Transcription factor VvbHLH92 negatively regulates salicylic acid mediated proanthocyanidins biosynthesis in grapevine

Abstract

Proanthocyanidins (PAs) are essential secondary metabolites in grapevine, which not only determine the fruit quality but also participate in plant resistance metabolism and plays a vital role in regulating plant stress tolerance. The previous research in our laboratory showed that the contents of proanthocyanidins tended to increase and decrease by SA treatment in grape leaves. However, the mechanism of negative regulation of proanthocyanidin biosynthesis has rarely been reported. In this study, we proposed to use ' Zaoheibao' grape cultivar as the test material to investigate the role of transcription factor VvbHLH92 in the PAs pathway through the bioinformatics analysis of the VvbHLH family, incubation with SA, overexpression and RNA interference of the gene, the yeast one-hybrid assay, and dual-luciferase. Bioinformatics and promoter analysis of the VvbHLH family revealed that VvbHLH92 may be involved in both flavonoid metabolism and regulation in response to SA. The accumulation of proanthocyanidins and the expression of VvLAR1, VvLAR2, VvANR, and VvANS were improved in grapevine leaves within three hours by 0.5 mmol·L⁻¹ SA solution. Nevertheless, the content of proanthocyanidin and its related genes were decreased at the three hours later, and the expression of VvbHLH92 was promoted. VvbHLH92 overexpression in grape leaves showed a significant reduction in the content of PA and the expression of VvANR and VvANS. At the same time, the content of (-)-epicatechin was reduced in the VvbHLH92 overexpressed leaves, on the contrary, the content of (+)-catechin was significantly increased. VvbHLH92-RNAi resulted in significantly increased content of PAs, significantly higher expression of VvANR and VvANS, and improved the content of (-)-epicatechin and decreased the content of (+)-catechin in grape leaves. The yeast one- hybrid assay and dual-luciferase assay showed that VvbHLH92 could directly inhibit the promoter activities of VvLAR1, VvLAR2, VvANR, and inhibit the promoter activity of VvANS. In summary, VvbHLH92 mainly reduced (-)-epicatechin synthesis by down-regulating the expression of VvANR and VvANS, then decreased PAs accumulation during SA-induced reduction of PAs.