Bi‐functional transcription factor <scp>SlbHLH95</scp> regulates fruits flavonoid metabolism and grey mould resistance in tomato

Flavonoids are polyphenolic secondary metabolites in tomato fruit with important roles in nutritional quality. Dissecting the transcriptional regulatory network modulating flavonoid metabolism is the first step to improve the nutritional quality of tomato fruits through molecular breeding technology. In this study, we identified a transcription factor SlbHLH95 as a key regulator in flavonoid metabolism through analysis of the MicroTom Metabolic Network (MMN) data set. Functional analyses revealed that knockout of SlbHLH95 increased the accumulation of naringenin, while the levels of rutin and nictoflorin decreased. Conversely, overexpression of SlbHLH95 resulted in an opposite pattern of accumulation of flavonoids. Transactivation assays showed that SlbHLH95 positively activated the expression of SlF3H and SlFLS, two key enzyme-encoding genes in the flavonoid pathway, while repressing the expression of SlCHS1. Electrophoretic mobility shift assays (EMSA) demonstrated that SlbHLH95 could directly bind to the promoters of SlF3H and SlFLS, although it could not bind to the promoter of SlCHS1. Furthermore, SlbHLH95 interacted with the transcription factor SlMYB12 and coordinately regulated the expression of SlF3H and SlFLS. Beyond its role in flavonoid metabolism, SlbHLH95 positively regulated the grey mould resistance in tomato fruits by repressing SlBG10. Overall, our findings revealed the important role of bi-functional SlbHLH95 in flavonoid metabolism and grey mould resistance in tomato fruits by acting as both a transcriptional activator and a repressor. This study provides new insights into strategies for improving fruit quality and enhancing fruit disease resistance through targeted genetic modulation.