DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.