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 SCF^COI1 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.

类黄酮是一类广泛存在于植物中的次生代谢物，具有多种功能，例如色素沉着、紫外线防护以及抵御病原体和食草动物（Naik等 ，2022）。已成功鉴定出许多参与类黄酮生物合成的结构基因和转录因子（Naik等 人，2022 年），这显着增强了我们对植物中类黄酮产生的分子机制的理解。

MBW （MYB-bHLH-WDR） 转录因子蛋白复合物是类黄酮生物合成的关键调节因子（Xu等 人，2015 年）。其中，MYB 转录因子作为 MBW 复合物的主要调节因子已被广泛研究，调节各种植物中类黄酮的产生（Xu等人 ，2015 年）。在番茄中，SlMYB12 还通过正向调节类黄酮生物合成基因，如 CHS、CHI、F3H 和 FLS1，在果实中类黄酮的积累中起关键作用（Zhang et al.， 2015）。

MYC2 是一种碱性螺旋-环-螺旋 （bHLH） 转录因子，在茉莉酸 （JA） 信号通路中至关重要 （Liuet al.， 2019）。在低 JA-Ile 水平下，JAZ 蛋白募集共阻遏蛋白 TOPLESS，阻止 MYC2 激活下游基因。随着 JA-Ile 水平的增加，JAZ 与 COI1 结合，导致其由 SCF^COI1 泛素连接酶复合物介导的降解 （Liuet al.， 2019）。随后，MED25 与游离 MYC2 相互作用，募集组蛋白乙酰化酶 HAC1，该酶调节组蛋白 H3 在 MYC2 靶基因启动子区域中的 Lys-9 乙酰化水平，从而激活它们的表达（Liu等人 ，2019 年）。据报道，在番茄果实中，SlMYC2 对类黄酮含量有正向调节作用（Zhang等人 ，2022 年）;然而，其潜在机制仍不清楚。我们发现 SlMYC2 在成熟后期 （从破碎 （Br） 阶段 Br + 7 到 Br + 15） 表现出相对较高的表达（图 S1a），表明它参与成熟。亚细胞定位测定显示 SlMYC2-GFP 定位于细胞核（图 S1b），表明它起转录因子的作用。为了研究 SlMYC2 在成熟中的功能意义，我们使用 CRISPR/Cas9 和一个 sgRNA 生成了两个 SlMYC2 敲除 （KO） 细胞系（图 1a）。参与类黄酮生物合成的关键结构基因，包括 SlCHS1、SlCHS2、SlF3H、SlF3′H 和 SlFLS，以及转录因子 SlMYB12，在 SlMYC2-KO 系的 Br + 7 个果实中显著下调（图 1b，图 S2 和 S3）。 此外，SlMYC2-KO 果实中黄酮类化合物（包括柚皮素、芦丁、芸香素、烟花苷和咖啡酸）以及类黄酮衍生物绿原酸的水平显著低于 WT 果实（图 1c），表明 MYC2 在番茄果实类黄酮积累中具有正调节作用。尽管类黄酮含量发生变化，但 SlMYC2-KO 果实的成熟开始、果实硬度和类胡萝卜素水平与 WT 果实相似（图 S4），表明 SlMYC2 特异性激活类黄酮生物合成，而不影响更广泛的成熟过程。

MYC2 对靶基因的激活依赖于其与介质亚基 MED25 的相互作用，该相互作用促进下游基因启动子区域中组蛋白 H3 （H3K9Ac） 的 Lys-9 乙酰化 （Breeze， 2019）。为了探索 SlMYC2 促进果实中类黄酮积累的分子机制，我们对 WT 和 SlMYC2-KO 系（数据集 S1 和 S2）之间的差异表达基因 （DEG） 以及先前报道的 WT 和 MED25-AS （马） 系之间的差异表达基因 （DEGs） 进行了联合分析（邓等 人，2023 年）。结果显示，在 SlMYC2-KO 和 马 系中同时下调的 79 个基因（图 1d，数据集 S3），表明这些基因可能受到 SlMYC2-MED25 复合物的正调控。

为了确定参与类黄酮代谢调节的 SlMYC2-MED25 复合物的直接靶标，我们对先前通过 ChIP-Seq 分析鉴定的 4442 个推定的 SlMYC2 靶基因进行了全面比较 （Du et al.， 2017） 和在本研究中发现的 79 个受 SlMYC2-MED25 复合物正调控的基因。该综合分析确定了 18 个基因的子集，这些基因是被 SlMYC2 激活的推定直接转录靶标（图 1e;表 S1）。值得注意的是，番茄类黄酮途径中的关键阳性转录因子 SlMYB12 （Solyc02g077790） （Zhang et al.， 2015） 是 SlMYC2 直接调控的 18 个基因之一（表 S1）。此外，ChIP-Seq 数据 （Du et al.， 2017） 和 EMSA 证实 SlMYC2 在体内和体外与 CACRYG 位点的 SlMYB12 启动子直接结合（图 1f，g）。

为了进一步说明 SlMYC2-MED25 复合物在 SlMYB12 表达中的调节作用，我们首先进行了酵母双杂交和分裂荧光素酶互补试验，并验证了 SlMYC2 和 MED25 之间的相互作用在体外和体内（图 S5）。使用生物素标记的 SlMYB12 启动子进行的 DNA 沉降测定表明，MED25 向 SlMYB12 启动子的募集依赖于 SlMYC2（图 1h）。此外，反式激活试验表明，与单独表达 SlMYC2 或 MED25 相比，本氏烟草叶原生质体中 SlMYC2 和 MED25 与 proSlMYB12-LUC 报告基因的共表达导致 SlMYB12 启动子的转录活性显着增加（图 1i），进一步说明了 SlMYC2-MED25 复合物在SlMYB12.这些数据表明，SlMYC2-MED25 复合物通过直接调节 SlMYB12 表达来影响番茄果实中类黄酮的积累。

SlMYC2-MED25 复合物通过募集组蛋白乙酰转移酶 HAC1 与靶基因的启动子区域结合，这增加了启动子区域的组蛋白 H3 乙酰化，导致染色质松弛和靶基因表达的激活（Liu et al.， 2019）。我们研究了两个组蛋白 H3 修饰标志物 H3K9Ac 和 H3K27Ac在 WT 和 SlMYC2-KO 果实的 SlMYB12 启动子区域的富集。与 WT 果实相比，SlMYC2-KO 果实中 H3K9Ac 和 H3K27Ac 的水平降低（图 1j），表明 SlMYC2-MED25 复合物通过调节启动子区域内的组蛋白乙酰化水平来激活 SlMYB12 的表达。

总之，通过将 slmyc2 和 MED25-AS （马） 系的转录组分析与 SlMYC2 的 ChIP-Seq 数据相结合，我们确定了关键转录因子 SlMYB12 是 SlMYC2-MED25 复合物调节类黄酮代谢的直接靶标（图 1k）。我们的研究阐明了 SlMYC2 调控番茄果实类黄酮代谢的分子机制，从而扩展了我们对 SlMYC2 在果实品质调控中的功能意义的理解。