The R2R3-MYB transcription factor CsMYB73 negatively regulates l-Theanine biosynthesis in tea plants (Camellia sinensis L.)
Introduction
Tea (Camellia sinensis L.) is a commercial crop that is cultivated globally [1]. Tea leaves are used to produce various types of tea with rich flavors and health benefits [2]. Theanine (γ-glutamylethylamine), mostly present as its l-enantiomer in tea plants, accounts for more than 50 % of the free amino acids (FAAs) [3] and has numerous prominent health effects in humans, such as enhancing relaxation, cognitive performance, and learning ability [4]. l-Theanine is also an important contributor to tea quality and is mainly responsible for the umami flavor in green tea [5]. Further, in plant growth, l-theanine has vital physiological functions not only in protein synthesis, but also as a precursor for the biosynthesis of numerous bioactive molecules [6]. In mature tea plants, theanine is mainly synthetized in the roots and is then transported to the young shoots and other organs [4]. l-Theanine content is high in young leaves, and decreases progressively during leaf maturation [7,8]. However, the molecular mechanisms that regulate l-theanine biosynthesis are not completely unraveled. Exploration of the regulatory mechanisms underlying l-theanine biosynthesis may provide a better understanding of this process and could contribute to the genetic improvement and breeding of the tea plants.
l-Theanine is biosynthesized in the nitrogen cycle, specifically, in the phenylalanine pathway [9]. Several core enzymes participate in theanine metabolism, and the expression levels of genes encoding pivotal enzymes largely influence the accumulation, distribution, and metabolism of theanine in tea plants [7]. Glutamine synthetase (GS, EC 6.3.1.2) is a pivotal enzyme in l-theanine biosynthesis, especially in the GS-glutamine-2-oxoglutarate amidotransferase (GS-GOGAT) (GOGAT, EC 1.4.7.1; 1.4.1.14) cycle [9,10]. Studies have shown that GS catalyzes the conversion of glutamate and free ammonium to glutamine in plants [[11], [12], [13]]. The glutamine synthetase proteins can be divided into cytoplasmic (GS1) and plastidic (GS2) isoforms [13]. GS1 is particularly important for assimilating ammonium from primary nitrogen uptake and nitrogen remobilization. Studies on overexpression of cytosolic GS suggested that it is efficient in improving plant growth and seed production [11]. In higher plants, GS2 is the major form that mainly be functional in reassimilating ammonium released during photorespiration. In general, only one gene encoding GS2 protein, while a small group up to five or six genes were known to encodes GS1 protein in various species [14]. In tea plant, the expression of CsGS1 and CsGS2 genes exhibited the highest levels in ‘Huangjinya’ with the highest theanine concentration against with that in ‘Anjibaicha’ (Baiye 1), and ‘Yingshuang’ [7], suggested that CsGS1 and CsGS2 genes may play curial roles in theanine metabolism.
Recently, several transcription factors (TFs) involved in nitrogen metabolism or the phenylalanine pathway, including R2R3-MYB [15], NAC [16], Dof [17], NF-YC [18], TyrR [19], and ERF [20], have been identified. The MYB superfamily regulates numerous physiological responses, including plant growth, hormone signaling, stress responses, and metabolite biosynthesis, at the transcriptional level [21,22]. MYB TFs contain conserved MYB domains and are classified into three major subfamilies based on the number of MYB domain repeats, known as R1-MYB, R2R3-MYB, and R1R2R3-MYB [23]. In plants, the R2R3-MYB subfamily is the largest [24]. To date, 138, 100, and 140 R2R3-MYB TFs have been identified in Arabidopsis [25], Citrus sinensis [26], and Camellia sinensis [27], respectively. Some R2R3-MYB members can directly or indirectly regulate the expression of key genes in the phenylalanine pathway. For example, two MYB TFs isolated from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, directly bind to the PpGS1b promoter in maritime pine (Pinus pinaster) to activate PpGS1b transcription [28]. Similarly, CmMYB1, an R2R3-type MYB TF identified from a unicellular red alga (Cyanidioschyzon merolae) is related to the expression of key nitrogen assimilation genes, including CmGS, CmNIR, and CmAMT [29]. While R2R3-MYB TFs regulate the expression of key genes in plant nitrogen cycles, few R2R3-MYB downstream genes have been well explored. In addition, the transcriptional regulation of tea R2R3-MYB TFs in l-theanine biosynthesis remains elusive.
In the present study, the tea leaves of two cultivars, ‘Baojing Huangjincha 1’ and ‘Baiye 1’, were used to determine the l-theanine and FAA contents in three developmental stages. The expression patterns of the l-theanine biosynthetic genes CsGS1 and CsGS2 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR). Moreover, an R2R3-MYB TF, designated as CsMYB73, was identified and functionally characterized. Further assays revealed that CsMYB73 negatively regulates l-theanine biosynthesis by directly binding to the promoters of CsGS1 and CsGS2 to repress their expression.
Section snippets
Plant materials
Two tea cultivars [C. sinensis (L.) O. Kuntz, ‘Baojing Huangjincha 1’ and ‘Baiye 1’ (also known as ‘White leaf no. 1’ [30])] were cultivated at the Gaoqiao Tea Garden, Hunan Academy of Agricultural Sciences. For each cultivar, leaves from 10 mature tea plants at three growth stages were collected on March 21, April 7, and May 3 (i.e., 1, 3, and 7 weeks after bud germination, respectively) in 2018, and the sampling standard was “one bud and two leaves.” The samples were immediately frozen in
l-Theanine and FAA contents, and expression levels of l-theanine biosynthetic genes in tea leaves at three developmental stages
The contents of l-theanine and total FAAs in the leaves of two tea cultivars, i.e. ‘Baojing Huangjincha 1’ and ‘Baiye 1’, were determined in three developmental stages. We used the “one bud and two young leaves” sampling standard, as shown in Fig. 1A. l-Theanine contents were the highest in leaves sampled on March 21 (1 week after bud germination) (Fig. 1B), with 21.00 ± 1.87 mg/g and 23.00 ± 0.28 mg/g in ‘Baojing Huangjincha 1’ and ‘Baiye 1’, respectively. By May 3 (7 weeks after bud
CsMYB73 is a TF that belongs to subgroup 22 of the R2R3-MYB family
In this study, we isolated an R2R3-MYB TF named CsMYB73 from C. sinensis, that is negatively involved in l-theanine biosynthesis by regulating key metabolic genes. Bioinformatic analysis revealed that CsMYB73 belongs to subgroup 22 of the R2R3-MYB and has a high sequence similarity with AtMYB73 (78 %) (Fig. 3A and 3B). Subgroup 22 R2R3-MYB TFs have well-known, vital roles in stress responses [49], hormone signal transduction [50,51], and plant growth [52]. Notably, subgroup 22 R2R3-MYB TFs have
Conclusions
We isolated and characterized CsMYB73 of C. sinensis, and found that it is a transcriptional repressor in l-theanine biosynthesis. As a nuclear-located protein, CsMYB73 specifically binds to MYBRs in the promoters of target genes, including CsGS1 and CsGS2, to suppress their expression. This study provided insights into the mechanism of l-theanine biosynthesis.
Author contribution statement
BW carried out the experiments. YL, DL, and XZ analyzed the results. ZP and JL helped with the experiments. KW, JH, and ZL designed this research. BW wrote and JH revised the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (U19A2030, 31470692, 31670691, and 31500567). We are grateful to Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, South China Agricultural University for providing vectors and to graduate student Hui Zhang (Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, South China Agricultural University) for providing advice in the experimental
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