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Flavan-3-ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan-3-ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan-3-ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI-MSI method to reveal a distinct distribution pattern of B-ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3'H, and CsF3'5'H localizes endoplasmic reticulum. Protein-protein interaction studies demonstrated direct associations between CsC4H, CsF3'H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3'5'H, the enzyme for B-ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b 5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3'5'H. Our findings suggest the existence of distinct modules governing the synthesis of different B-ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B-ring trihydroxylated flavan-3-ols in tea plants., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background and Purpose: Liver fibrosis is a critical risk factor for the progression from chronic liver injury to hepatocellular carcinoma. Clinically, there is a lack of therapeutic drugs for liver fibrosis. Previous studies have confirmed that GL-V9, a newly synthesized flavonoid derivative, exhibits anti-inflammatory activity, but whether it has anti-hepatic fibrosis actions remains unclear. This study aimed to investigate the anti-fibrotic activities and potential mechanisms of GL-V9., Experimental Approach: Bile duct ligation (BDL) and carbon tetrachloride (CCl 4 ) challenges were used to assess the anti-fibrotic effects of GL-V9 in vivo. Mouse primary hepatic stellate cells (pHSCs) and the human HSC line LX2 also served as a liver fibrosis model in vitro. Cellular functions and molecular mechanism were analysed using senescence-associated beta-galactosidase staining, real-time PCR, western blotting, immunofluorescence, and co-immunoprecipitation., Key Results: GL-V9 attenuated hepatic histopathological injury and collagen accumulation, as well as decreasing the expression of fibrotic genes in vivo. GL-V9 promoted senescence and inhibited the expression of fibrogenic genes in HSCs in vitro. Mechanistic studies revealed that GL-V9 induced senescence by upregulating GATA4 expression in HSCs. Further studies confirmed that GL-V9 stabilized GATA4 by promoting autophagic degradation of P62., Conclusion and Implications: GL-V9 exerted potent anti-fibrotic effects both in vivo and in vitro by stabilizing GATA4, thereby promoting the senescence of HSCs, and by avoiding its activation and ultimately inhibiting liver fibrosis. This action indicated that the flavonoid GL-V9 is a potential therapeutic candidate for the treatment of liver fibrosis., (© 2022 British Pharmacological Society.)

SUMMARY: Flavan‐3‐ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan‐3‐ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan‐3‐ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI‐MSI method to reveal a distinct distribution pattern of B‐ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3′H, and CsF3′5′H localizes endoplasmic reticulum. Protein–protein interaction studies demonstrated direct associations between CsC4H, CsF3′H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3′5′H, the enzyme for B‐ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3′5′H. Our findings suggest the existence of distinct modules governing the synthesis of different B‐ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B‐ring trihydroxylated flavan‐3‐ols in tea plants. Significance Statement: Our work reveals the mechanism of flavonoid diversity and efficient synthesis in tea plants from the perspective of metabolon. Different metabolons modules provide a new perspective on the scientific problem of why tea plants accumulate high levels of EGCG. The discovery of obligate electron shuttle proteins provides new directions for improving metabolic engineering. [ABSTRACT FROM AUTHOR]