Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity

18 Pages·2016·0.24 MB·English

by  Bronwyn J. Barkla

proteomes
Review
Identification of Abiotic Stress Protein Biomarkers by
Proteomic Screening of Crop Cultivar Diversity
BronwynJ.Barkla
SouthernCrossPlantSciences,SouthernCrossUniversity,Lismore2481,NSW,Australia;
[email protected];Tel.:[phone]
AcademicEditors:M.MargaridaOliveira,IsabelA.AbreuandJacekR.Wisniewski
Received:21June2016;Accepted:3September2016;Published:8September2016
Abstract: Modern day agriculture practice is narrowing the genetic diversity in our food supply.
This maycompromisetheabilitytoobtainhighyieldunderextremeclimacticconditions,threatening
food security for a rapidly growing world population. To identify genetic diversity, tolerance
mechanisms of cultivars, landraces and wild relatives of major crops can be identified and
ultimatelyexploitedforyieldimprovement. Quantitativeproteomicsallowsfortheidentification
ofproteinsthatmaycontributetotolerancemechanismsbydirectlycomparingproteinabundance
under stress conditions between genotypes differing in their stress responses. In this review, a
summary is provided of the data accumulated from quantitative proteomic comparisons of crop
genotypes/cultivars which present different stress tolerance responses when exposed to various
abioticstressconditions,includingdrought,salinity,high/lowtemperature,nutrientdeficiencyand
UV-Birradiation. Thisfieldofresearchaimstoidentifymolecularfeaturesthatcanbedeveloped
asbiomarkersforcropimprovement,howeverwithoutaccuratephenotyping,carefulexperimental
design, statistical robustness and appropriate biomarker validation and verification it will be
challengingtodeliverwhatispromised.
Keywords: proteomics;biomarkers;abioticstress;geneticdiversity;cropimprovement
1. Introduction
Itisestimatedthatthereare352,000documentedfloweringplantsintheworldandanywhere
from30,000to80,000ofthesecontainpartsthatareinsomeformsuitableforhumanconsumption[1].
Historically, however, only about 7000 plants have actually been used for food [2], and since the
industrialrevolution,theexploitationofplantspeciesdiversityhasprogressivelydecreased. Modern
agriculturehasbecomereliantonfewerandfewercropvarietiesforintensivefarmingpractices,with
todayonlyabout150speciesactivelycultivated[3],andlessthanadozenproviding80%ofourcaloric
intake[4]. Thispractice, tendingtowardsmonoculture, raisesseriousfoodsecurityissues. Aswe
loseordisregardspeciesdiversity,themajorcropsthatweincreasinglyrelyonbecomevulnerableto
environmentalchangesincludingbothabioticandbioticstressfactors,asthegeneticvariationforstress
toleranceisratherlimitedindomesticatedgenepoolswhichhaveaverynarrowgeneticbase[3,5].
GenealogicalanalysisofthegeneticdiversityofRussianwinterTriticumaestivumL.wheatunderlines
thisgeneticerosion,with96%ofallwinterwheatvarietiescultivatedattributedtobeingdescendants
ofonlytwocultivars,leavingithighlyvulnerable[6]. Thetopthreeglobalstaplecrops,corn,wheat
andrice,whichmakeupmorethan60%oftheworldwidefoodproduction[5],aregenerallyhighly
susceptibletostressatvariousstagesofdevelopment. Unfavourableenvironmentalconditionsresult
inunacceptabledecreasesinqualityandyield,affectingglobalannualagriculturalproduction.
Asexamples,in1982,1994and2002,unusuallyhightemperatures,combinedwithlowrainfall
acrosssouthernAustraliacausedmajorcropfailure,withlessthanhalfthenormalwheatproduction
attained[7].Morerecently,in2012,UScornproductiondropped13%ascomparedwithpreviousyears
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duetodevastatingdrought,despiteplantingthelargestacreageina75yearhistory(CropProduction
2012Summary,USDA/NASS;www.nass.usda.gov). Ingeneral,adverseenvironmentalconditions
havebeenestimatedtohaveloweredwheatandcornyieldsbyroughly6%and4%,respectively,over
aperiodof30years[8].
Thereismuchinteresttogeneticallyimprovecropplantstresstolerance. Globalclimatechange
andextremeweatherconditionshaveresultedingreatertemperaturefluctuations,anddrastically
alteredrainfallpatterns;resultingindroughtand/orfloodingwhichinturninfluencessoilsalinity.
Thecostofinorganicnitrogenispredictedtoriseduetodepletionofsuppliesandincreaseddemand,
necessitatingbreedingforbetternitrogenuseefficiencyincrops. Furthermore,asthelandavailable
foragriculturebecomeslimiting,farmersarecompelledtomakeuseofmarginal,poorqualitysoils,
whichcancontainunfavourablelevelsofnutrients[9].
Muchofourknowledgeonabioticstresstolerancehascomefromstudiesonthemodelplant,
Arabidopsisthaliana,duetotheearlysequencingofthegenomeandpolicieswhichdirectedsignificant
amounts of research funds to work on this plant [10]. It was believed that by adopting a single
model species, the plant research community would make discoveries faster and test hypotheses
morerapidly[11]. TheavailabilityofalargenumberofnaturalaccessionsofArabidopsisadaptedto
varyingenvironmentswasalsoanattractivefeatureofthismodel[12]. WhileArabidopsisresearch
hasadvancedplantfunctionalgenomicstoalargedegree,itisnottheidealmodelforallcropresearch,
asitisadicotyledonousweedwhilethethreemajorcropsareallmonocots. Moreover,itdoesnot
producefruit,andtherefore,specificissuesmaynotbeaddressedadequatelybyfocusingsolelyon
asingleplant. Recentlytherehasbeenadramaticimprovementingenomictechnologies,enabling
thesequencingofreferencegenomesforawidevarietyofplants,includingmanycrops(reviewed
in[13]),facilitatingdirectresearchontheplantofinterestandadditionallyprovidingaccesstothe
geneticdiversityavailablewithinaspecies.
Oneapproachthatisgainingwidespreadinterestinthecommunitywhichemphasizestheutility
ofthesenewlyobtainedcropgenomicresourcesistheexploitationofcultivars,landracesandwild
relativesofmajorcropswhicharenaturallymorestresstolerant. Thesecanbecomparedatthelevelof
eitherthetranscriptomeorproteometoidentifypotentialsourcesoftoleranceforvarietyimprovement.
This approach has been not only employed for the major crops but also minor, less utilized crops
havealsobeenstudiedinthismanner. Inthisparticularreview,theuseofquantitativeproteomicsto
understanddifferencesintolerancemechanismswithincropcultivarsandidentifypossibleabiotic
stressmarkersbygenotypecomparisonhavebeenaddressedandthedatahavebeensummarized
belowwithrespecttocropandstresstype. Moreover,therelevantexperimentaldesign,including
plantage,treatmenttypeandlength,tissue,extractionmethodandquantitativeapproachusedineach
caseislistedinTable1.
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Table1.Alistofpublishedquantitativeproteomicspapersinthefieldofcropplantresponsestoabioticstressfactorswhichemploydifferentgenotypesofcommon
cropcultivars.Columnsindicatethecropplant,stresscondition,genotypescompared,ageofplantatendofstresstreatment,durationofstresstreatment,tissue
studied,methodsofquantitativeproteomicsused,numberofbiologicalreplicates,typeofproteinsampling,andwhethertheproteomicresultswerefurthervalidated.
Crop Stress Genotypes PlantAge StressDuration PlantTissue Quant.Methoda #Biol.Reps.b ProteinExtraction Val.c Ref.
CE704–tolerant,
Corn Drought–dehydration 40days 6days Leaves iTRAQ/2DGE NS/3 totalprotein No [14]
2023–resistant
Drought–waterdeficit Lc–sensitive,Io–tolerant 3weeksold 4,6,8,10,12,14days Leaves 2DGE 3or5 totalprotein No [15]
W23–sensitive,
UV-Birradiation Cacahuacintle-tolerant, 28days 21days Leaves DIGE 3 totalprotein Yes [16]
ConfitePuneño–tolerant
Excalibur-tolerant,
adultplants
Wheat Drought RAC875-tolerant, cyclic Leaves iTRAQ 1 totalprotein No [17]
unknownage
Kukri-sensitive
Nesser–tolerant,
ABA 10dayoldseedlings 6h Roots iTRAQ 3 totalprotein Yes [18]
Opata–sensitive
roots,leavesand
Hanxuan10–tolerant,
Drought seedlings 48h sectionsincludingleaf 2DGE 3 totalprotein No [19]
ChineseSpring–sensitive
sheathandstem
CS–sensitive,
Salt200mM 7weeks 7weeks Shoots/Roots DIGE 3 mitochondria No [20]
AMP–tolerant
China-108,Yennon-78,
Drought,heat,salt,cold 4months N.S. Seeds 2DGE 1 totalprotein No [21]
Norin-61,Kantou-107
Shanrong3–tolerant,
Droughtandsalt seedlings 24h Shoots/Roots 2DGE 3 totalprotein No [22]
Jinan177–sensitive
Zhenshan97B–susceptible,
Rice Drought 50days 20days Leaves 2DGE NS totalprotein No [23]
IRAT109–tolerant
IR62266–tolerant,
Drought upto53days various Leaves 2DGE NS totalprotein No [24]
CT9993–sensitive
KDML105–unknown,
Drought NSG19–tolerant, 25days upto96h Leaves 1DGE 3 totalprotein No [25]
IR20–sensitive
Pokkali–tolerant,
Salinity100mM 28days 14days Roots 2DGE 3 totalprotein No [24]
IR29–sensitive
Chunyou58–tolerant
Nitrogendeficiency 4thleaf upto7days Leaves 2DGE 3 totalprotein No [26]
Yongyou6–sensitive
PI416937–tolerant,
Soybean Aluminium seedlings upto72h Roots DIGE NS totalprotein No [27]
Young–sensitive
Clark–tolerant,
UV-Birradiation 12days 9days Seedlings 2DGE 5 totalprotein No [28]
Magenta–sensitive
Jackson–sensitive,
Salinity150mM 21days upto144h Leaves 2DGE 3 totalprotein No [29]
Lee–tolerant
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Table1.Cont.
Crop Stress Genotypes PlantAge StressDuration PlantTissue Quant.Methoda #Biol.Reps.b ProteinExtraction Val.c Ref.
Morex–tolerant,
Barley Salinity50–250mM seedlings 13days Roots 2DGEIPG3–10 3 totalprotein yes [30]
Steptoe–sensitive
Morex–tolerant,
Salinity100–150mM seedlings upto16days Roots 2DGEIPG4–7 3 totalprotein yes [31]
Steptoe–sensitive
Afzal–tolerant,Line
Salinity300mM seedlings 4days Leaves 2DGE 3 totalprotein No [32]
527–sensitive
Afzal–tolerant,Line
Salinity300mM 7weeks 3weeks Leaves 2DGE NS totalprotein No [33]
527–sensitive
004223–tolerant,
Drought 6dayoldseedlings 3days Shoots 2DGE 3 totalprotein No [34]
004186–sensitive
Arta–tolerant,
Drought 33days 7days Leaves DIGE 3 totalprotein No [35]
Keel–tolerant
DH+-tolerant,
Boron seedlings 2weeks Roots iTRAQ 2 solubleprotein No [36]
DH—sensitive
15141–tolerant,
Drought Seedlings–unknownage 7days Leaves DIGE 3 totalprotein No [37]
15163–sensitive
102–tolerant,
Brassica Lowphosphorus 41days 26days Leaves/Roots 2DGE 3 totalprotein No [38]
105–sensitive
COC041-tolerant,
Peanut Drought–waterdeficit 74days 7days Leaves 2DGE 3 totalprotein Yes [39]
COC166-sensitive
Sugar K86-161-tolerant,
Drought 15weeks 21days Leaves 2DGE NS totalprotein Yes [40]
cane KhonKhan-sensitive
Frida-sensitive,
Strawberry Cold 8weeks+hardening2–48days 48h Crowns 2DGEandLFQP 3 totalprotein No [41]
Jonsok-tolerant
Sugarbeet Drought 71127219 157days N.S. Leaves 2DGE 10 totalprotein No [42]
Roma–tolerant,
Tomato Salinity100mM 24days 14days Leaves 2DGE 4 totalprotein No [43]
Supermarmande–sensitive
JG-62–tolerant,
Chickpea Drought 27days 1–6days Shoots 2DGE 2 nuclei No [44]
ICCV-2–sensitive
Chardonnay–tolerant,
DroughtandSalinity
Grape Cabernet Two-year-oldrootedcuttings upto16days Shoots 2DGE 3 totalprotein No [45]
(250mMfinal)
Sauvignon–sensitive
MbwazirumeWilliams
Banana Drought(sorbitol) PopoulouObino 4weekoldexplants 48days Leaves DIGE 6 totalprotein No [46]
L’EwaiCachaco
aQuantificationmethod;bNumberofbiologicalreplicates;cValidation;NS,Notstatedinmanuscript.
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2. DifferentialProteomicstoIdentifyGenotypeDifferencesinAbioticStressToleranceinCrops
2.1. Corn
2.1.1. WaterDeficit,DehydrationandDrought
The impact of drought on protein abundance in different corn cultivars has been addressed
at the seedling state by two groups. Benešová et al. [14] studied drought-induced changes in the
leafproteomeoftwomaizegenotypeswithcontrastingsensitivitytodehydrationbroughtaboutby
withholdingwater,usingbothiTRAQand2DGEapproaches. ResultsofiTRAQanalysisidentified
220proteinswhoselevelschangedduetodroughtstressinatleastonegenotypebyatleast2-fold,with
106ofthesedifferentiallyexpressedbetweengenotypes. Notonlydidthedroughttolerantcultivar,
CE704, show upregulation of a much larger number of proteins than the drought-sensitive 2023,
butmanyofthesewerealsoactuallydown-regulatedin2023. Ofparticularnoteweretheupregulation
ofprotective/detoxificationassociatedproteinsinCE704,includingtheantioxidantenzymesascorbate
peroxidase,superoxidedismutaseandtoalesserextentglutathionereductaseandcatalase,aswellas
proteinsinvolvedintranslationsuchasEF-TuM,amitochondrialtranslationelongationfactor,and
eIF3,atranslationinitiationfactor,inthedrought-tolerantgenotype.
In a similar study, Riccardi et al. [15] analysed protein changes in response to progressive
waterdeficitintotalproteinfrommaizeseedlingleafbladesfromtwodifferentgeneticallydistinct
lines (Io—drought-tolerant, Lc—drought-sensitive) and compared these to the hybrid of the two.
Ofthe78drought-responsiveproteinsdetectedby2DGE,38oftheseshoweddifferentialbehaviour
between the three genotypes. However, only a subset of those were sequenced. One protein, an
ABA/water-stress/ripeningrelatedprotein(ASR-protein),wasfoundexclusivelyinducedintheIo
tolerantgenotype,whilemalatedehydrogenasewasfoundexclusivelyintheLcgenotype. TheASR
proteinhasbeendescribedasadrought-responsiveprotein[47],butitsfunctionremainsunknown.
Riccardialsoinvestigatedtheproteomeoftheelongationzoneoftheleafbladeinthesamegenotypes
overmultipletimepoints[48]. Manyproteinsidentifiedintheirearlierstudywereidentifiedatone
ormoretimepointsinthisstudy. IncreasesinABA45andOSR40werelinkedtodifferencesinABA
accumulationbetweenthegenotypesandASR1wasidentifiedinbothstudiesasadroughtresponsive
protein[48].
2.1.2. UV-Irradiation
Casati et al. [16] surveyed responses of maize landraces differing in their tolerance to UV-B
radiationusingDifferentialinGelElectrophoresis(DIGE)ofleaftotalprotein. Responsiveprotein
profilesofalinedeficientinredanthocyaninpigments(W23)duetomutationinchalconesynthasewas
comparedwiththatoftwohighaltitudemaizelandraces(CacahuacintleandConfitePuneño). These
highaltitudelandracesshowedimprovedUV-BtoleranceduetonaturallyelevatedlevelsofUV-B
radiation-absorbing flavones as well as reduced expression of stress related proteins. The study
identified 53 proteins that were differentially regulated by UV-B radiation in W23 but showed
constitutively high expression in at least one of the high altitude adapted landraces. This was
narroweddowntotenproteinsthatshowedincreasesinabundanceinboththehighaltitudelines
as compared to W23 and included, ribosome recycling factor, ferridoxin, actin PEPc, translation
elongationfactorTu,26Sproteasomeregulatoryprotein,hydroxylpyruvatereductase,ADP-glucose
pyrophosphorylasesmallsubunit,succinyl-CoAsynthasebetasubunitandglutamate-1-semialdehyde
mutase. Theproteinsidentifiedhaddiversebiologicalfunction,whichimpliedthatenhancedUV-B
toleranceofthehighaltitudelineswasduetomultipleunrelatedfactors. Fromthislist,noneofthe
expectedROSscavengingenzymeswereidentifiedasdifferentiallyresponsive,butrecentviewshold
thatwidespread,oxidativedamageisrareundernaturalUV-Blevels[49].
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2.2. Wheat
2.2.1. Drought
Quantitative proteomics using iTRAQ technology was used to monitor protein changes in
total protein extracts from leaves of adult plants in response to cyclic drought conditions in three
wheat cultivars differing in tolerance based on grain yield; RAC875 (drought tolerant), Excalibur
(drought-tolerant),andKukri(drought-intolerant)[17]. Cyclicdroughtwasappliedbywithholding
wateruntiltheleasttolerantlinewiltedfollowedbyrewateringandthenrepeatingthistreatment.
Whileallthreecultivarsshowedincreasesinproteinsinvolvedinoxidativestressmetabolism,both
drought-tolerantcultivarsshowedco-ordinateddecreasesinCalvincycleenzymesafterlong-term
waterdeficit. However,ingeneral,thetwodrought-tolerantcultivarsshowedfewsimilaritiesintheir
protein response, which was attributed to their phenotypic differences in maintaining grain yield
when subjected to stress [17] suggesting distinct drought-tolerant mechanisms were employed in
eachspecies.
Using a similar iTRAQ-based quantitative proteomics approach but employing the wheat
varieties Opata M85, an elite drought-sensitive cultivar and Nesser, a drought-tolerant
cultivar, Alverezetal.[18], targeted differences in the seedling root proteome response to ABA.
BothABA-responsiveandcultivar-specificproteinchangeswereidentified. Majordifferencesincluded
proteinsinvolvedintranslationthatwereupregulatedbyABAinNesserbutdownregulatedinOpata,
aswereheatshockproteins. Otherproteinsinvolvedinsignallingandtransport,includingP-ATPase,
G-proteins,potassiumchannel,andcalnexinwerealsoselectivelyincreasedinthedrought-sensitive
cultivarbyABA[18]. UsingvalidationbyPCRmostproteinchangescorrespondedtogeneexpression
changesforanumberofselectedcandidates.
Inanotherstudy,Haoetal.[19]comparedadrought-tolerantwheatcultivarHanxuan10toa
drought-sensitivecultivarChineseSpringunderPEGinduceddroughtstressandrecovery. 2D-PAGE
identified16%oftheproteinsasbeingdifferentiallyexpressedbetweenthetwocultivarsinisolated
total protein extracts from seedling roots, leaves and an intermediate section between the two.
IncontrasttoresultsreportedbyAlvarezetal.[18],manystress-relatedheatshockproteinchaperone
familymemberswereshowntobeupregulatedinthedrought-tolerantcultivarandthereappearedto
beanincreaseinthephosphorylationleveloftheseproteins.
2.2.2. Salinity
Inoneofthefewstudiesthatfocusesoncultivar-specificproteinchangesrestrictedtoasubcellular
compartment,Jacobyetal.[20]analysedthemitochondrialroleinsalinitytolerancebycomparingthe
salinityresponsesofbreadwheat(CS)againstasalt-tolerantamphiploidAMP(wheat×Lophopyrumelongatum);
analysing samples from both shoots and roots. They identified a large number of mitochondrial
proteinsasresponsivetosalinityinbothgenotypes,includingenzymesinvolvedinreactiveoxygen
species detoxification. Two enzymes involved in one carbon metabolism, glycine decarboxylase,
andserinehydroxymethyltransferasewerespecificallyandsignificantlyincreasedinabundancein
thetolerantcultivar. Theseenzymes,workingintandem,areresponsiblefortheessentialprimary
metabolismreactionwhichinvolvestheinterconversionofglycineandserineusingtetrahydrofolate
as a cofactor, serving as the hub of all carbon metabolism in the plant [50]. Independent of stress,
cultivar specific differences in protein composition of the mitochondria were also identified, with
alteredabundanceofmanganesesuperoxidedismutase,serinehydroxymethyltransferase,aconitase,
malatedehydrogenase,andβ-cyanoalaninesynthasebetweenthegenotypes. Itwassuggestedthat
theseproteinscouldcontributetothehighersalt-toleranceofAMPandserveasmolecularmarkersfor
breedingsalt-tolerantwheatvarieties[20].
Theindependenteffectsofbothdroughtandsalinitystressonseedlingsofanotherbreadwheat
cultivarcv. Jinan177werecomparedtothesomatichybridstresstolerantwheatcv. ShanrongNo. 3
(SR3)using2DGE[22]. Whilethemajorityofboththerootandleafstress-responsivedifferentially
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abundant proteins were shared, whether the imposed stress was drought or salinity, there were
genotype,stress,andtissuespecificdifferences. Overall,salt-stressresultedinmorechangesinprotein
abundancethandiddroughtstress. Anumberoftheseproteinswereidentified,however,themajority
hadonlyasinglematchedpeptideandmultiplespotscorrespondedtothesameprotein. Thefunctions
oftheseproteinswerevariedandincludedthealphasubunitoftheF1-ATPase,andRubiscolarge
subunit[22].
2.2.3. Salinity,Drought,Heat,orCold
Inanotherwheatstudy,Kamaletal.[21]contrastedfourwheatcultivars,twoChinesecultivars
(China-108andYennon-78)andtwoJapanesecultivars(Norin-61andKantou-107),withunknown
stressphenotypes,toidentifygrainstress-induciblesolubleproteins. Allfourcultivarsshowedthe
common induction of 11 proteins under the various stress regimes, several of which have known
roles in stress responses including the ROS scavenging enzyme ascorbate peroxidase, as well as
a truncated cold acclimation protein, and a salt-tolerant protein, both with unknown functions.
BothJapanesecultivarsshowedthehighestnumberofuniquestress-inducibleproteinswith24and
19uniquelyresponsiveproteinsidentifiedinNorin-61andKantou107,respectively,comparedtoonly
one(China-108)andfour(Yeonnon-78)uniquelyexpressedproteinsintheChinesecultivars. Genotype
differencesdidnotshowspecificenrichmentofproteinsinvolvedinspecificcellularprocessesbut
ratherproteinsfromvariouspathwayswereidentified. However,withoutdirectknowledgeofthe
tolerancelevelofthedifferentcultivarsunderthevariousstresstreatments,itwasdifficulttomake
meaningfulconclusions.
2.3. Rice
2.3.1. Drought
Severalgroupshavestudiedtheeffectsofdroughtorwaterdeficitontheproteomeindifferent
cultivarsofrice. Jietal.[23]usedquantitativeproteomicstoinvestigateflagleafproteinsintworice
cultivars: Zhenshan97B, a lowland drought-susceptible variety and the upland, drought-tolerant
cultivarIRAT109. Thesensitivevarietyshowedageneraldecreaseinproteinabundance(15decreased
and2increased),whilethetolerantvarietyshowedtheoppositetrendwith14proteinsshowingan
increasedabundanceandonly6proteinsshowingadecreaseinabundance. Amongtheincreased
proteins in IRAT109 were the antioxidant enzymes, dehydroascorbate reductase, and superoxide
dismutase. Inonlyasinglecasedidtheresponsiveproteinscorrespondtothesameproteininboth
cultivars; thiswasanATPsynthasebetasubunitwhichwasdecreasedinZhenshanbutincreased
inIRAT109.
Another rice proteomic study compared protein abundance changes in three rice cultivars
differingindrought-tolerance;NSG19,atolerantvariety,ThaijasminericeKDML105,ofintermediate
tolerance,andIR20asensitivecultivar. Inthisstudypolyethyleneglycol(PEG6000)wasusedtoelicit
thewaterstressresponse[25]. Analysisofdrought-responsiveproteinsidentified53(8.5%oftotal)that
showedsignificantdifferencesinabundancebetweenthethreecultivars. Enrichmentforresponsive
proteinsinvolvedincellandDNArepairwereobservedinthetolerantline,aswellasanincreasein
theamountofcoronatine-insensitive1protein,showntohavearoleinstomatalclosing,whichmay
helptolimitevaporativewaterlossandthusconferdrought-tolerance.
2.3.2. Salinity
Salekdeh et al. [24] studied salt-responsive proteins in total protein extracts from roots of the
tolerantricecultivarPokkaliandthesensitivericecultivarIR29followinglong-termexposure.Analysis
of2Dgelsidentifiedtwoproteinswhichwereupregulatedexclusivelyinthetolerantcultivarunder
stressconditions,includinganabscisicacidstressripeningprotein,ASR1,andaproteininvolvedin
ligninsynthesis,caffeoyl-CoAO-methyltransferase. Theyalsoidentifiedhigherconstitutivelevelsof
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theROSscavengerascorbateperoxidaseinthesalt-tolerantcultivar,butthisproteinwasupregulated
inbothcultivarsundersalinitystress.
2.3.3. NitrogenDeficiency
Nitrogen-deficiency stress responsive proteins in two rice cultivars differing in nitrogen use
efficiency(Chunyou58—tolerantandYongyou6—sensitive)wasstudiedbyaproteomicsapproach
using2DGE[26]. Gelanalysisdetected31proteinspotsinthetwocultivarsthatshowedreproducible
changes, with four of these spots stress-inducible in both of the varieties. These shared proteins
includedribulose-1,5-bisphosphatecarboxylase/oxygenaseactivase,Rubiscolargesubunit,glycine
decarboxylase,aswellasatransposase.InChunyou58,only10proteinsshowedchangesinabundance
whileinthesensitivecultivarYongyou6,27proteinswerealtered,withmanyofthesebeingidentified
previouslyasnitrogendeficiency-relatedproteins,includingheatshockproteinGSTF14,fibrillin-like
protein,glutathioneS-transferaseandDegP2protease.
2.4. Soybean
2.4.1. Aluminium
Duressaetal.[27]carriedoutaquantitativeproteomicanalysisusing2D-DIGEofsoybeanroot
totalproteinunderaluminiumstresstocomparetheresponseofanaluminiumtolerant(PI416937)toa
sensitivecultivar(Young). Thealuminiumtolerantcultivarshowedamorerapidproteinresponseto
theonsetofthestress,withmoreproteinsrespondingatashorterexposuretime,thanthesensitive
cultivar.Up-regulatedproteinsinthetolerantcultivarincludedmalatedehydrogenase,enolase,malate
oxidoreductaseandpyruvatedehydrogenase. Theseenzymescontributetotheincreasedsynthesisof
organicacids,inparticularcitrate,whichhasbeenmeasuredinthiscultivarandshowntobesecreted
andimportantforbindinganddetoxifyingAl[27]. Anumberofproteinsinvolvedinstressprotection
werealsospecificallyenhancedinPI416937,andincludedthioredoxin,dehydroascorbatereductaseas
antioxidantsandcysteinesynthaseandisoflavonereductasewhichcontributetoAldetoxification.
2.4.2. UV-Irradiation
QuantitativeproteomicswasemployedbyXuetal.[28]tostudytheimpactofsolarultraviolet-B
(UV-B)radiationonthesoybeanleafproteometoinvestigatetheprotectiveroleofflavonoidsagainst
UV-B,usingtwoisolineswhichdifferintheirflavonoidcontent;astandardlinethathadmoderate
levels and magenta line that had reduced flavonoid levels making it susceptible to UV-B. Results
indicated that many more proteins were responsive to solar UV-B in the reduced flavonoid line
(29increased,22decreased)thaninthestandardline(8increased,14decreased)andthemajorityof
thesewerecategorizedasfunctioningincellularenergyreactions,includingmultipleproteinspots
fortheoxygenevolvingenhancerprotein1(OEE)ofphotosystemIIandsubunitsofPS1. Another
12 proteins were not altered by UV-B but showed different accumulation between the two lines
includingcarbonicanhydrase,andGCVH-protein,whichwerebothidentifiedbytwospotswhich
showedhigherabundanceinthestandardline. Ingeneral,theproteinsbelongedtodiversefunctional
groupsandtherewasnoobviouspathwayenrichment.
2.4.3. Salt
Salinitytoleranceoftwosoybeangenotypes;Jackson(salt-sensitive)andLee68(salt-tolerant),
wasinvestigatedusinga2DGEquantitativeproteomicapproachoftotalproteinsextractedfromleaves
ofseedlings[29]. Analysisofproteinabundancechangesshowed91proteinspotsoutofatotalof
800 reproducible spots were salt-responsive. Identification of 78 of these indicated enrichment in
proteinsinvolvedinfreeradicalscavenginginthesalt-tolerantline,includingascorbateperoxidase,
glutathione-S-transferase,ferritinlightchainprecursor,andproteindisulphideisomerase.
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2.5. Barley
2.5.1. Drought
Kausar et al. [34] investigated the changes in the protein profile of shoot total protein from
three-day-old barley seedlings from a drought-sensitive genotype 004186 and a drought-tolerant
genotype004223subjectedtoshort-termdroughtstress(bothPEGandwaterwithholdingexperiments).
Interestingly,plantsrespondeddifferentlytowaterstressappliedbywithholdingwatercomparedto
waterstressfromPEGtreatmentwithonlyafewresponsiveproteinssharedbetweenthesetreatments
(3outof35inthesensitivegenotype,and8outof36inthetolerantgenotype),suggestingthatPEG
applicationisnotnecessarilycomparativetodroughtbywaterwithholding. Highestfoldchangein
proteinabundanceinthesensitivecultivarwasobservedforaPtrTox-Abindingprotein1anda50S
ribosomalproteinL9. Whileinthetolerantcultivarproteinspotsforamethioninesynthaseanda
photosystem1reactioncentresubunitIIshowedthehighestfoldchanges. Keydifferencesbetweenthe
drought-tolerantandsensitivecultivarswereobservedinmetabolism,defense,andphotosynthetic
relatedproteins,whichwereincreasedinthetolerantcultivarsanddecreasedinthesensitivecultivars.
Inanotherstudyaimedatunderstandingbarleydrought-tolerance,Ashoubetal.[37]compared
the response of shoot total proteins of two Egyptian barley land races, accession number 15141,
whichisdrought-tolerant,andaccessionnumber15163,considereddrought-sensitive,usingDIGE.
Whiletherewereanumberofdrought-inducedproteinchangesthatweresharedbetweencultivars
includingincreasesinmethioninesynthase,ATPsynthasealphasubunit,aconitase,alanineglyoxylate
aminotransferase, ATP-dependentCLpprotease, HSP90, andproteindisulphideisomerase, there
were also cultivar specific changes. In the tolerant cultivar, these included Elongation factor
EF2, metalloprotease, HSP 70, and a stress responsive protein. In the sensitive cultivar leucine
aminopeptidase,lipoxygenase,sucrosesynthase,betainealdehydedehydrogenaseandNADP-malic
enzymeallwereincreasedwithnochangesinthetolerantcultivar.However,aschangeswererelatively
smallandproteinsbelongedtoawiderangeoffunctionalprocessgroups,noclearpictureemerged
whichwouldhelpexplainthedifferentresponsesofthetwocultivarstodroughtstress.
2.5.2. DroughtandHeat
Oneoftheonlyreportstouchingontheimportanceofstudyingtheeffectsofcombinedstresses
oncropplantswascarriedoutbyRollinsetal.[35],whichused2D-DIGEtechnologytostudythe
molecularbasisofstressresponsesinleaftotalproteinextractsfollowingsingleorcombinedexposure
todroughtandheattreatmentsintwodrought-adapted,geneticallydiversegenotypes;theSyrian
landraceselectionArtaandtheAustralianbarleycultivarKeel.Keeltendedtoshowhigheryieldunder
droughtandheatalone,whileArtatendedtoshowahigheryieldunderthecombinationtreatment.
From a total of 1005 protein spots, DIGE analysis identified 305 spots significantly differentially
regulatedbytheheattreatment,and473spotsdifferentbetweengenotypes,with35spotsdifferent
duetotheinteractionbetweentemperatureandgenotype. Surprisingly,nospotswerefoundtobe
significantlyregulatedbythedroughttreatmentdespitetheseverityofthestress(soilwatercontent
was reduced to 15%). Only a small proportion of these stress-responsive proteins were identified
bymassspectrometry. Oneprotein, glyceraldehyde-3-phosphatedehydrogenase, wasinducedby
heat,genotypeandacombinationofheatandgenotype. Asignificantenrichmentinproteinswith
roles in photosynthesis were differentially abundant between the genotypes and also responsive
totemperature,includingoxygen-evolvingenhancerprotein1and2,andchlorophylla/bbinding
protein. However,whiletheseproteinsincreasedinabundance,thephotosyntheticefficiencyshowed
asignificantreduction[35].
2.5.3. Salinity
Theassessmentofsalt-stressresponsesbetweencontrastingbarleycultivarswascarriedoutusing
2DGEfocusingonroottotalproteinsusingthebarleygenotypesSteptoe(salt-sensitive)andMorex
Proteomes2016,4,26 10of18
(salt-tolerant)[30]. Gelanalysisidentified760proteinspotsofwhich39weredifferentiallyexpressed
withmostshowingacultivar-ratherthanastress-specificresponseandmoreproteinswereaffectedby
thestressinthesensitivegenotypethaninthetolerantgenotype. Accordingtofunctionalannotation
oftheidentifiedproteins,enrichmentwasobservedforthoseinvolvedinoxidativestressresponses
witheightproteinsfunctioninginredoxregulation. TheseincludedMDAR,ascorbateperoxidase,and
lactoylglutathionelyase,whichwereselectivelydown-regulatedbysalinityinMorex,whileascorbate
peroxidasewasup-regulatedinSteptoe,highlightinggenotypedifferences.Inthisstudy,thevalidation
ofspotexpressionchangesforfouroftheidentifiedproteinswascarriedoutbyWesternblotanalysis
andresultsindicatedagoodagreementbetweenthetwoapproaches.
In a similar study by the same group, Witzel et al. [31] expanded on the results summarized
abovebyusinganarrowerpHrangeoftheIPGstripsemployedfor2DGEandtrackingtheexpression
of proteins over multiple time points. From 685 matched spots they could detect 91 which were
significantlydifferentiallyexpressedwithafold-changeofatleast2;amuchlargernumberthanin
theirpreviousstudy. Interestingly,inthisstudy,ahighernumberofproteinswereinducedbysalinity
in the tolerant genotype, while in their previous study more proteins were responsive to stress in
thesensitivegenotype[30]. Asidentifiedpreviously[30],therewasinvolvementofROSscavenging
proteinsbutalsoMEPpathwayenzymes,includinghydroxymethylbutenyl4-diphosphatesynthase
and4-diphosphocytidyl-2-C-methyl-D-erythritolkinasewereinducedinbothcultivars. Nodirect
comparisonsweremadebetweenthetwostudieswhichwouldhavebeenhelpful.
Rasoulniaetal.[32]andFatehietal.[33],alsousedquantitativeproteomics(2DGE)toinvestigate
salinityresponsivetotalproteinsindifferentbarleygenotypesbutinthiscasecomparingleafproteins
fromseedlingsofthesalt-tolerantAfzalcultivartothesalt-sensitiveL.527barleygenotypeexposedto
bothshorttermandlong-termstress. Undershort-termsalinitystress,atotalof117salt-responsive
proteins were detected in the two genotypes, with the identity of 13 reported in the manuscript.
Ingeneral,thetolerantgenotypeshowedahighernumberofpositivelyresponsiveproteinswhile
thesalt-sensitivegenotypeshowedahighernumberofproteinswithdecreasedabundance. Proteins
thatwereincreasedinthesalt-tolerantcultivarbutdecreasedinthesalt-sensitivecultivarincluded:
phosphoribulokinase, sedoheptulose 1,7-bisphosphatase, plastocyanin photosystem I subunit VII
proteins, oxygen-evolving enhancer protein 2, 2-cys peroxiredoxin, and glutathione-S-transferase.
Thefactthatmanyoftheseproteinsarechloroplastlocalizedandinvolvedinphotosynthesismay
suggest that differences in salinity tolerance between the cultivars are due to a reorganization of
photosyntheticmachinery.
Underlong-termsalinitystressatotalof44salt-responsiveproteinsinthetwogenotypeswere
identified[33]. Interestingly,proteinsthatwerespecificallyinducedinthetolerantcultivarundershort
termstresswereinducedinbothcultivarsunderlong-termstress;includingoxygenevolvingenhancer
protein. Inaddition,incontrasttotheshort-termstress,manyphotosyntheticandcarbonassimilation
relatedproteinswereinducedinbothgenotypes.
2.5.4. Boron
Pattersonetal.[36]usediTRAQlabellingtolookforboron-induceddifferencesinrootsoluble
protein abundances between two barley lines that are defined by the presence or absence of both
the4Hand6Hboron-toleranceloci,whichencodeananion-permeabletransporterandamemberof
theNIPaquaporinfamily, respectively[51,52]. Fourproteinsshowedanincreaseinabundancein
theB-tolerantplants. Threeoftheseproteinsareinvolvedinproductionofphytosiderophores,and
thefourthwasadefence-relatedprotein. Allfourproteinshavepreviouslybeendemonstratedtobe
increasedinexpressioninresponsetoirondeficiency[53],andmaybeindicatorsofageneralmineral
deficiencyresponse.