Cross-talk Regulations Between MYB,WRKY,and ERF On Persimmon Fruit Deastringency

Persimmon(Diospyros kaki)fruit can be divided into astringent and nonastringent types.Most of the cultivars are astringent type,which require the postharvest deastringency treatments.In present study,four cultivars of astringent type,i.e.' Mopanshi','Jingmianshi','Tonewase'and 'Gong cheng-shuishi ',were chosen and AHCA treatment was used to accelerate deastringency.Isolation and characteristics of transcription factors involved in fruit deastringency through RNA-seq and library screening by DkPDC2 promoter were carried out.Further analysis investigate the individual as well as the synergistic effects of different transcription factors,the results of which may enrich current knowledge about the mechanism of persimmon fruit deastringency.Main results are as follows:1.Based on the RNA-seq data,a DkMYB/DkERF cascade is involved in the fruit deastringency.AHCA treatment induced the accumulation of ethanol and acetaldehyde,which lead to the decrease of soluble tannin content and triggered the fruit deastringency.Nine AHCA-responsive transcription factors were isolated by RNA-seq and verified by real-time PCR.Further analysis by dual-luciferase,yeast one-hybrid and cis-element mutagenesis confirmed that DkMYB10 could physically bind and trans-activate the DkERF9 promoter,DkERF21 and DkERF22 could trans-activate the DkERF10 promoter by indirect way,and DkERF18,DkERF19 and DkMYB6 could physically bind and trans-activate the DkERF19 promoter.The potential roles of these TFs in fruit deastringency were analysed by investigating their transient over-expression(TOX)in persimmon fruit discs.Results showed that DkMYB6TOX,DkMYB10TOX,DkERF18TOX and DkERF19TOX were all effective in causing insolubilization of tannins,concomitantly with the up-regulations of the corresponding genes,and they work as a TF-TF regulatory cascade during the deastringency in the fruit.2.Yeast one hybrid library screening by using the DkPDC2 promoter indicated that DkWRKY/DkERF complex was involved in the fruit deastringency.Three ERFs,belonging to ERF IX subfamily,i.e.DkERF23/24/25,and two WRKYs,that could trans-activate DkPDC2 promoter,i.e.DkWRKYl/7 were identified and all of them were responsive to AHCA treatment.Further analysis through dual-luciferase,yeast two-hybrid and BiFC indicated that a synergistic trans-activation effect of combination of DkERF24 and DkWRKY1 on DkPDC2 promoter,and there were protein-protein interactions between DkERF24 and DkWRKY1.Similar results were observed in Arabidopsis homologous genes AtERF1 and AtWRKY53.In conclusion,a TF-TF regulatory cascade is involved in the persimmon fruit deastringency,and protein complex between different transcription factors regulated the deastringency.Such results may enrich the molecular mechanism of persimmon fruit deastringency as well as plant response to high CO2/hypoxia stress.