Transcription factor CsWRKY40 regulates L-theanine hydrolysis by activating the CsPDX2.1 promoter in tea leaves during withering

Abstract

L-Theanine is a crucial secondary metabolite in tea and positively determines the potential quality and health benefits of tea products. Previous work found that the content of L-theanine decreased during the withering process, although the specific mechanism is still unknown. Here, weighted gene co-expression network analysis (WGCNA) was performed based on transcriptome data obtained previously. The key hydrolysis gene CsPDX2.1 in L-theanine metabolism and seven candidate transcription factors were screened out. Among these transcription factors, CsWRKY40 displayed the strongest activation on the CsPDX2.1 promoter (373.18-fold) by binding to the W box element, based on dual luciferase assay and EMSA results. The CsWRKY40 protein was located in the nucleoplasm, whereas CsPDX2.1 was found in both the nucleoplasm and cytoplasm. Analysis of withering, water-retention, and water-loss treatments confirmed that water loss from tea leaves was the critical factor that affected ABA and L-theanine contents by activating the expression of CsWRKY40 and CsPDX2.1. Our results provide a new insight into the regulatory mechanism of L-theanine hydrolysis metabolism.

References

[1] Wurtzel ET, Kutchan TM . Plant metabolism, the diverse chemistry set of the future. Science. 2016; 353: 1232-6.

[2] Yang L, Wen KS, Ruan X et al. Response of plant secondary metabolites to environmental factors. Molecules. 2018; 23: 762.

[3] Isah T . Stress and defense responses in plant secondary metabolites production. Biol Res. 2019; 52: 39-64.

[4] Zhang S, Xuan H, Zhang L et al. TBC2health: a database of experimentally validated health-beneficial effects of tea bioactive compounds. Brief Bioinform. 2017; 18: 830-6.

[5] Wei CL, 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 U S A. 2018; 115: E4151-8.

[6] Dong CX, Li F, Yang T et al. Theanine transporters identified in tea plants (Camellia sinensis L.) . Plant J. 2020; 101: 57-70.

[7] Feng L, Gao MJ, Hou RY et al. Determination of quality constituents in the young leaves of albino tea cultivars. Food Chem. 2014; 155: 98-104.

[8] Narukawa M, Toda Y, Nakagita T et al. L-theanine elicits umami taste via the T1R1 + T1R3 umami taste receptor. Amino Acids. 2014; 46: 1583-7.

[9] Guo X, Song C, Ho CT et al. Contribution of L-theanine to the formation of 2,5-dimethylpyrazine, a key roasted peanutty flavor in oolong tea during manufacturing processes. Food Chem. 2018; 263: 18-28.

[10] Guo X, Ho CT, Schwab W et al. Aroma compositions of large-leaf yellow tea and potential effect of theanine on volatile formation in tea. Food Chem. 2019; 280: 73-82.

[11] Haskell CF, Kennedy DO, Milne AL et al. The effects of L-theanine, caffeine and their combination on cognition and mood. Biol Psychol. 2008; 77: 113-22.

[12] Zhao Y, Zhao BL . The neuroprotective effect of L-theanine and its inhibition on nicotine dependence. Chin Sci Bull. 2014; 59: 4014-9.

[13] Turkozu D, Sanlier N . L-theanine, unique amino acid of tea, and its metabolism, health effects, and safety. Crit Rev Food Sci Nutr. 2017; 57: 1681-7.

[14] Sasaoka K, Inagaki H, Kito M . Studies on biosynthesis of theanine in tea seedlings synthesis of theanine by homogenate of tea seedlings. Biosci Biotechnol Biochem. 1963; 27: 467-8.

[15] Ashihara H. Occurrence, biosynthesis and metabolism of L-theanine (gamma-glutamyl-L-ethylamide) in plants: a comprehensive review. Nat Prod Commun. 2015; 10: 803-10.

[16] Cheng SH, Fu X, Liao Y et al. Differential accumulation of specialized metabolite L-theanine in green and albino-induced yellow tea (Camellia sinensis) leaves . Food Chem. 2019; 276: 93-100.

[17] Zhang L, Ho CT, Zhou J et al. Chemistry and biological activities of processed Camellia sinensis teas: a comprehensive review. Compr Rev Food Sci Food Saf. 2019; 18: 1474-95.

[18] Chen QC, Shi J, Mu B et al. Metabolomics combined with proteomics provides a novel interpretation of the changes in nonvolatile compounds during white tea processing. Food Chem. 2020; 332: 127412.

[19] Yu XL, Li Y, He C et al. Nonvolatile metabolism in postharvest tea (Camellia sinensis L.) leaves: effects of different withering treatments on nonvolatile metabolites, gene expression levels, and enzyme activity . Food Chem. 2020; 327: 126992.

[20] Xu P, Su H, Zhao S et al. Transcriptome and phytochemical analysis reveals the alteration of plant hormones, characteristic metabolites, and related gene expression in tea (Camellia sinensis L.) leaves during withering . Plants (Basel). 2020; 9: 204.

[21] Liu ZW, Li H, Wang WL et al. CsGOGAT is important in dynamic changes of theanine content in postharvest tea plant leaves under different temperature and shading spreadings. J Agric Food Chem. 2017; 65: 9693-702.

[22] Fu XM, Cheng S, Liao Y et al. Characterization of L-theanine hydrolase in vitro and subcellular distribution of its specific product ethylamine in tea (Camellia sinensis) . J Agric Food Chem. 2020; 68: 10842-51.

[23] Sharma E, Joshi R, Gulati A . L-theanine: an astounding sui generis integrant in tea. Food Chem. 2018; 242: 601-10.

[24] Wang PJ, 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.

[25] Cao HL, Wang L, Yue C et al. Isolation and expression analysis of 18 CsbZIP genes implicated in abiotic stress responses in the tea plant (Camellia sinensis) . Plant Physiol Biochem. 2015; 97: 432-42.

[26] Cui X, Wang YX, Liu ZW et al. Transcriptome-wide identification and expression profile analysis of the bHLH family genes in Camellia sinensis . Functional Integrative Genomics. 2018; 18: 489-503.

[27] Wang YX, Liu ZW, Wu ZJ et al. Genome-wide identification and expression analysis of GRAS family transcription factors in tea plant (Camellia sinensis) . Sci Rep. 2018; 8: 3949.

[28] Wang PJ, Yue C, Chen D et al. Genome-wide identification of WRKY family genes and their response to abiotic stresses in tea plant (Camellia sinensis) . Genes Genom. 2019; 41: 17-33.

[29] Eulgem T, Rushton PJ, Robatzek S et al. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000; 5: 199-206.

[30] Rushton PJ, Somssich IE, Ringler P et al. WRKY transcription factors. Trends in Plant Sci. 2010; 15: 247-58.

[31] Sun C, Palmqvist S, Olsson H et al. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter. Plant Cell. 2003; 15: 2076-92.

[32] Cai M, Qiu D, Yuan T et al. Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance . Plant Cell Environ. 2008; 31: 86-96.

[33] Schluttenhofer C, Yuan L . Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol. 2015; 167: 295-306.

[34] Chen W, Hao WJ, Xu YX et al. Isolation and characterization of CsWRKY7, a subgroup IId WRKY transcription factor from Camellia sinensis, linked to development in Arabidopsis . Int J Mol Sci. 2019; 20: 2815.

[35] Chen W, Zheng C, Yao MZ et al. The tea plant CsWRKY26 promotes drought tolerance in transgenic Arabidopsis plants . Beverage Plant Reasearch. 2021; 1: 3.

[36] Wang Y, Shu Z, Wang W et al. CsWRKY2, a novel WRKY gene from Camellia sinensis, is involved in cold and drought stress responses . Biol Plant. 2016; 60: 443-51.

[37] Luo Y, Yu S, Li J et al. Molecular characterization of WRKY transcription factors that act as negative regulators of O-methylated catechin biosynthesis in tea plants (Camellia sinensis L.) . J Agric Food Chem. 2018; 66: 11234-43.

[38] Ai ZY, Zhang B, Chen Y et al. Impact of light irradiation on black tea quality during withering. J Food Sci Technol. 2017; 54: 1212-27.

[39] Liu ZW, Wu ZJ, Li H et al. L-theanine content and related gene expression: novel insights into theanine biosynthesis and hydrolysis among different tea plant (Camellia sinensis L.) tissues and cultivars . Front Plant Sci. 2017; 8: 498.

[40] Wang WD, Xin H, Wang M et al. Transcriptomic analysis reveals the molecular mechanisms of drought-stress-induced decreases in Camellia sinensis leaf quality . Front Plant Sci. 2016; 7: 385.

[41] Yu ZM, Yang ZY . Understanding different regulatory mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea (Camellia sinensis) provides new insights into the safe and effective alteration of tea flavor and function . Crit Rev Food Sci Nutr. 2020; 60: 844-58.

[42] Zeng LT, Wang XW, Liao YY et al. Formation of and changes in phytohormone levels in response to stress during the manufacturing process of oolong tea (Camellia sinensis) . Postharvest Biol Technol. 2019; 157: 110974.

[43] Liao YY, Yu Z, Liu X et al. Effect of major tea insect attack on formation of quality-related nonvolatile specialized metabolites in tea (Camellia sinensis) leaves . J Agric Food Chem. 2019; 67: 6716-24.

[44] Shang Y, Yan L, Liu ZQ et al. The mg-chelatase H subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition. Plant Cell. 2010; 22: 1909-35.

[45] Hellens RP, Edwards EA, Leyland NR et al. pGreen: a versatile and flexible binary Ti vector for agrobacterium-mediated plant transformation. Plant Mol Biol. 2000; 42: 819-32.

[46] Wu W, Wang MM, Gong H et al. High CO₂/hypoxia-induced softening of persimmon fruit is modulated by DkERF8/16 and DkNAC9 complexes . J Exp Bot. 2020; 71: 2690-700.