C2H2-Type Zinc Finger Proteins (DkZF1/2) Synergistically Control Persimmon Fruit Deastringency

Hypoxic environments are generally undesirable for most plants, but for astringent persimmon, high CO₂ treatment (CO₂ > 90%), also termed artificial high-CO₂ atmosphere (AHCA), causes acetaldehyde accumulation and precipitation of soluble tannins and could remove astringency. The multiple transcriptional regulatory linkages involved in persimmon fruit deastringency have been advanced significantly by characterizing the ethylene response factors (ERFs), WRKY and MYB; however, the involvement of zinc finger proteins for deastringency has not been investigated. In this study, five genes encoding C2H2-type zinc finger proteins were isolated and designed as DkZF1-5. Phylogenetic and sequence analyses suggested the five DkZFs could be clustered into two different subgroups. qPCR analysis indicated that transcript abundances of DkZF1/4 were significantly upregulated during AHCA treatment (1% O₂ and 95% CO₂) at day 1, DkZF2/5 at both day 1 and 2, while DkZF3 at day 2. Dual-luciferase assay indicated DkZF1 and DkZF2 as the activators of deastringency-related structural genes (DkPDC2 and DkADH1) and transcription factors (DkERF9/10). Moreover, combinative effects between various transcription factors were investigated, indicating that DkZF1 and DkZF2 synergistically showed significantly stronger activations on the DkPDC2 promoter. Further, both bimolecular fluorescence complementation (BiFC) and yeast two hybrid (Y2H) assays confirmed that DkZF2 had protein-protein interactions with DkZF1. Thus, these findings illustrate the regulatory mechanisms of zinc finger proteins for persimmon fruit deastringency under AHCA.