Functional Verification Of Persimmon 9-Lipoxygenase In Plant Senescence And Defense Response

Persimmon(Diospyros kaki L.) is a high-economic-value crop, which is rich in a variety of nutrients and minerals and high in medical and health care value. However, as a kind of climacteric fruit, persimmons soften and decay quickly after harvest, thereby a ecting their marketability and causing great economic losses. Therefore, it is critical to control fruit ripening and softening in the storage of persimmon fruit. Lipoxygenase has long been considered to associate with lipid peroxidation which caused the degradation of cell membrane, as a result, the senescence of fruit and leaves was started. To date, many studies have shown that the expression level of LOX genes was up-regulated during ripening and senescense or stresses induction. However, there has always been lack of direct genetic evidence and systematic research. In this study, expression patterns of persimmon‘Fupingjianshi’(Diospyros Kaki L)LOX genes were investigated, furthermore, the molecular regulation mechanisms of Dk LOX3 in senescence and defense response were verified. The main results were as follows.1. Different tissues from adult persimmon trees and young persimmon fruits were sampled to investigate the expression patterns of persimmon LOX gene family. In young fruits, DkLOX1 expressed specifically in fruit, while DkLOX3 and DkLOX4 were expressed in all tissues, i.e., fruits, leaves, flowers, calyces, and stems. DkLOX1 and DkLOX4 were expressed at a higher level than DkLOX3, and there were two expression peaks of DkLOX1 and DkLOX4 at 20 and 100 th days after full bloom, accompanying by the persimmon growth curve which presented a double “S”respectively. The expression of DkLOX3 was at particularly low levels in the developing stages. The results raised the possibility that DkLOX1 and DkLOX4 were involved in fruit development.2. The expression profiles of three persimmon LOX genes were examined in persimmon fruit under different stress treatments. The expression of DkLOX1 was slightly up-regulated and DkLOX4 was induced by mechanically damage and cold treatment. With the other treatments, the expression of the DkLOX1 and DkLOX4 decreased after harvest and displayed low transcript abundances during the whole storage duration. Compared to DkLOX1 and DkLOX4, the expression of DkLOX3 and LOX activity were enhanced after harvest, andthetranscriptabundanceofdklox3 reachedthehighestlevelaccompanyingwiththeethylenereleasingpeak.inaddition,comparedwithcontrol,dklox3 expressionwasenhancedrapidlyandremainedatahigherlevelwithwoundtreatment,moreover,dklox3expressionwasalsosignificantlyinducedwithhighcarbondioxidetreatmentat4 daysafterharvestandthendecreasedandmaintainatastablelevelpriortotheclimactericperiod.theseresultsprovideddirectgeneticevidencefordklox3playasignificateinfruitripeningandsofeningandstressresponse.3.therecombinantplasmiddklox3-pvbg2307 wasconstructed,andtransgenictomatoandarabidopsiswereobtainedviaagrobacterium-mediatedtransformation.tomatofruitswereharvestedatthematuregreenstageandstoredunderthesameconditions,itwasobservedthatmaturationandsenescencewasacceleratedandstorabilitywasreducedintransgenictomatofruits,accompaniedbyhighermalondialdehydecontentsandlycopeneaccumulation,advancedethylenereleasepeakandelevatedexpressionofethylenesynthesisgenes,includingacs2,aco1,andaco3.inaddition,dklox3 overexpressionpromoteddarkinducedtransgenicarabidopsisleafsenescencewithmorechlorophyllloss,increasedelectrolyteleakage,rosaccumulationandmalondialdehydecontents.overall,theseresultsprovideddirectevidencesforlipoxygenaseparticipatinginfruitripeningandsofteningandleafsenescence.4.seedsofthetransgeniclinesexhibiteda24%–55%highergerminationratesthanthatofwildtypeunderosmoticstress.furthermore,whenculturedverticallyonosmoticmedium,therootlengthofwtwasinhibitedmoreseverely,nearlylessthanhalfthelengthofthatobservedinthetransgeniclines.fordroughtstress,therosetteleavesofwildtypewereshriveledmoreseriously,24hafterre-watering,moretransgenicplantsresumednormalgrowththanwildtype.whenseedingsweretreatedwithnacl,thesuppressionoftherosetteleafgrowthofwtwasmoreseriousthanthatinthetransgeniclines.intransgenicplants,saltanddroughtstresstreatmentsledtoloweraccumulationofrosandupregulationofabaindependentstress-responsivegenerd29aandabadependentstress-responsivegenesexpression,includingrd22,rd29 b,nced3and,exceptforfry1,whichplaysanegativeroleinstressresponse.theresultssuggestedthatdklox3 playspositiverolesinstressresponsepossiblyviaregulatingreactiveoxygenspeciesaccumulationandstressresponsivegenesexpression.5.detachedleavesofthetransgeniclinesandwildtypewereinoculatedwithpstdc3000.ectopicexpressionofdklox3inarabidopsisresultedinenhancedresistancetopstdc3000.hypersensitiveresponsewasoccurredintransgenicleaves,accompaniedbymoreo2-、h2o2andcelldeathandlowerantioxidantenzymeactivityincludingpod,catandsodat12hand24 hafterinoculation.however,at48hand72 hafterinoculation,activitiesofantioxidantenzymeintransgeniclineswerehigherthanwildtype,whilerosaccumulationandthebacterialgrowthwerelessthanwildtype.quantificationofjasmonicacidandsalicylicacidshownthatsacontentsintransgeniclineswerehigherthanwildtype,theexpressionlevelofsamarkergenepr1andpr2 wasalsoupregulated.whilethereisnosignificantdifferenceinjacontentsandtheexpressionlevelofjamarkergenepdf1.2.alltheseresultssuggestedthatdklox3overexpressionreducedthesensitivitytopstdc3000 viaregulatingrosaccumulationandsabiosyntheticpathways.6.detachedleavesofthetransgeniclinesandwildtypewereinoculatedwithbotrytiscinerea.ectopicexpressionofdklox3 inarabidopsisresultedinenhancedresistancetobotrytiscinerea.afterinoculatedwithbotrytiscinerea,thereweremoreo2-、h2o2andlowerantioxidantenzymeactivityincludingpod,catandsodduringthefirst12 hafterinoculation.however,afterinoculation24 h,48hand72h,activitiesofantioxidantenzymeintransgeniclineswerehigherthanwildtype,whilerosaccumulationwaslessthanwildtype.quantificationofjasmonicacidandsalicylicacidshownthatbothsaandjacontentsandtheirmarkergenepr1,pr2andpdf1.2hadnosignificantdifference.thedatarevealedthatlipoxygenaseenhancedresistancetobotrytiscinereaprobablybyregulatingrosaccumulation,butnotsaorjametabolicpathway.