ABSTRACT
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 CO2 (95%, with 4% N2 and 1% oxygen) and high N2 (99% N2 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.
Subject(s)
Diospyros/genetics , Plant Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis Proteins/genetics , Carbon Dioxide/metabolism , DNA-Binding Proteins/genetics , Diospyros/metabolism , Electrophoretic Mobility Shift Assay , Fruit/genetics , Fruit/metabolism , Gene Expression Regulation, Plant , Gene Library , Oxygen/metabolism , Peptide Termination Factors/genetics , Plant Proteins/genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Protein Interaction Maps , Nicotiana/genetics , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
Plant responses to anaerobic environments are regulated by ethylene-response factors (ERFs) in both vegetative and productive organs, but the roles of other transcription factors (TFs) in hypoxia responses are poorly understood. In this study, eight TFs (DkbHLH1, DkMYB9/10/11, DkRH2-1, DkGT3-1, DkAN1-1, DkHSF1) were shown to be strongly up-regulated by an artificial high-CO2 atmosphere (1% O2 and 95% CO2). Dual-luciferase assays indicated that some TFs were activators of previously characterized DkERFs, including DkMYB10 for the DkERF9 promoter, DkERF18/19 and DkMYB6 for the DkERF19 promoter, and DkERF21/22 for the DkERF10 promoter. Yeast one-hybrid and cis-element mutagenesis confirmed these physical interactions with one exception. The potential roles of these TFs in persimmon fruit deastringency were analysed by investigating their transient over-expression (TOX) in persimmon fruit discs, which indicated that DkMYB6TOX, DkMYB10TOX, DkERF18TOX, and DkERF19TOX were all effective in causing insolubilization of tannins, concomitantly with the up-regulation of the corresponding genes. These results indicated that multiple TFs of different classes are responsive to high-CO2/hypoxia in fruit tissues, and that a TF-TF regulatory cascade is involved in the hypoxia responses involving the Group VII DkERF10, and DkERFs and DkMYBs.
Subject(s)
Carbon Dioxide/metabolism , Diospyros/metabolism , Oxygen/metabolism , Plant Proteins/metabolism , Transcription Factors/metabolism , Diospyros/genetics , Fruit/genetics , Fruit/metabolism , Gene Expression Regulation, Plant , Plant Proteins/genetics , Promoter Regions, Genetic , Tannins/metabolism , Transcription Factors/geneticsABSTRACT
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.
