CRISPR/Cas9-mediated SNAC9 mutants reveal the positive regulation of tomato ripening by SNAC9 and the mechanism of carotenoid metabolism regulation.

NAC transcriptional regulators are crucial for tomato ripening. Virus-induced gene silencing (VIGS) of (, Gene ID: 101248665) affects tomato ripening, and is involved in ethylene and abscisic acid (ABA) metabolic pathways. However, the function of in pigment metabolism in tomatoes remains unclear. This work seeks to discover the mechanism of involvement in pigment metabolism during tomato ripening by establishing a knockout model using CRISPR/Cas9 technology. The results indicated that fruit ripening was delayed in knockout (KO) mutants, and mutation significantly affected carotenoid metabolism. The chlorophyll (Chl) degradation rate, total carotenoid content, and lycopene content decreased significantly in the mutants. The transformation rate of chloroplasts to chromoplasts in mutants was slower, which was related to the carotenoid content. Furthermore, changed the expression of critical genes (, , , , , , , , and ) involved in pigment metabolism in tomato ripening. knockout also altered the expression levels of critical genes involved in the biosynthesis of ethylene and ABA. Accordingly, regulated carotenoid metabolism by directly regulating , , , and . This research provides a foundation for developing the tomato ripening network and precise tomato ripening regulation.