Dataset Information

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

ABSTRACT:

Background

Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized.

Results

The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45?°C (pH?8.0) and 40?°C (pH?7.0), respectively. CsAlaDC was stable under 30?°C (pH?7.0) and CsSDC was stable under 40?°C (pH?6.0-8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5'-phosphate. The specific activity of CsSDC (30,488?IU/mg) was 8.8-fold higher than that of CsAlaDC (3467?IU/mg).

Conclusions

Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

SUBMITTER: Bai P

PROVIDER: S-EPMC7923638 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Publications

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

Bai Peixian P Wang Liyuan L Wei Kang K Ruan Li L Wu Liyun L He Mengdi M Ni Dejiang D Cheng Hao H

BMC biotechnology 20210301 1

<h4>Background</h4>Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized.<h4>Resul ...[more]

PMID: 33648478

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Project description: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.

Project description:Tea green leafhopper [Empoasca (Matsumurasca) onukii Matsuda] is one of the most devastating pests of tea plants (Camellia sinensis), greatly impacting tea yield and quality. A thorough understanding of the interactions between the tea green leafhopper and the tea plant would facilitate a better pest management. To gain more insights into the molecular and biochemical mechanisms behind their interactions, a combined analysis of the global transcriptome and metabolome reconfiguration of the tea plant challenged with tea green leafhoppers was performed for the first time, complemented with phytohormone analysis. Non-targeted metabolomics analysis by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS), together with quantifications by ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QqQ MS), revealed a marked accumulation of various flavonoid compounds and glycosidically bound volatiles but a great reduction in the level of amino acids and glutathione upon leaf herbivory. RNA-Seq data analysis showed a clear modulation of processes related to plant defense. Genes pertaining to the biosynthesis of phenylpropanoids and flavonoids, plant-pathogen interactions, and the biosynthesis of cuticle wax were significantly up-regulated. In particular, the transcript level for a CER1 homolog involved in cuticular wax alkane formation was most drastically elevated and an increase in C29 alkane levels in tea leaf waxes was observed. The tea green leafhopper attack triggered a significant increase in salicylic acid (SA) and a minor increase in jasmonic acid (JA) in infested tea leaves. Moreover, transcription factors (TFs) constitute a large portion of differentially expressed genes, with several TFs families likely involved in SA and JA signaling being significantly induced by tea green leafhopper feeding. This study presents a valuable resource for uncovering insect-induced genes and metabolites, which can potentially be used to enhance insect resistance in tea plants.

Dataset Information

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

Background

Results

Conclusions

Publications

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

Similar Datasets

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