The MADS-RIPENING INHIBITOR–DIVARICATA1 module regulates carotenoid biosynthesis in nonclimacteric Capsicum fruits

Carotenoids play indispensable roles in the ripening process of fleshy fruits. Capsanthin is a widely distributed and utilized natural red carotenoid. However, the regulatory genes involved in capsanthin biosynthesis remain insufficient. Here, we identified the MADS-box transcription factor RIPENING INHIBITOR (MADS-RIN) in pepper (Capsicum annuum), which regulates ripening in climacteric tomato (Solanum lycopersicum) fruits, using weighted gene co-expression network analysis. We found MADS-RIN can directly bind to the promoters of carotenoid biosynthetic genes phytoene synthase 1 (PSY1) and capsanthin/capsorubin synthase (CCS) and the promoter of DIVARICATA1 to activate their expression, thereby regulating carotenoid biosynthesis directly or indirectly. The physical interaction between MADS-RIN and DIVARICATA1 enhances the transactivation effect on PSY1 and CCS. The self-transactivation of MADS-RIN demonstrates its capability to expedite the above process under specific conditions. Interestingly, chromatin immunoprecipitation sequencing assays revealed consistency and divergence of potential targets of MADS-RIN in climacteric tomato and nonclimacteric pepper fruits, suggesting potential conservation and variation of MADS-RIN in regulating ripening and carotenoid metabolism. The present study illustrates the regulatory mechanism of the MADS-RIN-DIVARICATA1 module in capsanthin biosynthesis in pepper, providing targets for breeding high-quality peppers. These findings enrich our understanding of the regulatory network of carotenoid biosynthesis and offer insights into the complex mechanisms of MADS-RIN in climacteric/nonclimacteric fruit ripening and carotenoid biosynthesis.