Regulation Of ERF And BZR Transcription Factors On Persimmon Fruit Ripening And Softening

Persimmon(Diospyros kaki L.)fruit softens easily during the ripening process.Once persimmon fruit are ripened,the soft texture creates difficulty in further handling or transportation,possibly resulting in a reduction of commercial value.It is important to understand the molecular mechanisms of persimmon fruit ripening,which is advantageous for offering a useful reference for persimmon breeding and extending fruit storage and shelf life.Persimmon is a climacteric fruit,and ethylene plays an essential role in persimmon fruit ripening.Brassinosteroids are a new group of plant hormones that regulate numerous processes including fruit ripening.ERF and BZR are critical components of the ethylene signaling pathway and brassinosteroids signaling pathway,respectively.However,the transcriptional regulatory mechanism underlying ERF and BZR in persimmon fruit ripening remains poorly understood.In the present study,two persimmon cultivars that showed differences in their fruit ripening and softening rates were used as material.In order to screen ripening-related genes,and investigate regulatory interactions of these genes during persimmon fruit ripening and further clarify the role of ethylene and brassinosteroids in the ripening process of persimmon fruit,q RT-PCR,western blot assay,dual luciferase assay,electrophoretic mobility shift assay and transient overexpression in persimmon fruit discs were performed.The main results were listed below:1.To screen persimmon ripening and softening-related genes,two persimmon cultivars that exhibit different fruit softening rates were used,and ethylene and 1-MCP treatments were conducted with persimmon fruit.‘Fupingjianshi' fruit had the fastest softening rate,and ‘Xinchangniuxinshi' fruit had better postharvest storage tolerance.Transmission electron microscopy showed that persimmon fruit softening was associated with middle lamella degradation and cell wall swelling and distortion.The cell wall was still relatively intact in ‘Xinchangniuxinshi' at 18 d,while the cell wall was distorted and disrupted in ‘Fupingjianshi' at 18 d.q RT-PCR displayed that the expression of Dk PG1,Dk PL1,Dk PE1/2,Dk?-GAL1,Dk EGase1,Dk XTH2/9/10/11,Dk MAN1,Dk EXP4,Dk ACS1/2 and Dk ACO1/2 was obviously induced after ethylene treatment and was suppressed by 1-MCP treatment.During ‘Fupingjianshi' and ‘Xinchangniuxinshi' fruit ripening,the relative expression of all these genes(excluding Dk MAN1 in ‘Xinchangniuxinshi')was upregulated following ethylene production in each cultivar,which means that these genes related to ethylene-mediated persimmon fruit ripening and softening.Among these genes,the expression of Dk PG1,Dk XTH2/9/10,Dk MAN1 and Dk EXP4 in ‘Fupingjianshi' was significantly higher than that in ‘Xinchangniuxinshi'.2.The regulation of ERF transcription factor on persimmon fruit ripening and softeningrelated gene were investigated.The promoters of sixteen structural genes related to persimmon fruit ripening(Dk PG1,Dk PL1,Dk PE1/2,Dk?-GAL1,Dk EGase1,Dk XTH2/9/10/11,Dk MAN1,Dk EXP4,Dk ACS1/2 and Dk ACO1/2)were isolated by genome walking.Potential ERF transcription factor binding sites were identified manually within all these structural gene promoters.Dual luciferase assays,site mutations and electrophoretic mobility shift assays indicated that Dk ERF8 and Dk ERF16 activate Dk XTH11 and Dk EXP4,respectively,by binding to their promoters.Dk ERF18 binds to the Dk ACS2 promoter,increasing its activity.Dk ERF8/18 protein was localized in nucleus,and Dk ERF16 protein was localized in nucleus and the cell membrane.Transient overexpression of Dk ERF8 promotes the conversion of acid soluble pectin to water soluble pectin in persimmon fruit.Moreover,transient overexpression of Dk ERF18 resulted in increased ethylene production.These results suggest that Dk ERF8/16/18 may be involved in persimmon fruit ripening by promoting cell wall modification and ethylene biosynthesis.3.The contents of brassinosteroids and castastarone gradually increased at earlier storage period,and the levels of 6-deoxocastastarone did not significantly change during the whole storage period.24-epibrassinolide(EBR)treatment accelerates persimmon fruit ripening.EBR treatment significantly increased both the content of water-soluble pectin and the activities of polygalacturonase,pectate lyase,and cellulase but significantly reduced the content of acidsoluble pectin and cellulose,resulting in rapid fruit softening.EBR treatment also promoted ethylene production and improved the expression of Dk PG1,Dk PL1,Dk PE2,Dk EGase1,Dk ACO2,Dk ACS1,and Dk ACS2.4.The regulation of BZR transcription factor on BR-responsive genes during persimmon fruit ripening were investigated.Four persimmon Dk BZRs sequences were isolated from persimmon RNA-Seq library.Among the four Dk BZRs genes,Dk BZR1/2 was expressed in persimmon fruit.Western blot assay showed that the protein level of Dk BZR1 was decreased,and the status of dephosphorylated Dk BZR2 protein gradually accumulated during the storage period.Dk BZR1/2 protein was localized in both nucleus and cytoplasm,and it accumulated in the nucleus after application EBR treatment.Dk BZR1 could suppress the transcription of Dk EGase1 and Dk ACS1 via binding to the BRRE motif in their promoters.Dk BZR2 could activate the transcription of Dk PL1 and Dk ACO2 via binding to E-box motif in their promoters.EBR treatment significantly improved the transcriptional regulation capacity of Dk BZR2 on Dk PL1 and Dk ACO2.Moreover,transient overexpression of Dk BZR2 promotes the conversion of acid soluble pectin to water soluble pectin in persimmon fruit and increased ethylene production.These results,together,demonstrate that Dk BZR1 and Dk BZR2 is repressor and activator of persimmon fruit ripening,respectively.