lumen. Apart from these, inner organellar membranes also contain multidrug resistance-associated protein (MRP) type ATP binding cassette (ABC) transporters, chloride channels (CLC) type ion channels, cation channels, and aquaporins [99].

Storage of nutrient minerals in the LV is important in buffering any variations in the supply of nutrients like when plants are grown in nutrient-rich conditions, they will deposit large quantities of such nutrients in vacuoles of vegetative tissues. This helps the plant to survive during subsequent periods of nutrient deficiency by remobilizing from the vacuolar store. Thus, the solute composition of vacuoles is highly dynamic and reflects changes in the environment and the plant developmental stage [101].

The vacuole is a major organelle in higher plants functioning as a store for metabolites, mineral nutrients, and toxicants. Studies have shown that in addition to its storage role, the vacuole also contributes to long-distance transport of metals, through the modulation of vacuolar sequestration capacity (VSC) which is basically controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them [102].

VSC regulates the long-distance transport of mineral nutrients in plants. Zhang et al. [103] isolated the two vacuolar membrane-localized metal transporters OsVIT1 and OsVIT2 in rice. Both *OsVIT1* and *OsVIT2* primarily function to sequester Fe/Zn into vacuoles across the vacuolar membrane. In rice plant, flag leaves show a high expression of *OsVIT1* and *OsVIT2*. *OsVIT1* and *OsVIT2* along with VSC play an important role in Fe and Zn long-distance translocation between flag leaves and seeds.

Studies have also suggested that the long-distance transport of nonessential toxic metals and their detoxification is regulated by VSC thus making the plants highly tolerant to metals [102]. Generally, the VSC of certain metal varies between different plant tissues to ensure proper metal distribution.

oxidizing agents which cause oxidative damage to biomolecules, like lipids and proteins and can eventually lead to cell death [112]. It has been shown that plant tolerance to metals is correlated with an increase in antioxidants and activity of radical scavenging enzymes [113]. Plants respond to oxidative stress by activating antioxidative defense mechanisms, which involves enzymatic and nonenzymatic antioxidants. The enzymatic components include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and enzymes of ascorbate glutathione cycle, while the nonenzymatic antioxidants include ascorbate and glutathione and atocoperol [3, 113]. These antioxidants play an important role in the elimination

An imbalance between the detoxification of the ROS products and the antioxidative system results in oxidative damage [113]. The tolerance of deleterious environmental stresses, such as heavy metals, is associated with the increased capacity to scavenge or detoxify activated

Studies using comparative analysis suggest that each heavy metal is accumulated differentially in root tissues. Heavy metal stress induces production of reactive oxygen species (ROS) which promotes cell death by apoptosis, necrosis, or mechanisms with both features. Methods

SOD, APX, and glutathione peroxidase (GPX) are the ROS scavenging antioxidant enzymes.

radical, peroxy radical, and singlet oxygen species. SOD acts as the first line of defense against

cell, plays an important role as an antioxidant either by participating in ascorbate-glutathione cycle or by directly quenching the ROS [115]. Ascorbate is also utilized by APXs as a reducing

Similar modulation of the antioxidant system upon exposure to heavy metal stress has been

Glutathione is an important antioxidant in plant cells which is involved in scavenging of free

Glutathione (GSH) is the precursor of phytochelatins (PC), which bind above the optimal concentrations of heavy metals [118]. It also serves as a substrate for GSTs which catalyzes the conjugation of GSH with xenobiotics like herbicides [119]. In silico studies and over expression of GSTs have shown to provide tolerance toward different heavy metals [120, 121]. Glutathione

In rice plants, the root tissue shows variable response of antioxidant enzymes during growth

Heavy metal stress in plants severely modulates the gene expression pattern. In plants, many genes are downregulated due to heavy metal stress. They are related to the energy metabolism, carbohydrate metabolism, lignin biosynthesis, phenylalanine metabolism, cell growth and death, lipid metabolism, biodegradation of xenobiotics, amino acid metabolism, etc. Among the

levels in plant tissues are known to accelerate under stress induced by heavy metals.

**9.2. Differential expression and modulation of genes during heavy metal stress**

to water and GPX breaks down H<sup>2</sup>

, which are formed as a result of oxidative stress along with ascorbate [117].

O2

O2

[114]. Ascorbate, which is present inside the

Heavy Metal and Mineral Element-Induced Abiotic Stress in Rice Plant

http://dx.doi.org/10.5772/intechopen.76080

O2 to H<sup>2</sup>

, hydroxyl

O and O<sup>2</sup>

.

165

They play a very important role in scavenging ROS like superoxide radical, H<sup>2</sup>

O2

staining are used to measure production of ROS.

and destruction of the reactive oxygen species [3].

ROS as it converts superoxide radical to H<sup>2</sup>

observed in studies of different rice cultivars [110, 116].

with different heavy metals of varying concentration.

agent to catalyze the conversion of H<sup>2</sup>

O2

oxygen species [113].

like H<sup>2</sup> O2

radicals and H<sup>2</sup>

Nonessential metals like Cd and As have adverse effects on plants, either through oxidative stress or competitive inhibition of essential mineral nutrient involved in any biological pathways. In order to protect the aerial parts which are associated with important biological processes like photosynthesis, plants have developed several mechanisms to regulate metal distribution between roots and shoots, VSC is one such mechanism.

In rice plant, *OsHMA3* is involved in Cd accumulation in the rice shoots. In rice root, OsHMA3 is localized to the vacuolar membrane, and it mediates the transport of Cd into vacuoles. In rice roots, VSC along with OsHMA3 plays a major role in the long-distance transport of Cd from roots to shoots [75, 76].

### **9. Biochemical processes modulated by heavy metal stress**

Both abiotic and biotic stresses have several effects on plant growth as well as productivity. Plant vigor and crop yields are strikingly influenced by these abiotic stresses. In order to combat these stresses, plants have evolved many responses. Plants have developed and used various strategies to cope with and also to adapt to these stress conditions. It depends on variation in protein relative abundance of stress-responsive proteins, resulting in changes in the whole proteome, transcriptome, and metabolome levels [104].

Expression patterns of these protein and transcript levels are influenced by the intensity and duration of stress apart from the usual post-translational regulatory mechanisms such as RNA stability and protein degradation [105]. In addition, the intensity and duration of stress can have a substantial effect on the complexity of the stress response. Recent progress in different areas of rice research such as analysis of interactome analysis, transcriptome, metabolome etc., have given us a better insight of abiotic stress response in rice plant [106–109]. The study of these differential changes in proteome profiles in response to abiotic stress is an approach to better understanding the physiology and molecular mechanisms that underlie rice stress responses. Most common response to all stresses is the induction of oxidative stress [110] and modulation of gene expression.

### **9.1. ROS production and modulation of antioxidant system during heavy metal stress**

In an environment of metal toxicity, the elevated activities of antioxidant enzymes and nonenzymatic constituents play important role in the plant tolerance to stress. Metal tolerance is enhanced by the plant's antioxidant resistant mechanisms. The harmful effects of heavy metals in plants are due to the production of ROS and induction of oxidative stress. Increased levels of reactive oxygen species such as singlet oxygen (1 O2 ), superoxide radical (O−2), hydrogen peroxide (H<sup>2</sup> O2 ), and hydroxyl radical (OH− ) result in oxidative stress [111]. ROS are strong oxidizing agents which cause oxidative damage to biomolecules, like lipids and proteins and can eventually lead to cell death [112]. It has been shown that plant tolerance to metals is correlated with an increase in antioxidants and activity of radical scavenging enzymes [113]. Plants respond to oxidative stress by activating antioxidative defense mechanisms, which involves enzymatic and nonenzymatic antioxidants. The enzymatic components include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and enzymes of ascorbate glutathione cycle, while the nonenzymatic antioxidants include ascorbate and glutathione and atocoperol [3, 113]. These antioxidants play an important role in the elimination and destruction of the reactive oxygen species [3].

Studies have also suggested that the long-distance transport of nonessential toxic metals and their detoxification is regulated by VSC thus making the plants highly tolerant to metals [102]. Generally, the VSC of certain metal varies between different plant tissues to ensure proper

Nonessential metals like Cd and As have adverse effects on plants, either through oxidative stress or competitive inhibition of essential mineral nutrient involved in any biological pathways. In order to protect the aerial parts which are associated with important biological processes like photosynthesis, plants have developed several mechanisms to regulate metal

In rice plant, *OsHMA3* is involved in Cd accumulation in the rice shoots. In rice root, OsHMA3 is localized to the vacuolar membrane, and it mediates the transport of Cd into vacuoles. In rice roots, VSC along with OsHMA3 plays a major role in the long-distance transport of Cd

Both abiotic and biotic stresses have several effects on plant growth as well as productivity. Plant vigor and crop yields are strikingly influenced by these abiotic stresses. In order to combat these stresses, plants have evolved many responses. Plants have developed and used various strategies to cope with and also to adapt to these stress conditions. It depends on variation in protein relative abundance of stress-responsive proteins, resulting in changes in the whole

Expression patterns of these protein and transcript levels are influenced by the intensity and duration of stress apart from the usual post-translational regulatory mechanisms such as RNA stability and protein degradation [105]. In addition, the intensity and duration of stress can have a substantial effect on the complexity of the stress response. Recent progress in different areas of rice research such as analysis of interactome analysis, transcriptome, metabolome etc., have given us a better insight of abiotic stress response in rice plant [106–109]. The study of these differential changes in proteome profiles in response to abiotic stress is an approach to better understanding the physiology and molecular mechanisms that underlie rice stress responses. Most common response to all stresses is the induction of oxidative stress [110] and

**9.1. ROS production and modulation of antioxidant system during heavy metal** 

In an environment of metal toxicity, the elevated activities of antioxidant enzymes and nonenzymatic constituents play important role in the plant tolerance to stress. Metal tolerance is enhanced by the plant's antioxidant resistant mechanisms. The harmful effects of heavy metals in plants are due to the production of ROS and induction of oxidative stress. Increased lev-

O2

), superoxide radical (O−2), hydrogen

) result in oxidative stress [111]. ROS are strong

distribution between roots and shoots, VSC is one such mechanism.

**9. Biochemical processes modulated by heavy metal stress**

proteome, transcriptome, and metabolome levels [104].

els of reactive oxygen species such as singlet oxygen (1

), and hydroxyl radical (OH−

metal distribution.

164 Rice Crop - Current Developments

from roots to shoots [75, 76].

modulation of gene expression.

**stress**

peroxide (H<sup>2</sup>

O2

An imbalance between the detoxification of the ROS products and the antioxidative system results in oxidative damage [113]. The tolerance of deleterious environmental stresses, such as heavy metals, is associated with the increased capacity to scavenge or detoxify activated oxygen species [113].

Studies using comparative analysis suggest that each heavy metal is accumulated differentially in root tissues. Heavy metal stress induces production of reactive oxygen species (ROS) which promotes cell death by apoptosis, necrosis, or mechanisms with both features. Methods like H<sup>2</sup> O2 staining are used to measure production of ROS.

SOD, APX, and glutathione peroxidase (GPX) are the ROS scavenging antioxidant enzymes. They play a very important role in scavenging ROS like superoxide radical, H<sup>2</sup> O2 , hydroxyl radical, peroxy radical, and singlet oxygen species. SOD acts as the first line of defense against ROS as it converts superoxide radical to H<sup>2</sup> O2 [114]. Ascorbate, which is present inside the cell, plays an important role as an antioxidant either by participating in ascorbate-glutathione cycle or by directly quenching the ROS [115]. Ascorbate is also utilized by APXs as a reducing agent to catalyze the conversion of H<sup>2</sup> O2 to water and GPX breaks down H<sup>2</sup> O2 to H<sup>2</sup> O and O<sup>2</sup> . Similar modulation of the antioxidant system upon exposure to heavy metal stress has been observed in studies of different rice cultivars [110, 116].

Glutathione is an important antioxidant in plant cells which is involved in scavenging of free radicals and H<sup>2</sup> O2 , which are formed as a result of oxidative stress along with ascorbate [117]. Glutathione (GSH) is the precursor of phytochelatins (PC), which bind above the optimal concentrations of heavy metals [118]. It also serves as a substrate for GSTs which catalyzes the conjugation of GSH with xenobiotics like herbicides [119]. In silico studies and over expression of GSTs have shown to provide tolerance toward different heavy metals [120, 121]. Glutathione levels in plant tissues are known to accelerate under stress induced by heavy metals.

In rice plants, the root tissue shows variable response of antioxidant enzymes during growth with different heavy metals of varying concentration.

#### **9.2. Differential expression and modulation of genes during heavy metal stress**

Heavy metal stress in plants severely modulates the gene expression pattern. In plants, many genes are downregulated due to heavy metal stress. They are related to the energy metabolism, carbohydrate metabolism, lignin biosynthesis, phenylalanine metabolism, cell growth and death, lipid metabolism, biodegradation of xenobiotics, amino acid metabolism, etc. Among the upregulated genes, the majority of affected genes are associated with the biosynthesis of secondary metabolites, specially flavonoid biosynthesis, lipid metabolism, amino acid metabolism, carbohydrate metabolism, biodegradation of xenobiotics, ascorbate, and aldarate metabolism, membrane transport especially multidrug resistance protein, major facilitator superfamily, ABC transporters, glutathione metabolism, MAPK (mitogen-activated protein kinases) signaling pathway, a large number of GST, etc. In a study using rice seedlings grown in metal supplemented media in comparison to control, genes which significantly modulated were filtered. It was found that 17 and 83 genes are commonly upregulated and downregulated under different heavy metal stress. One each of cytochrome P450, Proton-dependent oligopeptide transporter (POT) family protein, heat shock protein, and two NAC domain-containing proteins are commonly upregulated during heavy metals stress, they play important role in detoxification of heavy metals. On the other hand, one heavy metal-associated domain-containing protein, zinc finger protein, cytochrome P450, ring-H2 zinc finger protein, and catalase-1 are commonly downregulated during heavy metal exposure [122]. Plants have developed cellular mechanisms to tolerate and regulate the uptake of heavy metals [123]. However, molecular mechanisms and networks involved in the uptake and detoxification of heavy metals remain poorly understood. Phytochelatins (PCs), a class of cysteine-rich heavy metal-binding peptides, bind to heavy metals, and detoxify by vacuolar sequestration [123]. Sulfur homeostasis in plants results in the increased production of S-rich metal-binding peptides (such as GSH, PCs), which provide metal tolerance [116].

efflux transporters to remove toxic compounds from the cell [132]. Various methyltransferases are differentially modulated under different stresses. In plants, O-methyltransferases constitute a large family of enzymes that are involved in stress tolerance as reported by Lam et al. [133]. Specific methyltransferases catalyze the transfer of methyl groups which are involved in several pathways that lead to the accumulation of methylated inositols, quaternary amines, and tertiary sulfonium species, which play a significant role in stress tolerance [134]. Therefore, modulation of these transcripts must play a secondary role in different heavy metal toxicity. During different heavy metal stresses, these metals induce damage to the thylakoid membrane leading to increased lipid peroxidation and thus cause downregulation of peroxidases. These specific peroxidase family genes might play a key role in the enzymatic defense of plant cells by scavenging ROS during stress conditions [135]. Heat shock proteins (HSPs) in particular play important role in protecting plants against stress by re-establishing normal protein

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It has been observed that various transcription factors like WRKY, MYB family, zinc finger protein, RING-H2 finger protein, and basic leucine zipper (bZIP) are differentially expressed under heavy metal stress [122]. In plants, WRKY transcription factors are linked to various processes associated with different biotic and abiotic stresses and regulation of differential transcription a response to stress in plants [137]. Similarly, MYB TFs play very important roles in many physiological processes under normal or unfavorable growth conditions [138] and also in defense and stress responses [139]. During heavy metal stress various stress-related genes are transcriptionally regulated such as GSTs, dehydrin, sulfite oxidase (SO), L-ascorbate peroxidase, L-ascorbate oxidase, and germin-like proteins. GSTs are a superfamily of multifunctional, dimeric enzymes. It induces the conjugation of GSH a tripeptide glutathione to electrophilic xenobiotics and this is followed by sequestration of this complex into the vacuole for detoxification [140]. Recently, it has been reported that a particular class of GST gene family, that is, Lambda GST plays an important role during heavy metal stress [120, 121]. During drought stress, cold stress, and other defense processes dehydrins are produced in plants [141]. SO catalyzes the transformation of sulfites to the nontoxic sulfate. It has been reported earlier that sulfur is an essential nutrient that is taken up as sulfate by plants and chemical compounds which contain S, such as glutathione (GSH), phytochelatins (the polymers of GSH) play a prominent role in arsenic detoxification [142]. Similarly, L-ascorbate peroxidase plays an important role in defense against oxidative stress as it has been studied that APX is an important antioxidant enzyme

by converting into water. Germin-like proteins have been reported to

play a significant role in germination and defense response [143] during Cd toxicity.

Thus transcription factors play a significant role during different heavy metal stress response and indirectly modulate several genes responsible for stress. Further study of these TFs would help to understand the difference in the network of pathways during different heavy metal stresses.

Though different heavy metals are detoxified through similar mechanism their uptake from soil by root system differs for each metal. There are several families of transporters, which

conformation and thus cellular homeostasis [136].

which detoxifies H<sup>2</sup>

O2

**10. Conclusion and future prospective**

From the expression data, it was demonstrated that the upregulation of a unique cytochrome P450s in different heavy metal stresses is a major detoxification mechanism. In plants, cytochrome P450s plays a major role in the metabolism of several biosynthetic pathways such as flavonoids, coumarins, anthocyanins, isoflavonoids, phytoalexins, salicylic acid, jasmonic acid, and many others [124]. Previously, it has been reported that cytochrome P450 is involved in the metabolism of toxic compounds, indicating their role in heavy metal detoxification. These results indicate that metabolism of plant biosynthetic pathways are very much affected during metal exposure and different cytochrome P450s are involved in the metabolism of different heavy metals. A large number of transporter genes are differentially up- and downregulated under different heavy metal stresses, which include major facilitator genes, sulfate transporters, peptide transporters, nitrate transporters, ABC transporters, multidrug resistance proteins, zinc transporters, Nramp6, and multidrug and toxic compound extrusion (MATE) efflux family proteins. One of the essential nutrient required for plant growth is sulfur that enters the cell via sulfate transporters as inorganic sulfate, it may induce the production of S-rich metal-binding peptides (such as GSH, PCs) and thus provide defense against heavy metal stress [125]. It is clear from their study that each heavy metal has induced specific sulfate transporters. The peptide transporters [126] have been shown to transport nitrate and tripeptides such as glutathione which is a major component in sulfur metabolism and plant defense during stress [127]. It is suggested that nitrate transporter plays a role in root-to-shoot translocation of nitrate thus plays a role in Cd toxicity [128]. ABC transporter proteins play an important role in the transport of various substances like lipids, phytohormones, carboxylates, heavy metals, chlorophyll catabolites, etc. across various biological membranes [129]. It has been shown that Nramp proteins are conserved bivalent metal transporters [130]. NRAMP3 and NRAMP4 are reported to be responsible for Cd2+ efflux from the vacuole [131]. MATE proteins bind to a variety of potentially toxic compounds and function as proton-dependent efflux transporters to remove toxic compounds from the cell [132]. Various methyltransferases are differentially modulated under different stresses. In plants, O-methyltransferases constitute a large family of enzymes that are involved in stress tolerance as reported by Lam et al. [133]. Specific methyltransferases catalyze the transfer of methyl groups which are involved in several pathways that lead to the accumulation of methylated inositols, quaternary amines, and tertiary sulfonium species, which play a significant role in stress tolerance [134]. Therefore, modulation of these transcripts must play a secondary role in different heavy metal toxicity.

upregulated genes, the majority of affected genes are associated with the biosynthesis of secondary metabolites, specially flavonoid biosynthesis, lipid metabolism, amino acid metabolism, carbohydrate metabolism, biodegradation of xenobiotics, ascorbate, and aldarate metabolism, membrane transport especially multidrug resistance protein, major facilitator superfamily, ABC transporters, glutathione metabolism, MAPK (mitogen-activated protein kinases) signaling pathway, a large number of GST, etc. In a study using rice seedlings grown in metal supplemented media in comparison to control, genes which significantly modulated were filtered. It was found that 17 and 83 genes are commonly upregulated and downregulated under different heavy metal stress. One each of cytochrome P450, Proton-dependent oligopeptide transporter (POT) family protein, heat shock protein, and two NAC domain-containing proteins are commonly upregulated during heavy metals stress, they play important role in detoxification of heavy metals. On the other hand, one heavy metal-associated domain-containing protein, zinc finger protein, cytochrome P450, ring-H2 zinc finger protein, and catalase-1 are commonly downregulated during heavy metal exposure [122]. Plants have developed cellular mechanisms to tolerate and regulate the uptake of heavy metals [123]. However, molecular mechanisms and networks involved in the uptake and detoxification of heavy metals remain poorly understood. Phytochelatins (PCs), a class of cysteine-rich heavy metal-binding peptides, bind to heavy metals, and detoxify by vacuolar sequestration [123]. Sulfur homeostasis in plants results in the increased production of S-rich

166 Rice Crop - Current Developments

metal-binding peptides (such as GSH, PCs), which provide metal tolerance [116].

From the expression data, it was demonstrated that the upregulation of a unique cytochrome P450s in different heavy metal stresses is a major detoxification mechanism. In plants, cytochrome P450s plays a major role in the metabolism of several biosynthetic pathways such as flavonoids, coumarins, anthocyanins, isoflavonoids, phytoalexins, salicylic acid, jasmonic acid, and many others [124]. Previously, it has been reported that cytochrome P450 is involved in the metabolism of toxic compounds, indicating their role in heavy metal detoxification. These results indicate that metabolism of plant biosynthetic pathways are very much affected during metal exposure and different cytochrome P450s are involved in the metabolism of different heavy metals. A large number of transporter genes are differentially up- and downregulated under different heavy metal stresses, which include major facilitator genes, sulfate transporters, peptide transporters, nitrate transporters, ABC transporters, multidrug resistance proteins, zinc transporters, Nramp6, and multidrug and toxic compound extrusion (MATE) efflux family proteins. One of the essential nutrient required for plant growth is sulfur that enters the cell via sulfate transporters as inorganic sulfate, it may induce the production of S-rich metal-binding peptides (such as GSH, PCs) and thus provide defense against heavy metal stress [125]. It is clear from their study that each heavy metal has induced specific sulfate transporters. The peptide transporters [126] have been shown to transport nitrate and tripeptides such as glutathione which is a major component in sulfur metabolism and plant defense during stress [127]. It is suggested that nitrate transporter plays a role in root-to-shoot translocation of nitrate thus plays a role in Cd toxicity [128]. ABC transporter proteins play an important role in the transport of various substances like lipids, phytohormones, carboxylates, heavy metals, chlorophyll catabolites, etc. across various biological membranes [129]. It has been shown that Nramp proteins are conserved bivalent metal transporters [130]. NRAMP3 and NRAMP4 are reported to be responsible for Cd2+ efflux from the vacuole [131]. MATE proteins bind to a variety of potentially toxic compounds and function as proton-dependent During different heavy metal stresses, these metals induce damage to the thylakoid membrane leading to increased lipid peroxidation and thus cause downregulation of peroxidases. These specific peroxidase family genes might play a key role in the enzymatic defense of plant cells by scavenging ROS during stress conditions [135]. Heat shock proteins (HSPs) in particular play important role in protecting plants against stress by re-establishing normal protein conformation and thus cellular homeostasis [136].

It has been observed that various transcription factors like WRKY, MYB family, zinc finger protein, RING-H2 finger protein, and basic leucine zipper (bZIP) are differentially expressed under heavy metal stress [122]. In plants, WRKY transcription factors are linked to various processes associated with different biotic and abiotic stresses and regulation of differential transcription a response to stress in plants [137]. Similarly, MYB TFs play very important roles in many physiological processes under normal or unfavorable growth conditions [138] and also in defense and stress responses [139]. During heavy metal stress various stress-related genes are transcriptionally regulated such as GSTs, dehydrin, sulfite oxidase (SO), L-ascorbate peroxidase, L-ascorbate oxidase, and germin-like proteins. GSTs are a superfamily of multifunctional, dimeric enzymes. It induces the conjugation of GSH a tripeptide glutathione to electrophilic xenobiotics and this is followed by sequestration of this complex into the vacuole for detoxification [140]. Recently, it has been reported that a particular class of GST gene family, that is, Lambda GST plays an important role during heavy metal stress [120, 121]. During drought stress, cold stress, and other defense processes dehydrins are produced in plants [141]. SO catalyzes the transformation of sulfites to the nontoxic sulfate. It has been reported earlier that sulfur is an essential nutrient that is taken up as sulfate by plants and chemical compounds which contain S, such as glutathione (GSH), phytochelatins (the polymers of GSH) play a prominent role in arsenic detoxification [142]. Similarly, L-ascorbate peroxidase plays an important role in defense against oxidative stress as it has been studied that APX is an important antioxidant enzyme which detoxifies H<sup>2</sup> O2 by converting into water. Germin-like proteins have been reported to play a significant role in germination and defense response [143] during Cd toxicity.

Thus transcription factors play a significant role during different heavy metal stress response and indirectly modulate several genes responsible for stress. Further study of these TFs would help to understand the difference in the network of pathways during different heavy metal stresses.

### **10. Conclusion and future prospective**

Though different heavy metals are detoxified through similar mechanism their uptake from soil by root system differs for each metal. There are several families of transporters, which play important role in metal ion uptake from the soil as well as redistribution within the plant and its sequestration in various organelles and plant parts.

**References**

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Apart from the mineral elements described above, rice requires several other mineral elements for its growth. The transporters associated with the uptake of those mineral elements are yet to be identified in rice. Rice plant because of its distinct root anatomical characteristic requires a pair of influx and efflux transporters for the transport of mineral elements from the soil solution to the stele, thus help to surpass the two casparian strips present in the root exodermis and endodermis. As described above, some of the rice plant transporters associated with mineral uptake have been studied and characterized but there exists no clear understanding about their influx-efflux transporter pairs. More transporters associated with metal uptake and sequestration have to be identified and characterized using different ways like genetics (both forward and reverse), expression pattern, functional characterization in yeast or oocytes, phenotypic analysis using mutants, and so on in future. Each heavy metal modulates specific pathways in addition to common networks such as expression of a specific member of gene families including several transporters, different members of cytochrome P450 and transcription factors are modulated in different heavy metal stresses. In future, these complex responses have to be elucidated using various functional genomic approaches along with proteomic and metabolomic analyses. Also, many plant vacuolar membrane transporters and channels have been identified. Still, there is a dearth of knowledge about the regulation of these networks and how all these transporters and channels interact with each other in order to maintain a cytosolic ion homeostasis. Our understanding about the response of the rice plant to abiotic stress needs to be further refined.

Therefore, further studies on mineral transporters, antioxidant system, and differential gene expression regulated by heavy metals in rice is required to get deeper insight of the abiotic stress caused by heavy metals on rice plant which will in turn help to reduce the abiotic stress caused by heavy metals on rice plant ultimately leading to increased and better crop production for the benefit of mankind.

### **Acknowledgements**

We express our appreciation to Anna University for providing Anna Centenary Research Fellowship (ACRF) to Anitha Mani.

### **Author details**

Anitha Mani and Kavitha Sankaranarayanan\*

\*Address all correspondence to: skavitham@yahoo.com

Ion Channel Biology Laboratory, AU-KBC Research Centre, MIT Campus of Anna University, Chennai, Tamil Nadu, India

### **References**

play important role in metal ion uptake from the soil as well as redistribution within the plant

Apart from the mineral elements described above, rice requires several other mineral elements for its growth. The transporters associated with the uptake of those mineral elements are yet to be identified in rice. Rice plant because of its distinct root anatomical characteristic requires a pair of influx and efflux transporters for the transport of mineral elements from the soil solution to the stele, thus help to surpass the two casparian strips present in the root exodermis and endodermis. As described above, some of the rice plant transporters associated with mineral uptake have been studied and characterized but there exists no clear understanding about their influx-efflux transporter pairs. More transporters associated with metal uptake and sequestration have to be identified and characterized using different ways like genetics (both forward and reverse), expression pattern, functional characterization in yeast or oocytes, phenotypic analysis using mutants, and so on in future. Each heavy metal modulates specific pathways in addition to common networks such as expression of a specific member of gene families including several transporters, different members of cytochrome P450 and transcription factors are modulated in different heavy metal stresses. In future, these complex responses have to be elucidated using various functional genomic approaches along with proteomic and metabolomic analyses. Also, many plant vacuolar membrane transporters and channels have been identified. Still, there is a dearth of knowledge about the regulation of these networks and how all these transporters and channels interact with each other in order to maintain a cytosolic ion homeostasis. Our understanding about the response of the rice plant to abiotic stress needs to be further

Therefore, further studies on mineral transporters, antioxidant system, and differential gene expression regulated by heavy metals in rice is required to get deeper insight of the abiotic stress caused by heavy metals on rice plant which will in turn help to reduce the abiotic stress caused by heavy metals on rice plant ultimately leading to increased and better crop produc-

We express our appreciation to Anna University for providing Anna Centenary Research

Ion Channel Biology Laboratory, AU-KBC Research Centre, MIT Campus of Anna

and its sequestration in various organelles and plant parts.

168 Rice Crop - Current Developments

refined.

tion for the benefit of mankind.

Fellowship (ACRF) to Anitha Mani.

Anitha Mani and Kavitha Sankaranarayanan\*

University, Chennai, Tamil Nadu, India

\*Address all correspondence to: skavitham@yahoo.com

**Acknowledgements**

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**Chapter 10**

**Provisional chapter**

**Abiotic Stress Tolerance in Rice (***Oryza sativa* **L.): A**

**Abiotic Stress Tolerance in Rice (***Oryza sativa* **L.): A** 

DOI: 10.5772/intechopen.73571

Rice (*Oryza sativa* L.) is the main source of staple food for human population. Salinity is the major problem for agricultural production and it affects rice production globally. Different approaches have been developed and exploited to ameliorate the harmful effects of salinity on crop production. Development of salt-tolerant cultivars is the best option which ensures sustainable crop production. Genomics approaches have the potential to accelerate breeding process for the development of salt tolerant crop cultivars. Molecular mapping techniques are the most promising component of genomics. Molecular mapping approaches have greatly helped in the identification of genomic regions involved in salinity tolerance in different crop plants, including rice. Identified genomic regions associated with salinity tolerance accelerated molecular breeding efforts to develop salt-tolerant rice cultivars. Molecular mapping techniques (both linkage and association mapping) are the main components of genomics and these helped in the identification of genomic regions associated with salt-tolerance in rice. In this chapter, a detailed description of molecular mapping techniques, and major findings made by these techniques is presented. Future prospects of these techniques are also discussed. **Keywords:** genome-wide association studies, genomics, quantitative trait locus

Rice (*Oryza sativa* L.) belongs to family Poaceae and genus *Oryza*. Its genome size is approximately 430 Mb contained in 12 chromosomes. Large part of human population depends on it for staple food. Rice is a salt-susceptible crop. One third of world agricultural land is salt affected [1]. Salinity, both soil and water, has negative effect on rice production [2]. Elevated

> © 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

**Genomics Perspective of Salinity Tolerance**

**Genomics Perspective of Salinity Tolerance**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73571

Muhammad Saeed

Muhammad Saeed

**Abstract**

mapping, rice, salinity

**1. Introduction**

#### **Abiotic Stress Tolerance in Rice (***Oryza sativa* **L.): A Genomics Perspective of Salinity Tolerance Abiotic Stress Tolerance in Rice (***Oryza sativa* **L.): A Genomics Perspective of Salinity Tolerance**

DOI: 10.5772/intechopen.73571

Muhammad Saeed Muhammad Saeed

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73571

#### **Abstract**

Rice (*Oryza sativa* L.) is the main source of staple food for human population. Salinity is the major problem for agricultural production and it affects rice production globally. Different approaches have been developed and exploited to ameliorate the harmful effects of salinity on crop production. Development of salt-tolerant cultivars is the best option which ensures sustainable crop production. Genomics approaches have the potential to accelerate breeding process for the development of salt tolerant crop cultivars. Molecular mapping techniques are the most promising component of genomics. Molecular mapping approaches have greatly helped in the identification of genomic regions involved in salinity tolerance in different crop plants, including rice. Identified genomic regions associated with salinity tolerance accelerated molecular breeding efforts to develop salt-tolerant rice cultivars. Molecular mapping techniques (both linkage and association mapping) are the main components of genomics and these helped in the identification of genomic regions associated with salt-tolerance in rice. In this chapter, a detailed description of molecular mapping techniques, and major findings made by these techniques is presented. Future prospects of these techniques are also discussed.

**Keywords:** genome-wide association studies, genomics, quantitative trait locus mapping, rice, salinity

### **1. Introduction**

Rice (*Oryza sativa* L.) belongs to family Poaceae and genus *Oryza*. Its genome size is approximately 430 Mb contained in 12 chromosomes. Large part of human population depends on it for staple food. Rice is a salt-susceptible crop. One third of world agricultural land is salt affected [1]. Salinity, both soil and water, has negative effect on rice production [2]. Elevated

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

Na+ levels in agricultural lands are increasingly becoming a serious threat to the world agriculture. Plants suffer osmotic and ionic stress under high salinity due to the salts accumulated at the outside of roots and those accumulated at the inside of the plant cells, respectively.

Vegetative and reproductive growth potential of plant depends upon the process of photosynthesis. Increased sodium concentration in the leaf tissue negatively affects net photosynthesis and essential cellular metabolism [18, 21]. Chlorophyll content is important in photosynthesis. Reports suggest that there is no correlation between the chlorophyll content and photosynthesis under salinity stress. Net photosynthesis was reduced by a sodium concentration which did not affect chlorophyll content [18]. It implicates the disturbance by salinity stress of other cellular processes involved in photosynthesis. Sodium accumulation in the leaf also affects stomatal aperture and carbon dioxide fixation simultaneously [18] and thus it may be one of the reasons for reduced photosynthesis due to sodium accumulation. The most salt-

Abiotic Stress Tolerance in Rice (*Oryza sativa* L.): A Genomics Perspective of Salinity Tolerance

tions [22]. Rice plant evolved different mechanisms to cope salinity stress conditions. One of

Molecular mapping approaches are of two types, linkage mapping and association mapping,

In linkage mapping, bi-parental segregating populations are used. These populations include

near isogenic lines (NILs) and recombinant inbred lines (RILs). JoinMap [24], MapMaker [25] or QTL IciMapping [26] soft-wares are used for the construction of genetic linkage maps. WinQTLCartographer [27], QTL IciMapping [26], and QTLNetwork [28] programs are used for the identification of QTLs. Detailed information about input file requirements, statistical parameters thresholds, and the procedure to run the software are provided in the user

Association mapping uses natural populations for mapping purposes. In this technique, commercial crop cultivars can be employed for the assessment of QTLs. First reported in humans, association mapping is now widely used in plant sciences. Assessment of marker-trait associations is facilitated by controlling underlying population structure in the used plant material for mapping purposes [29]. STRUCTURE software is used for identifying sub-populations in the used plant germplasm [30]. TASSEL software is used for the identification of QTLs in this

In molecular mapping approaches, different types of DNA markers are used to identify QTLs. Amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), simple sequence repeats (SSRs), sequence tagged sites (STS), simple sequence length

ratio in the leaves and exhibited strongest yield reduc-

populations, introgression lines (ILs),

http://dx.doi.org/10.5772/intechopen.73571

183

/Na+

**3. Molecular mapping approaches: types and methodology**

these mechanisms is compartmenting salts within the plant body [23].

susceptible cultivars had lowest K<sup>+</sup>

on the basis of mapping population used.

backcross populations, doubled haploid (DH) lines, F2

**3.3. DNA markers used in molecular mapping approaches**

**3.1. Linkage mapping**

manuals of these softwares.

**3.2. Association mapping**

case [31].

Projected increase in human population demands a proportional increase in the food supply. This demand of increased food supply can be fulfilled only if we utilize all available land resources to their full potential. An associated phenomenon with the increase in human population is the decrease in world agricultural land area due to its use for human settlements. Due to these constraints, even marginal cultivable lands cannot be neglected. This urges that saline soils should be exploited to their full production potential. For good crop production on saline areas, different practices such as reclamation, agronomic adjustments, and biological amendments are used in combination. Considering sustainable crop production on these areas, the use of salt-tolerant crop cultivars seems to be most suitable option [3–5]. For development of salt-tolerant cultivars, genetic diversity with respect to salt tolerance in crops has to be evaluated. For genetic diversity assessment and identification of genomic regions associated with salt tolerance, molecular mapping approaches have made considerable contribution in different crop plants [6–14]. With the use of molecular mapping approaches, it has become possible to identify the chromosomal regions (quantitative trait loci, QTLs) associated with traits related to salt tolerance in rice. This chapter tries to cover effects of salinity on rice plant's growth and development, types of molecular mapping approaches, methodology involved in these approaches, and the achievements made through these approaches in salinity tolerance in rice to-date. It also highlights the future prospects of molecular mapping approaches. Thus, it will be a valuable resource for designing future research endeavors to genetically characterize salt tolerance mechanisms and develop salt-tolerant rice cultivars. It will also facilitate molecular breeding efforts for screening rice germplasm for salinity tolerance.

### **2. Effects of salinity on rice plant growth and development**

Salinity affects different morphological, biochemical, and physiological attributes of rice. Salinity has negative effect on percent relative-plant height, total tillers, root dry weight, shoot dry weight, and total dry matter [15]. Biochemical attributes of rice, affected by salinity, include chlorophyll content, proline content, hydrogen peroxide content, peroxidase (POX) activity, anthocyanins, Na<sup>+</sup> content, K<sup>+</sup> content, Ca++ content, total cations content [11, 16]. Physiological attributes of rice, which are affected by salinity, include relative growth rate, osmotic potential, transpiration use efficiency, senescence, Na<sup>+</sup> uptake, K<sup>+</sup> uptake, Ca++ uptake, total cations uptake, Na<sup>+</sup> /K<sup>+</sup> uptake, Na<sup>+</sup> uptake ratio, K<sup>+</sup> uptake ratio, Ca++ uptake ratio, Na<sup>+</sup> /K<sup>+</sup> uptake ratio, and total cations uptake ratio [11, 16, 17].

Rice shows different levels of salt tolerance at leaf and whole plant level [18, 19]. Similarly, behavior of rice plants towards salt stress may be different at vegetative and reproductive phases and this may not correlate with their mean level of relative resistance [20]. It is important to know the specific salt susceptible phase of a rice variety to have a better comparison of performance among varieties under salinity stress.

Vegetative and reproductive growth potential of plant depends upon the process of photosynthesis. Increased sodium concentration in the leaf tissue negatively affects net photosynthesis and essential cellular metabolism [18, 21]. Chlorophyll content is important in photosynthesis. Reports suggest that there is no correlation between the chlorophyll content and photosynthesis under salinity stress. Net photosynthesis was reduced by a sodium concentration which did not affect chlorophyll content [18]. It implicates the disturbance by salinity stress of other cellular processes involved in photosynthesis. Sodium accumulation in the leaf also affects stomatal aperture and carbon dioxide fixation simultaneously [18] and thus it may be one of the reasons for reduced photosynthesis due to sodium accumulation. The most saltsusceptible cultivars had lowest K<sup>+</sup> /Na+ ratio in the leaves and exhibited strongest yield reductions [22]. Rice plant evolved different mechanisms to cope salinity stress conditions. One of these mechanisms is compartmenting salts within the plant body [23].

## **3. Molecular mapping approaches: types and methodology**

Molecular mapping approaches are of two types, linkage mapping and association mapping, on the basis of mapping population used.

### **3.1. Linkage mapping**

Na+

182 Rice Crop - Current Developments

 levels in agricultural lands are increasingly becoming a serious threat to the world agriculture. Plants suffer osmotic and ionic stress under high salinity due to the salts accumulated at the outside of roots and those accumulated at the inside of the plant cells, respectively.

Projected increase in human population demands a proportional increase in the food supply. This demand of increased food supply can be fulfilled only if we utilize all available land resources to their full potential. An associated phenomenon with the increase in human population is the decrease in world agricultural land area due to its use for human settlements. Due to these constraints, even marginal cultivable lands cannot be neglected. This urges that saline soils should be exploited to their full production potential. For good crop production on saline areas, different practices such as reclamation, agronomic adjustments, and biological amendments are used in combination. Considering sustainable crop production on these areas, the use of salt-tolerant crop cultivars seems to be most suitable option [3–5]. For development of salt-tolerant cultivars, genetic diversity with respect to salt tolerance in crops has to be evaluated. For genetic diversity assessment and identification of genomic regions associated with salt tolerance, molecular mapping approaches have made considerable contribution in different crop plants [6–14]. With the use of molecular mapping approaches, it has become possible to identify the chromosomal regions (quantitative trait loci, QTLs) associated with traits related to salt tolerance in rice. This chapter tries to cover effects of salinity on rice plant's growth and development, types of molecular mapping approaches, methodology involved in these approaches, and the achievements made through these approaches in salinity tolerance in rice to-date. It also highlights the future prospects of molecular mapping approaches. Thus, it will be a valuable resource for designing future research endeavors to genetically characterize salt tolerance mechanisms and develop salt-tolerant rice cultivars. It will also facilitate molecular breeding efforts for

screening rice germplasm for salinity tolerance.

content, K<sup>+</sup>

tial, transpiration use efficiency, senescence, Na<sup>+</sup>

performance among varieties under salinity stress.

uptake, Na<sup>+</sup>

and total cations uptake ratio [11, 16, 17].

anthocyanins, Na<sup>+</sup>

/K<sup>+</sup>

uptake, Na<sup>+</sup>

**2. Effects of salinity on rice plant growth and development**

uptake ratio, K<sup>+</sup>

Salinity affects different morphological, biochemical, and physiological attributes of rice. Salinity has negative effect on percent relative-plant height, total tillers, root dry weight, shoot dry weight, and total dry matter [15]. Biochemical attributes of rice, affected by salinity, include chlorophyll content, proline content, hydrogen peroxide content, peroxidase (POX) activity,

attributes of rice, which are affected by salinity, include relative growth rate, osmotic poten-

Rice shows different levels of salt tolerance at leaf and whole plant level [18, 19]. Similarly, behavior of rice plants towards salt stress may be different at vegetative and reproductive phases and this may not correlate with their mean level of relative resistance [20]. It is important to know the specific salt susceptible phase of a rice variety to have a better comparison of

uptake, K<sup>+</sup>

content, Ca++ content, total cations content [11, 16]. Physiological

uptake ratio, Ca++ uptake ratio, Na<sup>+</sup>

uptake, Ca++ uptake, total cations

/K<sup>+</sup>

uptake ratio,

In linkage mapping, bi-parental segregating populations are used. These populations include backcross populations, doubled haploid (DH) lines, F2 populations, introgression lines (ILs), near isogenic lines (NILs) and recombinant inbred lines (RILs). JoinMap [24], MapMaker [25] or QTL IciMapping [26] soft-wares are used for the construction of genetic linkage maps. WinQTLCartographer [27], QTL IciMapping [26], and QTLNetwork [28] programs are used for the identification of QTLs. Detailed information about input file requirements, statistical parameters thresholds, and the procedure to run the software are provided in the user manuals of these softwares.

### **3.2. Association mapping**

Association mapping uses natural populations for mapping purposes. In this technique, commercial crop cultivars can be employed for the assessment of QTLs. First reported in humans, association mapping is now widely used in plant sciences. Assessment of marker-trait associations is facilitated by controlling underlying population structure in the used plant material for mapping purposes [29]. STRUCTURE software is used for identifying sub-populations in the used plant germplasm [30]. TASSEL software is used for the identification of QTLs in this case [31].

### **3.3. DNA markers used in molecular mapping approaches**

In molecular mapping approaches, different types of DNA markers are used to identify QTLs. Amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), simple sequence repeats (SSRs), sequence tagged sites (STS), simple sequence length polymorphism (SSLP), and single nucleotide polymorphism (SNP) [12, 32–35] are different types of DNA markers which are employed for genotyping in molecular mapping studies.

**Trait Plant material used Marker system** 

Seed germination (%); seedling root length; seedling dry matter; seedling vigor

/K<sup>+</sup>

concentration; shoot Na+

Survival days of seedlings; score of salt toxicity

concentration; fresh weight of shoots; tiller number per plant; plant height at the tillering

Plant height; panicle length; tillers per hill; spikelets per panicle; grain yield

Reduction rate of dry weight; reduction rate of fresh weight; reduction rate of leaf area; reduction rate of seedling height

Seedling height; dry shoot weight; dry root

Plant height; root length; shoot dry weight;

Days to seedlings survival; score on salt toxicity

concentration at seedling stage

Morphological and yield-related traits F2

Salt tolerance traits F2

/Na+

ratio in roots and shoots

concentrations in the roots and

concentration and Na/K

**Table 1.** QTLs identified through linkage mapping studies.

ratio; K<sup>+</sup>

to sheath Na+

) in roots and

), and calcium (Ca++)

weight; Na/K ratios in roots

symptoms on leaves; shoot K<sup>+</sup>

concentration; K<sup>+</sup>

) and potassium (K<sup>+</sup>

), potassium (K<sup>+</sup>

concentrations; ratio of leaf Na+

/K<sup>+</sup>

Pollen fertility; Na+

ratio in the flag leaf

accumulation traits

Sodium (Na+

ratio in roots; dry

concentration;

Salt tolerance rating; Na+

matter weight of shoots

of leaves; shoot K<sup>+</sup>

stage

shoot Na+

Leaf Na+

concentrations

Sodium (Na+

shoots; Na+

Na+ and K<sup>+</sup>

shoots

shoot fresh weight

Seedling survival days RILs population RFLP [32]

Shoot length; tiller number; shoot fresh weight Backcross inbred lines RFLP [37]

F2

BC2 F8

BC2 F8

Sodium and potassium uptake RILs AFLP, RFLP, SSR [33] Salt tolerance traits RILs RFLP, SSLP [44]

– 140 RILs SSR [47]

and F<sup>3</sup>

lines (BILs)

Physiological traits F2:4 population SSR, AFLP [51]

F2

F2

population

Doubled haploid (DH)

Abiotic Stress Tolerance in Rice (*Oryza sativa* L.): A Genomics Perspective of Salinity Tolerance

**used**

population SSR [34]

RILs population SSR [39]

Introgression lines SSR [41]

RILs, *F*2:9 SSR [42]

RILs SNP [35]

population SSR [12]

RILs RFLP, SSR [46]

RILs, F2:9 SSR [50]

population SSR [52]

population SSR [11]

populations RFLP [45]

 advanced backcross introgression lines (ILs)

Advanced backcross-inbred

introgression lines (IL) SSR [40]

RFLP [36]

http://dx.doi.org/10.5772/intechopen.73571

SSR [43]

SSR [48]

**Reference**

185
