Functional Analysis Of OsRopGAP5 On Flowering,Plant Height And Other Yield Related Traits In Rice

ROP(Rho-related GTPases from plants)family proteins play a plethora of roles in regulating plant development and immune response.Recently,data from our lab support a likely involvement of Arabidopsis ROP2 in modulating carbon and nitrogen balance(C/N balance).Considering its potential application to crop improvement,we investigated relationship between ROP signaling pathway and C/N balance in rice,by studying molecular functions of ROP2’s homologs in rice,as well as ROP regulators OsRopGAP(Rho GTPase activating protein)and OsRopGEF(Rho GTPase guanine nucleotide exchange factor).The aim of current study is to explore potential roles of ROP signaling pathway in regulating rice yield.Field test in Changsha showed that,when compared with wild type Nipponbare(WT),significant differences of plant height were observed in transgenic plants with altered expression of OsROP6,OsRopGAP 1 and OsRopGAP5,among which I mainly focused on elucidating function of OsRopGAP5.Besides exhibiting higher plant height,OsRopGAP5 overexpression plants also showed prolonged heading dates,longer flag leaves and panicles,larger number of branches,spikelets and grains per panicle,higher grain setting rates as well as higher yield compared with those of WT plants in further field tests.OsRopGAP5-RNAi plants,however,showed completely opposite phenotypes,further confirmed that OsRopGAP5 is involved with rice yield.Unfortunately,we did not observe any differences of carbon and nitrogen contents between WT and OsRopGAP5 overexpression plants,neither were any phenotypic differences between WT and OsRopGAP5 overexpression plants that grown under balanced or imbalanced C/N medium.In addition,OsRopGAP5 related transgenic plants showed obvious different phenotype with that of rice grown under nitrogen deficient condition in field tests.Thus,we excluded the possibility that OsRopGAP5 regulate rice yield by modulating C/N balance.Anatomical observation indicated that the increased plant height of OsRopGAP5 overexpression plants was largely due to increased cell division in internodes compared with WT plants.qRT-PCR and GUS analysis showed that OsRopGAP5 expressed most abundantly in culm and panicle,as well as in root,leaf blade and leaf sheath.Furthermore,using RNA in situ hybridization assay,we found that transcripts of OsRopGAP5 were increased during early developmental stage of panicle.All these data imply that OsRopGAP5 may regulate development of culm and panicle in rice.Using bioinformatic analysis,we found several hormones,stress and light responsive elements in promoter of OsRopGAP5,among which the light responsive elements are most abundant.Because photoperiod is a key regulator of flowering,we performed field experiments in different regions with different photoperiod.Interestingly,phenotypic differences between WT and OsRopGAP5 transgenic plants occurred only at short day conditions(SD).qRT-PCR analysis revealed that rice florigen genes heading date 3a(Hd3a)and rice flowering locus T1(RFT1)were up-regulated by SD treatment in both WT and OsRopGAP5 overexpression plants compared with long-day(LD)treatment.However,induction of these two genes by SD treatment was attenuated in OsRopGAP5 overexpression plants compared with WT plants.Whaf’s more,early heading data 1(Ehd1),the upstream regulator of Hd3a and RFT1,but not heading date 1(Hd1),showed similar expression patterns.These results suggest that high level of OsRopGAP5 could attenuate induction of Ehd1 by SD treatment,which results in relatively lower induction of Hd3a and RFT1 and thus late flowering phenotype of OsRopGAP5 overexpression plants.Several other lines of evidence hinted that phenotype of transgenic plants in SD condition might be mediated by Ghd7.By analyzing expression level of OsRopGAP5 in different photoperiods and light conditions,we found that:(1)OsRopGAP5 presents diurnal expression patterns under both SD and LD conditions.The oscillation peaks in the morning under LD condition,while before dawn under SD condition.(2)OsRopGAP5 shows higher expression levels under LD condition than under SD condition,especially during flowering inducing stage;(3)Expression of OsRopGAP5 is mildly down-regulated by red and blue light.Phylogenic tree analysis showed that homologs of OsRopGAP5 protein only exist in plant kingdom,especially in angiosperms.Furthermore,metabolomic profiling showed that contents of sugar,several organic acids and most amino acids were increased in OsRopGAP5 overexpression plants compared with those of WT plants,which were only found under SD condition but not under LD condition.The relationship of photoperiodic dependent changes of carbon and nitrogen metabolites and regulation of flowering and rice yield,however,is unclear based on current study.In summary,this study identified a ROP GTPase activating protein encoding gene OsRopGAP5,which is involved in regulating rice yield.Overexpression of OsRopGAP5 can increase plant height and other yield related traits under SD condition.We further found that OsRopGAP5 regulate flowering by affecting expression of Ehd1,while expression of OsRopGAP5 was under regulation of photoperiod.This work might be beneficial to the yield improvement of Nipponbare and other japonica varieties in low latitude regions.