CsMYB219 and CsMYB196 influence epigallocatechin gallate biosynthesis in tea plant (Camellia sinensis) by regulating CsSCPL1A gene expression

Epigallocatechin gallate (EGCG) is the most abundant and biologically active catechin in tea leaves and has been widely utilized in the development of functional foods. EGCG is catalyzed by serine carboxypeptidase-like 1A (CsSCPL1A) acyltransferases in tea plants. Although CsSCPL family genes are regulated by several transcription factors (TFs), systematic studies on their regulation by MYB TFs are lacking. This study integrates targeted metabolomics, transcriptomics, DNA-protein, and protein-protein interaction analyses to elucidate the transcriptional regulation of EGCG biosynthesis-related genes CsSCPL4 and CsSCPL5-1 by R2R3-MYB TFs. CsMYB219 and CsMYB196 can specifically bind to CsSCPL4 and CsSCPL5-1 promoters and activate their expression. CsMYB196 also interacted with CsTT8a and CsTTG1 to activate the transcription activity of CsSCPL4 and CsSCPL5-1 promoters by forming a MYB/bHLH/WD40 (MBW) complex. Promoter truncation assays delineated MYB-responsive cis-elements in CsSCPL4 (-613 to -1 bp with enhancers at -1967 to -1622) and CsSCPL5-1 (-503 to -296 bp). Silencing of CsMYB219 and CsMYB196 by virus-induced gene silencing (VIGS) assay significantly reduced the expression levels of CsSCPL4 and CsSCPL5-1 and EGCG content in tea leaves. Transient overexpression of CsMYB219 and CsMYB196 in tea leaves upregulated CsSCPL4 and CsSCPL5-1 expression and elevated EGCG content. These findings enhance our understanding of the regulatory network underlying EGCG biosynthesis in tea plants and provide a solid foundation for future genetic improvement of tea plant cultivars.