(Soybean, Chickpea, and Peanut): Omics Approaches to Enhance Abiotic Stress Tolerance

40 Pages·2011·0.72 MB·English

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39
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9 Grain Legumes (Soybean, Chickpea, and Peanut): Omics
10 Approaches to Enhance Abiotic Stress Tolerance
11
12 Q1 D.SrinivasReddy,PoojaBhatnagar-Mathur,VincentVadez,andKiranK.Sharma
13
14
Legumes rank third in world crop production, and abiotic stress is the major
15
constrainttocropproductivity.Biotechnologicalapplicationsincludingall“omics”
16
havebeenthedirectandpotentialapproachesforimprovingabioticstresstolerance
17
ingrainlegumesandrequiresknowledgeofstressresponseatmolecularlevel,which
18
includes gene expression to protein or metabolite and its phenotypic effects.
19 Genome-wideexpressionprofilingstudieshavebeencarriedoutinthelegumesto
20
identifythecandidategenesandregulatorynetworksamongabioticstressresponses.
21
Among the grain legumes, although soybean has been more intensively studied,
22
more recently, sensitive and tolerant varieties of chickpea and peanut have been
23
characterized under abiotic stress conditions. Nevertheless, proteomic studies in
24
response to abiotic stress in legumes are still very limited with only Medicago
25
truncatula andsoybean proteinreferencemaps available. SomeofthemajorQTL
26
controllingabioticstresstoleranceinlegumeshavebeenmappedforamajorQTLfor
27
salttoleranceinsoybeananddroughttolerance-relatedtraitsinpeanut.Although,
28
Agrobacterium-mediated gene transfer has been reported in all the major legume
29
crops,sofaronlyonelegume,thatis,soybean,hasbeencommercialized.Transgenic
30
technologiesforimprovedabioticstresstoleranceinvolvingregulatorygeneshave
31 proved more efficient than using single or multiple functional genes involved in
32
stresstolerance.Hence,thecurrentadvancesin“omics”technologiesandavailability
33
of the genome sequences of model legumes and soybean offer great potential to
34
improvethestresstoleranceofthelegumecrops.Thischapterattemptstoprovidea
35
detaileddiscussionaboutthedifferent“omics”approachesandtheirapplicationsfor
36
abioticstressresearchonmajorlegumes.
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39
40
39.1
41
Introduction
42
43
Legumesrepresentthemostutilizedplantfamilywith20000speciesandareamong
44
the most important crops worldwide, having major impacts on agriculture, the
45
ImprovingCropResistancetoAbioticStress,FirstEdition.
EditedbyNarendraTuteja,SarvajeetSinghGill,AntonioF.Tiburcio,andRenuTuteja
(cid:1)2012Wiley-VCHVerlagGmbH&Co.KGaA.Published2012byWiley-VCHVerlagGmbH&Co.KGaA.
j
994 39 GrainLegumes(Soybean,Chickpea,andPeanut):OmicsApproachestoEnhanceAbioticStress
1
environment, and human/animal nutrition and health [1]. Legumes rank third
2
behind cereals and oilseeds in world production [2] that accounts for 27% of the
3
world’sprimarycropproduction[1].Grainlegumesconstituteanimportantdietary
4
constituentforhumansandanimalsandthesealonecontribute33%ofthedietary
5
protein nitrogen (N) needs of humans [3] besides being a source of income and
6
livestockfeed.Theseperfectlymatchtherequirementsofsmall-scale,low-income
7
farmersinthedevelopingcountrieswheretheyaccountedfor61.3millionhectaresin
8
2002,comparedto8.5millionhectaresindevelopedcountries[2].Inorderofrank,
9
commonbeans(Phaseolusspp.),pea(PisumsativumL.),chickpea(CicerarietinumL.),
10
broadbean(ViciafabaL.),pigeonpea(CajanuscajanL.),cowpea(Vignaunguiculata
11
L.), and lentil (Lens esculentum L.) constitute the primary dietary legumes [4].
12
Moreover, grain legumes, predominantly soybean (Glycine max L.) and peanut
13
(ArachishypogeaeL.),arealsoamajorsourceforvegetableoil,providingmorethan
14
35%oftheworld’sprocessedvegetableoil.
15
Abioticstressisthemajorconstrainttocropproductivityinthesemiaridtropics
16
(SAT)thatincludepartsof55developingcountries,populatedbyabout1.4billion
17
people,wheregrainlegumesaremainlycultivated.Abioticstress,whichincludes
18
multiplestressessuchasdrought,salinity,waterlogging,hightemperature,chilling,
19
andsoonaretheprimarycausesofcroplossesworldwide,reducingaverageyieldsfor
20
mostmajorcropplantsbyover50%[5,6].Only10%oftheglobalarablelandcanbe
21 classifiedunderthenonstresscategory,whichimpliesthatcropsgrownontheother
22
90%ofarablelandsexperienceoneormoreenvironmentalstresses[7].Furthermore,
23
crops under abiotic stress are usually more susceptible to weeds, insects, and
24
diseases,whichconsiderablyincreasethelosses[8].
25
The grain legumes constitute important food and oilseed crops of the SAT, are
26
mostly grown in low-input, rain-fed agriculture, and suffer from drought due to
27 insufficient, untimely, and erratic rainfall in these climates that becomes major
28
constraintstocropproductivity.Severaloftheabioticstressesassociatedwithlegume
29
crops also directly affect symbiotic interactions and therefore limit their growth.
30 Waterdeficitscontinuetobethemajorabioticfactorthataffectcropyieldsglobally[9]
31
andarelikelytoworsenwiththeprojectedrapidexpansionofwater-stressedareasof
32
theworldencompassing3billionpeopleby2030[10].Moreover,inlegumessuchas
33
peanut (A. hypogaea), Brazil nuts (Bertholletia excelsa), and faba bean (V. faba),
34 aflatoxin contamination is a common occurrence during preharvest drought
35
stress[11,12].Inadditiontodrought,soilsalinityisanothermajorproblemaffecting
36
the total nitrogen uptake and soil nitrogen contribution [13] resulting in reduced
37
yields. Hence, there is a crucial need to increase the abiotic stress tolerance in
38
legumes,whichisamajorchallengeincropimprovementprogramsforenhancing
39
yield stability. Although conventional plant breeding and enhanced management
40
strategieshaveaddressedseveralconstraintsthatlimitcropproductivityorquality,
41
therearesituationswheretheexistinggeneticresourceslacktherequiredtraits.Yield
42
lossesduetoconstraintslikedroughtarehighlyvariableinnaturedependingonthe
43 stress timing, intensity, and duration. Moreover, location-specific environmental
44
stressfactorssuchashighirradianceandtemperaturemakebreedingfordrought
45
j
39.2 “OMICS”inLegumesandAbioticStress 995
1 tolerancedifficultthroughconventionalapproaches.Cutting-edge,knowledge-based
2
breedingpracticescomplementedadequatelybygenomicsandgenetictransforma-
3
tiontechnologiescouldleadtosimplerandmoreeffectivegene-basedapproachfor
4
improvingabioticstresstoleranceinthegrainlegumes.Applicationofbiotechno-
5 logicalapproacheshasapotentialtocontributeefficientlytosolveorreducethese
6
problems in the grain legumes, thereby contributing to sustainable agriculture,
7
especiallyintheSAT.
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39.2
11
“OMICS”inLegumesandAbioticStress
12
13
Biotechnologicalapproachessuchastissuesculture,invitromutagenesis,marker-
14
assisted breeding, and genetic transformation can speed up and overcome major
15
bottlenecksofclassicalplantbreedingduetothelackofnaturalsourcesofresistance
16
and sexual incompatibility. However, successful application of biotechnology to
17
abioticconstraintsrequiresagoodbiologicalknowledgeofboththetargetspecies
18
and the mechanisms underlying tolerance to these stresses. Mechanisms of
19
responsestostresscanbemeasuredatmanydifferentlevelsfromthewholeplant
20 tothemolecularlevel.Thetype,length,andseverityofthestresshavemoreinfluence
21
on the plant response to stress [14]. Since responses are controlled by the plant
22
genome,recenteffortshavefocusedonthemolecularresponseoftheplanttowater
23 deficits[15].Untilafewyears,theresearchonplantstressresponseswasfocusedon
24
model plants such as Arabidopsis, and not much work was done on the legumes.
25
However,sincesubstantialsimilaritiesexistbetweenthetwocrops,theknowledgeon
26
stress responses of Arabidopsis were used as source of information for legume
27 research. Nevertheless, there are also significant fundamental differences like all
28 physiological processes that differ and must be exploited to unravel the specific
29
mechanismsinvolvedinabioticstresstoleranceinthelegumes[16].Sincethelarge
30
genomesizeandthepolyploidyofsomelegumeshavehamperedthisgoal,recent
31
progressinlegumebiologyhasbeengreatlyenhancedbythedevelopmentofmodel
32
systemstoinvestigatethegeneticsofnodulationandotherimportantprocessessuch
33
asresistanceortolerancetostresses.Thetwomodellegume plantsystems,Lotus
34
japonicusandMedicagotruncatula,duetotheirsmallanddiploidgenomes,autog-
35 amousnature,shortgenerationtimes,andprolificseedproductionweretheobvious
36
choices[17,18].Sincethen,powerfulgeneticandgenomictoolshavebeendeveloped
37
that include genome sequencing [19],isolation ofexpressed sequence tags (ESTs)
38
[20, 21], and establishment of genetic and physical maps for each model species
39
[22,23].Theincreasingwealthofgeneticandgenomicdataandthehighdegreeof
40
syntenybetweenlegumegenomes[24,25]makethesetwospeciesvaluablemodels
41
for the molecular genetic study of the biotic and abiotic constraints that hamper
42
legume crop yields. Furthermore, the soybean genome sequence and the high
43
synteny between soybean and the model legumes have a potential to facilitate
44
positionalcloningandothergeneticproceduresforthesestudies.
45
j
996 39 GrainLegumes(Soybean,Chickpea,andPeanut):OmicsApproachestoEnhanceAbioticStress
1
While sequence information is invaluable and a necessary starting point, it is
2 insufficient to answer questions concerning gene function, regulatory networks,
3
and the biochemical pathways activated in response to stresses. To address these
4
questions,morecomprehensiveapproaches,includingquantitativeandqualitative
5
analysesofgeneexpressionproductsarenecessaryatthetranscriptomic,proteomic,
6
andmetabolomiclevels.Thiscomprehensiveknowledgeaboutthegenesinvolved
7
in stress response and tolerance will further allow a more precise use of marker-
8
assistedselection(MAS)andtransgenics[7].Sincethe“omics”involvesgenomics
9 andfunctionalgenomics,geneticengineering,transcriptomeprofiling,proteomics,
10
and metabolomics describing an organism’s genome contribution to its overall
11
phenotype, the recent progress made in these areas has considerably contributed
12
tobetterunderstandingofthemolecularandgeneticbasisofstressresponsethat
13
hasbeenanimportantbottleneckformolecularandtransgenicbreeding.Sofar,a
14 significant progress has been made in research on the abiotic stress tolerance of
15
major legumes including soybean, chickpea, and peanut as discussed in the
16
followingsections.
17
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19
39.3
20
Transcript“OMICS”
21
22 A eukaryotic cell contains (cid:2)15000–30000 distinct mRNAs with a prevalence
23 ranging from one to several thousands in a total mass of (cid:2)100000 mRNAs [26].
24
About50%ofthetranscriptpopulationismadeupofarelativelysmallnumber(some
25
hundreds) of abundant transcripts representing only 1% of the different mRNA
26
species, and the other half contains the “rare” mRNAs [27]. The set of all the
27
messengerRNAs(mRNAs)inacell/tissue/organismisreferredtoasthetranscrip-
28
tomeandinvestigationofpopulationsofmRNAsisthuscalled“transcriptomics.”
29 A genome-wide expression profiling is a powerful tool for studying genes
30
involved in various biological phenomena, identifying the candidate genes, and
31
revealing the molecular crosstalk of gene regulatory networks among abiotic
32
stressresponses.
33
Plantsundergoingabioticstressesingeneralfacedehydrationatthecellularlevel
34
andhencealmost50%ofthegenesactivatedbythesestressesincludingdrought,
35 salinity,orABAtreatmentarecommon.Cellularwaterdeficitinaplantstresstriggers
36 many changes in gene expression that in turn define its response to a particular
37 environmental condition. The induced genes in response to cellular water deficit
38
stress constitute different functional categories such as metabolism, transport,
39
signaling, transcription, hydrophilic proteins, and the unknown, including the
40
repressionofgenesinvolvedinplantgrowthanddevelopment,suchasphotosyn-
41
thesis-relatedgenes.Broadly,thegenesrespondingtoabioticstresscanbecatego-
42
rizedintotwoclassesbasedontheirresponseintermsoftimescaleorbasedontheir
43
involvement in tolerance; some respond immediately within seconds or minutes,
44
while others respond later,in hours,days, oreven weeks [28]. This allows for the
45
speculation that the early responsive genes may provide initial protection and
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39.3 Transcript“OMICS” 997
1 regulate gene expression by being involved in amplification of signals and signal
2
transduction.Theseincludevariousproteinkinasesandgenesencodingtranscrip-
3
tionfactors,whereasthegenesthatrespondlatermaybeinvolvedinadaptationto
4
stressconditions,suchasheatshockproteins,LEAproteins,ROSscavengerproteins,
5
andsoon[14,28].
6
The genomic approaches allow changes induced by abiotic stresses on a global
7
scaletobeanalyzedatthelevelofthewholeorganism.Muchmoreextensivegene
8
expressionstudieshavebeenperformedinArabidopsis,andtheresultingknowledge
9
canalsobeusedinlegumesthroughcomparativegenomics.Forexample,Ishitani
10 etal.[29]selected100–200genesfromtheArabidopsisdatabaseandshowedthatat
11
least3DREB-likegenes,thoughttobekeytranscriptionalregulatorsofdroughtand/
12
orcoldtolerance,werepresentincommonbean.Similarly,inArabidopsis,analysisof
13
thetranscriptomechangesoccurringduringcold,drought,andsaltstressinasurvey
14
of7000genesshowedasharedresponseforamajorityofcoldanddroughtstress-
15
regulatedgenes,supportingthehypothesisthatacommonsetofsignaltransduction
16
pathways are triggered during different stress responses [30]. Around 11% of the
17 stress-inducible genes are potential transcription factors further confirming the
18
relevanceofgeneregulationinstressadaptation[31].
19
The Arabidopsis model is likely to be very different from legumes in terms of
20 responses to stress in relation to grain filling, nitrogen utilization, fixation, and
21
transport, root architecture, and interactions, all physiological processes that are
22
fundamentally different in legumes. Hence, the usefulness of developing a
23
legume model has become increasingly relevant in recent years. Moreover, the
24
inductionofgeneexpressionbyenvironmentalstressmustbeexploitedtounravel
25
mechanisms dealing with abiotic stress tolerance in the agriculturally important
26
grainlegumes.Inlegumes,thegeneexpressionpatternsfollowing bioticstresses
27
have been more extensively studied than those following abiotic stresses. With
28
respecttoabioticstress,geneexpressionanalyseshavebeenmainlybasedonstudies
29 withclonedgenes[32].Significantprogressisbeingmadeatthegeneticandgenomic
30
levels using the model legume M. truncatula through macro- and microarray
31
analysis,reversegenetics,genomesequencing,andotherhigh-throughputtechni-
32
ques[33,34].Theanalysisofalmost200000ESTsofM.truncatula,isolatedfrom
33
many different libraries constructed from diverse stages and treatments, was
34
facilitated by searchable databases such as MtDB2 [35] and the TIGR Gene Index
35
(http://www.tigr.org).
36
Theadventofnext-generationsequencingplatforms[36],mostrecentlythe“third
37 generation” (also called “next–next generation” or NGS) sequencing systems will
38
enable plant genome to be sequenced within hours. The NGS approaches allow
39
decipheringthecell’stranscriptsonthesequencelevel,whichwilltrulyrevolutionize
40
the research of organisms that are not now in line for genomic sequencing. This
41
approachcouldcircumventtheproblemsposedbyextremelylargegenomessuchas
42
legumes. The next-generation sequencing not only is a dramatic advance over
43 capillary-basedsequencingbutalsopresentssignificantchallengesinassemblyand
44 sequenceaccuracyduetoshortreadlengths,method-specificsequencingerrors,and
45
theabsenceofphysicalclones.However,thepromiseofmuchlowersequencingcost
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998 39 GrainLegumes(Soybean,Chickpea,andPeanut):OmicsApproachestoEnhanceAbioticStress
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withthenowprovenconceptofnext-generationexpressedsequencetagsequencing
2
will allow assessment of plant genomes at least at the functional level [37]. At
3
ICRISAT,theseNGSapproaches arebeing usedtodevelopEST-basedmarkersto
4
maptheQTLforstressresponseingrainlegumes.Recentreportshavealsoshown
5
thattranscriptomictoolsareagoodoptionforlegumebreedingtoenvironmental
6
stressesasdiscussedinthenextsections.
7
8
9 39.3.1
10 Soybean
11
12 Amongthegrainlegumes,soybeanhasbeenmoreintensivelystudiedandaccording
13 tothelegumeinformationsystemdata,over1.3millionESTsweredevelopedfrom
14 differentcDNAlibraries,whichisthelargestinnumberamongtheindividualgrain
15 legumeESTs.TheavailabilityofalargenumberofESTandBACsequencesfacilitated
16 thediscoveryofnewSNPandSSRmarkersinsoybeantowardtheconstructionof
17 high-resolutiongeneticmaps.Besides,usingamodifiedcDNA-AFLPtechniquein
18 soybean,140differentiallyexpressedcDNAfragmentswereobtainedbycomparing
19 controlandisoosmotictreatedplantswheresomeoftheresponsivegenesencoded
20 foriontransporters,transcriptionfactors(TFs),andredoxenzymes[38].
21
22
39.3.2
23
Chickpea
24
25
Chickpea is the most important food legume of semiarid tropics (SAT) and taxo-
26
nomically one of the closest crops to the model legume Medicago. Sensitive
27
and tolerant varieties of chickpea have been characterized under abiotic stress
28
conditions,althoughverylittleisknownaboutthegenesinvolvedintheseresponses.
29
However,thecharacterizationofgenesinvolvedinthedifferentialbehaviorofthese
30
cultivars may constitute a good basis to extrapolate these results to other grain
31 legumes. Five differentially expressed cDNAs were identified using differential
32
display reverse transcriptase PCR (DDRT-PCR) under drought conditions with
33
drought-tolerant cv. ICCV2 and drought-susceptible cv. ILC3279 of chickpea [39].
34
Moreover,319uniqueESTsavailablefromdifferentlibrarieshavebeenanalyzedfor
35 differencesintranscriptprofilingduringdroughtstresstreatmentintwochickpea
36
varieties having contrasting levels of drought tolerance (C. arietinum cv.
37
PUSABGD72andICCV2).TheseESTswereclusteredinfourgroupsaccordingto
38
theirexpressionpatterns[40].
39 Atranscriptionalprofilingstudyinchickpeaunderdrought,cold,andhighsalinity
40
wascarriedoutusingcDNAmicroarrayapproachtolookatthegeneexpressioninthe
41 leaf, root, and/or flower tissues in tolerant and susceptible genotypes [41]. The
42
differentiallyexpressedtranscriptsinresponsetotheparticularstresswereanalyzed
43
andatranscriptionalchangeofovertwofoldwasobservedfor109,210,and386genes
44
afterdrought,cold,andhigh-salinitytreatments,respectively.Amongthese,2,15,
45
and 30 genes were consensually differentially expressed between tolerant and
j
39.3 Transcript“OMICS” 999
1
susceptible genotypes studied for drought, cold, and high salinity, respectively.
2
The differentially expressed genes coded for various functional and regulatory
3
proteins, highlighting the multiple gene control and complexity of abiotic stress
4
responsemechanisminchickpea.
5
TwononnormalizedcDNAlibrariesfromtheseedlingleavesofadrought-tolerant
6
chickpea cultivar under PEG-treated and nontreated conditions have been con-
7 structed where 92 differentially expressed genes were identified [42]. Most of the
8
upregulatedgeneswererelatedtodroughttolerance,whilethedownregulatedgenes
9
weremainlyinvolvedinthephotosynthesis.Asetofover2800chickpeaESTshave
10
beengeneratedfromalibraryconstructedaftersubtractivesuppressivehybridization
11
(SSH)ofroottissuefromtwocloselyrelatedchickpeagenotypespossessingdifferent
12
sourcesofdroughtavoidanceandtolerance,ICC4958(tester)andAnnigeri(driver),
13
respectively [43]. A total of 106 EST-based markers were designed from 477
14
sequenceswithfunctionalannotationsthatweretestedonC.arietinum.Forty-four
15
ESTmarkerswerepolymorphicwhenscreenedacrossnineCicerspecies(including
16
thecultigen)[44].ThechickpearootESTdatabasedevelopedinthesestudiesprovide
17
researcherswithamajornewresourcefordataminingassociatedwithroottraitsand
18
drought tolerance [43]. More recently, a total of 20 162 drought- and salinity-
19
responsive ESTs were generated from 10 different root tissue cDNA libraries of
20
chickpeaand177newEST-basedSSRmarkersweredeveloped[45].
21
Besides,SuperSAGEanalysisforgeneexpressioninchickpearootsinresponseto
22
droughtwascarriedoutresultinginsequencingof80238of26bptags[46].Among
23
thesetags,7532(43%)UniTagsweremorethan2.7-folddifferentiallyexpressedand
24
880(5.0%)wereregulatedmorethan8-folduponstressresultinginunambiguous
25
annotationof22%(3858)ofthesetags.Microarrayanalysisofthese3000annotated
26 UniTags confirmed 79% of the tag-based results, whereas RT-PCR confirmed the
27 SuperSAGE data in all cases. This is the first study to prove the potential of
28
SuperSAGE technology for molecular breeding in the nonmodel crops. However,
29
lackofavailabilityofachickpeareferencegenomelimitsthevalueofSuperSAGE
30
tags,asonlyafractionofthemcouldbeannotated.
31
32
39.3.3
33
Peanut
34
35
Inpeanut,differentialDDRT-PCRhasbeenusedtoidentifydifferentiallyexpressed
36
genesinpeanutgrownunderdroughtstressversusirrigationconditionswheresome
37 drought-responsive mRNA transcripts were identified based on expression pat-
38
tern [47, 48]. Besides, DDRT-PCR studies have been carried out with transgenic
39
peanuteventsoverexpressingrd29A:DREB1Atodetectthedifferentiallyexpressed
40
transcriptsunderabioticstress[49].Here,51differentiallyexpressedtranscriptswere
41 identifiedunderstresstreatments;amongthem35transcriptswerenewlyexpressed,
42
11wereupregulated,and5weredownregulated.IntheBLASTsearchofdifferentially
43 expressedpartialcDNAs,only17clonesshowedasignificantsimilaritytotheESTsin
44
thedatabase,indicatingthatthemajorityofthecDNAsclonedinthisstudymaybe
45
novel and needs further research to identify their role in stress response. These
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1000 39 GrainLegumes(Soybean,Chickpea,andPeanut):OmicsApproachestoEnhanceAbioticStress
1
results also suggested that the increased plant tolerance against drought stress in
2
transgenicpeanutmaynotbeattributableonlytotheexpressionofDREB1A-targeted
3 cold-responsive(COR)genesidentifiedinArabidopsis[49].
4
Inarecentstudy,sixdifferentcDNAlibrarieswereconstructedfromdeveloping
5
peanutseedsatthreereproductionstages(R5,R6,andR7)fromaresistantanda
6
susceptiblecultivatedpeanutgenotype,“Tifrunner”thatissusceptibletoAspergillus
7 infectionwithhigheraflatoxincontaminationandresistanttotomatospottedwiltvirus
8 (TSWV)and“GT-C20”thatisresistanttoAspergilluswithreducedaflatoxincontam-
9
ination and susceptible to TSWV. The developing peanut seed tissues of these
10 genotypeswerechallengedbyAspergillusparasiticusanddroughtstressinthefield
11
and 21777 high-quality EST sequences were generated from cDNA clones of 6
12
libraries [50]. Similarly, EST libraries for cultivated peanut were developed from
13
leavesofpeanutlineC34-24(resistanttoleafspotsandTSVW)andimmaturepodsof
14 peanutlineA13(toleranttodroughtstressandpreharvestaflatoxincontamination).
15 Atotalof1825ESTs,769fromtheC34-24and1056fromtheESTswereidentifiedand
16
44EST-derivedsimplesequencerepeat(SSR)markershavebeencharacterizedfor
17
cultivatedpeanut[51].Atotalof6264high-qualityESTsweregeneratedfromleaves
18
androotsofawildpeanutArachisstenosperma,and188microsatellitemarkershave
19
developedformtheseESTs[52].
20 Morerecently,nearly700geneswereidentifiedinsubtractivecDNAlibraryfrom
21
gradualprocessofdroughtstressadaptationinpeanut.Thisstudyalsoshowedthe
22
functionalimportanceofHSP70geneandkeyregulatorssuchasJumonjiindrought
23
stressresponse[53].Ahigh-densityoligonucleotidemicroarrayforpeanuthasalso
24
beendevelopedusing49205publiclyavailableESTsandtestedtheutilityofthisarray
25 forexpressionprofilinginavarietyofpeanuttissues[54].Over108putativelypod-
26 specific/abundant genes, as well as transcripts, whose expression was low or
27
undetected in pod compared to peg, leaf, stem, or root were detected. Several
28 transcripts that significantly overrepresented in the peanut pod included genes
29
responsible for seed storage proteins and desiccation (e.g., late-embryogenesis
30
abundantproteins,aquaporins,leguminB),oilproduction,andcellulardefense[54].
31
32
33
39.4
34
Prote“omics”
35
36 Since the 1990s, genomics has been the most active research field in biological
37
science generating a huge amount of information, while structural genomics has
38
emergedatthemethodologicalleveltounderstandgeneexpressionandfunction.A
39
complete knowledge of the proteins expressed by the genome of a cell, tissue, or
40 organismataspecifictimepoint(proteome)isnecessarytounderstandthebiologyof
41 acelloranorganism.Theproteomereflectstheactualstateofthecellortheorganism
42
andisanessentialbridgebetweenthetranscriptomeandthemetabolome.Proteins
43
act directly on biochemical processes, and thus must be closer to the phenotype,
44
comparedtoDNA-basedmarkers.Althoughresearchonplantresponsestostresson
45
the DNA or RNA level provided an important insight into stress tolerance, the
j
39.4 Prote“omics” 1001
1
proteomics approach is very important in evaluating stress responses since the
2
mRNAlevelsmaynotalwayscorrelatewithproteinaccumulation[55].Inaddition,
3 many proteins are modified by posttranslational modifications such as phosphor-
4 ylation, glucosylation, and ubiquitinylation, which significantly influence protein
5
functions.Proteomics,understoodasproteinbiochemistryonanunprecedentedand
6
high-throughputscale, isbecoming apromisingandactiveapproach inthispost-
7
genomic period. However, its application to plants is rather limited compared to
8
otherbiologicalsystems[56].
9
Compared to analysis of the transcriptome, analysis of the plant proteome in
10
response to abiotic and biotic stresses is still limited, although good technical
11 progress has been achieved in the separation of proteins and their identification
12
by mass spectrometry. Studies have evaluated changes in protein levels in plant
13
tissuesinresponsetostresses[57,58].However,thesestudieshavemainlyfocused
14
onnonlegumespeciessuchasArabidopsisandrice[57]andsomelegumesrecent-
15
ly [56]. As a result, only a handful of studies have been carried out in legumes,
16 althoughinthenextfewyearsthereshouldbeasignificantincreaseinthenumberof
17
legumespeciesandstressesanalyzed.Sofar,peahasbeenmoreintensivelystudied,
18
withtheanalysisofinducedproteinexpressioninrootsinresponsetosalt[59]andto
19
cadmiumstress[60].Recently,M.truncatilahasbeenthesubjectofseveralproteomic
20
studies that represent the most extensive proteomic description of M. truncatula
21
suspension cells to date and provide a reference map for future comparative
22
proteomics and functional genomics studies of biotic and abiotic stress
23
responses[61].
24
25
39.4.1
26
Soybean
27
28
Some reference maps of soybean that are available in the proteomics database
29
provide a starting point for ongoing functional genomics studies associated with
30
biotic/abioticstressinsoybean.TheSoybeanProteomeDatabaseisaimedtobeadata
31 repository for functional analyses of soybean responses to flooding injury that is
32
recognizedasamajorconstraintfortheestablishmentandproductionofthisplant.
33
Thelatestreleasecontains21referencemapsofsoybean(G.maxcv.Enrei)proteins
34
electrophoresized on two-dimensional polyacrylamide gels of which the samples
35
werecollectedfromseveralorgans,tissues,andorganelles.Thesereferencemaps
36 included7311detectedproteinsand532identifiedproteins,orproteinsforwhicha
37
sequenceorpeptide peak hasbeen determined.TheSoybean ProteomeDatabase
38
alsointegratesmultiple“omes,”wherean“omics”tablerevealsrelationshipsamong
39 106 mRNAs, 51 proteins, and 89 metabolites that vary over time under flooding
40 stress.Thetabulatedmetabolitesareanchoredtoametabolomenetwork.Aunified
41 temporal profile tag attached to the mRNAs, proteins, and metabolites facilitates
42 retrieval of the data based on the temporal expression profiles. A graphical user
43
interface based on dynamic HTML facilitates viewing of both the metabolome
44 network and the profiles of multiple “omes” in a uniform manner. The entire
45
databaseisavailableathttp://proteome.dc.affrc.go.jp/soybean/[62].
j
1002 39 GrainLegumes(Soybean,Chickpea,andPeanut):OmicsApproachestoEnhanceAbioticStress
1
39.4.2
2
Chickpea
3
4
Mostoftheearlierunderstandingofdehydration-responsivecellularadaptationin
5
chickpeahasevolvedfromtranscriptomeanalysisandthecomparativeanalysisof
6
dehydration-responsiveproteins,particularlyproteinsinthesubcellularfraction,is
7
limited.Bhushanetal.[63]haveinitiatedaproteomicsapproachtoidentifydehy-
8
dration-responsive ECM proteins in JG-62, a drought-tolerant variety of chickpea
9
wherethedehydration-responsivetemporalchangesinECMproteinsrevealed186
10 proteins with variance at a 95% significance level. The comparative proteomics
11 analysisledtotheidentificationof134differentiallyexpressedproteinsthatinclude
12 predictedandnoveldehydration-responsiveproteins.Thisstudy,forthefirsttime,
13
demonstratedthatovera100ECMproteinsarepresumablyinvolvedinavarietyof
14 cellularfunctions,namely,cellwallmodification,signaltransduction,metabolism,
15
and cell defense and rescue, and impinge on the molecular mechanism of dehy-
16
drationtoleranceinplants.Sincethenuclearproteinsconstituteahighlyorganized,
17
complex network that plays diverse roles during cellular development and other
18
physiologicalprocesses.Anotherstudyprovidedinsightsintothecomplexmetabolic
19
networkoperatinginthenucleusduringdehydrationinchickpea[64].Approximate-
20
ly, 205 protein spots were found to be differentially regulated under dehydration;
21 massspectrometryanalysisallowedtheidentificationof147differentiallyexpressed
22
proteins,presumablyinvolvedinavarietyoffunctionsincludinggenetranscription
23
and replication, molecular chaperones, cell signaling, and chromatin remodeling.
24
The dehydration-responsive nuclear proteome of chickpea revealed a coordinated
25
response,whichinvolvesboththeregulatoryandthefunctionalproteins.
26
27
39.4.3
28
Peanut
29
30
Inpeanutveryfewproteomicstudieswereconductedonstressresponse;inarecent
31
studywithselectedtolerantandsusceptiblepeanutgenotypesfromtheUSminicore
32 collectionwereanalyzedforchangesinleafproteinsunderwaterdeficitstress[65].A
33
totalof102proteinbands/spotswereanalyzedbymatrix-assistedlaserdesorption/
34 ionization–time-of-flightmassspectrometry(MALDI-TOFMS)andbyquadrupole
35 time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine
36 nonredundantproteinswereidentifiedthatimplicatedavarietyofstressresponse
37
mechanisms in peanut. It was observed that lipoxygenase and 1L-myo-inositol-1-
38
phosphatesynthase,whichaidininter-andintracellularstresssignaling,weremore
39 abundant in tolerant genotypes under water deficit stress. Here, the acetyl-CoA
40
carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance
41
along with a corresponding increase in epicuticular wax content in the tolerant
42
genotypes suggesting an additional mechanism for water conservation and stress
43
tolerance. In addition, there was a marked decrease in the abundance of several
44
photosyntheticproteinsinthetolerantgenotypealongwithaconcomitantdecrease
45 innetphotosynthesisinresponsetowaterdeficitstress.