Tea GOLDEN2-LIKE genes enhance catechin biosynthesis through activating R2R3-MYB transcription factor

Abstract

The biosynthesis of catechins, a major type of flavonoids accumulated in tea, is mediated by developmental cues and environmental stimuli. Light enhances but shading treatment reduces catechin accumulation in tea leaves. However, the transcription factors involved in light-mediated catechin biosynthesis remain to be identified. Two GOLDEN2 LIKE genes from tea plant (CsGLK1 and CsGLK2) were isolated and characterized in both tomato and tea plants. Transcripts of both CsGLK1 and CsGLK2 were affected by light intensity in tea plants. Overexpression of CsGLK1 and CsGLK2 promoted chloroplast development and carotenoid accumulation in tomato fruits. An integrated metabolomic and transcriptomic approach revealed that both catechin content and related biosynthetic genes were upregulated in CsGLK-overexpressing tomato leaves. Our further studies in tea plants indicated that CsGLKs directly regulate the transcription of CsMYB5b, a transcription factor involved in catechin biosynthesis. Suppression of CsGLKs in tea leaves led to the reduction of both CsMYB5b expression and catechin accumulation. Taken together, the results show that CsGLKs are involved in light-regulated catechin accumulation in tea plants by regulating expression of CsMYB5b and have great potential for enhancing the accumulation of both carotenoids and flavonoids in fruits of horticultural crops.

References

[1] Middleton E Jr, Kandaswami C . Effects of flavonoids on immune and inflammatory cell functions. Biochem Pharmacol. 1992; 43: 1167-79.

[2] Khan N, Mukhtar H . Tea polyphenols in promotion of human health. Nutrients. 2018; 11: 39.

[3] Xing L, Zhang H, Qi R et al. Recent advances in the understanding of the health benefits and molecular mechanisms associated with green tea polyphenols. J Agric Food Chem. 2019; 67: 1029-43.

[4] Yang CS, Wang X, Lu G et al. Cancer prevention by tea: animal studies, molecular mechanisms and human relevance. Nat Rev Cancer. 2009; 9: 429-39.

[5] Baliga MS, Meleth S, Katiyar SK . Growth inhibitory and antimetastatic effect of green tea polyphenols on metastasis-specific mouse mammary carcinoma 4T1 cells in vitro and in vivo systems. Clin Cancer Res. 2005; 11: 1918-27.

[6] Gupta S, Hastak K, Ahmad N et al. Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc Natl Acad Sci USA. 2001; 98: 10350-5.

[7] Singh HP, Ravindranath SD, Singh C . Analysis of tea shoot catechins: spectrophotometric quantitation and selective visualization on two-dimensional paper chromatograms using diazotized sulfanilamide. J Agric Food Chem. 1999; 47: 1041-5.

[8] Punyasiri PA, Abeysinghe ISB, Kumar V et al. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways. Arch Biochem Biophys. 2004; 431: 22-30.

[9] Wu LY, Fang ZT, Lin JK et al. Complementary iTRAQ proteomic and transcriptomic analyses of leaves in tea plant (Camellia sinensis L.) with different maturity and regulatory network of flavonoid biosynthesis . J Proteome Res. 2019; 18: 252-64.

[10] Rani A, Singh K, Ahuja PS et al. Molecular regulation of catechins biosynthesis in tea [ Camellia sinensis (L.) O. Kuntze] . Gene. 2012; 495: 205-10.

[11] Wu ZJ, Li XH, Liu ZW et al. De novo assembly and transcriptome characterization: novel insights into catechins biosynthesis in Camellia sinensis . BMC Plant Biol. 2014; 14: 277.

[12] Wang P, Zhang L, Jiang X et al. Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis . Planta. 2018; 247: 139-54.

[13] Zhang LQ, Wei K, Cheng H et al. Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages. Bot Stud. 2016; 57: 31.

[14] Wang P, Liu Y, Zhang L et al. Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins. Plant J. 2020; 101: 18-36.

[15] Wang W, Zhou Y, Wu Y et al. Insight into catechins metabolic pathways of Camellia sinensis based on genome and transcriptome analysis. J Agric Food Chem. 2018; 66: 4281-93.

[16] Nesi N, Jond C, Debeaujon I et al. The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell. 2001; 13: 2099-114.

[17] Bogs J, Jaffe FW, Takos AM et al. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 2007; 143: 1347-61.

[18] Liu C, Jun JH, Dixon RA . MYB5 and MYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula . Plant Physiol. 2014; 165: 1424-39.

[19] Wei K, Wang L, Zhang Y et al. A coupled role for CsMYB75 and CsGSTF1 in anthocyanin hyperaccumulation in purple tea. Plant J. 2019; 97: 825-40.

[20] Xie DY, Sharma SB, Wright E et al. Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J. 2006; 45: 895-907.

[21] Iwasa K . Physiological aspects of catechin biosynthesis in tea plants. Jpn Agric Res Q. 1976; 10: 89-93.

[22] Saijo R . Effect of shade treatment on biosynthesis of catechins in tea plants. Plant Cell Physiol. 1980; 21: 989-98.

[23] Liu L, Li Y, She G 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. 2018; 18: 233.

[24] Rossini L, Cribb L, Martin DJ et al. The maize Golden2 gene defines a novel class of transcriptional regulators in plants. Plant Cell. 2001; 13: 1231-44.

[25] Fitter DW, Martin DJ, Copley MJ et al. GLK gene pairs regulate chloroplast development in diverse plant species. Plant J. 2002; 31: 713-27.

[26] Waters MT, Wang P, Korkaric M et al. GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis . Plant Cell. 2009; 21: 1109-28.

[27] Nakamura H, Muramatsu M, Hakata M et al. Ectopic overexpression of the transcription factor OsGLK1 induces chloroplast development in non-green rice cells. Plant Cell Physiol. 2009; 50: 1933-49.

[28] Bravo-Garcia A, Yasumura Y, Langdale JA . Specialization of the Golden2-like regulatory pathway during land plant evolution. New Phytol. 2009; 183: 133-41.

[29] Brand A, Borovsky Y, Hill T et al. CaGLK2 regulates natural variation of chlorophyll content and fruit color in pepper fruit. Theor Appl Genet. 2014; 127: 2139-48.

[30] Powell AL, Nguyen CV, Hill T et al. Uniform ripening encodes a Golden 2-like transcription factor regulating tomato fruit chloroplast development. Science. 2012; 336: 1711-5.

[31] Nguyen CV, Vrebalov JT, Gapper NE et al. Tomato GOLDEN2-LIKE transcription factors reveal molecular gradients that function during fruit development and ripening. Plant Cell. 2014; 26: 585-601.

[32] Li G, Chen D, Tang X et al. Heterologous expression of kiwifruit (Actinidia chinensis) GOLDEN2-LIKE homolog elevates chloroplast level and nutritional quality in tomato (Solanum lycopersicum) . Planta. 2018; 247: 1351-62.

[33] Yasumura Y, Moylan EC, Langdale JA . A conserved transcription factor mediates nuclear control of organelle biogenesis in anciently diverged land plants. Plant Cell. 2005; 17: 1894-907.

[34] Liu Y, Roof S, Ye Z et al. Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proc Natl Acad Sci USA. 2004; 101: 9897-902.

[35] Mustilli AC, Fenzi F, Ciliento R et al. Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1. Plant Cell. 1999; 11: 145-57.

[36] Zhang D, Tan W, Yang F et al. A BIN2-GLK1 signaling module integrates brassinosteroid and light signaling to repress chloroplast development in the dark. Dev Cell. 2021; 56: 310-324.e7.

[37] Tang X, Miao M, Niu X et al. Ubiquitin-conjugated degradation of golden 2-like transcription factor is mediated by CUL4-DDB1-based E3 ligase complex in tomato. New Phytol. 2016; 209: 1028-39.

[38] Schroeder DF, Gahrtz M, Maxwell BB et al. De-etiolated 1 and damaged DNA binding protein 1 interact to regulate Arabidopsis photomorphogenesis. Curr Biol. 2002; 12: 1462-72.

[39] Wang S, Liu J, Feng Y et al. Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4. Plant J. 2008; 55: 89-103.

[40] Li H, Li Y, Deng H et al. Tomato UV-B receptor SlUVR8 mediates plant acclimation to UV-B radiation and enhances fruit chloroplast development via regulating SlGLK2. Sci Rep. 2018; 8: 6097.

[41] Wei C, Yang H, Wang S 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. 2018; 115: E4151-8.

[42] Jiang X, Huang K, Zheng G et al. CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis. Plant Sci. 2018; 270: 209-20.

[43] Zhao M, Zhang N, Gao T et al. Sesquiterpene glucosylation mediated by glucosyltransferase UGT91Q2 is involved in the modulation of cold stress tolerance in tea plants. New Phytol. 2020; 226: 362-72.

[44] Muir SR, Collins GJ, Robinson S et al. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nat Biotechnol. 2001; 19: 470-4.

[45] Butelli E, Titta L, Giorgio M et al. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol. 2008; 26: 1301-8.

[46] Kumar S, Pandey AK . Chemistry and biological activities of flavonoids: an overview. Sci World Journal. 2013; 2013: 162750.

[47] Bino RJ, Ric de Vos CH, Lieberman M et al. The light-hyperresponsive high pigment-2dg mutation of tomato: alterations in the fruit metabolome. New Phytol. 2005; 166: 427-38.

[48] Davuluri GR, van Tuinen A, Fraser PD et al. Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotechnol. 2005; 23: 890-5.

[49] Sun C, Deng L, Du M et al. A transcriptional network promotes anthocyanin biosynthesis in tomato flesh. Mol Plant. 2020; 13: 42-58.

[50] Yan S, Chen N, Huang Z et al. Anthocyanin fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit. New Phytol. 2020; 225: 2048-63.

[51] Zhang Q, Liu M, Ruan J . Metabolomics analysis reveals the metabolic and functional roles of flavonoids in light-sensitive tea leaves. BMC Plant Biol. 2017; 17: 64.

[52] Zheng C, Ma JQ, Ma CL et al. Regulation of growth and flavonoid formation of tea plants (Camellia sinensis) by blue and green light . J Agric Food Chem. 2019; 67: 2408-19.

[53] Wang P, Chen S, Gu M et al. Exploration of the effects of different blue LED light intensities on flavonoid and lipid metabolism in tea plants via transcriptomics and metabolomics. Int J Mol Sci. 2020; 21: 4606.

[54] Lu Z, Liu Y, Zhao L et al. Effect of low-intensity white light mediated de-etiolation on the biosynthesis of polyphenols in tea seedlings. Plant Physiol Biochem. 2014; 80: 328-36.

[55] Howles PA, Sewalt VJH, Paiva NL et al. Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux into phenylpropanoid biosynthesis. Plant Physiol. 1996; 112: 1617-24.

[56] Tanner GJ, Francki KT, Abrahams S et al. Proanthocyanidin biosynthesis in plants. J Biol Chem. 2003; 278: 31647-56.

[57] Xie DY, Sharma SB, Paiva NL et al. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science. 2003; 299: 396-9.

[58] Laloi C, Stackhowiack M, Pers-Kamczyc E . Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana . Proc Natl Acad Sci USA. 2007; 104: 672-7.

[59] Asada K . The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol. 1999; 50: 601-39.

[60] Asada K . Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol. 2006; 141: 391-6.

[61] Shapiguzov A, Vainonen JP, Wrzaczek M et al. ROS-talk - how the apoplast, the chloroplast, and the nucleus get the message through. Front Plant Sci. 2012; 3: 292.

[62] Winkel-Shirley B . Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol. 2002; 5: 218-23.

[63] Bernatoniene J, Kopustinskiene DM . The role of catechins in cellular responses to oxidative stress. Molecules. 2018; 23: 965.

[64] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25: 402-8.

[65] Want EJ, Wilson ID, Gika H et al. Global metabolic profiling procedures for urine using UPLC-MS. Nat Protoc. 2010; 5: 1005-18.

[66] Wang L, Tang W, Hu Y et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang . Plant J. 2019; 99: 359-78.

[67] Li Y, Deng H, Miao M et al. Tomato MBD5, a methyl CpG binding domain protein, physically interacting with UV-damaged DNA binding protein-1, functions in multiple processes. New Phytol. 2016; 210: 208-26.