A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Abstract

Flavan-3-ols and oligomeric proanthocyanidins (PAs) are the main nutritional polyphenols in green tea (Camellia sinensis), which provide numerous benefits to human health. To date, the regulatory mechanism of flavan-3-ol biosynthesis in green tea remains open to study. Herein, we report the characterization of a C. sinensis tryptophan-aspartic acid repeat protein (CsWD40) that interacts with myeloblastosis (MYB) and basic helix-loop-helix (bHLH) transcription factors (TFs) to regulate the biosynthesis of flavan-3-ols. Full length CsWD40 cDNA was cloned from leaves and was deduced to encode 342 amino acids. An in vitro yeast two-hybrid assay demonstrated that CsWD40 interacted with two bHLH TFs (CsGL3 and CsTT8) and two MYB TFs (CsAN2 and CsMYB5e). The overexpression of CsWD40 in Arabidopsis thaliana transparent testa glabra 1 (ttg1) restored normal trichome and seed coat development. Ectopic expression of CsWD40 alone in tobacco resulted in a significant increase in the anthocyanins of transgenic petals. CsWD40 was then coexpressed with CsMYB5e in tobacco plants to increase levels of both anthocyanins and PAs. Furthermore, gene expression analysis revealed that CsWD40 expression in tea plants could be induced by several abiotic stresses. Taken together, these data provide solid evidence that CsWD40 partners with bHLH and MYB TFs to form ternary WBM complexes to regulate anthocyanin, PA biosynthesis, and trichome development.

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.…”

Integrative Transcriptomic and Metabolic Analyses Provide Insights into the Role of Trichomes in Tea Plant (Camellia Sinensis)

“…To date, certain MBW component genes that regulate flavonoid biosynthesis have been identified in tea plants. For instance, Sun, et al [57] showed that R2R3-MYB CsAN1 could interact with bHLH TFs (CsGL3 and CsEGL3) and CsTTG1 (a WD-repeat protein) to form an MBW complex to regulate anthocyanin accumulation; Liu, et al [58] found that CsWD40 (homologous to AtTTG1 TF) partners with bHLH (CsGL3 and CsTT8) and MYB (CsAN2 and CsMYB5e) TFs to form WBM complexes to control anthocyanin and proanthocyanidin biosynthesis; finally, the overexpression of certain tea plant MYB genes, such as CsMYB5a, CsMYB5b, CsMYB5e, CsMYB5-1, and CsMYB5-2, could alter anthocyanin and proanthocyanidin accumulation in other plants [59][60][61]. We found that most of the DEGs that were involved in the MYB, NAC, and WRKY classes were significantly upregulated in trichomes since tea trichomes usually exhibit glossy characteristics and might not have the capability to synthesize and store flavonoids and phenylpropanoids, especially anthocyanins and proanthocyanidins (Figure 4).…”

“…The WBM complexes not only regulate flavonoid synthesis, but also modulate trichome development through different TFs competing to form WBM complexes [6,16,62]. Recently, Liu, et al [58] indicated that the tea plant CsWD40 interacted with bHLH and MYB TFs to form WBM complexes that could alter anthocyanin accumulation and regulate trichome development. Nonetheless, known key TFs, such as GL1, GL3, EGL3, and TTG1, which were involved in trichome development, were not determined to be expressed at a significant level, although TFs, such as TTG1 (TEA000080), were upregulated in trichomes.…”

The Effects of Ultraviolet A/B Treatments on Anthocyanin Accumulation and Gene Expression in Dark-Purple Tea Cultivar ‘Ziyan’ (Camellia sinensis)

“…Whereas C1 and MYB110 were downregulated by UV-B. C1 repressed DFR and UFGT expression levels [73], while MYB110 appeared to be the activator of anthocyanin biosynthesis in purple kiwifruit [74]. Therefore, the transcription factors mentioned above may be the key TFs to regulate anthocyanin synthesis induced by ultraviolet light in tea plant 'Ziyan'.…”