The specific regulatory-role of SlMADS-Macrocalyx (MC) in tomato fruit ripening

The rin mutant has long been the crucial material for investigating the SlMADS-RIN regulation role during tomato fruit ripening. Our previous study has proved that the rin mutant caused by formation of fusion protein RIN-MC, negatively regulates fruit ripening, predicting the unexplored regulatory role of MC on fruit ripening. However, current researches on MC role in tomato focus on florescence formation and abscission zone development. This study mainly explored the regulatory role and mechanism of MC in tomato fruit ripening. First, phylogenetic analysis indicated that MC protein was more closely related to FUL1 and FUL2 compared with other MIKC transcription factors in tomato family, and an EAR inhibitory domain was shown in MC protein. Second, the expression of MC was lower in root, stem and leaf, but higher in flower and fruit, and ethylene induced expression of MC in fruit. Third, the MC overexpression (OE) and CRISPR/Cas9 knockout (CR) lines showed similar phenotype which both delayed the initiation of tomato fruit ripening compared with the WT, as well the ethylene summit. Fourth, differentially expressed genes (DEGs) in OE-MC and CR-MC were similar, such as the expression levels of ACS2, ACS4, ACO1 ETR7, RIN and FUL1 decreased, while the expression of MADS1 and MYB70 both increased. Finally, MC protein significantly activated the promoters of LoxC and ACS2 genes with dual luciferase trans-activity analysis. These results indicate that both overexpression and knockout of the MC gene delayed the initiation of fruit ripening, showing the complex regulatory-role of SlMADS-MC in tomato fruit ripening.