A feedback loop comprising RhMYB114 and RhMYB16 regulates anthocyanin accumulation and tissue acidification in Rosa hybrida

Coloration in rose (Rosa hybrida) petals is primarily determined by anthocyanin accumulation in vacuoles, and vacuolar acidification plays a central role in controlling the accumulation of this pigment. Nevertheless, the regulatory interplay between anthocyanin accumulation and tissue acidification processes remains somewhat unclear. The present study characterized an activator RhMYB114 and a repressor RhMYB16, which functioned synergistically in anthocyanin accumulation and tissue acidification in rose. Transforming tobacco and roses by overexpression, the introduction of RhMYB114 resulted in an increase in anthocyanin levels and a noticeable decrease in pH in the petal cells of both rose and tobacco, whereas RhMYB16 introduction led to inverse effects. To further clarify the underlying the regulatory mechanisms, the yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and dual-luciferase (LUC) were employed. The results showed that RhMYB16 competed with RhMYB114, bound to RhbHLH3 or RhbHLH33, and inhibited its ability to induce the expression of genes related to anthocyanin biosynthesis and acidification. Our findings revealed a feedback mechanism for the regulation of anthocyanin synthesis and tissue acidification involving RhMYB114, which stimulated the transcriptional expression of RhMYB16, whose encoded protein RhMYB16, in turn, negatively regulated the transcriptional expression of RhMYB114. Therefore, this study underscores the pivotal roles of the RhMYB114–RhMYB16 loop in regulating anthocyanin synthesis and tissue acidification, offering insights into metabolic manipulation to enhance the aesthetic appeal of roses.