Journal of Tea Science Research, 2024, Vol.14, No.6, 335-343 http://hortherbpublisher.com/index.php/jtsr 339 4.2 Coordinated regulation by bHLH and WD40 proteins bHLH and WD40 proteins act as co-regulators with MYB transcription factors. bHLH proteins (such as CsbHLH and bHLH96) interact with MYBs to modulate the expression of flavonoid biosynthetic genes, while WD40 proteins (like CsTTG1) serve as scaffolds, stabilizing the transcriptional complexes. These interactions are crucial for the precise regulation of flavonoid pathway genes and for tissue- and stage-specific flavonoid accumulation (Wang et al., 2018; Zhu et al., 2020). 4.3 Molecular model of the MYB-bHLH-WD40 (MBW) transcriptional complex The MBW complex, composed of MYB, bHLH, and WD40 proteins, is a well-established regulatory module in flavonoid biosynthesis. In tea plants, this complex directly activates the transcription of key structural genes such as ANS, FLS, and UFGT, thereby controlling the biosynthesis of anthocyanins and other flavonoids. Functional studies have demonstrated that the co-expression of MYB and WD40 proteins enhances the transcription of target genes, confirming the cooperative nature of the MBW complex (Wang et al., 2018; Ye et al., 2023). 4.4 Functional diversity and genotype-specific regulation of transcription factors Flavonoid biosynthesis transcription factors are also extremely functionally diverse and regulated by genotype. For example, expression and functions of the MYB and bHLH factors could vary among tea cultivars, and these variations lead to differences in flavonoid content and composition. Environmental factors such as light, temperature, and hormone signals also modulate the activity of the transcription factors to ultimately lead to dynamic and context-dependent flavonoid biosynthesis regulation (Song et al., 2022). 5 Epigenetic Regulation and the Role of Non-coding RNAs 5.1 Influence of DNA methylation and histone modifications on gene expression Epigenetic mechanisms such as DNA methylation and histone modifications play a crucial role in regulating gene expression in plant secondary metabolism, including flavonoid biosynthesis. These modifications can alter chromatin structure, thereby activating or repressing the transcription of key biosynthetic genes. Although direct studies in tea plants are limited, evidence from broader plant research indicates that epigenetic regulation is a key layer of control in the biosynthesis of flavonoids and other secondary metabolites (Li et al., 2022). 5.2 Emerging roles of miRNAs and lncRNAs in flavonoid pathway regulation MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are being increasingly recognized as key flavonoid biosynthesis regulators. miRNAs may regulate structural genes and transcription factors, such as those within the MYB-bHLH-WD40 complex, by translational repression or cleavage of mRNAs. For example, it has been shown that miRNAs can regulate structural genes and regulatory proteins to modulate flavonoid accumulation and how plants respond to the environment (Yang et al., 2021). lncRNAs can work as transcriptional, post-transcriptional, and epigenetic regulators. Recent studies in plants such as Ginkgo biloba have identified lncRNAs that regulate flavonoid biosynthesis by acting either as precursors or as decoys of miRNAs, or directly influencing the expression of biosynthetic genes. Individual lncRNAs (such as lnc10 and lnc11) overexpression has been shown to promote flavonoid content and modulate related genes in transgenic plants (Liu et al., 2020; Li et al., 2023). 5.3 Interaction models between epigenetic and transcriptional regulation There is growing evidence that epigenetic modifications and non-coding RNAs interact with classical transcriptional regulatory networks. miRNAs and lncRNAs can modulate the activity of transcription factors, while epigenetic marks can influence the accessibility of DNA to these factors. This multi-layered regulation enables plants to finely tune flavonoid biosynthesis in response to developmental and environmental signals, integrating epigenetic, transcriptional, and post-transcriptional controls into a complex regulatory network (Yang et al., 2021).
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