CsbZIP1-CsMYB12 mediates the production of bitter-tasting flavonols in tea plants (Camellia sinensis) through a coordinated activator–repressor network

Abstract

Under high light conditions or UV radiation, tea plant leaves produce more flavonols, which contribute to the bitter taste of tea; however, neither the flavonol biosynthesis pathways nor the regulation of their production are well understood. Intriguingly, tea leaf flavonols are enhanced by UV-B but reduced by shading treatment. CsFLS, CsUGT78A14, CsMYB12, and CsbZIP1 were upregulated by UV-B radiation and downregulated by shading. CsMYB12 and CsbZIP1 bound to the promoters of CsFLS and CsUGT78A14, respectively, and activated their expression individually. CsbZIP1 positively regulated CsMYB12 and interacted with CsMYB12, which specifically activated flavonol biosynthesis. Meanwhile, CsPIF3 and two MYB repressor genes, CsMYB4 and CsMYB7, displayed expression patterns opposite to that of CsMYB12. CsMYB4 and CsMYB7 bound to CsFLS and CsUGT78A14 and repressed their CsMYB12-activated expression. While CsbZIP1 and CsMYB12 regulated neither CsMYB4 nor CsMYB7, CsMYB12 interacted with CsbZIP1, CsMYB4, and CsMYB7, but CsbZIP1 did not physically interact with CsMYB4 or CsMYB7. Finally, CsPIF3 bound to and activated CsMYB7 under shading to repress flavonol biosynthesis. These combined results suggest that UV activation and shading repression of flavonol biosynthesis in tea leaves are coordinated through a complex network involving CsbZIP1 and CsPIF3 as positive MYB activators and negative MYB repressors, respectively. The study thus provides insight into the regulatory mechanism underlying the production of bitter-tasting flavonols in tea plants.

References

[1] Lakenbrink, C., Lapczynski, S., Maiwald, B. & Engelhardt, U. H. Flavonoids and other polyphenols in consumer brews of tea and other caffeinated beverages. J. Agric. Food Chem. 48, 2848-2852 (2000).

[2] Yang, C. S. & Hong, J. G. Prevention of chronic diseases by tea: possible mechanisms and human relevance. Annu. Rev. Nutr. 33, 161-181 (2013).

[3] Gilbert, N. The science of tea’s mood-altering magic. Nature 566, S8-S9 (2019).

[4] Scharbert, S., Holzmann, N. & Hofmann, T. Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse. J. Agric. Food Chem. 52, 3498-508 (2004).

[5] Zhuang, J. et al. Evaluation of astringent taste of green tea through mass spectrometry-based targeted metabolic profiling of polyphenols. Food Chem. 305, 125507 (2020).

[6] Cui, L. et al. Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis). J. Exp. Bot. 67, 2285-2297 (2016).

[7] Zhu, J. et al. Comprehensive co-expression analysis provides novel insights into temporal variation of flavonoids in fresh leaves of the tea plant (Camellia sinensis). Plant Sci. 290, 110306 (2019).

[8] Liu, L. et al. Metabolite profiling and transcriptomic analyses reveal an essential role of UVR8-mediated signal transduction pathway in regulating flavonoid biosynthesis in tea plants (Camellia sinensis) in response to shading. BMC Plant Biol. 18, 233 (2018).

[9] Zheng, C. et al. Regulation of growth and flavonoid formation of tea plants (Camellia sinensis) by blue and green light. J. Agric. Food Chem. 67, 2408-2419 (2019).

[10] Li, W. et al. The effects of ultraviolet A/B treatments on anthocyanin accumulation and gene expression in dark-purple tea cultivar ‘ziyan’ (Camellia sinensis). Molecules 25, 354 (2020).

[11] Wang, Y. S. et al. Influence of shade on flavonoid biosynthesis in tea (Camellia sinensis (L.) O. Kuntze). Sci. Hortic. 141, 7-16 (2012).

[12] Ji, H. G. et al. Diverse metabolite variations in tea (Camellia sinensis L.) leaves grown under various shade conditions revisited: a metabolomics study. J. Agric. Food Chem. 66, 1889-1897 (2018).

[13] Zhao, J., Li, P., Xia, T. & Wan, X. Exploring plant metabolic genomics: chemical diversity, metabolic complexity in the biosynthesis and transport of specialized metabolites with the tea plant as a model. Crit. Rev. Biotechnol. 40, 667-688 (2020).

[14] Xia, En-Hua et al. Tea plant genomics: achievements, challenges and perspectives. Hortic. Res. 7, 7 (2020).

[15] Irmisch, S. et al. Flavonol biosynthesis genes and their use in engineering the plant antidiabetic metabolite montbretin A. Plant Physiol. 180, 1277-1290 (2019).

[16] Luo, P. et al. Disequilibrium of flavonol synthase and dihydroflavonol-4-reductase expression associated tightly to white vs. red color flower formation in plants. Front. Plant Sci. 13, 1257 (2016).

[17] Owens, D. K. et al. Functional analysis of a predicted flavonol synthase gene family in Arabidopsis. Plant Physiol. 147, 1046-1061 (2008).

[18] Stracke, R. et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J. 50, 660-677 (2007).

[19] Wang, N. et al. MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant J. 90, 276-292 (2017).

[20] Czemmel, S. et al. The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol. 151, 1513-1530 (2009).

[21] Wang, X. et al. Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis. Plant J. 101, 637-652 (2020).

[22] Cavallini, E. et al. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine. Plant Physiol. 167, 1448-1470 (2015).

[23] Ma, D., Reichelt, M., Yoshida, K., Gershenzon, J., Constabel, C. & Two, P. R2R3-MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar. Plant J. 96, 949-965 (2018).

[24] Lau, O. S. & Deng, X. W. Plant hormone signaling lightens up: integrators of light and hormones. Curr. Opin. Plant Biol. 13, 571-577 (2010).

[25] Sun, J., Qi, L., Li, Y., Chu, J. & Li, C. PIF4-mediated activation of YUCCA8 expression integrates temperature into the auxin pathway in regulating Arabidopsis hypocotyl growth. PLoS Gene 8, e1002594 (2012).

[26] Chen, M., Chory, J. & Fankhauser, C. Light signal transduction in higher plants. Annu. Rev. Genet. 38, 87-117 (2004).

[27] Legris, M., Nieto, C., Sellaro, R., Prat, S. & Casal, J. J. Perception and signaling of light and temperature cues in plants. Plant J. 90, 683-697 (2017).

[28] Nguyen, N. H. et al. MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis. Plant J. 84, 1192-1205 (2015).

[29] Solovchenko, A. & Schmitz-Eiberger, M. Significance of skin flavonoids for UV-B-protection in apple fruits. J. Exp. Bot. 54, 1977-1984 (2003).

[30] Zhu, D. et al. Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development. Plant Cell 20, 2307-23 (2008).

[31] Osterlund, M. T., Hardtke, C. S., Wei, N. & Deng, X. W. Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405, 462-466 (2000).

[32] Zhang, H. et al. Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulation. Plant J. 65, 346-358 (2011).

[33] Stracke, R. et al. The Arabidopsis bZIP transcription factor HY5 regulates expression of the PFG1/MYB12 gene in response to light and ultraviolet-B radiation. Plant Cell Environ. 33, 88-103 (2010).

[34] Zhang, X. et al. A PIF1/PIF3-HY5-BBX23 transcription factor cascade affects photomorphogenesis. Plant Physiol. 174, 2487-2500 (2017).

[35] Leivar, P. & Monte, E. PIFs: systems integrators in plant development. Plant Cell. 26, 56-78 (2014).

[36] Kim, J. et al. Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction. Plant Cell. 15, 2399-2407 (2003).

[37] Al-Sady, B., Ni, W., Kircher, S., Schäfer, E. & Quail, P. H. Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol. Cell 23, 439-446 (2006).

[38] Pedmale, U. V. et al. Cryptochromes interact directly with PIFs to control plant growth in limiting blue light. Cell 164, 233-245 (2016).

[39] Chen, D. et al. Antagonistic basic helix-loop-helix/bZIP transcription factors form transcriptional modules that integrate light and reactive oxygen species signaling in Arabidopsis. Plant Cell 25, 1657-1673 (2013).

[40] Shi, J. et al. Transcriptional responses and flavor volatiles biosynthesis in methyl jasmonate-treated tea leaves. BMC Plant Biol. 15, 233 (2015).

[41] Zhang, Q. et al. Transcriptome dynamics of Camellia sinensis in response to continuous salinity and drought stress. Tree Genet. Genomes 13, 78 (2017).

[42] Ahmad, M. Z. et al. Genome-wide analysis of serine carboxypeptidase-like acyltransferase gene family for evolution and characterization of enzymes involved in the biosynthesis of galloylated catechins in the tea plant (Camellia sinensis). Front. Plant Sci. 11, 848 (2020).

[43] Li, P. et al. Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume. Plant Biotechnol. J. 14, 1604-1618 (2016).

[44] Dinç, E. et al. Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus-and chloroplast-encoded proteins of higher plant chloroplasts. Plant Physiol. 157, 1628-1641 (2011).

[45] Xie, Z. et al. A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition. Plant J. 77, 954-961 (2014).

[46] Wei, C. et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality. Proc. Natl Acad. Sci. USA 115, E4151-E4158 (2018).

[47] Xu, Y. et al. Quantitative analyses of the bitterness and astringency of catechins from green tea. Food Chem. 258, 16-24 (2018).

[48] Narukawa, M., Kimata, H., Noga, C., & Watanabe, T. Taste characterization of green tea catechins. Int. J. Food Sci. Technol. 45, 1579-1585 (2010).

[49] Vu, T. T. et al. Characterization of Brassica napus flavonol synthase involved in flavonol biosynthesis in Brassica napus L. J. Agric. Food Chem. 35, 7819-7829 (2015).

[50] Neugart, S., Krumbein, A., & Zrenner, R. Influence of light and temperature on gene expression leading to accumulation of specific flavonol glycosides and hydroxycinnamic acid derivatives in kale (Brassica oleracea var. sabellica). Front. Plant Sci. 7, 326 (2016).

[51] Matus, J. T. et al. Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J. Exp. Bot. 60, 853-867 (2009).

[52] Jiao, Y., Lau, O. S. & Deng, X. W. Light-regulated transcriptional networks in higher plants. Nat. Rev. Genet. 8, 217-230 (2007).

[53] Kranz, H. D. et al. Towards functional characterization of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J. 16, 263-276 (1998).

[54] Li, M. et al. Functional characterization of tea (Camellia sinensis) MYB4a transcription factor using an integrative approach. Front. Plant Sci. 8, 943 (2017).

[55] Zoratti, L., Karppinen, K., Escobar, A. L., Häggman, H. & Jaakola, L. Light-controlled flavonoid biosynthesis in fruits. Front. Plant Sci. 5, 534 (2014).

[56] Menkens, A. E., Schindler, U. & Cashmore, A. R. The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem. Sci. 20, 506-510 (1995).

[57] Ni, M., Tepperman, J. M. & Quail, P. H. PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein. Cell 95, 657-667 (1998).

[58] Binkert, M. et al. UV-B-responsive association of the Arabidopsis bZIP transcription factor ELONGATED HYPOCOTYL5 with target genes, including its own promoter. Plant Cell 26, 4200-4213 (2014).

[59] Loyola, R. et al. The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signaling pathway in grapevine and mediate flavonol accumulation in response to the environment. J. Exp. Bot. 18, 5429-5445 (2016).