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.

Comparative analysis of tissue-specific transcriptomic responses to nitrogen stress in spinach (Spinacia oleracea)

“…Additionally, we found expression of serine decarboxylase (SDC; Spo13254) significantly upregulated in response to high N in roots. Most recently, SDC like gene showing remarkably high expression in young roots under sufficient N has been characterized in tea plants [69]. We performed a BLASTP homology search of the NCBI database.…”

Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

“…New genes were defined as those that are present in all members of a monophyletic group but absent from all outgroup species [ 20 ]. Our previous research has shown that AlaDC was a novel enzyme and SDC was the protein most closely related to AlaDC [ 16 ]. In this study, a BLAST search result showed that AlaDC gene was only found in tea plants, while the homologous genes of SDC were widely distributed in monocots and dicots (Fig.…”

“…Theanine is synthesized from glutamate (Glu) and ethylamine catalyzed by theanine synthetase (TS) mainly in the roots of tea plants [ 11 – 14 ]. Ethylamine is derived mainly from the alanine decarboxylation catalyzed by alanine decarboxylase (AlaDC) [ 12 , 14 – 16 ]. A great amount of theanine is accumulated in C. sinensis, but little or no theanine in other species [ 17 , 18 ].…”

“…A previous study revealed that the differential accumulation of theanine between C. sinensis and other species could be attributed to the availability of ethylamine [ 18 ], indicating that CsAlaDC was a crucial enzyme in theanine biosynthesis pathway. Up to now, the new gene AlaDC has been found and reported only in C. sinensis [ 16 ] but not in any other species. Based on the consistency of the distribution of theanine and new gene AlaDC among species, it seemed that the differential accumulation of theanine might be attributed to the origination of AlaDC .…”

“…Serine decarboxylase (SDC), belonging to the pyridoxal-5′-phosphate-dependent decarboxylase (PLP-deC) super family, has been found to be the protein most closely related to CsAlaDC [ 16 ]. Previous studies suggested that AlaDC [ 16 ] and SDC [ 19 ] catalyzed the decarboxylation of alanine (Ala) and serine (Ser), respectively. Ala is similar to Ser in the chemical structure with the side chain of Ala being a methyl (−CH 3 ) and that of Ser being a hydroxymethyl (−CH 2 OH).…”