Part IIISpecies-Specific Case Studies

Section IIIA Graminoids

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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28Wheat: Functional Genomics of Abiotic Stress ToleranceParamjit Khurana, Harsh Chauhan, and Neetika Khurana

Abiotic stresses such as extreme temperatures and water availability, high salt, anddeficiencies or toxicity of minerals severely affect productivity of cereal cropsworldwide. These stresses become even more disastrous in present environmentof climate change. Wheat is one of the most important cereal crops providingapproximately 20% percent of calories in human food. Wheat is grown in adverseenvironments, especially high temperature and low water availability, therebylimiting yield potential. There is ample variation available in abiotic stress tolerancein germplasm of wheat and its wild relatives. However, it has been relatively lessexploited due to poor understanding of wheat genome and its molecular basis ofstress response. Functional genomics is now widely seen as providing tools fordissecting abiotic stress response in various crop plants. Functional genomics involvemany related approaches such as global gene expression profiling, identification ofresponsive genes/alleles, followed by mutant analysis or transgenic approaches toassign the function of specific gene or its product protein. Since wheat genome is notsequenced, genome-wide collection of ESTs and full-length cDNAs is important forstructural and functional analysis of wheat genes responsive to abiotic stresses. Thischapter deals with the present knowledge of wheat functional genomics and pro-spects of molecular breeding for abiotic atress tolerance.

28.1Introduction

Wheat is an important crop globally and ranks second in production as a cereal crop.Being a staple food, demand for wheat in developing countries is expected to grow ataround 2.2% annually similar to the existing rate of production growth; thus, it hasbeen postulated that by 2020 one billion tonnes ofwheatwould be required to feed thepopulation [1]. Of the various factors influencing wheat crop productivity, abioticstresses play an important role. Climate change acceleration leading to globalwarming and higher CO2 has a major impact on wheat productivity and worldwheat production is predicted to decline by as much as 8% from the 2008/09 recordvolume (FAO, 2009). Thus, to develop improved wheat varieties, molecular genetic

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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.

tools and functional genomics need to be utilized and integrated into the existingbreeding practices. Though wheat genome has been refractory due to its largegenome size (16 000Mb), amplification of transposable elements (TEs), coupledwithduplication of chromosome segments, and polyploidization, a number of approacheshave been developed for wheat genomic research. Development of transgenic wheat,massive EST collections, and cDNA arrays along with comparative genomicsapproaches have opened new opportunities for wheat improvement. To identifyagronomically important genes, the function of genes must be known. Thus, of late,functional genomics is fast emerging as a major tool for wheat improvement.

28.2Functional Genomics Approaches

28.2.1Proteomics

For functional analysis of proteins, their structural analysis is necessary. Moritaet al. [2] developed a novel way of screening protein folding and function by usingwheat germ cell-free system. This was the first experimental evidence of theapplicability of the wheat germ cell-free protein synthesis system to high-throughputstructural analysis of functional proteins. In the same year, Majoul et al. [3] analyzedthe effect of high-temperature stress on wheat endosperm proteome by employingtwo-dimensional electrophoresis coupled with analysis by matrix-assisted laserdesorption/ionization mass spectrometry and tandem mass spectrometry andcharacterized heat-induced proteins. The same group in 2004 [4] analyzed thenonprolamin fraction that contains albumins and globulins and functionally iden-tified 42 proteins, some involved in carbohydrate metabolism and others associatedwith abiotic stresses and heat shock proteins. Using a similar approach, Bahrmanet al. [5] identified wheat leaf protein expression profile and found that most of themare involved in carbohydratemetabolism. For further enhancingwheat leaf proteomeresearch,Donnelly et al. [6] used a combination of protein and expressed sequence tagdatabase and partially characterized the wheat leaf proteome. They found that amajority of proteins were involved in energy production and primary or secondarymetabolism. Mamone et al. [7] undertook a more detailed characterization of wheatgliadin proteins providing insight into the complex nature of gliadin production.Techniques involving subproteome fractionation combined with two-dimensionalelectrophoresis and protein identification led to an effective separation of the highlyabundant gliadins and glutenins from much less abundant albumins and globu-lins [8]. An interactome of proteins associated with abiotic stress was developed byusing a yeast-two-hybrid GAL4 system and specific protein interactive assays [9]. Thisrevealed a networking of regulatory factors such as phospholipaseCandGTPbindingprotein, VRN1/2, that play vital roles during vernalization, flower initiation, andabscisic acid signaling along with various abiotic stress-related proteins. Gobaaet al. [10] using 1BL.1RS translocation lines provided valuable insight into the

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endosperm protoeome. They found that in the translocated genotypes the loss of adimeric a-amylase inhibitor may explain the dough stickiness associated with suchgenotypes. Upregulation of a certain c-gliadin with nine cysteine residues indicatedthe regulation involved in the synthesis of prolamines in the wheat endosperm.

Merlino et al. [11] analyzed soluble proteins by two-dimensional electrophoresis andMALDITOF in 112 recombinant inbred lines (�opata 85�� �synthetic W7984�) andidentified heat shock proteins, b-amylases, UDP-glucose pyrophosphorylases, perox-idases, and thioredoxins. Winning et al. [12] examined the implications of differentdrought treatments on the protein fractions in grains of winter wheat using protonnuclear magnetic resonance spectrosocopy followed by chemometric analysis. Theresults indicated that protein metabolism is highly influenced by multiple droughtevents. In a recent study on flooding stress and cell wall proteome of wheat roots, Konget al. [13] employed a gel-based proteomic and LC-MS/MS-based proteomic techniquesand found that most upregulated proteins belonged to the category of defense anddisease responses. However, downregulation of three proteins such as methioninesynthase, b-1,3-glucanases, and b-glucosidase suggested that wheat seedlings respondto flooding stress by restricting cell growth. Thus, the various proteomics studiesidentified several key proteins leading to a newer discipline called �metabolomics.�

28.2.2Metabolomics

Metabolomics includes a large number of metabolomic reactions that represents thedynamic changes from one condition to the other. Balmer et al. [14] analyzed theamyloplast proteome fromdevelopingwheat endosperm, and identified 289 proteinsinvolved in carbohydrate metabolism, plastid division, energetics, nitrogen andsulphur metabolism, nucleic acid-related reactions, synthesis of amino acids, iso-prenoids, fatty acids, transport, signaling, stress, and related processes. This studythus presents a broad metabolic capability of amyloplasts. On the basis of thisproteomic data, Dupont [15] organized the putative amyloplast proteins into pro-posed metabolic and biosynthetic pathways. An �amyloplast metabolic map� wasthus created emphasizing the role played by amyloplasts in endospermmetabolism.

28.2.3RNA Interference-Based Gene Silencing

RNAi silencing has emerged as a potential tool for functional genomics for hexaploidwheat. It is particularly advantageous for polyploid species such as wheat since itallows the silencing of all homologous gene copies [16]. Yan et al. [17] developed thefirst stable wheat transgenics using RNAi transformation. They reported the reduc-tion of VRN2 RNA by RNA interference resulting in reduced flowering time. Inanother study by Travella et al. [18], RNAi constructs expressing phytoene desaturase(PDS) and ethylene insensitive 2 (EIN2) were introduced by particle bombardment-mediated transformation in wheat and the endogenous target mRNA levels of threehomologous genes were seen to decrease due to RNAi silencing. This study also

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demonstrated that homozygous transgenic plants have a stronger reduction in targetmRNA and thus have severe phenotypic changes compared to heterozygous plants,thus also emphasizing a dosage effect of RNAi in hexaploid wheat. Uauy et al. [19]cloned a wild wheat allele encoding an NAC transcription factor (NAM-B1) andshowed that reduction in RNA levels of the TaNAM genes by RNA interferencedelayed senescence and decreased protein content, zinc, and iron in wheat grains.Yue et al. [20] report the silencing of highmolecular weight (HMW) glutenin subunitIDX5 that caused reduction in gluten content and dough quality. Thus, RNAi hasbeen identified as an effective tool to manipulate gene expression for studyingfunctional genomics in polyploids such as wheat.

28.2.4TILLING

Targeting-induced local lesions in genomes (TILLING) has been reported as anefficient tool for functional genomics in plants [21]. Slade et al. [22] demonstrated theuse of TILLING, a reverse genetic, nontransgenic approach for the identificationof 246 alleles of waxy locus encoding granule-bound starch synthase I (GBSSI), inboth hexaploid and tetraploid wheat. They also demonstrated that the triple homo-zygous mutant contains mutations in two waxy loci created through TILLING, and apreexisting deletion of the third homologue displayed a near-waxy phenotype. Sladeand Knauf [23] reviewed the advantages of TILLING over RNAi transformation,especially for identification of genetic variations in complex genomes required forfunctional genomic studies. However, till date LI-COR gene analyzers, which usefluorescently labeled primers, were used for TILLING techniques, which is anexpensive setup. But recently, a nondenaturing polyacryamide gel setup that usesethidiumbromidewas used byUauy et al. [24]. They developedmutant populations ofpasta and common wheat and each was characterized by TILLING multiple genesrevealing high mutation density in both populations. Thus, TILLING approachprovides evidence of the presence of many novel alleles for functional genomicstudies.

28.2.5Transcriptomics

For wheat functional genomics, any alterations in the transcriptome during devel-opmental processes or abiotic stresses would be very useful in assessing genefunctions. Wilson et al. [25] randomly picked EST clones from 35 cDNA librariesof different stages of wheat grains and plant development and functionally annotatedthem. High-density microarrays based on these libraries were also produced. In2005 [26], they presented the use of 9155 wheat unigene cDNA microarrays andreported changes in the wheat embryo transcriptome during maturation andgermination. As expected, accumulation of many mRNAs encoding proteinsinvolved in amino acid biosynthesis and metabolism, cell division and cell devel-opment, signal transduction, lipidmetabolism, energy production, protein turnover,

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respiration, initiation of transcription and translation, and ribosomal compositionwere observed. Also described were changes in abscisic acid signaling and mRNAsencoding viviparous1 (VP1), suggesting a detailed analysis of transcriptomics toenable manipulation and development of new wheat varieties with superior traits.

Lopato [27] enabled the importance and role of proteins involved in posttranscrip-tional processes such as splicing in the developing wheat grain. The wheat homo-logue ofAtRSZ33 splicing factor, designated TaRSZ38, was shown to be expressed inthe embryo and inmitotically active tissues of the endosperm by in situ hybridizationand immunodetection. By employing TaRSZ38 as the bait in a two-hybrid screen,they identified additional proteins that showed high homology to known splicingfactors, thus annotating TaRSZ38 protein to be involved in spliceosome formation.Laudencia-Chingcuanco et al. [28] studied global gene expression patterns by using8K wheat cDNA microarrays in developing caryopses and functionally annotatedgenes with respect to their expression in the respective developmental stages.Similarly, Wan et al. [29] investigated the transcriptome of wheat developingcaryopses by Affymetrix GeneChip� wheat oligonucleotide arrays, which has probesfor 55 052 transcripts. Gene expression of 14 550 transcripts was found to bedifferentially regulated at different development stages.

SAGE (serial analysis of gene expression) for the transcriptome analysis ofdeveloping caryopsis in wheat was employed by McIntosh et al. [30]. Pooleet al. [31] generated 71 930 long SAGE tags from 6 libraries of 2 wheat genotypesgrown under hot and dry conditions. By using single-copy puroindoline a and bgenes, this SAGE analysis revealed the presence of antisense transcripts that mayhave a role in gene regulation. The regulation by antisense transcripts was furtherconfirmed by Coram et al. [32] by developing a novel protocol to assay sense andantisense-strand transcription on the 55K Affymetrix GeneChip wheat genomearray, which is a 30 in vitro transcription (30IVT) expression array. Of the 110 sense–antisense transcript pairs, most were annotated as genes involved in energy pro-duction, suggesting that photosynthesis is likely to be regulated by antisensetranscripts. Schreiber et al. [33] compared gene expression profiles in barley andwheat by Affymetrix gene chips and reported their transcriptomes to be highlycorrelated and of use in analyzing functional genomics of these cereals.

28.2.6Transgenics

There has been an unprecedent increase in our knowledge of functional genomics inwheat by utilizing various newer approaches already discussed. However, thetraditional transgenic approach allows the introduction of gene of interest withselective modifications. Enhancing nutritional quality, resisting biotic and abioticstresses, and herbicide and pesticide resistance would enable to predict the functionof introduced genes [34]. There are various reports utilizing wheat as a system forraising transgenics for the characterization of a desired gene. Generating geneknockouts by the introduction of T-DNAs or transposons into wheat has been foundto be very useful for analyzing the gene functions [35].

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28.3Wheat Genomic Resources

28.3.1ESTs

Functional genomics exploits various genomic tools, one of thembeing ESTsequencedatabases, which has accelerated the pace of gene discovery. There are several publiclyavailable ESTdatabases for wheat where one can download all the available ESTdatafrom hexaploid wheat and other Triticeae species. One such site for downloadingwheat EST data is http://wheat.pw.usda.gov/NSF/curator/wheat_est.html, whereprocessed 50 EST data, 50 EST raw data, quality scores, 30 EST data, and trace filesare available for download. ESTcontig assembly is performed using a program Phrap(www.phrap.org/). It is necessary to assess the level of library redundancy. Sequencingof individual cDNA libraries was then carried out. The results provide unique ESTs toa specific library. According to the GenBank dbEST database (http://www.ncbi.nih.gov/dbEST/dbEST_summary.html), there are now over 1 067 304 Triticum aestivumESTs available to the public by the ITEC (International Triticeae EST Cooperative)effort and other sequencing projects (dbESTrelease 012910). Several studies availablemade use of these EST databases for comparative mapping revealing structural andfunctional relatedness [35–37]. Several groups worked on construction of cDNAlibraries generating ESTs and their utilization for selection of distinct sequencemotifunigenes, mapping with wheat aneuploid and deletion stocks, required for wheatgenomics and functional genomics studies [38, 39]. Several studies in2006 focused onthe digital expression analysis of these high-quality ESTs obtained fromvarious stagesof wheat growth and development and biotic and abiotic stress-related issues such astemperature, drought, photoperiod, moisture, and ABA [40–42]. A large number ofstress-responsive genes were identified and their putative functions were analyzedaccording to gene ontology. Comparison between different wheat genomes (B andD)was also reported on the basis of unique ESTs associated with various abiotic stressessuch as heat, cold, drought, salinity, and aluminum by Ramalingam et al. [43].

28.3.2Full-Length cDNA Resource

Construction of full-length cDNAs provides the putative annotation based onhomology search against several protein databases. One database that providesfull-length coding sequences for wheat along with their annotation is TriFLDB:Triticeae full-length CDS Data Base (http://trifldb.psc.riken.jp/index.pI). The cur-rent version provides 8530 putative full-length wheat cDNA sequence and theirannotations. Similarly, anotherwheat genetic resource database (KOMUGI) providesseveral tissue-specific, biotic and abiotic stress-related cDNA sequences (www.shigen.nig.ac.jp/wheat/komugi/ests/tissue). cDNA resource/library has also beenconstructed from young spikelets of hexaploid wheat and 24056 ESTs were obtainedfrom both ends of cDNA clones [44]. Till date, it has been found useful in mainly

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biotic stresses. There are studies describing the construction of full-length cDNAlibrary fromwheat stripe rust fungus [45]. Kawaura et al. [46] generated a high-quality,full-length cDNA resource for common wheat and 6162 clones were sequenced.About 10% of the clones were uniquely present in wheat while rest showed highhomology to those of rice. They analyzed their expression patterns in 28 differenttissues and abiotic stress treatments, the differential expression suggests thatmolecular selection occurred during the diversification of wheat and rice and isconsidered a valuable asset for functional genomics in wheat.

28.3.3Wheat Mutants

As a wheat genetics resource, reverse genetics approach includes the creation ofmutants by targeting specific gene to assess its function. A collection of severalknockout mutants in wheat has been generated to assess the function of genesinvolved in biotic stress. Several mutants were isolated in wheat that showedincreased resistance toward fungal causal agent of yellow rust, brown rust, blotchfungus such asMycosphaerella graminicola andPyrenophora tritici-repentis [47–51]. Forunderstanding fungal gene expression related to pathogenicity, Goswami et al. [52]constructed 3 cDNA libraries and the probable functions of 49 genes inferred. Onemutant showed reduced sporulation and delayed spread of Fusarium on wheat.Several studies also focused on the identification and molecular characterization ofwaxy mutants in wheat [53–55]. Three WAXY proteins (granule-bound starchsynthase I) have been characterized for the development of new waxy wheat lines.

Many vernalization-related mutants such as VRN1 and VRN2, the mvp mutants(maintained vegetative phase) of einkorn wheat (T. monococcum) that has null allelesof VRN1, have also been very useful in analyzing genes that control photoperiod andflowering-time genes [56–59]. The analysis indicated a genetic network of flowering-time genes in wheat leaves and the need for a more detailed molecular character-ization of themvpmutants.However, attention is now focused on creation ofmutantswith respect to abiotic stress. Thermotolerantmutants [60] and salt stressmutants [61]have already been characterized to study the genetic basis of stress tolerance inwheat.

28.3.4Introgression Lines

Introgression lines derived frommultiple inbred strains serve as a powerful resourcefor studying multiple quantitative trait loci by introducing specific traits. For thegeneration of biotic/abiotic stress-resistant wheat, development of alien addition/substitution lines, and thus introgression lines, become essential. However, thepairing homologous 1 (Ph1) locus restricts chromosome pairing and recombinationduring meiosis. Thus, to uncover the mechanism of the Ph1 locus, various Ph1 andPh2 mutants and introgression lines have been developed [62–64]. Several youngisogenic lines and their genetic analyses have also contributed to the detailedcharacterization of regulatory regions of VRN1 vernalization genes [65, 66]. For

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understanding themechanism of resistance for biotic stress tolerance, near-isogeniclines for wheat rust resistance gene Lr34/Yr18 were examined [67]. For abiotic stress,several RIL (recombinant inbred line) and NIL (near-isogenic line) populations wereanalyzed for the effect of heat stress onCMS (cellularmembrane thermostability) andGluD-1 [68, 69]. To understand the role of each chromosome during cold tolerance,20 isogenic lines of wheat were analyzed [70]. However, further studies are essentialfor understanding themechanismof resistance for biotic and abiotic stress tolerance.

28.4Wheat Functional Genomics for Various Stresses

28.4.1Drought Stress

Among several abiotic stresses, water stress is themost widespread in wheat. Severalstudies therefore focus on the accumulation of LEA (late embryogenesis abundant)proteins, osmolytes (proline, glycine, etc.), as adaptive mechanisms for protectionagainst drought stress. Pellegrineschi et al. [71] expressed the AtDREB gene (dehy-dration responsive element binding) under the control of a stress-induced promoterrd29 in wheat. The transgenics raised showed some tolerance to drought stress by a10 day delay in wilting. Selote et al. [72] demonstrated limited ROS (reactive oxygenspecies) accumulation in the leaves and roots of drought-acclimated wheat (T.aestivum cv.306) that caused minimal membrane damage. In another related study,Xu et al. [73] examined the transcript accumulation of wheat PP2Ac-1 (catalyticsubunit of protein phosphatase 2A) during water stress. Transgenic tobacco plantsoverexpressing TaPP2Ac-1 gene were found to be resistant to water deficit. A�feedback mechanism� was proposed to be operating under drought stress condi-tions inmitochondria involvingROSproduction by Pastore et al. [74]. The function ofwheat calreticulin (TaCRT) toward drought stress was demonstrated by TaCRT-overexressing tobacco (Nicotiana benthamiana) plants that showed enhanced droughtresistance by higherWUE (water use efficiency),WRA (water retention ability), RWC(relative water content), and lower MDR (membrane damaging ratio) under waterdeficit conditions [75]. Transcriptome profile of wheat �opata� roots under droughtstress revealed 394 transcripts differentially regulatedwith a fold change of at least 1.5between stressed and control roots [76]. The genes of importance were putativeglucanases and class III peroxidases. Kam et al. [77] identified 47 Q-type zinc fingerprotein genes from T. aestivum and analyzed their expression profile in differentorgans during leaf development and aging, drought stress, and ABA and sucrosetreatment. They concluded that 30 genes were predominantly expressed in roots and37 TaZFP genes responded to drought stress. Recently, Ergen et al. [78] comparedglobal expression profiles of drought-tolerant and -sensitive wild emmer wheatgenotypes at two different time points in two different tissues (root and leaf) usingthe Affymetrix GeneChip wheat genome array. The data revealed several uniquegenes and signal related pathways such as IP3, ethylene, ABA-dependent signaling,

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and a faster induction of ABA-dependent transcription factors in the tolerantgenotype, suggesting some unique transcriptome pathways in wild emmer wheatthat correlated with their ability to withstand drought conditions.

28.4.2Salinity Stress

Wheat is exposed to salt stress as it is mainly grown under irrigated and rain-fedconditions [79]. Naþ exclusion, Naþ compartmentalization, and Kþ uptake are someof the adaptive mechanisms reported in wheat. Colmer et al. [80] discussed differentsources of Naþ exclusion among various genomes that make up tetraploid wheat (T.durum),hexaploidbreadwheat (T.aestivum), andwild relatives suchasAegilopsspp.andThinopyrum spp. In the same year, Kawaura et al. [81] constructed a 22K wheat oligo-DNA microarray with 148 676 ESTs of common wheat. They analyzed 1811 geneswhose expression changed more than twofold in response to salt stress. Such globalgene profiling studies help to understand the mechanism of salt tolerance in wheat.

Using a comparative genomics approach, Huang et al. [82] utilized rice genomesequence and wheat ESTdata to identify and characterize a candidate gene forNax1responsible for low Naþ concentration in leaf blades. They reported two putativesodium transporter genes (TmHKT7A1–2) related to OsHKT7, out of whichTmHKT7A2 was expressed in roots and leaf sheaths of salt-tolerant durum wheatline 149, which correlated well with the physiological role of Nax1 in reducing Naþ

concentration.Another adaptive measure other than Naþ exclusion studied in wheat is its ability

to retain Kþ . Cuin et al. [83] used the noninvasive ion flux measuring technique tomeasure Kþ

flux from roots in two bread and two durum wheat genotypes,contrasting in their salt tolerance. Kawaura et al. [84] designed oligo-DNA micro-arrays from approximately 32 000 unique wheat genes. They observed 5996 genesdifferentially expressed when treated with salt for different intervals by more thantwofold. These genes were assigned functions using gene ontology (GO) andcategorized as transcription factors, transcriptional regulators, DNA binding func-tions, and some as early-responsive genes and late-responsive genes as transferaseand transporters. For creating novel germplasm for improving salt tolerance in breadwheat,Mullan et al. [85] chose Lophopyrum elongatum (tall wheat grass). They inducedrecombination of chromosome 3E from tall wheat grass with chromosome 3A and3D from bread wheat and using molecular marker analysis and genomic in situhybridizations provided a novel germplasm that could be deployed to enhance Naþ

exclusion in bread wheat, thus providing novel functional genes that also give aninsight into the mechanism of salt tolerance in wheat.

28.4.3Low-Temperature Stress

Low-temperature stress/cold stress to wheat plants produces many morphological,biochemical, and physiological changes. Thus, expression profile of differentially

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expressed genes under cold stress is desirable and advantageous. For the samepurpose, Gulick et al. [86] performed microarray analysis with cDNA inserts from1184 wheat ESTs that represented 947 genes. Transcriptome comparison of winterand spring wheat revealedmore than 300 genes expressed under cold stress of which65 were differentially regulated between the cultivars. To study the function of wheatalternative oxidase (AOX) genes under low temperature, Sugie et al. [87] producedtransgenic plants by introducing wheat aox1a under the control of CaMV 35 Spromoter in Arabidopsis thaliana. The results revealed that levels of reactive oxygenspecies decreased in transgenic plants under low-temperature stress and recovery oftotal respiration activity under low temperature occurred rapidly in the transgenicplants, suggesting thatAOX alleviates oxidative stress when the cytochrome pathwayof respiration is inhibited under abiotic conditions.

Monroy et al. [88] compared the gene expression inwinter wheat cultivar CDCClairandspringwheatcultivarQuantum,undercoldstressbya5740featurecDNAampliconmicroarray thatwas enrichedfor signaling and regulatorygenes.About450geneswerefound to be regulated by cold and among them about 130 were for signaling orregulatory gene candidates that includedvarious transcription factors, protein kinases,ubiquitin ligases, and GTP, RNA, and calcium binding proteins. Kobayashi et al. [89]isolatedWlip19; wheat lip19 homologue that encoded a bZIP-type transcription factor,which expressed under low temperature in seedlings andwas found inhigher amountin the freezing-tolerant cultivar. Transgenic tobacco plants expressingWlip19 showedenhanced tolerance to freezing and other abiotic stress as well. Under the control ofpromoter sequences of four wheat Cor/Lea genes, Wdhn13, Wrab17, Wrab18, andWrab19, expression of a GUS reporter gene was enhanced by Wlip19 expression inwheat callus and tobacco plants. Thus, it was concluded that WLIP19 acts as atranscription regulator of Cor/Lea genes under abiotic stress tolerance.

28.4.4High-Temperature Stress

The effect of global warming on various crop plants can be estimated by the yield loss,which is maximum in the case of wheat [90]. However, not many studies have beenreported detailing the expression analysis or assessing the function of genes that cancontribute to functional genomics in case of wheat under heat stress. Nonetheless,Gallie et al. [91] analyzed the translation elongation factors from wheat during heatshock treatment. They examined the protein levels and isoelectric state of elongationfactor (EF) 1a and 2 in the regulation of translation. EF2 expression level fromwheatseeds decreased slightly under heat stress; however, no changes were observed in thenumber or levels of isoforms observed. Campbell et al. [92] cloned two heat stress-inducible members of HSP101/ClpB family (TaHSP101 B and TaHSP101C) thatwere found to be inducible by heat stress treatments, dehydration, and ABA, thussuggesting role ofHSP101 in osmotic stress responses. Gulli et al. [93] isolated andcharacterized four cDNAs encoding HSP101 in durum wheat. The expressionanalysis revealed their differential regulation under heat stress. Thus, this datawould be useful in analyzing the functions of HSP101 family members.

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Genome-wide gene expression profiling using GeneChip wheat genome array oftwowheat genotypes, Chinese Spring (CS), susceptible to heat stress, and �TAM107,�tolerant to heat stress, was undertaken by Qin et al. [94] and found 6560 probe setsresponsive to heat treatment. These genes belong to heat shock proteins (HSPs),transcription factors, phytohormone biosynthesis/signaling, calcium and sugarsignal pathways, RNA metabolism, ribosomal proteins, primary and secondarymetabolism, and biotic and abiotic stresses. Ristic et al. [95] observed rubisco activase(RCA) expression in four genotypes of wheat and a positive correlation was foundamong the wheat 45–46 kDa RCA of different genotypes under heat stress, suggest-ing an important role played by endogenous levels of RCA as well.

Detailed transcriptome analysis has been carried out through suppression sub-tractive hybridization of heat-stressed and nonstressed tissues of wheat at threedifferent development stages, that is, young seedlings, prepollinated flowers, anddeveloping grains [96]. In all, 5500 ESTs were generated, out of which 3516 high-quality ESTs were submitted to GenBank. Their differential expression was con-firmed by cDNA macroarray and by Northern/RT-PCR analysis. Some of thetranscripts that showed high induction by high-temperature stress are stress-relatedproteins. Real-time PCR of selected genes gave further insight into their putativeroles that, however, needs functional validation through transgenic technology.

28.4.5Signaling Network

Plants respond to various abiotic stresses through their interconnection, whichcontributes to protection of the plant against unfavorable environmental conditions.There are various mechanisms by which plants respond to abiotic stresses such asdrought, salt, and cold stress.However, Ca2þ signaling is a universal early-responsivemechanism that involves calcium sensors, such as calmodulin, calmodulin-likeproteins, calcineurin B-like proteins, and calcium-dependent protein kinases [97].Wang et al. [98] functionally analyzed a novel Ca2þ -permeable channel gene TaTPC1from wheat. This putative membrane protein showed enhanced expression underhigh salinity, PEG, low temperature, and ABA. TaTPC1-overexpressing transgenicplants exhibitmore stomatal closing underCa2þ than the control plants, suggesting arole for the calcium channels in response to various abiotic stresses in wheat.Charron et al. [99] identified and characterized plant lipocalins and lipocalin-likeproteins. They analyzed the expression of wheat lipocalins under various abioticstress responses such as PEG-induced dehydration, salt, high-temperature, andfreezing stress that correlated with the capacity of the plant to develop freezingtolerance. Data mining also revealed that lipocalins are present in desiccation-tolerant red algaePorphyra yezoensis and the cryotolerant yeastDebaryomyces hansenii,suggesting the putative function as protection of the photosynthetic system againsttemperature stress.

Egawa et al. [100] isolated a DREB2 homologue Wdreb2, a candidate gene for atranscription factor of theCor/Lea genes; its detailed expression analysis indicated itsactivation in drought, cold, salt, and exogenous ABA treatment. They also showed

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three transcript forms ofWdreb2 (a, b, and c) through alternative splicing and theirdifferential expression. Faik et al. [101] reported the characterization of 34 wheatfasciclin-like arabinogalactan-proteins (FLAs) by expression and bioinformaticsanalysis. The wheat EST databases and RNA gel blots indicated that most of theTaFLA genes are expressed in reproductive organs and roots and two of them areupregulated by cold treatment in roots. This study laid the foundation for analysis ofthe function of each TaFLA protein in plant development during abiotic stressresponses. Stephenson et al. [102] identified 37 NY-F genes in wheat in the globalDNA databases. NF-Y is a trimeric complex that binds to the CCAAT-box, highlyconserved promoter element in eukaryotes. Quantitative RT-PCR revealed that someof them were predominantly expressed in the endosperm and three were activatedunder drought conditions, indicating a plant-specific biological role for this tran-scription factor family.

Xu et al. [103], for the first time, isolated a TaERF1 (T. aestivum ethylene responsivefactor 1) with a conserved DNA binding domain, a conserved N-terminal motif(MCGGAIL), and a putative phosphorylation site (TPDITS) in the C-terminal region,substrate for TaMAPK1 protein kinase. Transactivation assays of TaERF1 in tobaccoleaves revealed the activation ofGUS reporter gene drivenbyGCC-box indicating thatTaERF1 binds to the GCC-box and transactivates reporter gene expression. Expres-sion analysis of TaERF1 revealed its induction in drought, salinity, low-temperaturestress, and exogenous ABA, ethylene, and salicylic acid indicating its involvement inmultiple stress and signal transduction pathways. Expectedly, overexpression of theTaERF1 gene improved tolerance to abiotic stresses in transgenic plants. A study byBrini et al. [104] has shown that transgenic A. thaliana plants overexpressing one ofthe two wheat vacuolar Naþ /Hþ antiporter TNHX1 or Hþ pyrophosphatase TVP1showed resistance to high salt concentration and water deficit conditions. There isincreased osmotic adjustment in transgenic plants due to accumulation ofmoreNaþ

and Kþ in their leaf tissue than the control plants. Similarly, Kobayashi et al. [105]generated tobacco transgenic plants expressing Wdreb2 (wheat DREB2 homologue)with clearly improved freezing and osmotic stress tolerance in tobacco plants. TheGUS expression was enhanced by cold, drought, and ABA treatment under thecontrol of Cor/Lea promoter sequences in transgenic plants, indicating WDREB2could be a transcription factor that positively regulates wheat Cor/Lea genes undervarious abiotic stresses. Jung et al. [106] examined four wheatO-methyl transferases(OMT) genes, involved in primary and secondary metabolism. Their tissue-specificexpression and differential regulation in response to various abiotic stresses andhormones such as PEG, Cold, NaCl, UV-B, wounding, and methyl jasmonate,salicylic acid, ethylene, and ABA was studied that signifies functional diversity ofwheat OMTs in response to differential expression.

Three homologues of the DBF (dehydration-resposive element binding factors)gene family in wheat were isolated by Xu et al. [107] and designated as TaAIDFs a,b,c(T. aestivum abiotic stress-inducedDBFs).TaAIDFa transcript was upregulated underdrought, salinity, cold stress, and exogenous ABA. Also, overexpression of TaAIDFain transgenic Arabidopsis showed enhanced tolerance toward drought and osmoticstresses. Shaw et al. [108] identified 31 Dof (DNA binding with one finger) genes in

650j 28 Wheat: Functional Genomics of Abiotic Stress Tolerance

bread wheat and studied their expression analysis. While most of the TaDof genesexpressed in vegetative organs, they were also downregulated by drought andactivated by light and dark cycle. The data indicate the number of genes involvedin photosynthetic process or sucrose transport, suggesting its potential role in thephotosynthetic process. Kovacs et al. [109] compared the effects of cold, osmoticstress, and ABA on polyamine accumulation in wheat variety CS and in two-derivedchromosome 5A substitution lines (T. spelta 5A) and CS (Cheyenne 5A) with lowerand higher levels of freezing tolerance, respectively. The differential regulationprovides an insight into the involvement of polyamines in abiotic stress adaptationto plants and the possible regulatory role of chromosome 5A. Mao et al. [110]generated transgenic Arabidopsis plants overexpressing wheat TaSnRK2.4 (sucrosenonfermenting 1-related protein kinase 2) under the control of CaMV35S promoterresulting in enhanced tolerance to drought, salt, and freezing stresses.Morphologicaland physiological assays revealed decreased rate of water loss, enhanced higherrelative water content, strengthened cell membrane stability, improved photosyn-thesis and increased osmotic potential, indicating TaSnRK2.4 acts as a multifunc-tional regulatory factor in Arabidopsis.

A comprehensive study of CDPK was provided by Li et al. [111]. They isolated 14full-length cDNA sequences of CDPKs and analyzed their expression profile undervarious biotic and abiotic stresses such as cold, H2O2, salt, drought, powderymildew,abscisic acid, gibberellic acid, suggesting their role in multiple signal transductionpathways. Using knowledge of this crosstalk between biotic and abiotic stresssignaling pathways involving CDPKs, Li et al. [112] developed a model depictingpossible roles of wheat CDPK genes under various biotic and abiotic stress condi-tions. Thus, utilizing this knowledge of CDPK genes and other Ca2þ sensorsprovides a strong foundation for further functional characterization of genesinvolved in Ca2þ signaling-mediated stress tolerance in wheat plants.

28.5Wheat Functional Genomics for Plant Growth and Development

Berna et al. [113] studied the expression of germin-like proteins, which constitute aubiquitous family of plant proteins. They observed the expression of gf-2.8 gene inabiotic stresses such as heavy metal ions Cd2þ , Cu2þ , and Co2þ , polyamines, andbiotic stress such as wounding and TMV infection, thus suggesting its role in severalaspects of plant growth and development. Yao et al. [114] identified 18 expansin genesfromwheat thatwere expressed in leaf, root, and the developing seed. Fourb-expansingenes were expressed in the internode tissue in F1 hybrids, suggesting importantroles of expansin gene family in growth and development. Kulshreshtha et al. [115]isolated and functionally characterized aPHYgene (TaPHYC) fromwheat that sharedstructural similaritywith ricePHYCcontaining four exons and three introns. Reversetranscriptase-PCR (RT-PCR) analysis showed it to be a constitutively expressed genein all organs under light/dark conditions, but showed maximum upregulation in 3day-old dark-grown seedlings.

28.5 Wheat Functional Genomics for Plant Growth and Development j651

Zhao et al. [116] isolated 42 putative wheat MADS-box genes and their expressionanalysis revealed differential expression patterns in various organs and developmentstages such as primary root tips, whole spikelets (lodicules, palea, stamens, andpistil), leaf, and stem indicating their universal role in wheat growth and develop-ment. Paolacci et al. [117] functionally characterized 45MIKC-typeMADS-box genesby RT-PCR and northern hybridization revealing the putative functions of the genesby comparing expression patterns with functionally characterized ArabidopsisMADS-box genes. Recently, Kovalchuk et al. [118] isolated and characterized TaPR60gene from bread wheat, which encodes a small cysteine-rich protein with a hydro-phobic signal peptide that might direct the TaPR60 protein to a secretory pathway. Tounderstand the function of this gene, yeast two-hybrid screen of a cDNA libraryprepared from developing wheat grain was performed, where TaPR60 was used as abait and the interacting proteins were found to be involved in the proteolyticprocessing and secretion of TaPR60.

28.6Comparative Genomics

Global comparative sequence analysis at the level of both DNA and protein sequenceis performed with the aim of deriving structural, functional, and evolutionaryrelationships across several species [119]. Arabidopsis and rice are considered twomodel species suitable for comparative genomics. Arabidopsis is used as a model forall flowering plants, while rice is used as a model for genomes of cereals like wheat.Through comparative analysis, Mullan et al. [120] studied genes that control Naþ

accumulation, such as HKT1 and SOS1 in Arabidopsis; wheat orthologues wereidentified, characterized, and confirmed through similar intron–exon structure inArabidopsis and rice. On the basis of additional exons identified in the predictedNHX1 and SOS1 genes of rice and wheat compared to Arabidopsis, they suggestedevolutionary relationships among them. Boutrot et al. [121] performed comparativeanalysis of rice nonspecific lipid transfer protein (nsLTPs) and wheat ESTs and thusidentified 156 putative wheat LTPs, where the majority (91) were from �ChineseSpring� cultivar. Thus, plant nsLTPs were categorized on the basis of sequencesimilarity and/or phylogenetic clustering.

Recently, Brachypodium distachyon (L.) is emerging as a model system for cerealsbecause of its small genome, short life cycle, self-fertility, diploid accessions, andsimple growth requirements [122]. It is phylogenetically very similar to wheat andbarley, and thus various genomic resource studies involving the construction ofcDNA libraries, BAC libraries, EST sequences, linkage map, and the completegenome sequence are under development [123]. This group has also developed atransformation method with efficiency as high as 41%, which will play an importantrole in comparative genomics. Microcolinearity was found to be more conserved forthe Q gene region between wheat and rice than between wheat and Brachypodium,but phylogenetic analysis indicates Brachypodium is more closely related to wheatthan rice [124]. Its syntenic relationship with rice and wheat is being analyzed by

652j 28 Wheat: Functional Genomics of Abiotic Stress Tolerance

structural characterization of its genome and construction of a BAC-based physicalmap [125, 126].

28.7Conclusions

With utilization of wheat genomic resources along with functional genomicsapproaches, though slow yet a steady progress in wheat functional genomics isevident. Since complete wheat genome sequencing is not possible in near future,Brachypodium genome would serve as a platform for identification and functionalanalysis of genes of importance inwheat. In themeantime, availability ofmore ESTs/full-length cDNAs approachwould beused for allelemining andmolecular breeding.With the availability of cereal genomes such as rice, maize, sorghum, and so on, thefield of comparative genomics appears promising and complex genomes such aswheat may also benefit with the progress in the area of plant genomics. It is expectedthat the coming century will witness landmark discoveries and pathfinding leadsin our understanding of plant biology in general and help plant improvement inunprecedented ways.

Acknowledgment

This workwas supported by theDepartment of Biotechnology (DBT), Government ofIndia, New Delhi, India, and the Indo-Swiss Collaboration in Biotechnology (ISCB).

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