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forhighsugar,ascomparedtolowsugargenotypes(Papini-Terzietal.2007).Thedifferentiallyexpressedgenesidenti?edbelongedtoseveralfunctionalcategoriesincludingcalciumsignaling,stressresponses,transcription,andubiquitination.ThecategorieswiththehighestnumberofhitsincludedproteinkinasesfromtheSNF-relatedfamilyofkinases,auxinhormonesignaling,theCypfamilyofcytochromeP450monoxygenases,andotherstress-relatedgenes.
Theexpressionpro?leofgenesassociatedwithsignaltransductionwasalsoevaluatedinleavesfromhighsugarandlowsugarsugarcaneplantsfromanF1progenyselectedfromacrossbetweenthesugarcanevarietiesSP80–180andSP80–4966(Felix2006).Twenty-fourgenesweredifferentiallyexpressedbetweenhighandlowsugarplants.Fivehadhighertranscriptlevelsinhighsugarplants,includinganomega-3fattyaciddesaturaseputativelyinvolvedinmethyljasmonate(MeJa)signaling,aputativereceptor-likeserine/threoninekinase,andanMybdo-maintranscriptionfactor.Mostofthegeneshadhigherexpressionsinlowsugargenotypes,suchasthoseencodingthree14-3-3likeproteinsandanSNF1-relatedprotein.AhomologueofthisproteinphosphorylatestheenzymeSPSinvitro(Sug-denetal.1999),makingitaputativetargettointeractwith14-3-3proteins,whichinturnreducestheSPSactivity(Toroseretal.1998;Huberetal.1998).
Theef?ciencyandcontrolofcarbon?xationandallocation,whichisaffectedbysinkstrength(Wattetal.2005),mayberegulatedatthesourcetissues.Maetal.(2004)investigatedgeneexpressioninsourcetissuesusingESTanalyses,andmorerecently,serialanalysisofgeneexpression(SAGE)wasused(CalsaandFigueira2006).Sugarcane,aswellasmaizeandsorghum,wasconsideredtoop-erateundertheNADP-malicenzyme(NADP-ME)pathway(BowyerandLeegood1997),althoughahighlyexpressedphotosynthesis-relatedphosphoenolpyruvatecarboxykinase(PEPCK)hadalreadybeendetectedandvalidatedinmaizeleafbundlesheathcells(Furumotoetal.1999,2000).C4grassessuchassugarcane,maize,andsorghum,containanatomicalandphysiologicaladaptationstooptimizeCO2?xationforcarbohydratebiosyntheses(Brownetal.2005).Basically,threeC4photosyntheticprimarycarboncyclepathwayshavebeendescribedthatdifferinthefour-carbonorganicacidintermediatetransportedfromthemesophylltothebundlesheathcells(malateand/oraspartate),inthethree-carbonacidreturnedtothemes-ophyllcells(pyruvateoralanine),aswellasthedecarboxylationenzymepresentinthebundlesheathcells,whichcanbeeitherNADP-ME,NAD+malicenzyme(NAD-ME),or(PEPCK)(TaizandZeiger1998).ThecombinedSAGEandrealtimequantitativePCR(RT-qPCR)results(CalsaandFigueira2006)suggestedthatPEPCKdecarboxylationappearedtopredominateoverNADP-MEinmature?eld-grownsugarcaneleaves,incontrasttotheconventionalNADP:MEmodelacceptedforsugarcane,althoughbothmayoccur.
21.5.3.2ResponsestoEnvironmentalChallenges
Plantsreacttochangesintheenvironmentthroughanarrayofcellularresponsesthatareactivatedbystressstimuli,leadingtoplantdefenseand/oradjustmenttoadverseconditions.Physiologicalchangeselicitedbyexternalsignalscanbemodulatedby
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transcriptionalregulationresultingintheinductionorrepressionoftargetgenes.Studieshavebeenconductedtounraveltheresponsesofsugarcanetobioticandabioticstressandtheroleofphytohormonesintheseprocesses.Drought
Droughtisaconditionofspecialinterestwithrespecttosugarcane,sincewaterscarcityconditionspreventtheexpansionofthisculturetovastareasintropicalre-gions.Toidentifydifferentiallyexpressedgenesinresponsetohydricstress,cDNAmicroarraysrepresenting1,545geneswereused(Rochaetal.2007).Amongthedifferentiallyexpressedgenes,regulatorsofdrought-responsivegenessuchastheWRKYandMYCtranscriptionfactors(Abeetal.1997)wereidenti?ed.Coldanddroughtsignalingoverlapandmanyoftheresponsesaremediatedbythephyto-hormoneABA.Accordingly,lowtemperature-induced(LTI)proteinswereseentobeup-regulatedinresponsetolackofwaterinsugarcane,andgenesinducedbyABAhavealsobeenfoundtobeinducedbydrought,includingtwodelta-12oleatedesaturases,oneS-adenosylmethioninedecarboxylase,andaproteinphosphataseABI1/ABI2(TahtiharjuandPalva2001)thatregulatesstomatalclosure.Asugar-canetranscriptionfactorhomologoustoriceDREB2wasinducedandmayrepresentanimportanttranscriptionfactorfortheregulationofsugarcanedroughtresponses,sincetheover-expressionofDREB2AinArabidopsisledtothedevelopmentofplantstoleranttodrought(Sakumaetal.2006).Othergenesidenti?edincludeanS-adenosylmethioninedecarboxylase,knowntoaccumulateinresponsetosalinityanddrought(LiandChen,2000)andfattyaciddesaturases(FAD2),directlyrelatedtodroughttolerance(Zhangetal.2005;Imetal.2002).Cold
Coldstress,whichincludeslowtemperaturesabove(chilling)andbelow(freezing)0?C,causesseverelossesofmostcropplants,duetotheformationofextracel-lularice(XinandBrowse2000).Sugarcaneisconsideredtobeacold-sensitivecrop(TaiandLentini1998),andalthoughsugarcane?eldsarerestrictedtotrop-icalandsubtropicalregions,coldstressisnotunusualintheseareas,decreasingsugarproductivity.Nogueiraetal.(2003)evaluatedthegeneexpressionpro?leinsugarcaneplantletsexposedto4?C.Thirty-fourcold-induciblegenesand25cold-repressedgeneswerefound.Basedonthesedata,amodelofsugarcaneresponsetocoldstresswasproposed.Intheirmodel,severaltranscriptionfactors,suchasanABI3-interactingprotein2,anOsNAC6andanOCSBF-1,regulatethetranscriptionofproteinsinvolvedintheprotectionagainstoxidativestress,sugartransporters,proteindegradationandcellwallsynthesis.Thesegenescouldbegoodtargetsforstudyandtopossiblyimprovesugarcanecoldtolerance.PhosphorusDe?ciency
Phosphorus(P)isanessentialnutrientbecauseitisusedinalargenumberofbiologicalprocesses,fromnucleicacidsbiosynthesistotheregulationofenzyme
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activities.PlantstakeupPasinorganicphosphate(Pi),andhavedevelopedseveralstrategiestocopewiththelowavailabilityofPiinthesoil,whichisusuallyintherangefrom2to10mM(Raghothama1999).Sugarcaneisacropthatperformswellinacidsoils,indicatingtheuseofstrategiestoovercomePde?ciency.Toaccesstheexpressionpro?leofgenesinresponsetoPstarvation,Rochaetal.(2007)evaluatedsugarcaneplantletsgrownintheabsenceofP.Surprisingly,onlygenesrepressedduetoPstarvationwerefound.Theexpressionpro?leobtainedpointedtochangesinproteinN-glycosylationandredoxstatusduetoanalteredexpressionofanN-acetylglucosamine-1-phosphatetransferaseandtwothioredoxins.GeneshomologoustoanMYBtranscriptionfactorandanethyleneinsensitive-like(EIL)transcriptionfactorputativelyinvolvedintheethyleneresponsewerealsorepressed.Thesedataindicatedthatunderlowlevelsofthenutrient,sugarcanerootsmightbeunderseveremetabolicrestraint,inlinewiththeobservationsinArabidopsis,wheregenesrelatedtophotosynthesiswererepressedinresponsetoPistarvation(Wuetal.2003).
Herbivory
Insectpestsfrequentlychallengesugarcaneproductivity.Eventhough,overthelastfewdecades,highlyproductivesugarcanecultivarswithenhancedinsectpestre-sistancehavebeendevelopedinconventionalbreedingprograms,moderncultivarsappeartoretainalowerdegreeofresistancewhencomparedtowild-typegeno-types.Theavailabilityofinsect-controlgenesthatcouldbegeneticallyengineeredtoobtainpestresistantvarietiesisofsigni?cantinterest.Inasearchforsugarcaneorthologsofgenesthatarepotentialtargetsforthemanagementofinsectresis-tance,Falcoetal.(2001)identi?ed,amongtheSUCESTsequencesESTscodingforproteinaseinhibitors,alpha-amylaseinhibitors,lectins,chitinases,andpolyphenoloxidases.Inthisstudy,putativesystemicandconstitutivewoundresponseproteinswereidenti?ed.
ThesugarcaneborerDiatraeasaccharalisisthemajorsugarcanepestinBrazil,causingplantdeathduetoapicalbuddeath(deadheart)inuptofour-month-oldplantsanddamagetolateralbuddevelopment,aerialrooting,weightloss,andstalkbreakageinolderplants.Theattackalsoallowsforinfectionbyoppor-tunisticfungi,whichresultsinproductionlossesforboththesugarandalcoholindustries(Bragaetal.2003).Theexpressionpro?leofavarietyhighlysus-ceptibletotheborerwasobtainedinresponsetotheinsectattackusingcDNAmicro-arrays(Rochaetal.2007).Theexpressiondataindicatedastronginduc-tionofapathogenesis-relatedproteinsimilartothaumatin,24haftertheonsetofthisstress.Theseproteinsareimportantforplantdefensemechanismsandmaypresentanti-fungalaction,endo-?1,3-glucanaseactivity,andtrypsinor?-amylaseinhibitoryactivity(Grenieretal.1999;Francoetal.2002).Furthercharacteriza-tionofthissugarcanethaumatin-likeproteinshouldbecarriedouttode?neitsactivityandthedefensemechanismthatitmaytriggeragainstthesugarcanestalkborer.
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EndophyticBacteria
InBrazil,thelong-termcontinuouscultivationofsugarcanewithlowNfertilizerinputs,withoutapparentdepletionofthesoil-Nreserves,ledtosuggestionsthatN2-?xingbacteriaassociatedwiththeplantsmightbethesourceofagronomicallysigni?cantNinputsforthiscrop.Yearsofstudyledtotheconclusionthatthedia-zotrophsthatinfectedtheinterioroftheplants,suchasthe‘endophyticdiazotrophs’wereresponsiblefortheincreasednitrogencontributiontoBraziliansoils(Boddeyetal.2003).DiazotrophicacetobacterswerealsoisolatedfromsugarcanerootsorsoilcollectedfromfourregionsinQueensland,Australia(LiandMacrae1991).However,biologicalnitrogenutilizationseemstoberestrictedtosomecultivarsandregions.InSouthAfricaforinstance,itwasshownthatbiologicalnitrogen?xationdidnotcontributetothenitrogendemandofacommerciallygrowncultivar(Hoefslootetal.2005).
InBrazil,sugarcaneculturebene?tsconsiderablyfromitsassociationwithN2-?xingendophyticbacteria(Herbaspirillumseropedicae/Herbaspirillumrubrisub-albicansandGluconacetobacterdiazotrophicus).Unlikerhizobium/leguminosaesymbiosis,wherethebacteriaarerestrictedtonodules,Herbaspirillumspp.andG.diazotrophicusareendophytic,andcolonizetheintercellularspacesandvasculartissuesofmostplantorgans,withoutcausingdamagetothehost(JamesandOlivares1998;Rheinhold-HurekandHurek1998).Thesebacteriapossiblypromoteplantgrowthbynitrogen?xationandalsobytheproductionofplanthormones(Sevillaetal.2001).Despitethenon-pathogenicaspectsofthisinteraction,plantsshouldlimitbacterialgrowthinsidetheirtissuestoavoiddiseasedevelopment(Olivaresetal.1997).Itisbelievedthatsugarcaneplantsrecognizethesemicroorganismsandac-tivatedefenseresponsesuntiltheestablishmentofanef?cientassociation(Vina-greetal.2006).UsingcDNAmicro-arrays,fourresistancegeneanalogswerefoundtoberesponsivetotheendophyticassociation(Rochaetal.2007).Plantdiseaseresistancegenesmediatespeci?crecognitionofpathogensviathepercep-tionofavirulencegeneproducts(reviewbyEllisetal.2000).TworesistancegeneanalogswereinducedonaccountoftheassociationwithbothHerbaspirillumandGluconacetobacterdiazotrophicus.InoculationwithGluconacetobacteralsoledtotheinductionofasalicylicacidbiosynthesisgene.Salicylicacidaccumulatesinplanttissuesinresponsetopathogenattack,andisessentialfortheinductionofsystemicacquiredresistanceandforsomeresponsesmediatedbyresistancegenes(Gaffneyetal.1993;Delaneyetal.1994;Muretal.1997).Theexpres-sionofaPP2Cand?vetranscriptionfactorswasalteredwhentheplantswerecultivatedinassociationwithendophyticbacteria.Amongstthese,thereweretwozinc-?ngertranscriptionfactors,oneofwhichwasupregulatedbyinoculationwitheitherGluconacetobacterorHerbaspirillum.Apossibleroleforphosphatasesandzinc-?ngertranscriptionfactorsinresponsetoendophyticbacteriahasalsobeenpointedoutbytheinsilicoanalysisofESTs,thatidenti?edaSAScorrespondingtothesecategories,exclusivelyorpreferentiallyexpressedinthecDNAlibrariesconstructedfromplantsinoculatedwithGluconacetobacterandHerbaspirillum(Vargasetal.2003).
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21.5.3.3PhytohormoneSignaling
Hormonessuchasmethyl-jasmonate(MeJA)andabscisicacid(ABA)arekeyregu-latorsofmechanismsthatintegrateplantresponsestointernalandexternalstimuli.Geneexpressionchangesinresponsetothesehormoneshavebeenevaluatedinsugarcane(Boweretal.2005;DeRosaetal.2005;Rochaetal.2007).MeJainducedseveralgenesencodingproteinhomologuesrelatedtophytohormonesignaling,in-cludinganMYBtranscriptionfactorandareceptor-likeproteininsugarcaneroots(Boweretal.2005)andazinc?ngerprotein,aheatshockfactor,andaproteinkinaseinyoungsugarcaneleaves(DeRosaetal.2005).Inasurveyofmostofthesugarcanehomologuesforknowngenesrelatedtohormonesignaling,Rochaetal.(2007)foundthatMeJainducedtheexpressionofproteinkinases,anMYBtranscriptionfactor,andanNACprotein,andrepressedanotherproteinkinase.ABAinducedtheexpressionofgenesencodinghomologuestotworeceptorSer/Thrki-nases,aphosphataseandasmallGTPase,whileaproteinkinasehomologuewasrepressed.Schl?gletal.(2006)evaluatedtheexpressionpro?leofthewholesetofknownb-ZIPtranscriptionfactorsinresponsetoABAandMeJa.TwobZIPswereinducedbyABAandfourwererepressed,whiletwootherswereinducedbyMeJa.21.5.3.4TransposonExpression
RetrotransposonsmobilizethemselvesthroughanRNAintermediateandarenowconsideredoneofthemajorforcesdrivinggenomeexpansioninplants(Pieguetal.2006),whiletransposonsusuallymoveusingeitheracut/pasteoracopy/pastemechanism.Recently,ahypothesisontheimpactoftransposableelements(TE)ongenomicstructure,generegulation,andevenonfunctionhasbeenproposed(CasacubertaandSantiago2003;Kashkushetal.2003;BundockandHooykaas2005).Twenty-onedifferentfamiliesofTEswereidenti?edintheSUCESTcollec-tion,ofwhich54%correspondtoclassicaltransposonsand46%toretrotransposons(Rossietal.2001).Furtherstudiestovalidatetheexpressionpro?leoftheTEfam-iliesidenti?edweredeveloped,whichcon?rmedthatthecallusisthetissuewithmoreexpressedTEfamilies(Araujoetal.2005).AlthoughithasbeenproposedseveraltimesthattissueculturesomaclonalvariationcouldbearesultofTEactivity,thisisthe?rstreportthatdemonstratesthatcallusisindeedatissuewheredifferentTEsareexpressedatthesametime.Focusonparticularsugarcanefamilieshigh-lightedtheexistenceoflineagesofelementswithdiverselevelsofrepresentationinthegenome(Rossietal.2004).
21.6GeneticEngineering
Genetictransformationhasbeenextensivelyusedtoproducecommercialvarietiesofanumberofdifferentcropssuchassoybeans,corn,andcotton,expressingtraitssuchasherbicideandinsectresistance,resultinginimprovementsinthefarmers’