SlMYC2‐SlMYB12 module orchestrates a hierarchical transcriptional cascade that regulates fruit flavonoid metabolism in tomato

Flavonoids are a class of secondary metabolites widely present in plants that serve various functions, such as pigmentation, UV protection and defence against pathogens and herbivores (Naik et al., 2022). Numerous structural genes and transcription factors involved in flavonoid biosynthesis have been successfully identified (Naik et al., 2022), which has significantly enhanced our understanding of the molecular mechanisms underlying flavonoid production in plants. MBW (MYB–bHLH–WDR) transcription factor protein complexes are crucial regulators of flavonoid biosynthesis (Xu et al., 2015). Among these, MYB transcription factors have been extensively studied as major regulators of the MBW complex, modulating flavonoid production in various plants (Xu et al., 2015). In tomato, SlMYB12 also plays a crucial role in the accumulation of flavonoids in the fruit by positively regulating flavonoid biosynthesis genes, such as CHS, CHI, F3H and FLS1 (Zhang et al., 2015). MYC2, a basic helix–loop–helix (bHLH) transcription factor, is crucial in the jasmonic acid (JA) signalling pathway (Liu et al., 2019). At low JA-Ile levels, JAZ proteins recruit the co-repressor TOPLESS, preventing MYC2 from activating downstream genes. With increased JA-Ile levels, JAZ binds to COI1, leading to its degradation mediated by the SCFCOI1 ubiquitin ligase complex (Liu et al., 2019). Subsequently, MED25 interacts with free MYC2, recruiting the histone acetylase HAC1, which regulates the acetylation level of Lys-9 of histone H3 in the promoter regions of MYC2 target genes, thereby activating their expression (Liu et al., 2019). In tomato fruits, SlMYC2 has been reported to positively regulate flavonoid content (Zhang et al., 2022); however, the underlying mechanisms remain unclear. We found that SlMYC2 displayed relatively high expression during the late ripening stages (from breaker (Br) stages Br + 7 to Br + 15) (Figure S1a), indicating its involvement in ripening. A subcellular localization assay showed that SlMYC2-GFP localized to the nucleus (Figure S1b), suggesting that it functions as a transcription factor. To investigate the functional significance of SlMYC2 in ripening, we generated two SlMYC2 knockout (KO) lines using CRISPR/Cas9 with one sgRNA (Figure 1a). Key structural genes involved in flavonoid biosynthesis, including SlCHS1, SlCHS2, SlF3H, SlF3′H and SlFLS, along with the transcription factor SlMYB12, were significantly downregulated in Br + 7 fruits of SlMYC2-KO lines (Figure 1b, Figures S2 and S3). Moreover, the levels of flavonoids, including naringenin, rutin, eriodictyol, nicotiflorin and caffeic acid, as well as that of the flavonoid derivative chlorogenic acid were significantly lower in SlMYC2-KO fruits than in WT fruits (Figure 1c), suggesting a positive regulatory role of MYC2 in flavonoid accumulation in tomato fruits. Despite changes in flavonoid content, ripening onset, fruit firmness and carotenoid levels in SlMYC2-KO fruits remained similar to those in WT fruits (Figure S4), indicating that SlMYC2 specifically activates flavonoid biosynthesis without affecting broader ripening processes. The activation of target genes by MYC2 relies on its interaction with the mediator subunit MED25, which promotes the acetylation of Lys-9 of histone H3 (H3K9Ac) in downstream gene promoter regions (Breeze, 2019). To explore the molecular mechanism by which SlMYC2 promotes flavonoid accumulation in fruits, we conducted a combined analysis of differentially expressed genes (DEGs) between the WT and SlMYC2-KO lines (Data sets S1 and S2), as well as the previously reported DEGs between the WT and MED25-AS (Ma) lines (Deng et al., 2023). The results revealed 79 genes that were simultaneously downregulated in both the SlMYC2-KO and Ma lines (Figure 1d, Data Set S3), suggesting that these genes might be positively regulated by the SlMYC2-MED25 complex. To identify the direct targets of the SlMYC2-MED25 complex involved in flavonoid metabolism regulation, we performed a comprehensive comparison of 4442 putative SlMYC2 target genes previously identified through ChIP-Seq analysis (Du et al., 2017) and the 79 genes found to be positively regulated by the SlMYC2-MED25 complex in the present study. This integrated analysis identified a subset of 18 genes that were putative direct transcriptional targets activated by SlMYC2 (Figure 1e; Table S1). Notably, SlMYB12 (Solyc02g077790), a key positive transcription factor in the tomato flavonoid pathway (Zhang et al., 2015), was among the 18 genes directly regulated by SlMYC2 (Table S1). Moreover, ChIP-Seq data (Du et al., 2017) and EMSA confirmed the direct binding of SlMYC2 to the SlMYB12 promoter at the CACRYG sites in vivo and in vitro (Figure 1f,g). To further illustrate the regulatory role of the SlMYC2-MED25 complex in SlMYB12 expression, we first performed yeast two-hybrid and split-luciferase complementation assays and verified the interaction between SlMYC2 and MED25 both in vitro and in vivo (Figure S5). DNA pull-down assays conducted using a biotin-labelled SlMYB12 promoter showed that the recruitment of MED25 to the SlMYB12 promoter was dependent on SlMYC2 (Figure 1h). Moreover, transactivation assays demonstrated that the co-expression of SlMYC2 and MED25 with the proSlMYB12-LUC reporter in Nicotiana benthamiana leaf protoplasts resulted in a significant increase in the transcriptional activity of the SlMYB12 promoter compared to the expression of SlMYC2 or MED25 alone (Figure 1i), further illustrating the role of the SlMYC2-MED25 complex in transcriptional activation in SlMYB12. These data indicated that the SlMYC2-MED25 complex influences flavonoid accumulation in tomato fruits by directly regulating SlMYB12 expression. The SlMYC2-MED25 complex binds to the promoter regions of target genes by recruiting histone acetyltransferase HAC1, which increases histone H3 acetylation in the promoter regions, leading to chromatin relaxation and activation of target gene expression (Liu et al., 2019). We investigated the enrichment of two histone H3 modification markers, H3K9Ac and H3K27Ac, in the SlMYB12 promoter region of both WT and SlMYC2-KO fruits. Decreased levels of both H3K9Ac and H3K27Ac were found in SlMYC2-KO fruits compared to those in WT fruits (Figure 1j), suggesting that the SlMYC2-MED25 complex activates the expression of SlMYB12 by modulating histone acetylation levels within the promoter region. In conclusion, by combining analysis of the transcriptomes of slmyc2 and MED25-AS (Ma) lines with ChIP-Seq data of SlMYC2, we identified the key transcription factor SlMYB12 as a direct target of the SlMYC2-MED25 complex in regulating flavonoid metabolism (Figure 1k). Our study elucidates the molecular mechanism by which SlMYC2 regulates flavonoid metabolism in tomato fruits, thereby extending our understanding of the functional significance of SlMYC2 in fruit quality regulation. This work was supported in part by the National Natural Science Foundation of China (Nos 32172271, 32002029, 32160073), the Applied Basic Research Category of Science and Technology Program of Sichuan Province (2021YFQ0071; 2022YFSY0059-1; 2021YFYZ0010-5-LH), the Technology Innovation and Application Development Program of Chongqing (cstc2021jscx-cylhX0001), the Natural Science Foundation of Sichuan Province, China (2024NSFSC1301, 2024NSFSC0398) and the Institutional Research Funding of Sichuan University (2022SCUNL105). The authors declare no competing interests. M.L. and H.D. planned and designed the research; H.D., M.W., Y.W., X.X. and Z.G., performed experiments. H.L., N.H. and Y.G. analysed data. M.L. and H.D. wrote the manuscript and D.G. helped improve the manuscript. The RNA-Seq data of this study are openly available at the National Genomics Data Center (Beijing Institute of Genomics, Chinese Academy of Sciences) at https://ngdc.cncb.ac.cn/gsa/ (reference number: CRA015972). Published RNA-Seq and ChIP-Seq data are deposited in the National Genomics Data Center under accession numbers CRA003758 and CRA000222, respectively. Figure S1 The expression levels of SlMYC2 vary across different tissues and subcellular localization analysis of SlMYC2. Figure S2 The expression levels of CHS1, CHS2, HCT, CH3, F3H, F3′H, FLS and MYB12 were obtained from the qPCR data. Figure S3 RNA-seq analysis of WT and SlMYC2-KO fruits. Figure S4 SlMYC2 does not affect tomato fruit ripening process and carotenoid accumulation. Figure S5 Verification of interaction between SlMYC2 and MED25. Table S1 Putative transcriptional targets of the SlMYC2–SlMED25 complex by combining RNA-seq and ChIP-seq data. Data Set S1 Differentially expressed genes (DEGs) between slmyc2 and WT fruits. Data Set S2 Gene Expression (TPM) in slmyc2 and WT Fruits. Data Set S3 The gene locus numbers in the venn diagram of Figure S3a. Data Set S4 The kyoto encyclopedia of genes and genomes (KEGG) analysis of DEGs between slmyc2 and WT fruits. Data Set S5 Gene expression levels (TPMs) in the heat maps of Figure 1b and Figure S4e. Data Set S6 List of primers used in this study. Data Set S7 Flavonoid content and reference standard detection. Data Set S8 Carotenoid content and reference standard detection. Data Set S9 Statistical analysis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.