| Tomato(Solanum lycopersicum)is the world’s most produced and consuming vegetables.Tomato quality including flavor quality,nutrients and appearance impacts the economic benefits of tomato production.As the sixth plant hormone,brassinosteroids(BRs)has been shown to be involved in plant growth and development,stress response and other physiological processes.However,relatively little is known as regard to the function of BRs in fruit development,and the existing studies are relatively fragmentary and not systematic enough.Therefore,the systematic study on the role of BRs in tomato fruit has a strong practical significance.In this paper,the transgenic plants overexpressing BRs biosynthetic gene(OE-CYP85A1)or the constitutively active BZR1(OE-bzr1-D)and the ethylene receptor mutant Nr were used to study the role of BRs in tomato fruit ripening,and to explore the interactions beteen BR and ethylene.The main findings are as follows:1.In wildtype tomato fruit,the expression of BRs biosynthetic genes were analyzed and it was found that BRs positively regulate tomato fruit ripening.The accumulation of BRs was initially found after the tomato fruit breaker(BK).The BRs biosynthetic genes CPD and CYP85A1 showed significant upregulation after tomato fruit breaker while DET2 and DWARF4 showed no significant changes.At the same time,the BRs content in wildtype tomato fruit was measured,and the content of three BRs compounds TY,NOR-CS and CS was found to be significantly increased.CS is the active BRs component among them,whose content showed an upward trend.Consistent with the increase in bioactive BRs,we found that the ratio of dephosphorylated to phosphorylated forms of BZR1 protein,the core transcription factor downstream of BRs signaling pathway increased steadily from mature green(MG).In order to further study the role of BRs,we obtained BRs biosynthetic gene overexpressing plants OE-CYP85A1,which compared with wild types have accelerated fruit ripening process,increased carotenoid accumulation and,higher ethylene evolution rate and fruit softening rate.At the same time,ethylene synthesisgenes(ACO4,ACS4)and carotenoid synthesis genes(PSY1,PDS,ZDS,CRTL1,LCY1)were upregulated in OE-CYP85A1 plants.The experimental results show that BRs accumulation is increased in the process of tomato fruit ripening after fruit breaker,while BRs are able to regulate the ripening of tomato fruit.2.The interaction between BRs and ethylene in tomato fruit ripening was studied,and it was found that BRs could regulate tomato fruit ripening independently of ethylene after the tomato fruits breaker stage.The BRs biosynthetic gene overexpressing plant(OE-CYP85A1)was crossed with ethylene receptor mutant Nr to obtain OE-CYP85A1/Nr plants.We found that the carotenoid content of OE-CYP85A1/Nr was significantly increased as compared to CR/Nr.BRs can partially rescue the delayed ripening of tomato fruits caused by Nr.Furthermore,we found that the transcripts of carotenoid synthesis genes(PDS,ZDS,CRTL1,CRTL2,LCY1)in the OE-CYP85A1/Nr plants were all increased.The results suggested that BRs may have an independent pathway to regulate fruit ripening.To further study the relationship between BRs and ethylene,we determined the expression of BRs biosynthetic genes in CR/Nr.We found that there was no significant difference in BR biosynthesis genes CPD,DET2,and DWARF4 in CR/Nr,while CYP85A1 expression was down-regulated.To determine whether the BR content was reduced,we further measured the BRs content.We found that the CS content was decreased as compared to the wild type.The results show that BR promotes the accumulation of carotenoids and can partially rescue the delayed fruit ripening caused by Nr mutation.BR can regulate tomato fruit ripening independent of ethylene.It is speculated that ethylene can positively regulate the expression of BRs biosynthesis gene CYP85A1.3.BRs signaling pathway transcription factor BZR1 is involved in regulating tomato fruit ripening.To further study the ability of the transcription factor BZR1 in the BRs signaling pathway to regulate the ripening of tomato fruit,we constructed the transgenic plants overexpressing the constitutive active mutant form of BZR1(OE-bzr1-D).We found that fruit ripening was accelerated in the OE-bzr1-D,as indicated by significantly increased carotenoid content,and upregulation of carotenoid synthesis genes(PSY1,PDS,CRTL1,CRTL2,LCY1)and ethylenesynthesis genes(ACS2,ACS4).Because BRs biosynthesis gene was negatively feedback regulated by BZR1 in vegetative tissues,we studied whether BZR1 can perform similar functions in tomato fruit.We found that the expression of BR biosynthetic gene was inhibited at BK to BK+5,but enhanced at BK+7.The results show that BZR1 in the BRs signaling pathway positively regulates the accumulation of tomato carotenoids and the ripening of tomato fruit through promoting the expression of carotenoid synthesis genes,and ethylene synthesis genes.BZR1 can feedback regulate BR biosynthetic genes in the fruit,but it is speculated that BRs biosynthesis is also regulated by the developmental process. |
