Characterization of the transcriptional regulator CsbHLH62 that negatively regulates EGCG3"Me biosynthesis in Camellia sinensis.

Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) in tea (Camellia sinensis (L.) O. Kuntze) is a major source of O-methylated catechin and renowned for a wide range of health effects. However, the transcriptional regulation mechanisms of EGCG3"Me biosynthesis remain unclear. In the present work, the basic Helix-Loop-Helix (bHLH) transcription factor, designated as CsbHLH62, belonging to GBOF group of bHLH families, was isolated and characterized from Camellia sinensis. CsbHLH62 contains an Open Reading Frame of 1662 bp and encodes a polypeptide of 553 amino acids. Subcellular location and transcriptional activity analysis showed it as a nucleus protein and possessed transcriptional inhibition activity. Furthermore, the expression of CsbHLH62 was decreased during EGCG3"Me accumulation. More importantly, E-box motifs (5'-CANNTG-3') were found in the promoters of CCoAOMT, CsLAR, and CsDFR, and further transient expression assays showed that CsbHLH62 repressed the transcription of CCoAOMT, CsLAR, and CsDFR. Collectively, these results suggest that CsbHLH62 acts as a transcriptional repressor that might be negatively affecting the accumulation of EGCG3"Me. These findings provide novel insights into the regulatory mechanism of EGCG3"Me biosynthesis, which might help to breed high EGCG3"Me-content tea plants.