Mechanism Of DkZAT2 Regulating Postharvest Deastringency Of Persimmon Fruit

Persimmon(Diospyros kaki)fruit can be generally divided into astringent and nonastringent types.The most persimmon cultivars in China are astringent type and their mature fruit require the artificial treatments after harvesting.In present study,six cultivars of persimmom('Yidutuoshi',Lintongguaganshi','Meipishi','mopanshi'and 'Yaoxianwuhuashi')used as materials were treated with high CO2(AHCA)treatment for 1 d.Based on RNA-seq analysis,key transcription factors associated with deastringency were cloned,and explored the synergistic trans-activation effects of the effective transcription factors,and enrich the current transcriptional regulation mechanism of persimmon fruit deastringency.The main results are as follows:1.Based on the analysis of RNA-seq results,43 TFs were selected and 23 were newly isolated from persimmon.The DkERF29,DkZAT2 and DkASR1 were able to regulate the promoter of DkPDC2,indicated by dual-luciferase assay.EMSA analysis indicated that DkERF29 could physically bind to promoter of DkPDC2.Compared to all other transcription factors that acted as activators on DkPDC2 promoter,DkERF29 showed the most significant regulation(6.51-fold).The potential roles of DkERF29,DkZAT2 and DkASR1 in fruit deastringency were verified by their transient overpression(TOX)in persimmon fruit discs,in which all of these transcription factors decreased the content of soluble tannins.2.Exploring the synergistic effects between transcription factors that effectively regulate the promoter of DkPDC2.Five transcription factors of DkERF29,DkZAT2,DkASR1 and the reported transcription factors DkERF9 and DkERF19 were screened.The synergistic effect between the two was verified by the dual luciferase system and the results showed combination of DkERF29,together with DkERF19 or DkZAT2 respectively,generated higher activations.BiFC assay further confirmed these protein-protein interactions between DkERF29 with DkERF19 and DkZAT2 respectively.Moreover,BiFC assays indicated that these interactions main occurred at nucleus.3.Investigations of deastringency related transcription factors in five cultivars with different responses to AHCA treatment.It was found that the gene expression of DkZAT2 showed obvious regularity in the five cultivars.In responsive AHCA-trcated cultivars,gene expressions were significantly up-regulated;in non-responsive AHCA-treated cultivars,gene expressions were less affected.Further verifying the ability of the above five transcription factors to respond to oxygen,the gene expression of 0-8 days was detected under normoxic conditions,and the expression of DkERF29 was higher than other transcription factors,while the DkZAT2 gene was almost non-expressed.Therefore,it is speculated that DkZAT2 is a major transcription factor in response to hypoxia signaling during deastringency.