High CO2/hypoxia-induced softening of persimmon fruit is modulated by DkERF8/16 and DkNAC9 complexes.

Most persimmon (Diospyros kaki) cultivars are astringent and require postharvest de-astringency treatments such as 95% CO2 (high CO2 treatment) to make them acceptable to consumers. High CO2 treatment can, however, also induce excessive softening, which can be reduced by adding 1-methylcyclopropene (1-MCP). Previous studies revealed that genes encoding ETHYLENE RESPONSE FACTORS (ERFs) DkERF8/16/19 could trans-activate cell wall degrading enzyme xyloglucan endotransglycosylase/hydrolase gene (DkXTH9) associated with persimmon fruit softening. In this study, the RNA-seq data between three treatments (high CO2, CO2+1-MCP and control) were compared. 227 differentially expressed genes (DEGs), with 17 transcription factors (TFs), were predicted to be related to persimmon post-deastringency softening. Dual-luciferase assays indicated that DkNAC9 activated the DkEGase1 promoter 2.64-fold. Synergistic effects on transcription from the DkEGase1 involving DkNAC9 and previously reported DkERF8/16 were identified. Electrophoretic mobility shift assay (EMSA) indicated that DkNAC9 could physically bind to the DkEGase1 promoter. Bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation imaging assays (LCI) indicated protein-protein interactions between DkNAC9 and DkERF8/16. Based on these findings, DkNAC9 is a direct transcriptional activator of DkEGase1, which can cooperate with DkERF8/16 to enhance post-deastringency softening.