Concluding statement and research hypothesis

Alllivingorganismsexperiencevariouskindsofbioticandabioticstress.Inresponse,plants

haveevolvedaspectacularrangeofdefensesystemswhichmaybestructural,chemicaland/or

moleculartotoleratetheseadverseconditions.Insomeinstances,thecellularlevelsof

compoundsorproteinsthatareindispensableformetabolicprocessesandsurvivalunder

normalconditionsareincreasedtoactasdefensivecompoundsunderadverseconditions

(MosolovandValueva,2011).Anexampleofoneoftheseproteingroupsaretheproteinase

inhibitors.

1.8.1WhystudyKunitzproteinaseinhibitorsinwhiteclover?

Worldwide,whiteclover(TrifoliumrepensL.)isrecognizedforitshighforagequality.White

clovercanbeconsideredasoneofthepremiumpasturespeciesasitfixesatmospheric

nitrogeninasymbioticinteractionwithRhizobia,andtherebycanreducefertilizercost.Ithas

thepotentialtoregeneratefromstolonsaftergrazingandtherebyreducesthecostofseeding

pastureseveryyear.Astheplantspecieshasmanysuchadvantages,effortshavebeenmade

toincorporateitintothegrazinglandsofEurope,somepartsofwestAsiaandNorthAmerica.

However,theproportionofthisspeciesintheestablishedgrazinglandsisquitesmall

(Hopkinsetal.,1985).ThereasonsbehindthiscouldbehighorlowsoilpH,nutrientdeficiency

inthesoil,choiceofthewrongcultivarand/oroverexploitationoffertilizersandpesticides.As

damagebypestsanddiseasesisregularlyobserved,theyarealsopartlyresponsibleforthe

failureorpoorgrowthofclover.Thecropisalsovulnerabletodroughtthatreducesitsgrowth

rateandproductivity(Parsonsetal.,2011).

Bioticandabioticfactorsthatincreasethetoleranceofplantstopests,diseases,extremeheat

anddroughtarecriticaltothefuturesuccessofwhiteclovercultivation(Rhodesetal.,1994).

Henceidentifyinguniquegenesforachievingenhancedtolerancetobioticandabioticstresses

isamajorresearchfocus.Sinceplantgrowthanddevelopmentisinfluencedbyenvironmental

cues,itisperhapsnotsurprisingthatexposuretounfavorableconditionscausesmany

physiologicalandmorphologicalchanges,andthesecanaffectplantproductivityatevery

developmentalstage.Thoughmanygenesinvolvedintheinteractionsofplantstoinsect

damage,waterdeficit,andhighorlowtemperaturetolerancehavebeenidentifiedoverthe

interveningyears,thereremainsamajorgapintermsofpreciseinformationbecausemost

tissueorganizationandthereforethereisadifferenceinstressresponse(SmithandSmet,

2012).Suchstudieswithmodelplantsarefundamental,butitisalsoimportanttoidentify

genesthatareinvolvedintolerancemechanismsincropplantsofagriculturalimportance.

Theinvolvementofproteinaseinhibitorsintermsofmediatingplanttoleranceagainst

unfavorablegrowthconditionsisofconsiderableresearchinterest.Assummarized,Kunitz

proteinaseinhibitorsareimportantinthiscontextasitiswellestablishedthatKPIsplayan

importantroleinplantdefensemechanismsagainstinsectherbivoryindifferentplantspecies.

Sincetheinitialachievementofobtainingincreasedpesttolerancebytheintroductionofa

genecodingfortrypsinproteinaseinhibitor(Hilder,1987),researchershavebeenusing

differentPIstoincreasetoleranceagainstinsectsindifferentcropplants.Butthesignificance

ofproteinaseinhibitorsintheresponsesofplantstowaterdeficit,highpHandotherstress

conditionshasreceivedlessattention.

Untilnow,noinformationisavailableabouttheoccurrenceofKPIgenefamilyinwhiteclover.

Itisnecessary,therefore,toinitiallyisolateKPIsequencesfromwhitecloverandthento

determinewhetherthegenefamilyisdifferentiallyresponsivetoarangeofdevelopmental

andenvironmentalcues.

1.8.2Hypothesis

Consideringthisresearchcontext,thegeneralizedhypothesistobetestedstatesthat:

ThetranscriptionofmembersoftheKunitzProteinaseInhibitor(KPI)genefamilyinwhite

cloverisnotonlyresponsivetoendogenouslygeneratedcuesinresponsetobioticstressbut

alsothosegeneratedduringplantgrowthanddevelopmentandinresponsetosubͲoptimal

growthconditionscausedbyabioticstresses.

1.9Researchobjectives

Totestthehypothesis,thefollowingfourobjectiveswereset–

ͲIdentifycDNAswhicharecomplementarytogenesthatencodeKunitzproteinaseinhibitor

proteinsinwhiteclover.

ͲInvestigatethetranscriptionalregulationoftheseKPIgenesatdifferentdevelopmental

stagesoftheplant.

ͲDeterminethetranscriptionalregulationofKPIsunderbioticstressessuchaswoundingor

ͲInvestigatetheinvolvementofKPIsatthetranscriptionallevelunderabioticstressessuchas

osmoticstressandnutrientdeficiency.

ͲIdentificationoftheregulatoryelementsinthepromoterregionoftheidentifiedKPIgenes.

Theaboveobjectiveswillprovidevitalinformationonspecificity,functionandexpressionof

KPIgenesinwhitecloveratthetranscriptionallevel.Theseresultswillprovideafoundationfor

futurestudiestobegintounderstandthesignificanceofthefunctionsoftheencodedproteins

Chapter2