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Rice response to simultaneous biotic and abiotic stresses PDF

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i Rice response to simultaneous biotic and abiotic stresses Ritu Shree Jain Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biology Faculty of Biological Sciences December 2013 i ii Thecandidateconfirms that theworksubmittedis his/herownandthat appropriate credit has been givenwherereferencehas beenmadetotheworkofothers. This copyhas beensuppliedontheunderstandingthat it is copyright material andthat noquotationfrom thethesis maybepublishedwithout properacknowledgement. Assertionof moral rights(optional): Theright ofRituShreeJain tobeidentifiedas Authorofthis workhas beenassertedby herinaccordancewiththeCopyright,Designs and Patents Act 1988. ©<2013TheUniversityof Leeds andRituShree Jain ii iii Acknowledgements IwouldliketothankmysupervisorProf.Urwinforconstant guidanceand support throughout myPhD,for believinginme andhelpingmerealisemypotential. Iextendmysincere gratitudetoDrCatherine.J. Lilleyforherpatience,valuable guidanceandsupport at all crucial times. Myspecial thanks toProf.HowardAtkinson forhis encouragement. Itakethis opportunityto express mydeep gratitudetomyparents andmybrotherfor all theloveandsupport theyhaveprovided. Ithankthem forbelievinginme and encouragingmeto fulfil mydreams. Iam highlygrateful ofthewonderful peopleinthe Nematologylab,especiallyDrKate warner,DrNicolaAtkinson andthetechnicians –Fiona,JennieandBevforconsistent support andenthusiasm inlast four years. Withouttheirknowledgeand assistance growingricewouldhave beenatoughjob. Iwill also liketothankDrRosanneCurtis andDrJohnP Carrforprovidingthebiological material requiredformyresearch. Iam also thankful toDrDannyCoyne andstaffof IITAandAfricarice, Ibadan,formaking thefieldtrial happen. Last but not theleast, Iwill liketothankmyfriends Suruchi,Mirella,Mona,Shiraz, Tanya andSarvagya forlisteningtoall mycomplains andprovidingvaluableemotional andsometimes scientific input.AndAditya Ingrolefortakingcareofmeandstanding bymein goodandbadtimes. iii iv Abstract With the predicted climate change and an ever-growing population there is increasing pressuretodevelop crop plants withimprovedstress responses, increased yieldandhigh nutritive value. We have explored transcriptomic changes in the leaves and roots of rice plants (Oryza sativa japonica cv Nipponbare) in response to drought and the root-knot nematode Meloidogyne graminicola. A glasshouse model was developed to mimic conditions experienced by rice plants in the field. The plant responses under simultaneous biotic and abiotic stress were dominated by the drought element accompanied by a unique set of genes that were only responsive to the simultaneous stress. Highlighted within this group were novel members of stress-responsive gene families for example cytochrome P450, wall-associated kinases, lipid transfer protein- like proteins and new candidate genes that may play important roles in the response of rice to multiple stresses. The genes that were differentially regulated between the multiple and the drought stress treatment were explored using loss-of-function mutants. The loss-of-function mutant for peroxidase precursor gene (per) showed improved growth and yield compared to the wildtype Nipponbare plants. The experiments conducted in growth rooms were validated in a field study. Both Nipponbare rice plants, and the popular lowland indica rice cv IR64 were grown under prolonged vegetative drought stress accompaniedbycyst nematodeorroot-knot nematodeinfection. Reduction of phytate, an anti-nutrient, has been adopted as a major strategy to improve the nutritional value of crop plants. Nematode susceptibility of low phytate Arabidopsis plants was studied to determine the effect of reduced phytate content on the plant’s defenceresponse. The study has provided insight into the genome-wide transcriptional changes in rice under a combined biotic and abiotic stress. It has led to better understanding of the stress responses in plants that will be advantageous in developing crop varieties with improved yield andnutritivevalue. iv v Table of Contents ACKNOWLEDGEMENTS.......................................................................................................................3 ABSTRACT........................................................................................................................................4 TABLEOFCONTENTS..........................................................................................................................5 LISTOFFIGURES..............................................................................................................................10 LISTOFTABLES................................................................................................................................13 1 CHAPTER...................................................................................................................-1- INTRODUCTION...................................................................................................................-1- 1.1. RICE...................................................................................................................................-2- 1.2. DROUGHTSTRESS.................................................................................................................-4- 1.2.1. Plantmorpho-physiologicalresponsetowardsdroughtstress...................................-5- 1.2.2. Modelsystemstostudyricedroughtresponseinthelaboratory..............................-6- 1.2.3. Plantmolecularresponsetowardsdroughtstress......................................................-7- 1.3. RICENEMATODEPROBLEMS.................................................................................................-11- 1.3.1. Lifecycleoftheroot-knotnematodes.......................................................................-12- 1.3.2. Lifecycleofcystnematodes......................................................................................-13- 1.3.3. Plantresponsetowardsnematodeinfection............................................................-14- 1.4. SIMULTANEOUSSTRESSESEXPERIENCEDBYPLANTSINTHEFIELD....................................................20 1.4.1. Moleculareventsinsimultaneousbioticandabioticstress..........................................22 1.5. ENGINEERINGCROPSFORFOODSECURITY..................................................................................24 1.6. PROJECTOVERVIEW...............................................................................................................25 2 CHAPTER.....................................................................................................................27 ESTABLISHINGAGLASSHOUSEMODELTOMIMICSEVEREVEGETATIVEDROUGHTANDROOT-KNOTNEMATODE, MELOIDOGYNEGRAMINICOLAINFECTIONINRICE..........................................................................27 2.1. INTRODUCTION.....................................................................................................................27 2.1.1. Themodelmonocotplant-Rice....................................................................................27 2.1.2. Bioticstress-Root-knotnematode,M.graminicola.....................................................28 2.1.3. Abioticstress-waterscarcity........................................................................................29 2.2. MATERIALSANDMETHODS.....................................................................................................32 2.2.1. Riceseedsterilisation....................................................................................................32 2.2.2. Ricegrowthconditions..................................................................................................32 2.2.3. MolecularcharacterisationofMeloidogynegraminicola..............................................32 v vi 2.2.4. HatchingMeloidogynegraminicolajuvenilesforinfection...........................................37 2.2.5. InfectingriceplantswithMeloidogynejuveniles..........................................................37 2.2.6. ThelifecycleofMeloidogynegraminicolaat28°Cinaglass-house............................37 2.2.7. Extractionofeggsfromrootsofinfectedplants...........................................................38 2.2.8. Relativewatercontent..................................................................................................38 2.2.9. Volumetricsoilmoistureestimate.................................................................................38 2.2.10. Gravimetricestimationofevapotranspiration............................................................39 2.2.11. Determinationofpermanentwiltingpoint.................................................................39 2.2.12. TherelationshipbetweenRWCandsoilmoisturecontent.........................................39 2.2.13. Inducinggradualdroughtstress..................................................................................40 2.2.14. EffectofinitialinoculumdensityofM.graminicolaonrice........................................40 2.2.15. Optimisationofsimultaneousdroughtandnematodestress.....................................40 2.2.16. Expressionanalysisofstressresponsivegenes...........................................................41 2.2.17. Impositionofsimultaneousdroughtandnematodestress........................................43 2.3. RESULTS...............................................................................................................................47 2.3.1. MolecularcharacterisationofM.graminicolaisolates.................................................47 2.3.2. M.graminicolalifecycleat28°C...................................................................................47 2.3.3. InitialinoculumdensityofM.graminicolaonrice........................................................48 2.3.4. Expressionofpathogenesisrelatedgenes....................................................................48 2.3.5. PermanentwiltingpointofNipponbare........................................................................52 2.3.6. Relativewatercontentofplantsandsoilmoisturecontent.........................................52 2.3.7. Optimisationofsimultaneousdroughtandnematodestresstreatment.....................52 2.3.8. Simultaneousdroughtandnematodestressmodel.....................................................53 2.4. DISCUSSION..........................................................................................................................65 2.4.1. Whysimultaneousbioticandabioticstress?................................................................65 2.4.2. OptimisationofRKNinfection.......................................................................................65 2.4.3. Optimisationofdroughtstressregime..........................................................................66 2.4.4. OptimisationofsimultaneousdroughtandRKNinfection............................................69 3 CHAPTER.....................................................................................................................72 TRANSCRIPTOMICRESPONSEOFRICETOSIMULTANEOUSBIOTICANDABIOTICSTRESS.............................72 3.1. INTRODUCTION.....................................................................................................................72 3.1.1. Microarraytechnologyasatooloftranscriptomicanalysis..........................................72 3.1.2. Ricearrays......................................................................................................................74 3.1.3. Transcriptomicstudyofdroughtanddehydrationstressinrice..................................75 3.1.4. Transcriptomicstudyofnematodestressinrice...........................................................76 3.1.5. Transcriptomicstudyofsimultaneousbioticandabioticstressesinrice.....................77 vi vii 3.2. MATERIALSANDMETHODS.....................................................................................................80 3.2.1. Stresstreatments...........................................................................................................80 3.2.2. Tissuesampling..............................................................................................................81 3.2.3. RNAextraction...............................................................................................................81 3.2.4. Samplepreparationformicroarrayhybridisation.........................................................81 3.2.5. Microarraydataanalysis................................................................................................82 3.2.6. ValidationofmicroarrayresultsbyquantitativeRT-PCR..............................................83 3.2.7. Functionalanalysisofgenesdifferentiallyregulated....................................................84 3.3. RESULTS...............................................................................................................................88 3.3.1. RNAqualitycontrol........................................................................................................88 3.3.2. Qualitycontrolofthemicroarray..................................................................................88 3.3.3. Identificationofdifferentiallyexpressedgenes............................................................92 3.3.4. ValidationofmicroarrayresultsusingqPCR................................................................109 3.3.5. Functionalannotationofthedifferentiallyexpressedgenes......................................109 3.4. DISCUSSION........................................................................................................................118 3.4.1. Microarrayexperimentanddataanalysis...................................................................118 3.4.2. Identificationofdifferentiallyexpressedgenes..........................................................119 3.4.3. Transcriptomicresponsetodroughtstress.................................................................120 3.4.4. Transcriptomicresponsetonematodestress.............................................................123 3.4.5. Transcriptomicchangesinresponsetosimultaneousdroughtandnematodestress 126 4 CHAPTER...................................................................................................................131 FUNCTIONALANALYSISOFCANDIDATEGENESINRICE...................................................................131 4.1. INTRODUCTION...................................................................................................................131 4.1.1. Ricereversegenetics...................................................................................................131 4.1.2. Evaluatingmutantstress-responsiveness....................................................................133 4.2. MATERIALANDMETHODS.....................................................................................................136 4.2.1. Selectionofcandidategenesandricemutants...........................................................136 4.2.2. Growthconditionsandstresstreatment.....................................................................136 4.2.3. Datacollection.............................................................................................................136 4.2.4. Statisticalanalysis........................................................................................................137 4.3. RESULTS.............................................................................................................................144 4.3.1. Growthparameters.....................................................................................................144 4.3.2. Yieldparameters..........................................................................................................146 4.3.3. Nematodesusceptibility..............................................................................................147 4.4. DISCUSSION........................................................................................................................148 vii viii 5 CHAPTER...................................................................................................................152 THEEFFECTOFSIMULTANEOUSSTRESSONNIPPONBAREANDIR64RICECULTIVARSINFIELDCONDITIONS.152 5.1. INTRODUCTION...................................................................................................................152 5.1.1. Droughtstressonthericecrop...................................................................................153 5.1.2. Nematodeinfestationinthericecrop.........................................................................153 5.1.3. Chlorophyllparametersasameasureofstressinplants............................................154 5.1.4. Hormonalcrosstalkbetweenbioticandabioticstressresponses..............................155 5.2. MATERIALSANDMETHODS...................................................................................................157 5.2.1. Fieldpreparation..........................................................................................................157 5.2.2. Experimentaldesign....................................................................................................157 5.2.3. Datacollection.............................................................................................................158 5.2.4. Expressionanalysisofstressresponsivegenes...........................................................159 5.2.5. Statisticalanalysis........................................................................................................159 5.3. RESULTS.............................................................................................................................166 5.3.1. Quantifyingseverityofdroughtimposed....................................................................166 5.3.2. Effectofstresstreatmentsonchlorophyllcontentandmaximumquantumefficiencyof photosystemII..........................................................................................................................167 5.3.3. Hormonalsignallingduringstresstreatmentsinthefield..........................................168 5.3.4. Effectofstressregimesongrowthandyieldparameters...........................................169 5.4. DISCUSSION........................................................................................................................181 5.4.1. Molecularstressresponse...........................................................................................181 5.4.2. Responseofricetowardsseveredroughtstress.........................................................182 5.4.3. Responseofricetonematodeinfection......................................................................183 5.4.4. Responseofrice tosimultaneousdroughtandnematodeinfection.........................184 6 CHAPTER...................................................................................................................186 REDUCTIONOFPHYTATEBYDOWN-REGULATIONOFARABIDOPSISTHALIANAMIPSANDIPK1GENESALTERS SUSCEPTIBILITYTOCYSTNEMATODES........................................................................................186 6.1. INTRODUCTION...................................................................................................................186 6.2. MATERIALSANDMETHODS...................................................................................................191 6.2.1. Plantmaterialandgrowthconditions.........................................................................191 6.2.2. Nematodecollection....................................................................................................191 6.2.3. Nematodeassay...........................................................................................................191 6.2.4. Genotypingofthemips1-2mutant.............................................................................194 6.2.5. Growthanalysesofmutants........................................................................................194 6.2.6. Statisticalanalyses.......................................................................................................194 viii ix 6.3. RESULTS.............................................................................................................................195 6.3.1. EffectoflowphytatecontentongrowthanddevelopmentofArabidopsismutants 195 6.3.2. EffectofdisruptionofthefirstandlaststepsofphytatesynthesisonH.schachtii parasitism..................................................................................................................................200 6.3.3. EffectofdisruptionofthefirstandlaststepsofphytatesynthesisonMeloidogyne graminicolaparasitism..............................................................................................................201 6.4. DISCUSSION........................................................................................................................207 6.5. CONCLUSION......................................................................................................................211 7 CHAPTER...................................................................................................................213 GENERALDISCUSSION..........................................................................................................213 7.1. RELEVANCEOFCROPPLANTSININVESTIGATINGPLANTSTRESSRESPONSES....................................213 7.2. THENEEDFORAREALISTICSTRESSMODELINMOLECULARSTUDIES..............................................214 7.3. SIMULTANEOUSMULTIPLESTRESSESINDUCESPECIFICSUITEOFGENES..........................................216 7.4. PLANTRESPONDDIFFERENTLYTOASETOFSTRESSESINFIELDCOMPAREDTOTHESAMESTRESSES IMPOSEDINCONTROLLEDENVIRONMENT..........................................................................................217 7.5. THEORDERINWHICHSTRESSESOCCURALTERSPLANTRESPONSE.................................................219 7.6. MANIPULATINGNUTRITIONCOMPONENTOFCROPPLANTSCANAFFECTSTRESSRESPONSES.............221 REFERENCES....................................................................................................................223 LISTOFABBREVIATIONS..................................................................................................................257 ix x List of Figures FIGURE1-1LIFECYCLEOFAROOT-KNOTNEMATODE.............................................................................................18 FIGURE1-1LIFECYCLEOFACYSTNEMATODE.......................................................................................................19 FIGURE2-1GROWTHCONDITIONSFORRICEPLANTSDURINGTHEEXPERIMENT...........................................................34 FIGURE2-2STANDARDCURVESANDAMPLIFICATIONPLOTSFORTHEPRIMERPAIRUSEDTOAMPLIFYNORMALISINGGENE TUMOURHOMOLOGUEPROTEIN(TPH).....................................................................................................45 FIGURE2-3THEDISSOCIATIONCURVEANDAMPLIFICATIONPLOTSFORTHEPRIMERPAIRUSEDTOAMPLIFYTHENORMALISING GENETUMOURHOMOLOGPROTEIN(TPH)INTHETREATMENTSAMPLES..........................................................46 FIGURE2-4THE18SRDNAFRAGMENTAMPLIFIEDFROMTHEGENOMICDNAOFSINGLENEMATODESOFM.GRAMINICOLA COLONY...............................................................................................................................................49 FIGURE2-5.SEQUENCEIDENTITYOFTHE18SRDNASEQUENCEAMPLIFIEDFROMASINGLEM.GRAMINICOLAJUVENILEWITH THE18SRDNASEQUENCEOFM.GRAMINICOLA(GENBANK:AF442196.1)USINGCLUSTALW...........................50 FIGURE2-6LIFECYCLEOFM.GRAMINICOLAAT28ºCONRICECVNIPPONBAREINSOIL...............................................51 FIGURE2-7PHENOTYPICCHARACTERISATIONOFRICEPLANTSUNDERDIFFERENTREGIMESOFM.GRAMINICOLAINFECTION.55 FIGURE2-8EXPRESSIONANALYSISOFPATHOGENESISRELATEDGENE(PR1B)INAMATURELEAFOFTHERICEPLANTSUNDER MELOIDOGYNEGRAMINICOLAINFECTIONAT2DPIAND4DPI.........................................................................56 FIGURE2-9VARIATIONSINSOILMOISTUREANDPOTWEIGHTOFSTRESSEDANDCONTROLPLANTSDURINGTHECOURSEOF DROUGHTINDUCTION.............................................................................................................................57 FIGURE2-10THERELATIVEWATERCONTENTOFRICELEAVESDURINGTHECOURSEOFSEVEREDROUGHTSTRESSINDUCTION. ..........................................................................................................................................................58 FIGURE2-11RELATIONSHIPBETWEENRELATIVEWATERCONTENTANDMOISTURELOSSDURINGTHECOURSEOFSEVERE DROUGHTSTRESS...................................................................................................................................59 FIGURE2-12THEEXPERIMENTALSETUPANDNON-DESTRUCTIVESTRESSINDICATORSFORDROUGHTSTRESSANDNEMATODE INFECTIONINRICE..................................................................................................................................60 FIGURE2-13EFFECTOFSIMULTANEOUSROOT-KNOTNEMATODEWITHDROUGHTSTRESSUNDERDIFFERENTMOISTURE REGIMESONROOTSIZEOFTHERICEPLANTSATTILLERINGSTAGE.....................................................................61 FIGURE2-14 EXPRESSIONANALYSISOFSTRESSRESPONSIVEGENESINROOTSANDLEAVESOFRICEPLANTSUNDERDROUGHT STRESS,NEMATODEINFECTIONANDSIMULTANEOUSMULTIPLESTRESS.............................................................62 FIGURE2-15.PHENOTYPEOFRICEPLANTSUNDERDROUGHTSTRESS,NEMATODEINFECTIONANDSIMULTANEOUSDROUGHT ANDNEMATODESTRESS..........................................................................................................................63 FIGURE2-16.PHENOTYPICCHARACTERISATIONOFRICEPLANTSUNDERDROUGHTSTRESS,NEMATODEINFECTIONAND SIMULTANEOUSDROUGHTANDNEMATODESTRESS.......................................................................................64 FIGURE2.17SCHEMATICREPRESENTATIONOFTHEEXPERIMENTALSET-UPFORSTRESSTREATMENTSDESIGNEDFORTHE MICROARRAYANALYSIS...........................................................................................................................71 FIGURE3-1THEEXPERIMENTALPLANFORDETERMININGTRANSCRIPTOMICRESPONSESINRICEUNDERSIMULTANEOUSBIOTIC ANDABIOTICSTRESS...............................................................................................................................79 x

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simultaneous biotic and abiotic stress were dominated by the drought element accompanied by a unique set of genes that were only responsive to the
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