The MrHY5-mru-miR396-MrGRF4 module regulates UV-B-induced quercetin biosynthesis in Chinese bayberry (Morella rubra cv. Biqi)

Flavonols are important secondary metabolites that protect plants against UV-B stress. Although plant miRNAs can fine-tune abiotic stress responses, their role in regulating the biosynthesis of UV-B-induced flavonols remains unclear. Treatment with UV-B radiation markedly increased the levels of quercetin glycosides in Chinese bayberry fruits (Morella rubra cv. Biqi). The expression of the flavonol-related transcription factors, MrMYB12 and MrHY5, and quercetin biosynthesis genes was induced by UV-B. Using degradome sequencing and qRT-PCR, we identified an mru-miR396-MrGRF4 module in which mru-miR396 was downregulated and MrGRF4 was upregulated by UV-B. Assays in tobacco showed that mru-miR396 suppresses the expression of MrGRF4. Transient overexpression of mru-MIR396b or silencing MrGRF4 increased flavonol accumulation in "Biqi" leaves. Conversely, silencing mru-miR396 or overexpressing MrGRF4 decreased flavonol content. When untargeted by miRNA, MrGRF4 represses the promoter activities of MrF3H, MrF3'H, MrFLS1, and MrFLS2, reducing the positive effect of MrMYB12 on quercetin biosynthesis in tobacco. In addition, MrGRF4 could bind to the promoter of MrMYB12 and directly inhibit its expression. Transient co-overexpression of MrGRF4 and MrMYB12 in "Biqi" and tobacco leaves attenuated quercetin biosynthesis compared with overexpression of MrMYB12 alone. Furthermore, MrHY5 was found to directly suppress the expression of mru-miR396. Transient expression of MrHY5 increased the content of flavonols by upregulating MrMYB12, MrGRF4, flavonol biosynthesis genes, and downregulating mru-miR396, while silencing had opposite effects. Overall, MrMYB12 acts as a key activator of quercetin biosynthesis, while the MrHY5-mru-miR396-MrGRF4 cascade weakens the regulatory effect of MrMYB12, preventing excessive quercetin accumulation under UV-B. These results provide evidence for a mechanism that balances the biosynthesis of secondary metabolites in plants in response to UV-B.