Occurrence of KPIs in white clover

KunitzProteinaseInhibitorsarewidespreadintheplantkingdomandhavebeendescribed

frommanyplantspecies.MorethanonehomologueofKPIshasbeenidentifiedindifferent

plantspecies:forexample,twoinchickpea(HernandezͲNistaletal.,2009),11insoybean

(JofukuandGoldberg,1989),21inpotato(Heibgesetal.,2003aandb)and31inpoplar

(Philippeetal.,2009).AssumingthatKunitzProteinaseInhibitorsareamultigenefamilyin

whiteclover,thefirstgoalofthestudywastoidentifythemembersofthisgenefamily.

Inwhiteclover,onegenewasidentifiedusingtheAgReseachESTdatabasewhichwaslateron

amplifiedbygeneͲspecificprimersandnamedasTrͲKPI1.Degenerateprimersetswereusedto

findfurtherTrͲKPIgenesfromwhitecloverandeightpartialsequenceswereidentified.Later,

usinggenomewalkerand3’RACEtechnology,threefurtherfulllengthgeneswereisolated

andnamedasTrͲKPI2,TrͲKPI4andTrͲKPI5.Thoughrepetitiveattemptsweremadetoclone

theupstreamanddownstreamregionsforthefourotherpartialsequencesidentified(TrͲKPI3, TrͲKPI6,TrͲKPI7,andTrͲKPI8;Figure3.1B),thisproveddifficultasthesegenesareintronͲless

andtheyshareahighdegreeofidentity(forexample,TrͲKPI3shares97%identitywithTrͲKPI7;

KPIfamilyinwhiteclover.Aswhitecloverisanallotetraploid(2n=4x=32)outͲcrossingplant

species(AtwoodandHill,1940),itmightbespeculatedthatthesehighlyidenticalgenesarise

fromthetwodifferentparents(forexampleTrͲKPI1andTrͲKPI6orTrͲKPI8).Asmentioned

previously,amongthelegumes,soybeanhas11membersandMedicagohas12members

whicharebothdiploidplantspecies.Therefore,itislikelythatwhiteclovermighthavemore

than8TrͲKPIgenesinthegenome.However,inthisstudy,thefourfulllengthidentifiedgenes

werethefocusoffurtheranalysis.

BLASTsearchingwithtranslatedsequencesoftheidentifiedTrͲKPIsusingtheNCBIandJCVI

(againstM.truncatulaGBrowse)databasesgavehitstoonlyKunitzͲtypeTrypsinInhibitor

proteins.AllfouridentifiedTrͲKPIsgavespecifichitstotheSTI(SoybeanKunitzTrypsin

Inhibitor)superͲfamilyconserveddomain,stronglyindicatingthatthewhitecloversequences

belongtotheKPIgroup.NosequencefromanyothergenefamilywasidentifiedbyBLAST

searchingagainstGenBankandtheJCVIdatabase,againconfirmingthattheyaretheorthologs

ofKPI.Inordertoconfirmtheidentityofthesefourisolatedgenes,atleastninepublished

homologuesfromdifferentleguminousspecieswereselectedfromtheNCBIdatabaseand

includedinthepresentstudyforsequencecomparison.Thegenesshowedhighsequence

similaritywithKPIsfromP.sativum,C.arietinumandM.truncatula.

TheidentitiesofthefourfulllengthTrͲKPIproteinsvariedfrom40to64%witheachother.

ThishighvariationraisesthepossibilityofdiversifiedrolesforthemembersoftheTrͲKPI

familyinwhiteclover,asithasbeenreportedthatformoredistantlyrelatedproteinsina

superfamilyfunctionalvariationandsubstratespecificityisdiverse(Toddetal.,2001).

However,existenceofhighdissimilarityamongthemembersofKPIsisnotuncommoninother

plantspecies.Forexample,researchwiththeP.trichocarpaKunitzgenefamilysuggeststhat

31KPIsshareabout96to38%identitytoformsixdifferentcladesandalsosuggeststhat

tandemduplicationsmighthaveplayedanimportantroleintheexpansionofthisgenefamily

(Philippeetal.,2009).Poplarsarelonglivedtreespecies,andthereforealargeanddiverseKPI

family(31members)mightbecrucialforitssurvivalagainstmultiplestresses(Philippeetal.,

2009).However,soybeanandMedicagoareannualplantsandsothepresenceofmorethan

10memberscouldindicatetheimportanceofdifferentfamilymembersindifferentaspect

duringtheshorterlifecycleoftheseplants.Whencomparedtoannuallegumespecies

(soybeanandMedicago),whitecloverisastoloniferousperenniallegumeandtherefore,the

functionandoccurrenceofsomeoftheTrͲKPIsmightbequitedissimilar.Thusitcanbe

thathaveevolvedinwhitecloverorotherperennialplantsasaresultofmoreprolonged

exposuretodifferentbioticorabioticstresses.

TheidentifiedTrͲKPIscontainfourconservedcysteineresidueswhichisoneoftheidentifying

charactersoftheKPIfamily.AccordingtoOlivaetal.,(2011),plantKunitzproteinaseinhibitors

especiallyfromtheLeguminosaecanbeclassifiedbasedontheircysteineresiduesintothree

groups:KunitzPIswithfourormorecystineresidues(formingtwodisulfidebonds),twoor

threecysteineresidues(formingonedisulfidebond)andoneornocystineresidues.TrͲKPI1

andTrͲKPI2have5cysteineresiduesandTrͲKPI4andTrͲKPI5havefourcysteineresidues.

Therefore,accordingtothisclassificationallofthesefourproteinsfallintothesamegroup.

ThereactivesiteloopofTrͲKPIsisdevoidofLysorArgwhicharepresentinthereactiveloopof

STI.However,IIeandSer,whicharepresentinothertrypsininhibitorreactivesiteresidues

(MajorandConstabel,2008)ispresentinthereactivesiteofTrͲKPI1andTrͲKPI2.Thepresence

ofGluͲIIeinTrͲKPI4andThrͲThrinTrͲKPI5reactivesiteloopdoesnotmatchwithanyofthe

wellidentifiedKPIproteins,andthiscouldindicatefunctionaldivergenceofthesefourgenes.

Therefore,thefunctionalcharacteristicsofTrͲKPIscannotbeenvisagedonthebasisoftheir

sequencesimilarity.ThesubͲcellularlocalizationpredictionprogramme(TargetP1.1Server;

Emanuelsonetal.,2000),predictedthattheTrͲKPIsarelocatedinthesecretarypathway,in

commonwithKPIsfromotherplantspecies.Therefore,vacuolelocalizationisapossiblity,

whichisthecompartmentthatconfinesavarietyofchemicalcompoundsaswellasproteins,

includingbothenzymesandenzymeinhibitorsthatareinvolvedinstorageandactiveplant

defense(HollanderͲCzytkoetal.,1985).

Therefore,todeterminewhethertheseproteinsareactiveornot,additionalbiochemical

analysiswithawiderangeofcommerciallyavailableproteinasesisnecessary.Thehigh

inequityattheactivesitefoundinthisstudyalsoraisesthepossibilitythatsomeofthe

identifiedTrͲKPIsarenottargetingtheproteinasesatallinplantaasithasbeenreportedby

McCoyandKortt(1997)thatsomeproteinsoftheKPIfamilydonotactasinhibitors.Glaczinski

etal.,(2002)showedintubersfromcv.ProvitathataKunitztypeproteinaseinhibitorwhich

hadseveralaminoacidsubstitutionswhencomparedwiththeKPIsandnosequencesimilarity

withatuberinvertase,didnotinhibitanyoftheproteinasesbutshowinhibitoryactivity

againstinvertase.ThereisalsoapossibilitythatsomeofthesegenesarepostͲtranscriptionally

example,selectivetranslationofmRNAwasobservedinmaizerootsunderoxygendeprivation

wheremanynormalcellularmRNAsaresynthesizedbutshowedaminimalleveloftranslation

(BaileyͲSerres,1999).