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.
Highlights
Tea is the most widely consumed non-alcoholic beverage in the world [1,2]
A novel gene coding for CsAlaDC was identified from the roots of tea plants by analyzing amino-acid contents and the differentially expressed genes in roots between the nitrogen starvation and control groups
The enzymatic activity of CsAlaDC protein was measured in vitro, and the results illustrated that the target protein could catalyze the decarboxylation of Ala despite of its high similarity with serine decarboxylase (SDC) from other species
Summary
Tea is the most widely consumed non-alcoholic beverage in the world [1,2]. An important reason is the most widely consumed non-alcoholic beverage inprovided the worldby [1,2]. EC 1.4.1.2) pathway [32,33] Another precursor of theanine, ethylamine, is derived alanine (Ala). [32,33] Another precursor of theanine, ethylamine, is derived from alanine decarboxylation decarboxylation catalyzed by AlaDC [26]. Studies proposed that alanine andstudy acetaldehyde might be the the might be the of Previous ethylamine in plant tissues [34,35]. CsAlaDC plays a vital role in the occurrence of theanine in tea plants by producing ethylamine ethylamine from Ala [26,36,37]. The specific gene coding the protein remains to be reported remains to be reported in tea plants or any other species. After expression and purification the target gene was cloned from the root tissues of tea plants.
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