CRISPR/Cas9-mediated mutagenesis of transcriptional repressor SlMYB32 improves flavonols and flavanones accumulation in tomato fruit

• SlMYB32 was identified as a repressor regulating phenylpropanoid pathway • CRISPR/Cas9-mediated mutagenesis of SlMYB32 results in flavonol-rich tomato with 1 mg g -1 FW rutin Flavonols and flavanones are important bioactive compounds with multiple pharmacological activities and health benefits. Transcriptional activation of flavonol and flavanone biosynthesis has been studied extensively, while little is known about the negative regulators. CRISPR/Cas9 gene-editing technology, with the advantage of precise genetic modification, is a desirable tool for breeding biofortified materials and exploring potential molecular mechanisms. In this study, a transcriptional repressor, SlMYB32, was characterized in tomato fruit. Phenotype and metabolome analysis confirmed that knockout of SlMYB32 resulted in increased accumulation of flavonols and flavanones, especially about 1 mg g -1 FW of quercetin 3- O -rutinoside (rutin). Transcriptome analysis indicated that expression of key genes SlPAL6, Sl4CL3 and Sl4CL4 as well as five candidate SlUGT s were significantly up-regulated in slmyb32 mutants. Dual-luciferase and EMSA assay indicated SlMYB32 could bind to and repress promoter activities of SlPAL6 and Sl4CL3 . Expression of 27 transcription factors belonging to twelve families was significantly changed in slmyb32 mutants, among which two SlMYBs, two SlNACs, two SlAP2s and one SlWRKY were clustered with known flavonoid regulators. Our results provide new insights into improving bioactive compounds in fruit and understanding negative regulatory mechanisms in flavonol and flavanone biosynthesis.