MsMYB206-MsMYB450-MsHY5 complex regulates alfalfa tolerance to salt stress via regulating flavonoid biosynthesis during the day and night cycles.

Flavonoids are the major secondary metabolites participating in many biological processes of plants. Although flavonoid biosynthesis has been extensively studied, its regulatory mechanisms during the day and night cycles remain poorly understood. In this study, three proteins, MsMYB206, MsMYB450, and MsHY5, were found to interact with each other, in which MsMYB206 directly transactivated two flavonoid biosynthetic genes, MsFLS and MsF3'H. The expression patterns of MsMYB206, MsMYB450, MsFLS, and MsF3'H were fully consistent at regular intervals across day/night cycles that were higher at night than in the daytime. On the contrary, both gene expression levels and protein contents of MsHY5 increased in the daytime but decreased at night, and the lower expression of MsHY5 at night led to strengthened interaction between MsMYB206 and MsMYB450. The MsMYB206-overexpression plants were more salt-tolerant and their flavonoid contents were higher than the WT during the day/night cycles. This study revealed one mechanism interpreting the fluctuating flavonoid contents during day/night cycles regulated by the MsMYB206/MsMYB450/MsHY5-MsFLS/MsF3'H module that also contributed to salt tolerance in alfalfa.