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. 2022 Feb 19:9:uhac024.
doi: 10.1093/hr/uhac024. Online ahead of print.

Identification of a WRKY transcriptional activator from Camellia sinensis that regulates methylated EGCG biosynthesis

Yong Luo  1 , Xiang-Xiang Huang  2 , Xiao-Feng Song  2 , Bei-Bei Wen  3 , Nian-Ci Xie  2 , Kun-Bo Wang  2 , Jian-An Huang  2 , Zhong-Hua Liu  2
Affiliations

Identification of a WRKY transcriptional activator from Camellia sinensis that regulates methylated EGCG biosynthesis

Yong Luo et al. Hortic Res. .

Abstract

Naturally occurring methylated catechins, especially methylated EGCG in tea leaves are known to have many health benefits. Although the genes involved in methylated EGCG biosynthesis have been studied extensively, the transcriptional factors controlling methylated EGCG biosynthesis are still poorly understood. In the present study, a WRKY domain-containing protein termed CsWRKY57like was identified, which belongs to group IIc of the WRKY family, and contains one conserved WRKY motif. CsWRKY57like was found to localize in the nucleus, function as a transcriptional activator, and its expression positively correlated with methylated EGCG level. In addition, CsWRKY57like activated the transcription of three genes related to methylated EGCG biosynthesis, including CCoAOMT, CsLAR, and CsDFR by specifically interacting with their promoters via binding to the cis-acting element (C/T)TGAC(T/C). Further assays revealed that CsWRKY57like physically interacts with CsVQ4, and participates in the metabolic regulation of O-methylated catechin biosynthesis. Collectively, we conclude that CsWRKY57like may positively impact the biosynthesis of methylated EGCG in the tea plant, which comprehensively enriches the regulatory network of WRKY TFs associated with methylated EGCG and provide a potential strategy for the breeding of specific tea plant cultivars with high methylated EGCG .

Keywords: Camellia sinensis; Methylated EGCG; WRKY; transcriptional regulation.

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Figures

Figure 1
Figure 1
The contents of EGCG3″Me and the expression of CsWRKY57like. (a) The chemical structure of EGCG3″Me [7]. (b) The contents of EGCG3″Me cited by previous studies [9]. (c) The relative expression level of CsWRKY57like by qRT-PCR. **represents a significant difference compared to ‘Fuding Dabaicha’ (P < 0.01).
Figure 2
Figure 2
Amino acid sequence and phylogenetic relationship analysis of CsWRKY57like. (a) Multiple sequence alignment of CsWRKY57like with other WRKY proteins. Red lines and asterisks indicate the WRKY motifs and zinc-finger structures, respectively. (b) Phylogenetic analysis of CsWRKY57like and Arabidopsis WRKYs.
Figure 3
Figure 3
Characterization of CsWRKY57like. (a) Analysis of subcellular localization of CsWRKY57like in tobacco. Bars, 25 μm. (b) Transcriptional activities of CsWRKY57like in yeast cells. (c) The transcriptional activity of CsWRKY57like in Nicotiana benthamiana leaves. The pBD-Empty vector and pBD-VP16 served as the negative and positive control, respectively. **represents a significant difference (P < 0.01) compared with pBD-Empty and pBD-VP16.
Figure 4
Figure 4
CsWRKY57like binds to the W-box element of CsLAR-pro, CsDFR-pro, and CCoAOMT-pro in vitro and in vivo. (a) In vitro binding of CsWRKY57like to CsLAR-pro, CsDFR-pro, and CCoAOMT-pro assessed by EMSA. (b) In vivo binding of CsWRKY57like to CsLAR-pro, CsDFR-pro, and CCoAOMT-pro assessed by ChIP-PCR analysis. **P < 0.01.
Figure 5
Figure 5
Dual-luciferase transient expression of CsWRKY57like regulates the expression of CsLAR, CsDFR, and CCoAOMT. **P < 0.01.
Figure 6
Figure 6
CsWRKY57like interacts with CsVQ4. (a) Transcriptional activities of CsVQ4 in yeast cells. (b) The interaction between CsWRKY57like and CsVQ4 by Y2H assay. (c) The interaction between CsWRKY57like and CsVQ4 in N. benthamiana leaf epidermal cells by bimolecular fluorescence complementation (BiFC) assay. Bar, 30 μm.
Figure 7
Figure 7
A model for the mechanism by which CsWRKY57like affects O-methylated catechin biosynthesis in tea plants (Camellia sinensis L.).
See this image and copyright information in PMC

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