Climate Change and Plant Abiotic Stress Tolerance || Metabolome Analyses for Understanding Abiotic Stress Responses in Plants to Evolve Management Strategies

27

Metabolome Analyses for Understanding Abiotic Stress

Responses in Plants to Evolve Management Strategies

Usha Chakraborty, Bhumika Pradhan, and Rohini Lama

Abstract

Abiotic stress responses are of the utmost importance for plants because theycannot survive unless they are able to cope with environmental changes, such ashigh and low temperatures, drought, flooding, salinity, freezing, change in pH,strong light, UV, and heavy metals. Plants respond to various stresses at differentlevels, including molecular and cellular levels, as well as by modifying theirmetabolomes. Hence, studies on plant responses to stresses can be conducted atany of these levels to provide an understanding of the mechanisms involved. Thepresent chapter focuses on the metabolomic approach to understand the responsesof plants to different abiotic stresses, which can then be utilized to evolve strategiesto combat such stress. Osmoprotectant metabolites, such as proline, glycinebetaine, and polyamines, as well as carbohydrates, play important roles in theprotection of plants against osmotic disbalances due to abiotic stresses. In addition,oxidative stresses are also overcome by an array of antioxidants, such as phenols,ascorbate, carotenoids, and a-tocopherol, as well as antioxidative enzymes.Signaling cascades activated during abiotic stresses lead to overexpression ofprotein kinases and stress proteins, and also involve molecules such as jasmonicacid and salicylic acid. Protein kinases and protein phosphatases that are encodedby large gene families often act in tandem to perform the phosphorylationand dephosphorylation leading to their activation and inactivation involved instress signaling in plants. Analysis of microRNAs and transcriptomes hasprovided sufficient understanding of the gene expression levels during periodsof stress. Hence, taken together, all these results can be utilized for identifyinggenes and/or metabolites overexpressed in tolerant species during periods ofstress, and can be utilized to achieve higher tolerance and survivability duringstresses.

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Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

27.1

Introduction

Abiotic stress responses are of the utmost importance for plants because theycannot survive unless they are able to cope with environmental changes, such ashigh and low temperatures, drought, flooding, salinity, freezing, change in pH,strong light, UV, and heavy metals. Due to increases in urbanization, deforestation,and other anthropogenic activities, plants face threats for their survival in terms ofavailability of quality water, proper growing space, soil pH, and other favorableenvironmental conditions for growth. It is therefore necessary in the coming yearsto grow plants (which include all forest plants, and economically importantmedicinal and crop plants) that show maximum tolerance to all or most of theabove environmental conditions. The tolerant plants must have the followingimportant features to survive the above challenges: low water-use efficiency, fastgrowth, high productivity, tolerance to changes in the climatic conditions, and so on.Plants can show various strategies, such as stress avoidance and stress tolerance, tocombat the above climatic changes. Stress avoidance includes various protectivemechanisms that delay or prevent the negative impact of a stress factor on a plantthat are stable and inherited, whereas stress tolerance is the ability of a plant toacclimate to a stressful condition, and acclimation is plastic and reversible [1]. Whenthe adverse climatic condition no longer prevails, the induced physiological changesor modifications during acclimation, which are diverse, are usually lost.Water, temperature, and other stresses lead to metabolic toxicity in the plant,

disorganization of the cell membrane system, production of reactive oxygen species(ROS), photosynthetic inhibition, and alteration in the acquisition of nutrients [2].Mild abiotic stresses have been found to be tolerated by plants by alterations inmolecular mechanisms ensuring plant survival and reproduction, as evident by theplants’ ability to evolve and rearrange their genomic expression in response tochanges in their environment, although survival under such stressful conditionsrequires expenditure of energy and metabolism. The adverse effect of abioticstresses is reflected on plant health, habitus, and production, and it is estimated thatabiotic stresses account for 70% reduction in the yield for the major crops [3].Various tolerance mechanisms have been studied and continuous research isongoing at every level to study the different tolerances achieved by plants at thecellular, molecular, metabolomic and genomic level. Mechanisms that operate insignal perception, transduction, and downstream regulatory factors are now beingexamined, and an understanding of cellular pathways involved in abiotic stressresponses provide valuable information on such responses. At the molecular level,it has been suggested that abiotic stress tolerance can be achieved through genetransfer by altering the accumulation of osmoprotectants, production of chaper-ones, superoxide radical-scavenging mechanisms, and exclusion or compartmenta-lization of ions by efficient transporter and symporter systems (see reviews [2,4–9]).In the postgenomic era, comprehensive analyses using three systematic

approaches or “omics” have increased our understanding of the complex molecularregulatory networks associated with stress adaptation and tolerance: (i) “transcrip-

728 27 Metabolome Analyses for Understanding Abiotic Stress Responses in Plants

tomics” for the analysis of coding and non-coding RNAs, and their expressionprofiles, (ii) “metabolomics,” which is a powerful tool to analyze a large number ofmetabolites, and (iii) “proteomics” in which protein and protein modificationprofiles offer an unprecedented understanding of regulatory networks. Proteincomplexes involved in signaling have been analyzed by a proteomics approach[10,11]. The first step in switching on any molecular response is the perception ofstress and then to relay information about it through a signal transduction pathway.To perceive the initial stress signal, a sensor molecule is required that initiates (orsuppresses) a cascade to transmit the signal intracellularly and, in many cases,activate nuclear transcription factors to induce the expression of specific sets ofgenes [12].

27.2

Metabolite Changes During Abiotic Stresses

In this chapter, we focus on the metabolomics of plant responses to various abioticstresses like water stress (drought, flooding), salt (osmotic or ionic) stress, extremetemperatures (high, low, chilling, and freezing), and the involvement of antioxidantmechanisms. Plants show various alterations or changes in their metabolism,which include the production of stress metabolites also termed osmolytes orcompatible solutes (e.g., proline, soluble carbohydrates, starch, glycine betaine,raffinose, and polyamines), that are able to stabilize proteins and cellular structuresand/or to maintain cell turgor by osmotic adjustment, redox metabolism to removeexcess levels of ROS and re-establish the cellular redox balance, as well as stresshormones and kinases during stress perception and tolerance [9,13–15].The advent of various sophisticated technologies such as mass spectroscopy and

bioinformatics has made metabolite profiling along with transcriptome analysispossible, and thus we are now able to pinpoint the exact points of metabolicadjustments during various stresses such as water deficit (dehydration and highsalinity) and extreme temperature (cold and heat), and analyze the final steps ofabiotic stress signal transduction pathways comprehensively [16].

27.2.1

Proline and Glycine Betaine

The two major organic osmolytes glycine betaine and proline accumulate in avariety of plant species during various environmental stresses. The accumulation ofthe amino acid proline in tissues of several plant species is usually regarded as aresponse of the plant tissues to water and other kinds of stresses [17–20]. Theprotective role of proline, especially against damage by drought, high salinity, andheavy metals, as has been reported by several authors [21–24], may be due to itsability to act as an osmolyte, a ROS scavenger, and a molecular chaperonestabilizing the structure of proteins, thereby protecting cells from damage causedby stress [1].

27.2 Metabolite Changes During Abiotic Stresses 729

Proline levels are determined by the balance between biosynthesis andcatabolism (Figure 27.1) [24]. In the biosynthetic pathway of proline in the cytosolor chloroplasts, glutamate semialdehyde (GSA) is produced from glutamate, by D1-pyrroline-5-carboxylate synthetase (P5CS). GSA is then non-enzymatically con-verted to pyrroline-5-carboxylate (P5C), the immediate precursor of proline.Mitochondrial proline is degraded by proline dehydrogenase and P5C dehydrogen-ase (P5CDH) to glutamate. It has been suggested that proline can also besynthesized from ornithine as an alternative pathway that is transaminated first byornithine aminotransferase (OAT) producing GSA and P5C, which is thenconverted to proline [25,26]. The ornithine pathway is important during seedling

Figure 27.1 Model proposed for proline

metabolism in higher plants by Szabados and

Savour�e [24]. In meristematic and embryonic

cells, housekeepingprolinebiosynthesisoccurs

inthecytosol,andismediatedbyP5CSandP5CR

enzymes. Under stress conditions, P5CS1

accumulates in the chloroplasts, leading to

enhanced proline biosynthesis in the plastids.

Prolinedegradationoccurs in themitochondria,

where proline is oxidized to P5C and glutamate

through sequential action of proline

dehydrogenase and P5CDH. The ornithine

pathway uses arginine, and produces P5C and

glutamate in mitochondria. Mitochondrial P5C

canbe recycled toproline in thecytosolbyP5CR.

Enzymes are depicted as ellipses and

transporter proteins as octagons. BAC, basic

amino acid transporter involved in arginine and

ornithine exchange; Glu, glutamate; G/P,

mitochondrial glutamate/proline antiporter;

KG, a-ketoglutarate; P, mitochondrial proline

transporter; Pi, inorganic phosphate; ProT,

plasma membrane proline transporter; ?,

predicted transporters. Reproduced from [24]

with kind permission by Elsevier.


development and in some plants for stress-induced proline accumulation.However, quite recently the significance of this pathway and OAT in prolinebiosynthesis has been questioned, because proline levels were not affected inArabidopsis oat knockout mutants [24]. Funck et al. [27] have suggested that OATfacilitates nitrogen recycling from arginine through P5C, which is converted toglutamate by P5CDH.It is not clear whether there exists a direct correlation between the accumulation

of proline and abiotic stress tolerance; however, several examples show there existsa relation. It has been shown that high proline levels can be characteristic of saltand cold-hypersensitive Arabidopsis (Arabidopsis thaliana) mutants [28,29]. Accord-ing to Choudhary et al. [30], proline content is also high in drought-tolerant ricevarieties, but is not correlated with salt tolerance in barley (Hordeum vulgare)[31,32]. Several workers have demonstrated that the metabolism of proline in plantsunder different kinds of stresses is complex, and the accumulation is veryimportant to impart tolerance towards the external environmental conditions withrespect to growth, development, and stress responses of the plant [33–36].Stress conditions stimulate proline synthesis, while proline catabolism is

enhanced during recovery from stress. Overexpression of P5CS in tobacco andpetunia led to increased proline accumulation, and enhanced salt and droughttolerance [33,37], whereas Arabidopsis P5CS1 knockout plants were impaired instress-induced proline synthesis and were hypersensitive to salinity [35]. Consis-tently, proline dehydrogenase antisense Arabidopsis accumulated more proline, andshowed enhanced tolerance to freezing and high salinity [38]. It has also beensuggested that, in an alternative pathway, mitochondrial P5C can be produced byd-OAT from ornithine [36]. Overexpression of Arabidopsis d-OAT has been shownto enhance proline levels, and to increase the stress tolerance of rice, cotton, andtobacco [26,39,40], even though Arabidopsis plants deficient in d-OAT accumulatedproline in response to stress and showed a salt stress tolerance similar to the wild-type [27]. Thus, it is clear that proline has multiple roles in plant metabolism, bothduring the normal state as well as during stress (Figure 27.2).An amino acid derivative by nature, glycine betaine is naturally synthesized in

many plants, with some exceptions in crop species like potato or tomato, which areunable to accumulate glycine betaine [41]. It is a quaternary ammonium compoundthat occurs abundantly in response to dehydration stress [42–45]. In chloroplasts,where glycine betaine is found in abundance, it plays a vital role in the adjustmentand protection of the thylakoid membrane and maintains the efficiency ofphotosynthesis [46,47]. In chloroplasts, serine is converted to ethanolamine, whichgives rise to choline and betaine aldehyde [48,49]. Choline monooxygenase convertscholine to betaine aldehyde, which is then converted to glycine betaine by theaction of betaine aldehyde dehydrogenase and has been identified in plantsshowing glycine betaine accumulation (Figure 27.3). Direct N-methylation ofglycine also produces glycine betaine along with other pathways in the case ofplants [50]. Abou El-Yazied [51] suggested that glycine betaine functions as acompatible solute during salt and drought stress, and is elevated to regulate theintracellular osmotic balance. In addition, positive effects of a foliar spray of glycine


Figure 27.2 Multiple functionsof proline in plants bothunder normal conditions andduring stress

[24]. AOS, active oxygen species; CAT, catalase; PER, peroxidase; APOX, ascorbate peroxidase; GR,

glutathione reductase. Reproduced from [24] with kind permission by Elsevier.

Figure 27.3 Biosynthetic pathway of glycine betaine in higher plants. Reproduced from [42]

with kind permission by Elsevier.


betaine on yield and yield component in plants grown under water-limitedenvironments have been reported in different crops, such as wheat, common bean,rice, and sunflower [41,51–53]. Exogenous supply of glycine betaine (foliar spray)has also been shown to have an effective role in ameliorating the effects of waterstress on turgor potential and yield of two sunflower lines [53]. On the other hand,it has been suggested that exogenously applied glycine betaine has no effect on theyields of cotton [54].Both proline and glycine betaine are thought to have positive effects on enzyme

and membrane integrity along with adaptive roles in mediating osmoticadjustment in plants grown under stress conditions. Therefore, a better under-standing of the mechanisms of action of exogenously applied glycine betaine andproline is expected to aid their effective utilization in crop production in stressenvironments [42].

27.2.2

Carbohydrates

Under abiotic stress (drought, salinity) plants respond with the activation ofsynthesis or inhibition of catabolism of osmolytes such as sugars – trehalose,raffinose, galactinol, sorbitol, mannitol, fructans, saccharopine, sugar alcohols, andpolyols [55]. The changes in the concentration of carbohydrates, such as glucose,sucrose, sorbitol, galactose, raffinose, stachyose, polysaccharides, starch, and totalsugars, is important because they are directly related to physiological processessuch as photosynthesis, translocation, and respiration, and during abiotic stressthey accumulate as compatible solutes, as they do not interfere with thebiochemical reactions. Soluble sugars are highly sensitive to environmentalstresses, which act on the supply of carbohydrates from source organs to sinkorgans. Sucrose and hexoses both play dual functions in gene regulation asexemplified by the upregulation of growth-related genes and downregulation ofstress-related genes. Although coordinately regulated by sugars, these growth andstress-related genes are up- or downregulated through hexokinase-dependent and/or hexokinase-independent pathways [56]. Drought, salinity, low temperature, andflooding, in general, increased soluble sugar concentrations, whereas high lightirradiance (photosynthetically active radiation, UV-B), heavy metals, nutrientshortage, and ozone decreased sugar concentrations [57–59]. Singh [60] showedthat a greater accumulation of sugar lowers the osmotic potential of cells andreduces loss of turgidity in tolerant genotypes during abiotic stresses. Dkhiland Dendon [61] further suggested that another possible role of sugar may be as areadily available energy source. Glucose has been reported to induce a largenumber of genes involved in various stress responses, thus indicating the role ofsoluble sugars in environmental stress responses [62]. The alterations in thebalance between the source and the sink organs may be the signal for thesechanges that could have induced the change in the metabolism [63]. It was shownthat an artificial increase in leaf carbohydrate content modified gene expression forenzymes of photosynthetic metabolism [64]. Soluble sugars do not only function as


metabolic resources and structural constituents of cells, they also act as signalsregulating various processes associated with plant growth and development [65,66].Sugar signaling pathways interact with stress pathways in a complex network tomodulate metabolic plant responses [67,68]. Soluble sugars may either act directlyas negative signals or as modulators of plant sensitivity and thus they can also playimportant roles in cell responses to stress-induced remote signals [56]. Never-theless, sugar changes do not follow a static model, and vary with genotype andstress factor [69]. In addition, it has also been reported that not all soluble sugarsplay similar roles in events associated with the metabolism of stressed plants[59,70]. Sucrose and glucose either act as substrates for cellular respiration or asosmolytes to maintain cell homeostasis [63], while fructose seems not to be relatedto osmoprotection, but to secondary metabolite synthesis [56]. Hilal et al. [71]demonstrated that fructose might be related to synthesis of erythrose-4-phoshate,which acts as a substrate in lignin and phenolic compound synthesis. Thus, understress conditions the metabolism of soluble sugars is a dynamic processsimultaneously involving degrading and synthetic reactions [56].Among various sugars, mannitol is a well-characterized sugar alcohol. It is a

significant photosynthetic product in some plant species and has been shown toincrease during abiotic stress [72]. It had been suggested to act as an osmoprotec-tant by reacting with damaging hydroxyl radicals to form mannitol radicals, whichare then converted to mannose in the presence of oxygen [73,74]. Shen et al. [75]transformed tobacco with a construct in which the mt1D enzyme was targeted tochloroplasts. The resulting transgenic plants accumulated mannitol at concentra-tions from 2.5 to 7.0mmol g�1 fresh weight. The presence of mannitol in thechloroplasts resulted in enhanced resistance to oxidative stress induced by thepresence of methyl viologen. Such resistance was due to an increased capacity toscavenge hydroxyl radicals. In the seeds of mannitol-accumulating Arabidopsisplants, the concentration of mannitol reached 10 mmol g�1 dry weight [76].Mannitol-expressing seeds were able to germinate in medium supplemented withup to 400mM NaCl, whereas control seeds ceased to germinate at 100mM NaCl.Fructans are polyfructose molecules that are produced by many plants and

bacteria, and furthermore these may play a role in adaptation to osmotic stress dueto their highly soluble nature [77]. Meer et al. [78] modified non-fructan-storingpotato plants by introducing the microbial fructosyl transferase gene and theregenerated potato plants accumulated fructans. Drought treatment resulted in33% more fresh weight in transformed fructan-accumulating plants than in thecontrol plants [79].Soluble sugars seem to assume a dual role with respect to ROS. Soluble sugars

can be involved in, or related to, ROS-producing metabolic pathways. In reverse,soluble sugars can also feed NADPH-producing metabolic pathways, such as theoxidative pentose phosphate pathway, which can contribute to ROS scavenging[80,81]. The relationship between soluble sugars and ROS production or betweensoluble sugars and ROS responses is not a straightforward positive correlation,since, as discussed above, high sugar levels can correspond to activation of someROS-producing pathways and decrease of other ROS-producing pathways, and


both high sugar level and low sugar level can result in the enhancement of ROSresponses. Moreover, soluble sugars other than fructose, glucose, and sucrose aredetected at significant levels in plants. Hexoses, such as mannose, and disacchar-ides, such as trehalose, may play important roles in relation to the three mainsoluble sugars. Thus, the positive effects of trehalose on stress responses in plants,including decrease of photooxidative damage, have been ascribed to interactionswith sucrose metabolism, which is significantly modified by trehalose treatment[82]. There is proof to show that abiotic stresses induce sugar signal transductionthat is related to enzyme activities and gene expression (Figure 27.4). Thephosphoinositol cascade is activated by abiotic stress-induced changes in sugarlevels. Activities of enzymes such as sucrose synthase and sucrose phosphatesynthase are upregulated, while sucrose transport is downregulated [62,83–87].

27.2.3

Polyamines

Polyamines can be considered as one of the oldest groups of substances known inbiochemistry [88]. Putrescine, spermidine, and spermine are the most common

Figure 27.4 Abiotic stressors cause the

accumulation of ROS in plant cells. MAPK

cascades respond to the resulting oxidative

burstandmayregulateROSaccumulation.Such

activation of MAPK components may be

mediated by hormone-related pathways. The

kinase modules depicted are supported by

biochemical and in plant analyses inA. thaliana.

Republished from [87] with permission of

Annual Reviews.


polyamines in higher plants [1]. Putrescine can be produced from either ornithineby the action of ornithine decarboxylase or arginine by arginine decarboxylase.Putrescine, in turn, is converted to spermidine by spermidine synthase and then tospermine by spermine synthase. These are found to be protonated at normalcellular pH for which initially their biological function was associated with thecapability of binding different macromolecules carrying negative charge (i.e., DNA,RNA, chromatin, and proteins), thus confirming them as substances with astructural role [89].Several groups have hence confirmed that polyamines not only stabilize

macromolecular structures, but also act as regulatory molecules in manyfundamental cellular processes, including cell division, differentiation andproliferation, cell death, DNA and protein synthesis, and gene expression [90–93].The importance of polyamines in plants during various physiological processes aswell as abiotic and biotic stress responses has also been reported by several groups[92–94]. Alc�azar et al. [92] suggested that changes in plant polyamine metabolismoccur in response to a variety of abiotic stresses. However, the physiologicalsignificance of increased levels of polyamine in plants during abiotic stressresponses is still not clear [92,93,95]. They have also been implicated in protectingmembranes and alleviating oxidative stress [96–98], but their specific function instress tolerance is not well understood.Early studies on plant polyamine research pointed to their involvement in

responses to different environmental stresses. During the last few years, genetic,transcriptomic, and metabolomic approaches have unraveled key functions ofdifferent polyamines in the regulation of abiotic stress tolerance. Analyses oftransgenic plants and of mutants involved in polyamine metabolism clearly showeda positive role of polyamines in stress tolerance. Plants deficient in ADC1 or ADC2had reduced putrescine levels and were hypersensitive to stress [99,100], whereasconstitutive or stress-induced overexpression of ADC led to higher putrescinelevels and enhanced drought and freezing tolerance [89,101,102]. Nevertheless, theprecise molecular mechanism(s) by which polyamines control plant responses tostress stimuli are largely unknown. Recent studies indicate that polyaminesignaling is involved in direct interactions with different metabolic routes andintricate hormonal cross-talk. Thus, genetic manipulation of crop plants with genesencoding enzymes of polyamine biosynthetic pathways may provide better stresstolerance to crop plants [95].

27.3

Stress Hormones

The five classical phytohormones, auxin, cytokinin, ethylene, gibberellins, abscisicacid (ABA), and the recently identified brassinosteroids, jasmonic acid, and salicylicacid, are chemical messengers present in trace quantities; their synthesis andaccumulation are tightly regulated. Two groups of hormones are recognized: the“stress hormones,” which include ABA, salicylic acid, jasmonic acid, and ethylene,


and the “positive growth regulators,” which include auxins, cytokinins, gibber-ellins, and brassinosteroids. Depending on the context, they are subject to positiveor negative feedback control and often are affected by cross-talk due toenvironmental inputs. Phytohormones move throughout the plant body via thexylem or phloem transport stream. They move short distances between cells or aremaintained in their site of synthesis to exert their influence on target cells wherethey bind transmembrane receptors located at the plasma membrane orendoplasmic reticulum, or interact with intracellular receptors. The downstreameffects of hormonal signaling include alterations in gene expression patterns and insome cases non-genomic responses. Changes in plant hormone concentrationsand tissue sensitivity to them regulate a whole range of physiological process thathave profound effects on growth and development. The phytohormones affect allphases of the plant life cycle and the responses to environmental stresses, bothbiotic and abiotic. They are essential for the ability of plants to adapt to abioticstresses by mediating a wide range of adaptive responses [103–106] and, accordingto Santner and Estelle [103], they often rapidly alter gene expression by inducing orpreventing the degradation of transcriptional regulators via the ubiquitin–protea-some system. Hormonal signaling is critical for plant defenses against abiotic andbiotic stresses [107–109].

27.3.1

ABA

ABA is a central regulator of many plant responses to environmental stresses,particularly osmotic stresses [110–113], and hence one of the most studied topics inthe response of plants to abiotic stress, especially water stress, is ABA signaling andABA-responsive genes. ABA synthesis is one of the fastest responses of plants toabiotic stress, triggering ABA-inducible gene expression [114] and causing stomatalclosure, thereby reducing water loss via transpiration [115], and eventuallyrestricting cellular growth. Numerous genes associated with ABA de novobiosynthesis and genes encoding ABA receptors and downstream signal relayshave been characterized in A. thaliana (reviewed in [116]). In maize, at least 10viviparous mutants have been identified, most of which (vp2, vp5, vp7, vp9, w3, y3,and y9) were blocked in the biosynthesis of the carotenoid precursors for de novoABA synthesis; in rice (Oryza sativa), four phs mutants, defective in phytoenedesaturase (OsPDS), f-carotene desaturase (OsZDS), carotenoid isomerase (OsCR-TISO), and lycopene b-cyclase (b-OsLCY), were found to affect the biosynthesis ofcarotenoid precursors of ABA [117]. ABA signaling can also be very fast withoutinvolving transcriptional activity; a good example is the control of the stomatalaperture by ABA through the biochemical regulation of ion and water transportprocesses [112]. ABA is a key phytohormone in plant responses to water deficit;therefore, elucidation of the mechanism of ABA signal transduction significantlycontributes to the establishment of a suitable strategy for elevation of planttolerance to abiotic stresses [118]. Agarwal and Jha [119] suggested that ABAs arenot only involved in regulating stomatal opening, growth, and development, but are

27.3 Stress Hormones 737

also involved in coordinating various stress signal transduction pathways in plantsduring abiotic stresses, and that both ABA-dependent and ABA-independent signaltransduction pathways from stress signal perception to gene expression involvedifferent transcription factors such as DREB/CBF (dehydration-responsive elementbinding/C-repeat binding factor), MYC (myelocytomatosis oncogene)/MYB (mye-loblastosis oncogene), AREB/ABF (ABA-responsive element-binding protein/ABA-binding factor), NAC (NAM, ATAF, and CUC), and their corresponding cis-actingelements DRE (dehydration-responsive element), MYCRS/MYBRS, ABRE (ABA-responsive element), and NACRS. ABA also plays an important role during plantadaptations to cold temperatures. Cold stress induces the synthesis of ABA and theexogenous application of ABA improves the cold tolerance of plants [120].

27.3.2

Salicylic Acid

Salicylic acid has been recognized as a regulatory signal mediating plant responsesto abiotic stresses such as drought [121,122], chilling [123,124], heavy metaltolerance [125–127], heat [128,129], and osmotic stress [130]. It functions as anendogenous signal that mediates local and systemic plant defense responsesagainst pathogens, and thus it has been the focus of intensive research. In thissense, salicylic acid appears to be, just like in mammals, an “effective therapeuticagent” for plants [131].Treating plants with exogenous salicylic acid improved thermotolerance and heat

acclimation in several crops [123,132,133]. Using an Arabidopsis transgenic lineexpressing the salicylate hydroxylase gene (NahG) it was demonstrated thatsalicylic acid is required for ozone tolerance by maintaining the cellular redox stateand allowing defense responses [134]. However, by using Cvi-0, an Arabidopsisgenotype that accumulated high levels of salicylic acid, it was shown that salicylicacid activates an oxidative burst and a cell death pathway leading to ozonesensitivity [135].

27.3.3

Jasmonic Acid and Ethylene

Jasmonic acid levels in plants rapidly and transiently increase in response towounding, water deficit, mechanical stimulation, and elicitors, and it also mediatessome of the UV-induced defense responses [136].Abiotic stresses directly trigger jasmonic acid biosynthesis, which functions as a

signal in the cascade of plant reactions to oxidative stress-generating stimuli. Inaddition, jasmonic acid synthesis and signaling are interlinked by a positivefeedback loop whereby jasmonates stimulate their own synthesis [137,138].Jasmonic acid is also involved in the response to wounding. Jasmonic acidbiosynthesis is initiated by wound-mediated release of a-linolenic acid fromchloroplastic membranes, followed by the activity of several chloroplast-locatedenzymes, including 13-lipoxygenase. The combination of jasmonic acid deficiency


and ethylene insensitivity resulted in a novel growth phenotype characterized bymassive cell expansion around wounds, suggesting that both jasmonic acid andethylene may repress local growth after wounding and/or herbivore attack [139].Recent findings of Mahouachi et al. have suggested an interaction between

jasmonic acid and ABA in plants under drought stress [140]. According to theauthors, the pattern of accumulation of jasmonic acid is compatible with a triggeringsignal upstream ABA. The phytohormone ABA acts in all adaptive responses toenvironmental stresses, while gibberellin and ABA play essential and oftenantagonistic roles in regulating plant growth, development, and stress responses.The transduction of their signal occurs through several types of receptors [141].Ethylene has long been regarded as a stress hormone [142]. However, the roles of

ethylene signaling in abiotic stress responses are still not clear, but it is known thatethylene signaling is important in regulating plant growth and stress responses,and it functions through its receptors. In a study by Cao et al. [143], theytransformed a tobacco type II ethylene receptor homolog gene NTHK1 intoArabidopsis and found that the resulting transgenic plants, with NTHK1 mRNAand protein expression, were salt sensitive as was seen from the severe epinastyphenotype, high electrolyte leakage, and reduced root growth under salt stress.It is apparent from the evidence obtained so far by different workers that ABA,

jasmonic acid, and ethylene signaling pathways interact to regulate diverse stressresponses. The nature of the interaction between these pathways appears to dependon the type of stress experienced by the plant.

27.4

Antioxidants

One of the earliest signals in many abiotic stresses involves ROS and reactivenitrogen species (RNS), which modify enzyme activity and gene regulation[115,144]. The main source of ROS production in plants are chloroplasts andperoxisomes through photorespiration during light [145] and in mitochondriaduring darkness [146]. The chloroplast is one of the major producers of superoxide(O2

�) and hydrogen peroxide (H2O2) in plants. In chloroplast thylakoids, thereaction centers of Photosystems I and II are the major sites of generation of ROS[147].ROS signaling in response to abiotic stresses and its interactions with hormones

has been thoroughly reviewed [148]. ROS and RNS form a coordinated networkthat regulates many plant responses to the environment; there are a large numberof studies on the oxidative effects of ROS on plant responses to abiotic stress, butonly a few studies documenting the nitrosative effects of RNS [149]. ROS are anatural consequence of aerobic metabolism and plants have mechanisms to dealwith them under normal conditions, controlling the formation and removal rates.Under stress conditions, cell homeostasis is disrupted and ROS production canincrease significantly, putting a heavy burden on those antioxidative mechanisms,some of which are activated in order to eliminate the excess ROS [150]. Under

27.4 Antioxidants 739

certain stress conditions (like excess light, cold, heat, drought, heavy metals, etc.)the production of ROS can exceed the capacity of the plant’s defense mechanisms,an imbalance in intracellular ROS content is established, and this results inoxidative stress [95]. The ROS comprising O2

�, H2O2,1O2, HO2

�, OH�, ROOH,ROO�, and RO� are highly reactive and toxic, and cause damage to proteins, lipids,carbohydrates, and DNA, which ultimately results in cell death. Accumulation ofROS as a result of various environmental stresses is a major cause of loss of cropproductivity worldwide [151–156]. ROS may have damaging, protective, orsignaling functions, which are expressed at appropriate times. Stress-induced ROSaccumulation is counteracted by enzymatic antioxidant systems that include avariety of scavengers, such as superoxide dismutase, peroxidases, catalase, andglutathione reductase, as well as non-enzymatic low-molecular-weight metabolites,such as ascorbate, glutathione, a-tocopherol, carotenoids, and flavonoids[22,150,157].

27.5

Stress Proteins and Protein Kinases

Elucidating the function of proteins expressed by genes in stress-tolerant and-susceptible plants would advance our understanding of plant adaptation andtolerance to environmental stresses, but also may provide important informationfor designing new strategies for crop improvement [112]. Protein kinases andprotein phosphatases often act in tandem to perform the phosphorylationand dephosphorylation process, and much work is ongoing to unravel themolecular mechanisms involved in the expression of several of the large genefamilies that encode protein kinases and phosphatases involved in stress signalingin plants. Another large group of stress proteins are the heat shock proteins that areexpressed mainly during temperature stress, but also other stresses.Ca2þ is one of the most important second messengers in response to

extracellular stimuli in plants [158,159]. Signal molecules such as inositol trispho-sphate, diacylglycerol, inositol hexaphosphate, cADP-ribose, or ROS induceincreased levels of intracellular Ca2þ under stress conditions. Two protein kinasesare postulated to be the targets of the Ca2þ signal in plants. One is SnRK3, whoseactivity is dependent on the Ca2þ-binding calcineurin B-like (CBL) proteins [160].Arabidopsis has 25 SnRK3-type kinases, according to genome information, of whichthe best characterized is salt overly sensitive 2 (SOS2)/CIPK24/SnRK3.11, whichwas identified as an essential factor in the salinity stress response [161]. Inconjunction with SOS3/ScaBP8/CBL10 Ca2þ-binding protein, SOS2 activates theplasma membrane Naþ/Hþ antiporter (SOS1) required for salinity tolerance[162,163]. The other protein kinase is calcium-dependent protein kinase (CDPK).CDPKs comprise a large family of Ser/Thr kinases in plants and protozoans, andare calcium-binding, and it has been reported that CDPK transcripts are elevatedafter race-specific defense elicitation and hypo-osmotic stress [164]. Earlier reportshave also indicated CDPK transcript elevation after exposure of Arabidopsis to cold,


salt, and drought [165,166]. Arabidopsis has more than 30 CDPK genes, and severalof them have been shown to function in abiotic stress and ABA responses [160].CPK3 and CPK6 regulate the ABA response in guard cells [167], and CPK4, CPK11,and CPK32 positively regulate the ABA response [168,169]. In addition, CPK4 andCPK11 phosphorylate AREB/ABF transcription factors in an ABA-dependentmanner [169].Mitogen-activated protein kinase (MAPK) cascades function as major cellular

signaling components in eukaryotes. Therefore, plant genes for MAPK cascadeshave been examined to determine whether they are involved in various stressresponses. MAPK signaling pathways have been found to be involved in abioticstress responses in plants and this cascade links external stimuli with severalcellular responses. The cascade generally consists of MAP4Ks, MAP3Ks, MAP2Ks,and MAPKs. During stress, the stimulated plasma membrane activates MAP3Ks orMAP4Ks, which may act as adaptors linking upstream signaling steps to the coreMAPK cascades. MAP3Ks are also Ser/Thr kinases phosphorylating two aminoacids in the S/T–X–S/T motif of the MAP2K activation loop. MAP2Ks phosphor-ylate MAPKs on threonine and tyrosine residues at a conserved T–X–Ymotif [170].Arabidopsis genome information allowed identification of 20 MAPKs, 10 MAP2Ks,and 60 MAPK Kinase Kinases [171]. These MAPK components appear to functionin several different signaling processes, so they might not constitute simplecascades, but rather networks, making it difficult to identify the function of eachcomponent. Teige et al. [172] reported that an A. thaliana cascade that responds tosalt, drought, and cold may include the components MEKK1, MKK2, MPK4, and/or MPK6. Initially, this work showed that whereas mkk2 mutants had reducedtolerance to salinity, transgenic lines that overexpressed MKK2 exhibited enhancedtolerance to salt and cold. Stressor-specific induction of MAPK genes and increasedMAPK activity have been detected when plants are subjected to touch, cold, salinity,genotoxic agents, UV irradiation, ozone, and oxidative stress [173].MAPK cascades are induced by ROS, but may also regulate ROS levels by

affecting antioxidative enzymes, such as catalase activity. It has been shown inseveral studies that MAPK activity may be mediated through hormones, notably thestress-responsive hormones such as ABA, jasmonic acid, salicylic acid, andethylene [87].

27.6

Stress-Responsive Gene Expression

In the postgenomic era, comprehensive analyses using three systematicapproaches or “omics” have increased our understanding of the complex molecularregulatory networks associated with stress adaptation and tolerance: transcrip-tomics, metabolomics, and proteomics (see Section 27.1). Along with morphologi-cal and physiological studies on the responses of plants to stress conditions, severalmolecular mechanisms from gene transcription to translation as well asmetabolites are currently being investigated. Recent advances in genomic research,

27.6 Stress-Responsive Gene Expression 741

particularly in the field of proteomics, have created an opportunity for dissectingquantitative traits in a more meaningful way. Proteomics is a powerful tool forinvestigating the molecular mechanisms of the responses of plants to stresses andit provides a path toward increasing the efficiency of indirect selection for inheritedtraits [174]. Understanding the function of genes is a major challenge of thepostgenomic era. While many of the functions of individual parts are unknown,their function can sometimes be inferred through association with other knownparts, providing a better understanding of the biological system as a whole. High-throughput “omics” technologies are facilitating the identification of new genesand gene functions. In addition, network reconstructions at the genome scale arekey to quantifying and characterizing the genotype to phenotype relationship [175].There is a need to provide established or new varieties with genotypes havingenhanced or faster induction of expression of genes at the crossroad of permissivegrowth under stress conditions. This group of permissive genes includes aquaporinisoforms able to optimize water fluxes [176]. The stress-responsive genes can beclassified into two classes: early and delayed response genes [177]. The former areinduced quickly and transiently, while the latter are activated more slowly and theirexpression is sustained. The early-response genes encode transcription factors thatactivate downstream delayed-response genes. Regulatory proteins involved indrought stress such as proton antiporters TNHX1 and a proton pyrophosphataseTVP1 have been shown to improve salt and drought stress tolerance in Arabidopsis[178,179]. ERECTA is another gene regulating transpiration efficiency affectingstomatal closure, while the plant is able to maintain biomass production [180].SlERF5 is highly expressed in response to the harpin protein coded in the Hrpgene clusters in many Gram-negative phytopathogens; the overexpression ofSlERF5 is involved in the induction of the dehydration-responsive genes throughthe ABA-mediated abiotic stress response [181]. In rice, GF14 genes contain cis-elements in their promoter regions that are responsive to abiotic stress andpathogen attack. The 14-3-3s family genes are also subject to regulation by certaintranscript factors [182]. In rice, the RO-292 gene is upregulated in roots by salt ordrought stresses and by blast fungus infection [183]. Gene expression profiling hasallowed the identification of hundreds of genes induced when plants are exposed tostress [184–187]. The availability of the complete genome sequence of some modelplants, such as O. sativa and A. thaliana, has allowed the development of whole-genome tiling microarrays. This constitutes a powerful new technology that hasalready made possible the identification of several unannotated transcriptsresponsive to abiotic stress [188,189].

27.7

Role of MicroRNAs in Abiotic Stress

MicroRNAs (miRNAs) are known to play important regulatory roles in plants bytargeting mRNAs for cleavage or translational repression [190]. First discovered in2002, several groups of scientists have found plant miRNAs by cloning small RNAs


in Arabidopsis [191–193]. According to the miRNA Registry, 731 miRNA genes havebeen identified in various plants, including 117 from Arabidopsis, 178 from rice (O.sativa), 97 from Zea mays, and the rest from Populus trichocarpa, Saccharumoffcinarum, Sorghum bicolor, Medicago truncatula, and Glycine max [194]. While ithas been known that abiotic stresses, such as drought, salinity, and extremetemperatures, regulate the expression of thousands of genes in plants at bothtranscriptional and post-transcriptional levels, currently many miRNAs have beenpredicted and some have been confirmed experimentally to be involved in a varietyof abiotic stress responses.miRNAs generally target miR398, which was discovered in Arabidopsis and

rice (O. sativa), and is encoded by three loci (MIR398a, MIR398b, andMIR398c) in Arabidopsis. miR398 targets two closely related Cu/Zn-superoxidedismutase (SOD) genes, cytosolic CSD1 and chloroplast-localized CSD2[190,195,196], which under oxidative stress are overexpressed as a line of plantdefense. CSD1 and CSD2 mRNA levels were increased in response to highlight, Cu, Fe, and so on, and miR398 was downregulated under oxidative stressconditions. The lack of CSD1 and CSD2 expression in unstressed plantsdepends on miR398-mediated post-transcriptional regulation, and the stressinduction of CSD1 and CSD2 mRNA is mediated by the downregulation ofmiR398. It was also shown by Sunkar et al. [197] that transgenic A. thalianaplants overexpressing a miR398-resistant form of CSD2 accumulate more CSD2mRNA than plants overexpressing a regular CSD2, and are consequently muchmore tolerant to high light, heavy metals, and other oxidative stresses. Sunkarand Zhu [198] reported that the expression of miR393 was strongly upregulatedby ABA treatments, while miR397b and miR402 are slightly upregulated, andmiR389a downregulated, by ABA stress treatment.

27.8

Conclusion

Various abiotic stresses affect plants and are causes of stagnation or reduction incrop productivity. With the rapid increase in population, projected to be 9.4 billionby 2050, worldwide food production needs to be significantly increased to gear upto meet the demands in the coming years. There is evidence that yields of paddyand wheat have shown a declining trend in many parts of South-East Asia due toincreasing water stress [199]. Related to water stress are other abiotic stresses suchas elevated temperature and salinity that also have negative impacts on plants. Inthe Indian subcontinent, it is projected that by 2050 the average temperatures mayrise by 2.5 �C, which is an alarming situation for agriculture. Responses of plants toabiotic stresses are complex and affect several components. Such stresses not onlyaffect cell water potential, induce closure of stomata, and decrease photosynthesis,nitrate assimilation, and various anabolic enzyme reactions, but also induce thegeneration of ROS, such as superoxide radicals, hydrogen peroxide, and hydroxylradicals, which in turn cause lipid peroxidation and consequently membrane

27.8 Conclusion 743

injury, protein degradation, enzyme inactivation, pigment bleaching, and disrup-tion of DNA strands.In this context, metabolomic approaches for detailed analyses of metabolites

before, during, and after stresses can give meaningful inputs to develop crops withenhanced tolerance to different stresses. Although several technological advanceshave been made in the recent past in the field of biotechnology, one of the majorchallenges is the widening gap between the rate of development of new techno-logies and their deployment in applied breeding programs for crop improvement[200]. While many genes for different stresses have been cloned and characterizedin models as well as some crop plant species, and in some cases successfuldevelopment of transgenics has also been reported (e.g., rice for LOS5/ABA3 [201]and AP37 [202]), to date no reports of a released transgenic variety for droughttolerance have been published, even though transgenic crops have been widelyadopted globally [203]. Thus, the success of biotechnology for developing abioticstress-tolerant cultivars has been rather limited, indicating several inherentcomplexities [200]. It would therefore seem imperative to look for othertechnologies that would help plants overcome abiotic stresses and withstand globalclimate changes without affecting their productivity.

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