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 metabolomic analyses 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 SlUGTs were significantly up-regulated in slmyb32 mutants.  Dual-luciferase and EMSA assays indicated SlMYB32 could bind to and repress promoter activities of SlPAL6 and Sl4CL3.  Expression of 27 transcription factors belonging to 12 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.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a globally quarantine disease infecting nearly all Citrus cultivars.  Citron C-05 has been identified with complete and active resistance to Xcc.  However, the mechanism underlying Citron C-05’s resistance to Xcc remains elusive.  We identified a gene cluster on chromosome 8 of the citrus genome comprising five pathogenesis-related 4-like genes.  PR4A was upregulated in Citron C-05 leaves under Xcc infection, exhibiting the highest expression among these PR4-like genes.  In addition, PR4A expression was higher in leaves of disease-resistant genotypes than susceptible genotypes under Xcc invasion.  Bimolecular fluorescence complementation (BiFC) and Split-Luc assays indicated that CmWRKY75, a positive regulator of PR4A, interacted with pthA4 and upregulated expression of PR4A in Citron C-05 leaves.  Regulatory function for the expression of CmPR4A was localized to a 516-nucleotide region upstream of the translational start site, which was designated ProCmPR4A-P516.  Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange, and three transgenic lines of OE-CmPR4A exhibited resistance to  (Pst DC3000) in Arabidopsis.  Furthermore, CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait, BiFC and Split-Luc assays further verified their interaction.  Transient overexpression of CmSMU2 in sweet orange increased resistance to Xcc.  Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A, indicating that they may synergistically enhance resistance to Xcc in citrus.  These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.