High-CO2 treatment (95% CO2, 1% O2) triggers deastringency in persimmon fruit via a cascade of interactions between several different classes of transcription factors leading to activation of genes required for anaerobic metabolism.

Identification and functional characterization of hypoxia-responsive transcription factors is important for understanding plant responses to natural anaerobic environments and during storage and transport of fresh horticultural products. In this study, yeast one-hybrid library screening using the persimmon (Diospyros kaki) pyruvate decarboxylase (DkPDC2) promoter identified three ethylene response factor (ERF) genes (DkERF23/DkERF24/DkERF25) and four WRKY transcription factor genes (DkWRKY/ DdkWRKY5/DkWRKY6/DkWRKY7) that were differentially expressed in response to high CO 2 (95%, with 4% N 2 and 1% oxygen) and high N 2 (99% N 2 and 1% oxygen). Yeast one-hybrid assays and electrophoretic mobility shift assays indicated that DkERF23, DkERF24, DkERF25, DkWRKY6, and DkWRKY7 could directly bind to the DkPDC2 promoter. Dual-luciferase assays confirmed that these transcription factors were capable of transactivating the DkPDC2 promoter. DkERF24 and DkWRKY1 in combination synergistically transactivated the DkPDC2 promoter, and yeast two-hybrid and bimolecular fluorescence complementation assays confirmed protein-protein interaction between DkERF24 and DkWRKY1. Transient overexpression of DkERF24 and DkWRKY1 separately and in combination in persimmon fruit discs was effective in maintaining insolubilization of tannins, concomitantly with the accumulation of DkPDC2 transcripts. Studies with Arabidopsis (Arabidopsis thaliana) homologs AtERF1 and AtWRKY53 indicated that similar protein-protein interactions and synergistic regulatory effects also occur with the DkPDC2 promoter. We propose that an ERF and WRKY transcription factor complex contributes to responses to hypoxia in both persimmon fruit and Arabidopsis, and the possibility that this is a general plant response requires further investigation.

Persimmon fruit are unique in accumulating proanthocyanidins (tannins) during development, which cause astringency in mature fruit. In ‘Mopanshi’ persimmon, astringency can be removed by treatment with 95% CO2, which increases the concentrations of ethanol and acetaldehyde by glycolysis, and precipitates the soluble tannin. A TGA transcription factor, DkTGA1, belonging to the bZIP super family, was isolated from an RNA-seq database and real-time quantitative PCR indicated that DkTGA1 was up-regulated by CO2 treatment, in concert with the removal of astringency from persimmon fruit. Dual-luciferase assay revealed that DkTGA1 had a small (less than 2-fold), but significant effect on the promoters of de-astringency-related genes DkADH1, DkPDC2 and DkPDC3, which encode enzymes catalyzing formation of acetaldehyde and ethanol. A combination of DkTGA1 and a second transcription factor, DkERF9, shown previously to be related to de-astringency, showed additive effects on the activation of the DkPDC2 promoter. Yeast one-hybrid assay showed that DkERF9, but not DkTGA1, could bind to the DkPDC2 promoter. Thus, although DkTGA1 expression is positively associated with persimmon fruit de-astringency, trans-activation analyses with DkPDC2 indicates it is likely to act by binding indirectly DkPDC2 promoter, might with helps of DkERF9.