Rice response to simultaneous biotic and abiotic stresses

271 Pages·2014·9.29 MB·English

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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
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Thecandidateconfirms that theworksubmittedis his/herownandthat appropriate
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Assertionof moral rights(optional):
Theright ofRituShreeJain tobeidentifiedas Authorofthis workhas beenassertedby
herinaccordancewiththeCopyright,Designs and Patents Act 1988.
©<2013TheUniversityof Leeds andRituShree Jain
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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.
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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.
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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
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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
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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
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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
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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
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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
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