High-CO/Hypoxia-Responsive Transcription Factors DkERF24 and DkWRKY1 Interact and Activate Promoter.

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 () pyruvate decarboxylase () promoter identified three ethylene response factor (ERF) genes (//) and four WRKY transcription factor genes (//) that were differentially expressed in response to high CO (95%, with 4% N and 1% oxygen) and high N (99% N 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 promoter. Dual-luciferase assays confirmed that these transcription factors were capable of transactivating the promoter. DkERF24 and DkWRKY1 in combination synergistically transactivated the promoter, and yeast two-hybrid and bimolecular fluorescence complementation assays confirmed protein-protein interaction between DkERF24 and DkWRKY1. Transient overexpression of and 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 () homologs and indicated that similar protein-protein interactions and synergistic regulatory effects also occur with the 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.