CDPK6 phosphorylates and stabilizes MYB30 to promote hyperoside biosynthesis that prolongs the duration of full-blooming in okra

The flowers of okra (Abelmoschus esculentus) open and wilt within only a few hours, and this is accompanied by accumulation of hyperoside, a secondary metabolite in the flavonoid pathway. However, little is known about the relationship between flavonoids and flowering. Here, we found that exogenous application of hyperoside extended the duration of the full-blooming period by more than 3-fold, and this was accompanied by a 14.7-fold increase in the expression of CALCIUM-DEPENDENT PROTEIN KINASE6 (AeCDPK6). Gene expression profiling indicated that the transcription factor AeMYB30 was co-expressed with AeCDPK6, and detailed protein interaction and phosphorylation experiments together with yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated an interaction between AeMYB30 and AeCDPK6. AeCDPK6 specifically phosphorylated AeMYB30S191, leading to increased protein stability and prevention of degradation. Furthermore, AeMYB30 directly bound to the promoter of AeUF3GaT1, a key enzyme in the hyperoside biosynthesis pathway. Analysis of transgenic plants showed that AeCDPK6 was required for the hyperoside-induced phosphorylation of AeMYB30 to enhance its stability and transcriptional activity. Ectopic expression of AeCDPK6 promoted hyperoside accumulation and prolonged the full-blooming period in an AeMYB30-dependent manner. Our results indicate the role of AeCDPK6-AeMYB30 in the molecular mechanism by which hyperoside regulates the period of full blooming in okra, a plant with a short duration of flowering.