30Wheat and Rice Crops: �Omics� Approachesfor Abiotic Stress ToleranceSuja George, Ajay Parida, and Monkombu S. Swaminathan

Abiotic stresses are the principal causes of crop failure, reducing average yields ofmostmajor crops bymore than 50%. Rice andwheat are the twomost important foodcrops in the world, together occupying about 28% of all crop area. A considerableamount of crop biomass forwhich genetic potential exists in the present-day cultivarsin wheat and rice is not harvested under field conditions, primarily because of thesensitivity of these crops to various stresses. To meet human needs by 2050, grainproductionmust increase at an annual rate of 2%on an area of land overwhat is beingproduced at present. Modern biotechnology has a lot to offer in the field of cropimprovement in the present scenario both in understanding the mechanisms ofstress and stress tolerance in plants and in developing crop plants better equipped forharsher environmental conditions. A great deal of research has been carried out inthe recent past in the field of plant abiotic stress tolerance encompassing genomics,transcriptomics, proteomics, and metabolomics. These �omic� technologies inves-tigate different facets of a given scientific issue such as abiotic stress tolerance, butcomplement each other. Integration of phenotypic, genetic, transcriptomic, prote-omic, and metabolomic data will enable accurate and detailed gene network recon-struction. This chapter discusses the recent �omic� studies in wheat and rice in thefield of abiotic stress tolerance.

30.1Introduction

Abiotic stresses such as drought, high salinity, low and high temperature, submer-gence, and so on are frequently encountered by plants in bothnatural and agriculturalsystems. In many cases, several classes of abiotic stress challenge plants in combi-nation. For example, high temperatures and scarcity of water are commonlyencountered in periods of drought and can be exacerbated by mineral toxicities thatconstrain root growth. Abiotic stresses are the principal causes of crop failure,reducing average yields of most major crops by more than 50% [1].

Rice (Oryza sativa) is one of themost important food crops in theworldwith almosthalf of the world�s population depending on it as their staple food. More than 90% of

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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the world�s rice is grown and consumed in Asia, which is home to 60% of the Earth�spopulation. Rice accounts for 35–75% of the calories consumed by more than 3billion Asians. It is grown on about 154million hectares annually or on about 11% ofthe world�s cultivated land. Rice is cultivated in 42million hectares (mha) under fourmajor ecosystems, namely, irrigated (19mha), rain-fed lowland (14mha), flood-prone (3mha), and rain-fed upland (6mha) ecosystems. A considerable amount ofrice biomass for which genetic potential exists in the present-day cultivars is notharvested under field conditions, primarily because of the sensitivity of this crop tovarious stresses [2].

Wheat (Triticum aestivum L.) was the first domesticated crop and is the youngestpolyploid species among the agricultural crops. Together with rice and maize, wheatprovides 60% of the calories and proteins for our daily life. Unlike rice and maize,which prefer tropical environments, wheat occupies 17%of all crop area (in 2002, 210million hectares). The trade value of wheat exceeds that of any other cereal species,including rice and maize ($31 billion of world trade in 2001 versus $13 and $19billion for rice and maize, FAOSTAT database: http://apps.fao.org/default.jsp).Wheat cultivators lose about 25% annually due to various biotic (pests) and abioticstresses [3].

The world population is continuing to rise, but the gains in agricultural outputprovided by the Green Revolution have reached a plateau by now [4]. Manyexplanations have been offered for this, including deteriorating irrigation infrastruc-ture, soil degradation, stagnant technology for rain-fed farms, and the technologicalfatigue being reached on irrigated farms. Adverse regional climate changes caused bythe combined effects of atmospheric brown clouds (ABC) and greenhouse gases(GHG) add to this crisis. To meet human needs by 2050, grain production mustincrease at an annual rate of 2% on an area of land over what is being produced atpresent [2]. Modern biotechnology has a lot to offer in the area of crop improvementin the present scenario both in understanding the mechanisms of stress and stresstolerance in plants and in developing crop plants better equipped for harsherenvironmental conditions.

Since 2000, plant science has moved forward into the stage of postgenomics.A great deal of research has been carried out in the recent past in the field of plantabiotic stress tolerance encompassing genomics, transcriptomics, proteomics, andmetabolomics. The availability of whole genome sequences, microarrays, micro-RNA libraries, and so on has opened up research and result avenues that did notexist a decade ago. Completed in 2003, the Human Genome Project was a 13-yearproject. However, with the next-generation sequencing technologies from com-panies such as Roche, Illumina, and Applied Biosystems, sequencing of the wholegenome of higher organisms is being donewith unprecedented speed. Researchersall over the world have been enthusiastically exploiting the latest technologies andbioinformatics tools to provide better insights into various facets of abiotic stresstolerance.

This chapter explores the progress of research in the field of genomics, tran-scriptomics, proteomics, and metabolomics in wheat and rice, the most importantcereal crops, in the recent past.

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30.2Genomics

Major genomics initiatives have generated valuable data for the elucidation of theexpressed portion of the genomes of higher plants. The genome sequencing ofArabidopsis thaliana was completed in 2000 (The Arabidopsis Initiative), while thefinished sequence for rice was published in 2005 (project IRGS). The relatively smallgenome size of thesemodel organismswas a key element in their selection as thefirstplant genomes to be sequenced with extensive coverage. On the other hand, wheat isan allohexaploid having three homologous genomes. These genomes have beendesignated as A, B, and D, with the coding regions of the homologous genes sharingmore than 90% homology. Wheat genome is one of the largest among crop specieswith a haploid size of 16.7 billion bp [5], which is 110 and 40 times larger thanArabidopsis and rice, respectively [6]. The large size, combined with the highpercentage (over 80%) of repetitive noncoding DNA, presents a major challengefor comprehensive sequencing of the wheat genome. Although common wheatgenetic maps withmolecular markers and cytological maps with deletionmutants ofthe chromosome segments have been constructed, the number of mapped DNAmarkers for bothmap-based cloning and anchoring of the genomepositions remainsrestricted [7].

In 2005, the International Wheat Genome Sequencing Consortium (IWGSC) wascreated with the purpose of sequencing the complete genome of bread wheat. In themeantime,asignificantinsightintotheexpressedportionofthewheatgenomehasbeengained through large-scale generation and analysis of ESTs. cDNA libraries preparedfromdifferent tissuesexposedtovariousstressconditionsanddevelopmentalstagesarevaluable tools to obtain the expressed and stress-regulated portion of the genome.

To identify genes involved in cold acclimation and associated stresses, a large-scaleEST sequencing approach was undertaken by the Functional Genomics of AbioticStress (FGAS) project [8]. As part of this project, 73 521 quality-filtered ESTs weregenerated from 11 cDNA libraries constructed from wheat plants exposed to variousabiotic stresses and at different developmental stages. In addition, 196 041 ESTs forwhich trace files were available from the National Science Foundation wheat ESTsequencing program and DuPont were also quality filtered and used in the analysis.Assembly of the resolved ESTs generated a 75 488 unique sequence set (31 580contigs and 43 908 singletons/singlets). Digital expression analyses indicated that theFGAS data set is enriched in stress-regulated genes. Over 43% of the uniquesequence set was annotated and classified into functional categories according toGene Ontology.

In a similar study [7], a comprehensive collection of ESTs was prepared fromvarious tissues that develop during the wheat life cycle and from tissues subjected tostress. The study also examined their expression profiles in silico. By grouping theESTs of recombinant clones randomly selected from the full-length cDNA library, theresearchers were able to sequence 6162 independent clones with high accuracy.About 10% of the clones were found to be wheat unique genes, without anycounterparts within the DNA database. Wheat clones that showed high homology

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to those of rice were selected and their expression patterns in various tissuesthroughout the wheat life cycle and in response to abiotic stress treatments wereinvestigated. Their results showed each clone�s expression in various tissues andstress treatments and its variability in wheat and rice as a result of theirdiversification.

Comparative genomics is a powerful tool for investigating plant evolution at thewhole genome level. In a comparative analysis of wheat and rice genomes, 4485expressed sequence tags that were physically mapped in wheat chromosome binswere comparedwith the public rice genome sequence data using BLAST [9]. This ricegenome view of homologous wheat genome locations based on comparativesequence analysis revealed numerous chromosomal rearrangements between thetwo species over the course of evolution.

In the case of rice, the availability of the whole genome has made it possible to dogenome-wide analysis of specific classes of genes. Most of these analyses are quiteexhaustive, including identification of all the genes in a specific class, transcriptprofiling, phylogenetic analysis, regrouping based on the phylogenetic analysis or thepresence or absence of protein domains, identification of splice variants andintronless variants, expression in transgenic systems, proposing new functions, andso on. These studies often present a comprehensive viewof the specific class of genes.There are many reports of genome-wide identification and characterization of genesfunctioning in abiotic stress tolerance in rice.

Genome-wide analysis resulted in identification of 79 glutathione S-transferases(GSTs) genes in the rice genome [10]. A phylogenetic analysis grouped the GSTproteins into seven classes. Sequence analysis together with the organization ofputative motifs indicated the potential diverse functions of GST gene family mem-bers in rice. The authors proposed that tandem gene duplications have contributed amajor role in expansion of this gene family. Microarray data analysis revealed tissue/organ- and developmental stage-specific expression patterns of several rice GSTgenes. At least 31 GST genes showed response to plant hormones auxin andcytokinin. Furthermore, expression analysis showed the differential expression ofquite a large number of GSTgenes during various abiotic stress (20), arsenate stress(32), and biotic stress (48) conditions. Many of the GST genes were commonlyregulated by developmental processes, hormones, and abiotic andbiotic stresses. Thetranscript profiling suggested overlapping and specific role(s) of GSTs during variousstages of development in rice. The study also provides evidence for the role ofGSTs inmediating crosstalk between various stress and hormone response pathways [10].

A similar whole-genome analysis revealed 103 genes encoding WRKY transcrip-tion factors in rice. Among them, the majority of rice WRKY genes (77.7%) werelocated in duplicated regions; 45.6% of WRKY genes were fragmentally duplicatedand 35% of them were tandemly duplicated. These results suggested that genomeduplicationsmight be regarded as amajormechanism for expansion of this family inthe rice genome. Under abiotic (cold, drought, and salinity) stresses and variousphytohormone treatments, 54WRKYgenes exhibited significant differences in theirtranscript abundance; among them three genes were expressed only under stressedconditions. Among the stress-inducible genes, 13 genes were regulated only by

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abiotic stresses, another set of 13 genes were responsive to only phytohormonetreatments, and the remaining 28 geneswere regulated by both factors, suggesting aninteraction between abiotic stress and hormone signaling [11].

DREB2s (dehydration-responsive element binding protein 2s) are transcriptionfactors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in waterand heat shock stress responses and tolerance. A study analyzed all five DREB2-typegenes in rice (OsDREB2s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E, andOsABI4) to determine which of them contribute to plant stress responses. Theexpression patterns of these genes under abiotic stress conditions were studied andthe subcellular localization and transcriptional activation activity of their translationalproducts in protoplasts were examined. Only OsDREB2A and OsDREB2B showedabiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear-specific localization and thehighest transactivation activity. OsDREB2Bhas functionaland nonfunctional forms of its transcript similar to its orthologues in the grass family,and the functional form of its transcript was markedly increased during stressconditions. The splicing mechanism of OsDREB2B was analyzed with transgenicrice that expressed thenonfunctional transcript,which revealed that thenonfunctionalform is not a precursor of the functional form, indicating that stress-induciblealternative splicing of pre-mRNA is an important mechanism for the regulation ofOsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showedenhanced expression of DREB2A target genes and improved drought and heat- shockstress tolerance. These results pointed out the key role of OsDREB2B in stress-responsive gene expression in rice [12]. Another database search identified 29C3HC4-type RING finger family genes in rice. A comprehensive expression analysis of thesegeneshas beenperformedusingmicroarraydata obtained from27 tissuesor organs ofthree rice genotypes, Minghui 63, Zhenshan 97, and Shanyou 63. Expression analysisof C3HC4-type RING finger genes under abiotic stresses suggested that 12 genes aredifferentially regulated by hormones or stress in rice seedlings [13].

Heat shock proteins (Hsps) constitute an important component in the heat shockresponse of all living systems. Among the various plant Hsps (i.e., Hsp100, Hsp90,Hsp70, and Hsp20), Hsp20 or small Hsps (sHsps) are expressed in maximalamounts under high temperature stress. The characteristic feature of the sHsps isthe presence of alpha-crystallin domain (ACD) at the C-terminus. sHsps cooperatewith Hsp100/Hsp70 and cochaperones in ATP-dependent manner in preventingaggregation of cellular proteins and in their subsequent refolding. A database searchrevealed the presence of 40 alpha-crystallin domain containing genes in rice.Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. Theadditional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Adetailed scrutiny of 23 sHsp sequences resulted in categorizing these proteins in arevised scheme of classification, constituting 16 cytoplasmic/nuclear, 2 ER, 3mitochondrial, 1 plastid, and 1 peroxisomal genes. Expression analysis based onmicroarray and RT-PCR showed that 19 sHsp genes were upregulated by high-temperature stress. Besides heat stress, expression of sHsp genes was up- ordownregulated by other abiotic and biotic stresses [14].

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Several studies have pointed out the involvement of WRKY transcription factorgene family in a range of biological processes, including abiotic stress. A whole genefamily WRKYs expression study was carried out in rice. Twenty-four members of therice WRKYgene family (22% of the total) were differentially regulated in response toat least one of the stress conditions tested. The existence of nine OsWRKY geneclusters comprising phylogenetically related and unrelated genes that were signif-icantly coexpressed suggested that specific sets of WRKY genes might act incoregulatory networks. By identifying Arabidopsis orthologues of the coexpressedOsWRKY genes, it was also shown that specific coregulatory networks were con-served between the two model species. It is possible to highlight novel clusters ofplant genes contributing to the same biological processes or signal transductionpathways using such data [15].

Similar studies have also been conducted in wheat, although limited by theunavailability of the whole-genome sequence. From 960 174 ESTs of T. aestivum,117 putative AP2/ERF family genes were identified [16]. On the basis of the modelspecies A. thaliana, the AP2/ERF transcription factors from T. aestivum wereclassified into five subfamilies with the following number of members: DREB(57), ERF (47), AP2 (9), RAV (3), and Soloist (1). Using the available EST informationas a source of expression data, the putative AP2/ERF family genes from T. aestivumwere detected innine kinds of tissues. Transcripts of the geneswere shown to bemostabundant in leaves, followed by roots and seeds, and least abundant in stem.

Enzymatic methylation, which is catalyzed by the large number of O-methyl-transferases (OMTs), is one of the important reactions in the flow of primary andsecondary metabolism. The structural and expressional divergence of genes encod-ingO-methyltransferase has been studied inwheat [17].Wheat OMTgenes TaOMT3,TaOMT4, and TaOMT5 were analyzed using a bioinformatics approach for theirgenomic organization, tissue-specific expression, responses to abiotic stresses andhormones, and cis-elements.

30.3Transcriptomics

With the ever-increasing availability of genomic sequences and the introduction ofmicroarray technology, enabling the high-throughput analysis of gene expression,transcriptome profiling has rapidly become a favorite tool with many researchers.The study of plant transcriptomes has led to important discoveries and to anaccumulation of profiling data covering a wide range of different tissues, develop-mental stages, perturbations, and genotypes. Querying a large number ofmicroarrayexperiments can provide insights that cannot be gained by analyzing singleexperiments.

In an effort to elucidate genome-level responses to drought and high-salinitystress in rice, a 70-mer oligomer microarray covering 36 926 unique genes or genemodels was used to profile genome expression changes in rice shoot, flag leaf, andpanicle under drought or high-salinity conditions [18]. Patterns of gene expression

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in response to drought or high-salinity stress within a particular organ type showedsignificant overlap, but comparison of expression profiles among different organsshowed largely organ-specific patterns of regulation. Both stresses appeared to alterthe expression patterns of a significant number of genes involved in transcriptionand cell signaling in a largely organ-specific manner. This study identified thatpromoter regions of genes induced by both stresses or induced by one stress inmore than one organ type were relatively enriched in two cis-elements (ABRE coreand DRE core) known to be associated with water stress. Further computationalanalysis that indicated that novel promoter motifs are present in the promoters ofgenes involved in rehydration after drought led the authors to propose that ricemight possess a mechanism that actively detects rehydration and facilitates rapidrecovery.

The rice genome encodes a total of 10 genes that contain the highly conservedMTase catalytic domains found in DNAmethyltransferases (MTases). A microarray-based gene expression profile of all 10MTases during 22 stages/tissues that included14 stages of reproductive development and five vegetative tissues together with threestresses, cold, salt, and dehydration stress, revealed specific windows of MTaseactivity during panicle and seed development. One of the MTases was activated inyoung seedlings in response to cold and salt stress [19].

Microarray-based transcriptome profiling has been successfully used to analyzethe differences in gene expression between salt-sensitive and -tolerant rice cultivars.The expression profiles of 1194 salinity-regulated cDNAs in rice salt-sensitive cultivarIR64 and Pokkali, a well-known, naturally salt-tolerant relative, were analyzed usingmicroarrays [20]. The study revealed that salinity tolerance of Pokkali may be due toconstitutive overexpression of many genes that function in salinity tolerance.Analysis of genome architecture revealed the presence of these genes on all thechromosomes with several distinct clusters. A few genes were mapped on one of themajor quantitative trait loci, Saltol, on chromosome 1 and were found to bedifferentially regulated in the two contrasting genotypes. The study revealedthat a set of known abiotic stress-inducible genes, including CaMBP, GST, LEA,V-ATPase, OSAP1 zinc finger protein, and transcription factor HBP1B, wereexpressed at high levels in Pokkali even in the absence of stress.

Mitochondrial responses to abiotic stresses at the early stages of wheat develop-ment after imbibition under normal and low temperature, high salinity, and highosmotic potential stress have been evaluated by transcriptome profiling [21]. Micro-array analysis of the mitochondrial transcriptome revealed stress specific in tran-script levels in the case ofmost genes, but few groups of genes were found to respondcommonly to different stresses. Under continuous stresses for 3 days, 13 genesshowed low-temperature-specific responses with up- or downregulation, while 14and 23 genes showed responses specific to high salinity and high osmotic potential,respectively. On the other hand, 13 genes showed common responses. Among thenuclear-encoded mitochondrial-targeted genes, MnSOD and AOX increased theirtranscript amounts. These results also point out toward common and differentregulatory mechanisms that can sense different abiotic stresses and modulate bothnuclear and mitochondrial gene expression in wheat.

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In order to reveal differences in global expression profiles of drought-tolerant and-sensitive wild emmer wheat genotypes, a shock-like dehydration process wasdeployed to compare transcriptomes at two time points in root and leaf tissues [22].The comparison of transcriptomes revealed several unique genes or expressionpatterns such as differential usage of IP3-dependent signal transduction pathways,ethylene- and abscisic acid (ABA)-dependent signaling, and preferential or fasterinduction of ABA-dependent transcription factors by the tolerant genotype thatdistinguish contrasting genotypes indicative of distinctive stress response pathways.This comparison of transcriptomes in the absence of and after dehydration indicatesthat gene networks involved in drought response especially in root tissues may havebeen lost in the selection processes generating modern bread wheat.

T. aestivum �Opata� is an elite hard red spring wheat that has been used as a parentof the ITMI (International TriticeaeMapping Initiative) mapping population and alsoin the production of synthetically derived hexaploid wheats, some of which (followingselection) show increased drought tolerance relative to Opata. The response of Opataroots to water withholding was described using physiological variables and oligonu-cleotide microarrays [23]. They identified 190 transcripts whose expression increasedfollowing water limitation. In addition to previously characterized markers of abioticstress and many genes of unknown function, they were able to identify multipleputative glucanases and class III peroxidases as being particularly responsive to stress.A comparison of these data with microarray analyses of Opata�s more drought-tolerant, synthetic-derived Progeny revealed a relatively high correlation betweenresponsive transcripts in the two genotypes, despite differing physiological responses.Someof the transcripts thatwere differentially expressed betweenOpata and themoretolerant synthetic-derived genotype under stress included a class III peroxidase, anAP2 family transcription factor, and several transcripts of unknown function.

There are a few software applications that have been developed to query largemicroarray gene expression databases using a Web-browser interface. GENEVES-TIGATOR, a database and Web-browser data mining interface for AffymetrixGeneChip data, is an example.Users can query the database to retrieve the expressionpatterns of individual genes throughout chosen environmental conditions, growthstages, or organs. Reversely, mining tools allow users to identify genes specificallyexpressed during selected stresses, growth stages, or in particular organs. UsingGENEVESTIGATOR, the gene expression profiles of more than 22 000 Arabidopsisgenes can be obtained, including those of 10 600 currently uncharacterizedgenes [24]. More recently, Genevestigator rice and barley gene expression databaseshave been released that contain quality controlled and well-annotated microarrayexperiments using ontologies [25]. The databases comprise experiments frompathology, plant nutrition, abiotic stress, hormone treatment, genotype, and spatialor temporal analysis, but are expected to cover a broad range of research areas asmoreexperimental data become available. The transcriptome meta-analysis of the modelspecies rice is expected to deliver results that can be used for functional genomics andbiotechnological applications in cereals.

In addition to the whole transcriptome profiling studies, a plethora of researchreports are recently available on transcript profiling of individual genes or specific

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class of genes functioning in abiotic stress tolerance in wheat and rice. These studiescomplement the whole transcriptome profiling studies and in many cases are morein-depth when individual gene�s function is concerned. Many of them have served toreveal novel functions for already known genes and help in elucidating theirinteractions with other genes and pathways.

A recent review analyzed the role of APETALA 2/ethylene response elementbinding protein (AP2/EREBP) family transcription factors in abiotic stress tolerance.O. sativa subsp. Japonica has 163 gene loci assigned to this transcription factor. AP2/EREBP transcription factors have been implicated in hormone, sugar, and redoxsignaling in context of abiotic stresses such as cold and drought. It was suggested thatAP2/EREBP transcription factors integratemetabolic, hormonal, and environmentalsignals in stress acclimation and retrograde signaling [26].

Many transcription factors involved in abiotic stress tolerance in an ABA-depen-dantmanner have been characterized. TwogroupAbZIP transcription factors in rice,OsABF1 andOsABF2 (O. sativaABA-responsive element binding factor), were foundto be expressed in various tissues in rice and induced by different types of abioticstress treatments, such as drought, salinity, cold, oxidative stress, and ABA [27, 28].MYBS3 is a singleDNAbinding repeatMYB transcription factor previously shown tomediate sugar signaling in rice. A recent study revealed that MYBS3 also plays acritical role in cold adaptation in rice. Transgenic rice constitutively overexpressingMYBS3 tolerated 4 �C for at least 1week and exhibited no yield penalty in normalfieldconditions. Transcription profiling of transgenic rice overexpressing or underexpres-sing MYBS3 led to the identification of many genes in the MYBS3-mediated coldsignaling pathway. MYBS3 was found to repress the well-known DREB1/CBF-dependent cold signaling pathway in rice, and the analysis revealed that therepression appears to act at the transcriptional level. DREB1 responded quickly andtransiently whileMYBS3 responded slowly to cold stress, which suggests that distinctpathways act sequentially and complementarily for adapting short- and long-termcold stress in rice [29].

Differences in expression pattern of two abiotic stress-inducible genes incontrasting rice genotypes varying in their salt stress sensitivity were studied [30].Expression levels of two genes, Rab16A and SamDC, and corresponding proteins,in the seeds, at the background level (dry or water-imbibed state) and ABA-imbibedconditions in rice genotypes M-1-48 (salt sensitive), Nonabokra (salt tolerant), andGobindobhog (aromatic) were analyzed. An extremely low abundance of Rab16A orpractically undetectable SamDC transcripts were observed in M-1-48 and Gobin-dobhog seeds under control conditions, induced only after exogenous ABAtreatment, whereas they were expressed at a much higher level even in dry andwater-imbibed seeds of Nonabokra and lesser induced by ABA. The RAB16A andSAMDC protein expression in the three varieties were also identical to the geneexpression patterns. Thus, the expression was found to be stress inducible in M-1-48 and Gobindobhog, while constitutive in Nonabokra. Their results indicated thatthe difference in expression profiles of the two genes is partly responsible forincreased salt tolerance in Nonabokra.

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In an effort to characterize previously uncharacterized rice genes, a large popu-lation of Arabidopsis plants were transformed with rice full-length cDNAs to isolatethe rice genes that improve the tolerance of plants to environmental stress [31]. Onesalt-tolerant line identified, R07047, expressed a rice gene,OsSMCP1,which encodesa small protein with a single C2 domain, a Ca2þ -dependent membrane targetingdomain. Line R07047 showed enhanced tolerance to high salinity, osmotic, dehy-drative, and oxidative stresses. Furthermore, R07047 showed improved resistance toP. syringae. In vivo localization studies revealed the plastid localization of the protein.Overexpression of OsSMCP1 was found to induce overexpression of several nuclear-encoded genes, including the stress-associated genes, in transgenic Arabidopsis [31].

In similar experiments, the role of a previously unknown zinc finger transcriptionfactor called DST (drought and salt tolerance) that negatively regulates stomatalclosure by direct modulation of genes related to H2O2 homeostasis in drought andsalt tolerance was analyzed [32]. A DREB1B gene (dehydration-responsive elementbinding factor) from rice, differentially regulated at the transcriptional level byosmotic stress, oxidative stress, salicylic acid, ABA, and cold, was characterized byoverexpression in tobacco plants and analysis of transgenic plants [33]. Another studyrevealed the involvement of specific MAP kinase kinase (mitogen-activated proteinkinase) in different abiotic stress signaling [34]. Two rice calmodulin promotersOsCaM1 and OsCaM3 were characterized by fusion with GUS reporter gene andtransformation into tobacco [35].

A study in two wheat recombinant inbred lines contrasting in their salt toleranceexamined the expression levels under salt or drought stress of 10MYB transcriptionfactor genes [36]. FourMYB genes were found to be consistently upregulated in theseedling roots of both genotypes under short-term salt treatment. ThreeMYBgeneswere found to be upregulated in both genotypes under long-term salt stress. OneMYB gene was upregulated in both genotypes under both short- and long-term saltstress. Of these salt upregulated MYB genes, one MYB gene (TaMYBsdu1) wasmarkedly upregulated in the leaf and root of wheat under long-term drought stress.In addition, TaMYBsdu1 showed higher expression levels in the salt-tolerantgenotype than in the susceptible genotype under salt stress, indicating that it isan important regulator involved in wheat adaptation to both salt and droughtstresses.

A novel wheat NAC transcription factor gene (TaNAC4) was found to share highhomology with rice OsNAC4 gene. TaNAC4 transcript in wheat leaves was inducedby the infection of strip rust pathogen and also by exogenously applied methyljasmonate (MeJA), ABA, and ethylene. Environmental stimuli, including highsalinity, wounding, and low temperature, also induced TaNAC4 expression [37].A pectin methylesterase inhibitor (PMEI) was found to be upregulated underhydrogen peroxide treatments in wheat [38]. Dehydration-responsive element bind-ing factors (DBFs) belong to the AP2/ERF superfamily and play vital regulatory rolesin abiotic stress responses in plants. Three novel homologues of theDBFgene familyin wheat (TaAIDa-c, T. aestivum abiotic stress-induced DBFs) were isolated byscreening a drought-induced cDNA library [39]. Overexpression of TaAIDFa activatedCRT/DRE-containing genes under normal growth conditions and improved drought

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and osmotic stress tolerances in transgenic Arabidopsis plants. Vacuolar Hþ -trans-locating pyrophosphatase (V-PPase) is a key enzyme related to both plant growth andabiotic stress tolerance. V-PPase genes TaVP1, TaVP2, and TaVP3 were identifiedfrom wheat [40]. TaVP2 was observed to be mainly expressed in shoot tissues anddownregulated in leaves under dehydration. Its expression was upregulated in rootsunder high salinity. TaVP1 was relatively more ubiquitously and evenly expressedthanTaVP2. Its expression level in rootswas highest among the tissues examined andwas inducible by salinity stress. These results indicated that the V-PPase geneparalogues in wheat are differentially regulated spatially and in response to dehy-dration and salinity stresses.

Sucrose nonfermenting 1-related protein kinase 2 family members play essentialroles in response to hyperosmotic stresses in Arabidopsis, rice, and maize. A studycharacterized the function of TaSnRK2.4, an SNF1-type serine/threonine proteinkinase of wheat, in drought, salt, and freezing stresses inArabidopsis [41]. TransgenicArabidopsis overexpressing TaSnRK2.4 had enhanced tolerance to drought, salt, andfreezing stresses, which were simultaneously supported by physiological results,including decreased rate of water loss, enhanced higher relative water content,strengthened cell membrane stability, improved photosynthesis potential, andsignificantly increased osmotic potential. The results indicated that TaSnRK2.4 isinvolved in the regulation of enhanced osmotic potential, growth, and developmentunder both normal and stress conditions and imply that TaSnRK2.4 is a multifunc-tional regulatory factor in Arabidopsis.

A novel aquaporin gene fromwheat, TaNIP (T. asetivum L. nodulin 26-like intrinsicprotein), was characterized in a study [42]. TaNIP was identified and cloned throughthe GeneChip expression analysis of a salt-tolerant wheat mutant RH8706-49 undersalt stress. The overexpression of TaNIP in transgenic Arabidopsis produced highersalt tolerance than wild-type plants. Under salt stress treatment, TaNIP overexpres-singArabidopsis accumulated higher Kþ , Ca2þ , and proline contents and lower Naþ

level than thewild-type plants. The overexpression of TaNIP in transgenicArabidopsisalso upregulated the expression of a number of stress-associated genes. Their resultssuggested that TaNIP plays an important role in salt tolerance.

30.4Evaluation of the Role of MicroRNAs in Abiotic Stress

MicroRNAs (miRNAs) are small single-stranded RNAs with a length of about 21 nt;these noncoding RNAs regulate developmental and stress responses in plants bycleaving mRNAs. Most of the physiological processes are controlled by miRNAs inseveral organisms including plants. A huge database exists on miRNAs identifiedfrom diverse species. However, the processes of data mining of miRs in most of thespecies are still incomplete. AlthoughmanymiRNAs have been identified in rice andwheat, relatively little is known about their role in abiotic stress.

Cloning and identification of approximately 40 newputativemiRs is reported froma basmati rice variety [43]. About 23 sequences were derived from rice exposed to salt

30.4 Evaluation of the Role of MicroRNAs in Abiotic Stress j705

stress, while 18 were derived from rice infected with tungro virus. A few of theseputative miRs were common to both. Their results showed that at least two of thesemiRswere upregulated in response to both abiotic and biotic stresses. ThemiR targetpredictions indicated that most of the putative miRs target specific metabolicprocesses. The upregulation of similar miRs in response to two entirely differenttypes of stresses suggests a converging functional role of miRs in managing variousstresses. Recently, 18 cold-responsive rice miRNAs were identified using micro-arrays [44]. The existence of hormone-responsive elements in the upstream regionsof these cold-responsive miRNAs indicated the importance of hormones in thisdefense systemmediated bymiRNAs. Their findings confirm the role of miRNAs asubiquitous regulators in rice.

Deep sequencing of small RNA libraries is an effective approach to uncover rareand lineage- and species-specificmiRNAs in any organism.A study constructed threesmall RNA libraries from control rice seedlings and seedlings exposed to drought orsalt stress and then subjected them to pyrosequencing [45]. A total of 58 781, 43 003,and 80 990 unique genome-matching small RNAs were obtained from the control,drought, and salt stress libraries, respectively. Twenty-three new miRNAs, mostlyeach derived from a unique locus in rice genome, were identified. Six of the newmiRNAs are conserved in other monocots.

In wheat, 12 miRNAs responsive to heat stress have been identified [46]. Anotherstudy identified 2076 small RNAs in a small RNA library from leaf, root, and spike ofwheat. These small RNAsmapped to noncoding regions, the CDS region of protein-coding genes, and 50 UTR and 30 UTR regions. The expression of small RNAs inwheat seedling leaves, roots, and spikes were analyzed by Northern blot, whichindicated that some small RNAs were responsive to abiotic stress treatments,including heat, cold, salt, and dehydration [47].

30.5Generation of Transgenic Wheat and Rice Plants Tolerant to Abiotic Stress

Transgenic rice plants overexpressing ZFP245, a cold- and drought-responsive genethat encodes a zinc finger protein, were found to display high tolerance to cold anddrought stresses. The transgenic plants did not exhibit growth retardation, butshowed growth sensitivity against exogenous abscisic acid, increased free prolinelevels, and elevated expression of rice pyrroline-5-carboxylate synthetase and pralinetransporter genes under stress conditions. Overproduction of ZFP245 enhanced theactivities of reactive oxygen species scavenging enzymes under stress conditionsand increased the tolerance of rice seedlings to oxidative stress. It was postulatedthat ZFP245 may contribute to the tolerance of rice plants to cold and droughtstresses by regulating proline levels and reactive oxygen species scavengingactivities [48].

OsbZIP72, a member of the basic leucine zipper (bZIP) transcription factorfamily, is an ABRE binding factor in rice. Transgenic rice overexpressing OsbZIP72showed a hypersensitivity to ABA, elevated levels of expression of ABA response

706j 30 Wheat and Rice Crops: �Omics� Approaches for Abiotic Stress Tolerance

gene such as LEAs, and an enhanced ability of drought tolerance. These resultssuggested that OsbZIP72 plays a positive role in drought resistance through ABAsignaling [49]. Transgenic rice overexpressing OsbZIP23, another member of thebZIP family, showed significantly improved tolerance to drought and high-salinitystresses and sensitivity to ABA. GeneChip and real-time polymerase chain reactionanalyses revealed that hundreds of genes were up- or downregulated in the riceplants overexpressing OsbZIP23, indicating that OsbZIP23 is a major player ofthe bZIP family in rice for conferring ABA-dependent drought and salinitytolerance [50].

The DREB transcription factors, which specifically interact with C-repeat/DRE(A/GCCGAC), play an important role in plant abiotic stress tolerance by controllingthe expression of many cold and drought-inducible genes in an ABA-independentpathway. Three novel DREB genes, OsDREB1E, OsDREB1G, and OsDREB2B, wereisolated from rice [51]. Transgenic rice plants analysis revealed that overexpression ofOsDREB1G and OsDREB2B in rice significantly improved their tolerance to waterdeficit stress, while overexpression of OsDREB1E could only slightly improve thetolerance towater deficit stress, suggesting that theOsDREBsmight participate in thestress response pathway in different manners.

Overexpression of TERF1 (encoding a tomato ethylene response factor) in trans-genic rice resulted in an increased tolerance to drought andhigh salt [52]. The authorsassociated the enhanced tolerance with the accumulation of proline and the decreaseinwater loss. Furthermore, TERF1was found to effectively regulate the expression ofstress-related functional genes Lip5, Wcor413-l, OsPrx, and OsABA2, as well asregulatory genes OsCDPK7, OsCDPK13, and OsCDPK19, under normal growthconditions. cis-Acting elements such as DRE/CRT and GCC box exist in TERF1targeted gene promoters. Similarly, overexpression of a trehalose-6-phosphate phos-phatase gene OsTPP1 conferred stress tolerance in rice and resulted in the activationof stress-responsive genes [53].

An aluminum (Al3þ ) tolerance gene TaALMT1 was overexpressed in transgenicwheat under maize ubiquitin promoter [54]. The transgenics showed increasedTaALMT1 expression, malate efflux, and Al3þ resistance compared to untrans-formed controls. Some T2 lines showed greater Al3þ resistance than ET8, an Al3þ -resistant reference genotype. Increased drought tolerance was reported in transgenicwheat expressing Vigna aconitifolia D1-pyrroline-5-carboxylate synthetase (P5CS)cDNA that encodes the key regulatory enzyme in proline biosynthesis under thecontrol of a stress-induced promoter complex (AIPC) [55]. Drought stress tolerancewas accompanied by accumulation of proline in transgenic plants.

30.6Proteomics

The transcriptome analyses of gene expression at the mRNA level have contributedgreatly to our understanding of abiotic stress tolerance in plants.However, the level ofmRNA does not always correlate well with the level of protein, the key player in the

30.6 Proteomics j707

cell [56]. Therefore, it is insufficient to predict protein expression level fromquantitative mRNA data. This is mainly due to posttranscriptional regulationmechanisms, such as nuclear export and mRNA localization, transcript stability,translational regulation, and protein degradation. Proteome studies aim at thecomplete set of proteins encoded by the genome and thus complement the tran-scriptome studies.

Several researchers have used the proteomics approach to identify specific proteinsinvolved in rice stress response. The proteome response of the plasma membrane(PM) to environmental stresses was studied using a subcellular proteomics approach,monitoring changes in abundance of PM-associated protein in response to salini-ty [57]. The proteome was extracted from a root plasma membrane-rich fraction of arice salt-tolerant variety, IR651, grown under saline and normal conditions. Compar-ative two-dimensional electrophoresis revealed that 24 proteins were differentiallyexpressed in response to salt stress. Most of the proteins identified were involved inseveral important mechanisms of plant adaptation to salt stress, including regulationof PM pumps and channels, membrane structure, oxidative stress defense, signaltransduction, protein folding, and the methyl cycle. These results point to thesuitability of proteomics approach in identification of stress-regulated proteins.

In another study, a proteomic approach was adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposedto different temperatures, such as 5 or 10 �C, and samples were collected afterdifferent time courses. The researchers were able to identify some novel proteins,such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like,drought-inducible late-embryogenesis abundant, and a fibrillin-like protein [58].Another group of researchers also used proteomics approaches to get new insightsinto chilling stress responses in rice. Rice cultivar Nipponbare was treated at 6 �C for6 or 24 h and then allowed to recover for 24 h. The temporal changes in total proteinsin rice leaves were examined using two-dimensional electrophoresis [59]. Theresearchers were able to identify 85 differentially expressed proteins using massspectrometry analysis that were involved in several processes including signaltransduction, RNA processing, translation, protein processing, redox homeostasis,photosynthesis, photorespiration, and metabolisms of carbon, nitrogen, sulfur, andenergy. Interestingly, gene expression analysis of 44 different proteins by quantitativereal-time PCR showed that the mRNA level was not correlated well with the proteinlevel. This underlines the importance of proteomics in identification of key compo-nents in stress tolerance. The same group had previously used proteomic success-fully to investigate the salt stress-responsive proteins in rice cv. Nipponbare roots.They were able to identify six novel stress-responsive proteins involved in regulationof carbohydrate, nitrogen, and energy metabolism, reactive oxygen species scaveng-ing, mRNA and protein processing, and cytoskeleton stability [56].

Several studies have suggested a critical role of protein phosphorylation in saltstress response in plants. A study analyzed the differential expression of ricephosphoproteome under salt stress [60]. Seventeen differentially upregulated andeleven differentially downregulated putative phosphoproteins were identified. Thesame group further identified 31 salt stress differentially regulated proteins, the

708j 30 Wheat and Rice Crops: �Omics� Approaches for Abiotic Stress Tolerance

majority of which have not been reported in the literature. Thus, proteomics isindeed a valuable tool in providing new insight into plant response to abioticstress.

Cell wall proteins (CWPs) are important both formaintenance of cell structure andfor responses to abiotic and biotic stresses. To determine differentially expressedCWPs in wheat under flooding stress, gel-based proteomic and LC–MS/MS-basedproteomic techniques were used [61]. Eighteen proteins were found to be signifi-cantly regulated in response to flood by gel-based proteomics and 15 proteins by LC–MS/MS-based proteomics. Among the flooding downregulated proteins, most wererelated to the glycolysis pathway and cell wall structure and modification. However,themost highly upregulated proteins in response toflooding belong to the category ofdefense and disease response proteins. Among these differentially expressed pro-teins, only methionine synthase, beta-1,3-glucanases, and beta-glucosidase wereconsistently identified by both techniques. The downregulation of these threeproteins suggested that wheat seedlings respond to flooding stress by restrictingcell growth to avoid energy consumption; by coordinating methionine assimilationand cell wall hydrolysis, CWPs played critical roles in flooding responsiveness. Theimplication of different drought treatments on the protein fractions in grains ofwinter wheat was examined using 1H nuclear magnetic resonance spectroscopyfollowed by chemometric analysis [62]. Principal component trajectories of the totalprotein content and the protein fractions of flour, as well as the 1H NMR spectra ofsingle wheat kernels, wheat flour, and wheat methanol extracts, were analyzed toelucidate the metabolic development during grain filling. The results from both the1H NMR spectra of methanol extracts and the 1H HR-MAS NMR of single kernelsshowed that a single drought event during the generative stage had as strong aninfluence on protein metabolism as two consecutive events of drought. In contrast, adrought event at the vegetative growth stage had little effect on the parametersinvestigated.

30.7Metabolomics

Even after the completion of thewhole-genome sequencing inmany plants, networksof gene tometabolite are largely unknown. To reveal the function of genes involved inmetabolic processes and gene-to-metabolite networks, the metabolomics-basedapproach is regarded as a direct way. In particular, integration of comprehensivegene expression profile with targetedmetabolite analysis is shown to be an innovativeway for identification of gene function for specific product accumulation inplants [63].

Metabolomics represents the exhaustive profiling of metabolites contained inorganism. Proteomics and transcriptomics are both considered to be a flow of mediaconcerning genetic information. In contrast, metabolomic should be thought asbeing concerned with phenotype [64]. Perturbations including environmentalchange, physical stress, abiotic stress, nutritional stress, mutation, and so on lead

30.7 Metabolomics j709

to changes in the metabolome. Analysis of these changes serves to fine-tune ourknowledge on plant response to environmental changes, physical stress, abioticstress, nutritional stress, mutation, and so on.

Capillary electrophoresis–mass spectrometry (CE–MS) and capillary electro-phoresis diode–array detection (CE–DAD) were used to analyze the dynamicchanges in the level of 56 basic metabolites in rice foliage at hourly intervals overa 24 h period [65]. They found that in response to environmental stress,glutathione and spermidine fluctuated synchronously with their regulatorytargets.

Overexpressing YK1gene, the homologue of theHC toxin reductase (HCTR) gene,in transgenic rice was accompanied by an increase in the amounts of NAD(P)(H).Besides HCTR activity, YK1 also possessed dihydroflavonol-4-reductase activity [66].The overexpression of YK1was found to induce the activation of enzymes in theNADsynthetic pathway, which resulted in an increase in the amount of NAD(P)(H). Theseresults implied that the coupled increase in DFR activity and amounts of NAD(P)(H)may contribute to biotic and abiotic stress tolerance. A metabolite profiling of YK1transgenic rice was done by CE/MS [67]. They analyzed several metabolites ofglycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway.In addition, the concentrations of sugars and ions were quantified. Their resultsindicated that in YK1 overexpressing plants, the concentrations of cis-aconitate,isocitrate, and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, theamounts of free amino acids and metals were altered, whereas sugars in seeds werekept constant. While the overexpression of YK1 was associated with only slightchanges in the amounts of severalmetabolites analyzed, glutathione derivatives weresubstantially increased in suspension cultured cells.

Impact of nitrogen (N) and sulfur (S) deficiency on N and S remobilization fromsenescing canopy tissues during grain filling in winter wheat was studied usingtranscriptomic and metabolomic approaches [68]. Nuclear magnetic resonance(NMR) metabolite profiling revealed significant effects of suboptimal N or S supplyin leaves but not in developing grain. Analysis of amino acid pools in the grain andleaves revealed a strategy whereby amino acid biosynthesis switches to the produc-tion of glutamine during grainfilling. Glutaminewas found to accumulate in thefirst7 days of grain development, prior to conversion to other amino acids and protein inthe subsequent 21 days. Transcriptome analysis indicated downregulation of theterminal steps in many amino acid biosynthetic pathways. Their results indicatedthat vegetative tissue N has a greater control over the timing and extent of nutrientremobilization than S.

30.8Conclusions and Perspectives

The progress of �omic� technologies during the past decade has been spectacular.After sequencing the complete genome of Arabidopsis in 2002, the technology has

710j 30 Wheat and Rice Crops: �Omics� Approaches for Abiotic Stress Tolerance

catapulted, significantly reducing the time and cost required to sequence thecomplete genome of a higher plant. The next-generation sequencing technologieshave generated an information explosion that scientists all over the world areearnestly exploring. Genomics, transcriptomics, proteomics, and metabolomicsinvestigate different facets of a given scientific issue such as abiotic stress tolerance,but complement each other. Integration of phenotypic, genetic, transcriptomic,proteomic, and metabolomic data will enable accurate and detailed gene networkreconstruction. This will ultimately result in the elucidation of the molecular path-ways involved in complex phenotypic traits. A better understanding of genetic andcellular mechanisms behind abiotic stress tolerance would facilitate generation oftransgenic plants with desired traits with little or no undesired/unforeseen effects.

Acknowledgment

The authors gratefully acknowledge the financial assistance from Department ofBiotechnology (DBT), India.

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