Gibberellin promotes theanine synthesis by relieving the inhibition of CsWRKY71 on CsTSI in tea plant (Camellia sinensis)

Abstract

Theanine is a crucial indicator of tea quality, and its significance is closely tied to the economic value of tea. There have been many reports on the regulation mechanism of theanine synthesis and accumulation, but the mechanism by which gibberellin regulates theanine synthesis in tea plants is poorly understood. Previous studies have shown that the content of theanine experiences significant changes in the growth stages of tea shoots, displaying a strong correlation with gibberellin. This study confirmed that gibberellin significantly promoted the expression of the major gene of theanine synthesis, known as CsTSI. Additionally, the study identified CsWRKY71 as a transcription factor that mediated the regulation by gibberellin of theanine synthesis in tea plants. CsWRKY71 was localized in the nucleus and had a typical WRKY domain. It was a member of subclass IIC and its expression was significantly suppressed following exogenous GA₃ treatment. Further assays, such as the electrophoretic mobility shift assay, dual luciferase and asODN (antisense oligodeoxynucleotide) interference, demonstrated that CsWRKY71 significantly interacted with the promoter of CsTSI, which inhibited theanine synthesis by binding to the cis-acting element (C/T)TGAC(T/C) of the CsTSI promoter. Overall, the addition of exogenous gibberellin alleviated the inhibition of CsTSI by down-regulating the expression of CsWRKY71, ultimately facilitating the rapid biosynthesis of theanine. This study elucidated the molecular mechanism of CsWRKY71-mediated gibberellin regulation of theanine synthesis in tea plant. The findings not only enhance our understanding of the regulatory processes involved in theanine synthesis in tea plants, but also provide important references for maintaining the characteristics of high theanine in the tea plant.

Acknowledgements

This work was supported by grants from the National Key R&D Program of China (2022YFD1600801), the Natural Science Foundation of Hunan Province (2023JJ30317, 2024JJ5215), the Natural Science Foundation of Changsha (kq2208086), and the Hunan Innovative Province Construction Project (2021NK1020).

Author contributions

W.Li, F.X. and Y.S. designed this research. F.X., L.Z., C.D., X. J., H.L., W.Y., and W.Luo performed the experiments and analyzed the data. F.X. wrote the manuscript. W.L. conceived the research and finalized the manuscript. All authors reviewed and approved the manuscript.

Data availability

All relevant data in this study are provided in the article and its supplementary files.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary Data

Supplementary data are available at Horticulture Research online.

References

[1] Chen T, Ma J, Li H. et al. CsGDH2.1 negatively regulates theanine accumulation in the late-spring tea plants (Camellia sinensis var. sinensis). Hortic Res. 2022;10:uhac245

[2] Schallier A, Vermoesen K, Loyens E. et al. L-Theanine intake increases threshold for limbic seizures but decreases threshold for generalized seizures. Nutr Neurosci. 2013;16:78-82

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

[4] Zhu B, Qiao S, Li M. et al. Strong biosynthesis and weak catabolism of theanine in new shoots contribute to the high theanine accumulation in albino/etiolated tea plant (Camellia sinensis). Beverage Plant Res. 2023;3:1-9

[5] Liu S, Li J, Huang J. et al. New advances in genetic engineering for L-theanine biosynthesis. Trends Food Sci Technol. 2021;114:540-51

[6] 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;115:4151-8

[7] Xie N, Huang X, Zhou J. et al. The R2R3-MYB transcription factor CsMYB 42 regulates theanine biosynthesis in albino tea leaves. Plant Sci. 2023;336:111850

[8] Li F, Dong C, Yang T. et al. Seasonal theanine accumulation and related gene expression in the roots and leaf buds of tea plants ( Camellia sinensis L.). Front Plant Sci. 2019;10:1397

[9] LiuZ WuZ,LiH. 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;8:1-11

[10] Ku K, Choi J, Kim J. et al. Metabolomics analysis reveals the compositional differences of shade grown tea (Camellia sinensis L.). J Agric Food Chem. 2010;58:418-26

[11] Liu J, Zhang Q, Liu M. et al. Metabolomic analyses reveal distinct change of metabolites and quality of green tea during the short duration of a single spring season. J Agric Food Chem. 2016;64:3302-9

[12] Yu Y, Kou X, Gao R. et al. Glutamine synthetases play a vital role in high accumulation of theanine in tender shoots of albino tea germplasm "Huabai 1". J Agric Food Chem. 2021;69:13904-15

[13] Nan C, Joe B. Analysis by high-performance liquid-chromatography of free amino-acids extracted from needles of drought-stressed and shaded Pinus ponderosa seedlings. Physiol Plantarum. 1990;79:23-30

[14] Li F, Dong C, Yang T. et al. The tea plant CsLHT1 and CsLHT 6 transporters take up amino acids, as a nitrogen source, from the soil of organic tea plantations. Hortic Res. 2021;8:178

[15] Deng W, Fei Y, Wang S. et al. Effect of shade treatment on theanine biosynthesis in Camellia sinensis seedlings. Plant Growth Regul. 2013;71:295-9

[16] Guo J, Zhu B, Chen Y. et al. Potential ’accelerator’ and ’brake’ reg-ulation of theanine biosynthesis in tea plant (Camellia sinensis). Hortic Res. 2022;9:uhac169

[17] Wen B, Li J, Luo Y. et al. Identification and expression profiling of MYB transcription factors related to L-theanine biosynthesis in Camellia sinensis. Int J Biol Macromol. 2020;164:4306-17

[18] Cheng H, Wu W, Liu X. et al. Transcription factor CsWRKY40 regulates L-theanine hydrolysis by activating the CsPDX2.1 pro-moter in tea leaves during withering. Hortic Res. 2022;9:uhac025

[19] Wen B, Luo Y, Liu D. et al. The R2R3-MYB transcription factor CsMYB 73 negatively regulates L-theanine biosynthesis in tea plants (Camellia sinensis L.). Plant Sci. 2020;298:110546

[20] Heerah S, Katari M, Penjor R. et al. WRKY 1 mediates transcrip-tional regulation of light and nitrogen signaling pathways. Plant Physiol. 2019;181:1371-88

[21] Rushton P, Somssich I, Ringler P. et al. WRKY transcription factors. Trends Plant Sci. 2010;15:247-58

[22] Teng R, Wang Y, Lin S. et al. CsWRKY13, a novel WRKY transcrip-tion factor of Camellia sinensis, involved in lignin biosynthesis and accumulation. Beverage Plant Res. 2021;1:1-9

[23] Fan X, Yang Y, Li M. et al. Transcriptomics and targeted metabolomics reveal the regulatory network of Lilium davidii var. unicolor during bulb dormancy release. Planta. 2021;254:59

[24] Zhao H, Mallano A, Li F. et al. Characterization of CsWRKY29 and CsWRKY37 transcription factors and their functional roles in cold tolerance of tea plant. Beverage Plant Res. 2022;2:1-13

[25] Xie Z, Zhang Z, Zou X. et al. Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleu-rone cells. Plant J. 2006;46:231-42

[26] Zhang Z, Xie Z, Zou X. et al. A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol. 2004;134:1500-13

[27] Mauceri A, Abenavoli M, Toppino L. et al. Transcriptomics reveal new insights into molecular regulation of nitrogen use efficiency in Solanum melongena. J Exp Bot. 2021;72:4237-53

[28] Li W, Xiang F, Su Y. et al. Gibberellin increases the bud yield and theanine accumulation in Camellia sinensis ( L.) Kuntze. Molecules. 2021;26:3290

[29] Chen W, Zheng C, Yao M. et al. The tea plant CsWRKY26 promotes drought tolerance in transgenic Arabidopsis plants. Beverage Plant Res. 2021;1:1-11

[30] Luo Y, Huang X, Song X. et al. Identification of a WRKY transcrip-tional activator from Camellia sinensis that regulates methylated EGCG biosynthesis. Hortic Res. 2022;9:uhac024

[31] Bulley S, Wilson F, Hedden P. et al. Modification of gibberellin biosynthesis in the grafted apple scion allows control of tree height independent of the rootstock. Plant Biotechnol J. 2005;3:215-23

[32] Anzala F, Paven M, Fournier S. et al. Physiological and molecular aspects of aspartate-derived amino acid metabolism during ger-mination and post-germination growth in two maize genotypes differing in germination efficiency. J Exp Bot. 2006;57:645-53

[33] El-Yazal M, El-Yazal S, Rady M. Exogenous dormancy-breaking substances positively change endogenous phytohormones and amino acids during dormancy release in ’Anna’ apple trees. Plant Growth Regul. 2014;72:211-20

[34] Yuan X, Fang R, Zhou K. et al. The APETALA 2 homolog CaFFN regulates flowering time in pepper. Hortic Res. 2021;8:208

[35] Ma Y, Xu D, Li L. et al. Jasmonate promotes artemisinin biosyn-thesis by activating the TCP14-ORA complex in Artemisia annua. Sci Adv. 2018;4:eaas9357