Isolation and Characterization of Key Genes that Promote Flavonoid Accumulation in Purple-leaf Tea (Camellia sinensis L.)

Abstract

There were several high concentrations of flavonoid components in tea leaves that present health benefits. A novel purple-leaf tea variety, ‘Mooma1’, was obtained from the natural hybrid population of Longjing 43 variety. The buds and young leaves of ‘Mooma1’ were displayed in bright red. HPLC and LC-MS analysis showed that anthocyanins and O-Glycosylated flavonols were remarkably accumulated in the leaves of ‘Mooma1’, while the total amount of catechins in purple-leaf leaves was slightly decreased compared with the control. A R2R3-MYB transcription factor (CsMYB6A) and a novel UGT gene (CsUGT72AM1), that were highly expressed in purple leaf were isolated and identified by transcriptome sequencing. The over-expression of transgenic tobacco confirmed that CsMYB6A can activate the expression of flavonoid-related structural genes, especially CHS and 3GT, controlling the accumulation of anthocyanins in the leaf of transgenic tobacco. Enzymatic assays in vitro confirmed that CsUGT72AM1 has catalytic activity as a flavonol 3-O-glucosyltransferase, and displayed broad substrate specificity. The results were useful for further elucidating the molecular mechanisms of the flavonoid metabolic fluxes in the tea plant.

Transcriptomic Analysis Reveals the Molecular Adaptation of Three Major Secondary Metabolic Pathways to Multiple Macronutrient Starvation in Tea (Camellia sinensis)

“…Taken together, both structural and regulatory genes in the catechin, l-theanine, and caffeine biosynthesis pathways were regulated by more than one TF family. Up to now, only a few TFs belonging to the MYB, bHLH, WD40, and WRKY families have been characterized functionally in tea [72][73][74][75]. Hence, based on our findings, it is hypothesized that those identified TFs are not only responsive to nutrient starvation but also may act as regulators of three major metabolites in tea.…”

A coupled role for CsMYB75 and CsGSTF1 in anthocyanin hyperaccumulation in purple tea

“…Although it is well known from the literature that the MYB‐bHlH‐WD40 complex (MBW) complex can regulate anthocyanin biosynthesis, we reasoned that TFs alone are unlikely to explain their preferential accumulation as compared with catechins, as both classes of flavonoids share a common biosynthetic pathway. Thus, it is known that the MBW complex also regulates proanthocyanidin accumulation in a range of plants, including tea (He et al ., ). In tea, however, there is often a negative correlation between anthocyanin and catechin accumulation (Kerio et al ., ; Lai et al ., ), suggesting that there must be a downstream flux switch in the two closely related pathways.…”

“…Functions for PAP1 ‐like MYB genes in regulating flavonoid accumulation are well known (Zhao and Dixon, ; Schaart et al ., ). Specifically, overexpression of the CsMYB75 homolog CsMYB6A (98% identity in amino acid sequence) in transgenic tobacco activated the expressions of almost all flavonoid pathway genes, resulting in an increase in both flavonol and anthocyanin contents (He et al ., ). It therefore follows that the role of CsMYB75 in the purple phenotype is to activate the overall flux into the flavonoid pathway in tea.…”

“…First, normal tea accumulates high contents of catechins but not anthocyanins (Wei et al ., , ). Second, a negative correlation between anthocyanin and catechin accumulation is widely present in purple tea, suggesting a potential competition relationship (He et al ., ; Kerio et al ., ; Lai et al ., ). Third, anthocyanins are distributed in the vacuoles (Zhao and Dixon, ), whereas tea catechins are mainly located in the chloroplasts and vessel walls (Liu et al ., ).…”

“…Third, anthocyanins are distributed in the vacuoles (Zhao and Dixon, ), whereas tea catechins are mainly located in the chloroplasts and vessel walls (Liu et al ., ). Finally, the PAP1 ‐like MYB gene was proposed to play a key role in controlling anthocyanin biosynthesis in tea plants (He et al ., ; Wei et al ., ); however, overexpression of the gene in transgenic tobacco ( Nicotiana tabacum ) activated the expression of almost all flavonoid‐related structural genes, resulting in increases of both flavonols and anthocyanins, which was not consistent with the case in tea plants (He et al ., ). These results suggest the potential presence of an anthocyanin‐specific gene in the transportation system, and as such it was of interest to determine the molecular basis of the hyperaccumulation of anthocyanins in purple tea.…”