Identification of a Novel Gene Encoding the Specialized Alanine Decarboxylase in Tea (Camellia sinensis) Plants

Abstract

Theanine, a unique amino acid in Camellia sinensis, accounts for more than 50% of total free amino acids in tea and has a significant contribution to the quality of green tea. Previous research indicated that theanine is synthesized from glutamic acid (Glu) and ethylamine mainly in roots, and that theanine accumulation depends on the availability of ethylamine which is derived from alanine (Ala) decarboxylation catalyzed by alanine decarboxylase (AlaDC). However, the specific gene encoding AlaDC protein remains to be discovered in tea plants or in other species. To explore the gene of AlaDC in tea plants, the differences in theanine contents and gene expressions between pretreatment and posttreatment of long-time nitrogen starvation were analyzed in young roots of two tea cultivars. A novel gene annotated as serine decarboxylase (SDC) was noted for its expression levels, which showed high consistency with theanine content, and the expression was remarkably high in young roots under sufficient nitrogen condition. To verify its function, full-length complementary DNA (cDNA) of this candidate gene was cloned from young roots of tea seedlings, and the target protein was expressed and purified from Escherichia coli (E. coli). The enzymatic activity of the protein for Ala and Ser was measured in vitro using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). The results illustrated that the target protein could catalyze the decarboxylation of Ala despite of its high similarity with SDC from other species. Therefore, this novel gene was identified as AlaDC and named CsAlaDC. Furthermore, the gene expression levels of CsAlaDC in different tissues of tea plants were also quantified with quantitative real-time PCR (qRT-PCR). The results suggest that transcription levels of CsAlaDC in root tissues are significantly higher than those in leaf tissues. That may explain why theanine biosynthesis preferentially occurs in the roots of tea plants. The expression of the gene was upregulated when nitrogen was present, suggesting that theanine biosynthesis is regulated by nitrogen supply and closely related to nitrogen metabolism for C. sinensis. The results of this study are significant supplements to the theanine biosynthetic pathway and provide evidence for the differential accumulation of theanine between C. sinensis and other species.

Keywords: Camellia sinensis; alanine decarboxylase; enzymatic activity; ethylamine; gene expression; nitrogen metabolism; theanine.

Figure 1

Biosynthetic pathway of theanine (A), and decarboxylation of Ser (B). AlaDC, alanine decarboxylase; TS, theanine synthetase; SDC, serine decarboxylase.

Figure 2

Effects of nitrogen starvation on content of amino acids involved in theanine biosynthetic pathway (A–C) in the root of tea plants. ZC108 refers to tea cultivar “Zhongcha 108”, and ZC302 refers to tea cultivar “Zhongcha 302”. Error bars represent the standard errors across three biological replicates. Different letters above the bars indicate significant differences at p < 0.05.

Figure 3

Effects of nitrogen starvation on gene expression levels of pCsAlaDC in the roots of tea plants. * represents a significant difference at p < 0.05.

Figure 4

Cloning and sequence analysis of pCsAlaDC from tea plants. (A) Prediction of the gene structure using online software Splign. The genome sequence of the gene was obtained from the “Shuchazao” genome. (B) Phylogenetic relationship of pCsAlaDC from the tea plants with amino-acid decarboxylases from other species. The sequences of amino-acid decarboxylases from seven plant species were obtained from the UniProt database. The alignment was conducted using the ClustalW method, and the phylogenetic analysis was performed using the neighbor-joining method with MEGA7 software; pCsAlaDC is denoted by a dot. SDC, serine decarboxylase; TYDC, tyrosine decarboxylase; DCE, glutamate decarboxylase; KDC, lysine decarboxylase; SPE or ADC, arginine decarboxylase. At, Arabidopsis thaliana; Os, Oryza sativa; Gm, Glycine max; Tc, Theobroma cacao; Ga, Gossypium arboreum; Vv, Vitis vinifera; Jc, Jatropha curcas.

Figure 5

Expression, purification, and enzymatic activity of pCsAlaDC protein. (A) Gel image of pCsAlaDC protein expressed in Escherichia coli. Lanes 1–3 represent the marker, target protein, and total protein. (B) Measurement of AlaDC activity of the target protein. The assays were conducted by detecting the formation of ethylamine in the reaction mixture containing alanine after 2 h and 24 h of reaction at 35 °C. Error bars indicate the standard errors across three replicates. (C, D) Chromatogram for the decarboxylation of Ala (C) and Ser (D) catalyzed by pCsAlaDC. C-a~d (or D-a~d), the reaction buffer containing Ala (or Ser), ethylamine (or ethanolamine), pCsAlaDC, pCsAlaDC, and Ala. Peak I represents substrate Ala (or Ser), peak II represents the production ethylamine (or ethanolamine).

Figure 6

Expression of CsAlaDC in tea plants. (A) Expression of CsAlaDC in several tissues of tea cultivars. (B) Nitrogen treatment effect on the expression levels of CsAlaDC in the roots of tea plants. The reported concentrations (1 mM and 0.1 mM) refer to the level of NH₄NO₃ treatment for tea seedlings. The relative expressions of the gene were quantified using qRT-PCR and normalized to GAPDH. Error bars indicate the standard errors over three biological replicates.