Phosphorylation of the strawberry MADS-box CMB1 regulates ripening via the catabolism of abscisic acid.

Research on the ripening of fleshy fruits has relied on techniques that measure transcriptional changes. How ripening is linked to posttranslational modifications such as protein phosphorylation remains less studied. Here, we characterize the MADS-box SEPALLATA 4 (SEP4) subfamily transcription factor FaCMB1, a key negative regulator controlling strawberry ripening, whose transcript and protein abundance decrease progressively with fruit development and are repressed by abscisic acid (ABA). Transient RNAi or overexpression of FaCMB1 significantly altered the fruit ripening process and affected the content of endogenous ABA and ripening-related quality. Transcriptome sequencing (RNA-seq) analysis suggested that manipulation of FaCMB1 expression levels affected the transcription of FaASR (ABA-, stress-, ripening-induced), while FaCMB1 can repress the gene expression of FaASR by directly binding to its promoter. Furthermore, FaASR inhibited the transcriptional activity of FaCYP707A4, a key ABA 8'-hydroxylase enzyme involved in ABA catabolism. We show that FaCMB1 can be phosphorylated by the kinase FaSTPK, and Phos-tag assays indicated that the phosphorylation level of FaCMB1 increases during fruit ripening. This phosphorylation of FaCMB1 affects the binding ability of FaCMB1 to the FaASR promoter and alleviates its transcriptional repression. In conclusion, we elucidated a feedback regulatory path involving FaCMB1-FaASR-FaCYP707A4-ABA. During the fruit ripening process, an increase in ABA content led to a decrease in FaCMB1 transcript and protein levels, which, combined with increased phosphorylation levels, collectively impaired the transcriptional repression of FaASR by FaCMB1. Meanwhile, the increased transcriptional level of FaASR further repressed the expression level of FaCYP707A4, leading to ABA accumulation and fruit ripening.