Cloning and Expression Analysis of CsTDIFL and Its Regulation on Amino Acid Synthesis in Tea Plants （Camellia sinensis）

doi:10.13560/j.cnki.biotech.bull.1985.2025-0918

Abstract

Abstract:

Objective This study aimed to investigate the regulatory role of the TDIF signaling peptide in nitrogen metabolism and amino acid accumulation in tea plants, thereby laying a foundation for further research into the molecular mechanisms of TDIF peptide influencing tea physiological quality and amino acid metabolism. Method Using the tea cultivar ‘Baojing Huangjincha 2’ as plant material, the CsTDIFL gene was cloned and analyzed by bioinformatics. Tissue-specific expression profiles of CsTDIFL and its receptor gene CsTDR, along with their expression dynamics under exogenous TDIF peptide treatment, were examined using quantitative real-time polymerase chain reaction (RT-qPCR). High-performance liquid chromatography (HPLC) and enzyme activity assays were applied to evaluate the effects of TDIF on amino acid content and key nitrogen metabolism enzymes. Functional validation was validated through transient suppression of CsTDIFL expression with antisense oligonucleotides (asODNs). Result CsTDIFL encoded an extracellular protein containing a typical CLE motif and a signal peptide, with high expression in young leaves, whereas CsTDR expression was predominant in stems. Exogenous TDIF treatment induced significant up-regulation of both CsTDIFL and CsTDR within 4 hours, followed by a marked decline toward baseline levels. Treatment with 10 μmol/L TDIF notably suppressed theanine and glutamate accumulation and reduced glutamine synthetase (GS) and glutamate synthase (GOGAT) activities. After 24 h of asODN interference, CsTDIFL transcript levels decreased by approximately 78.24% compared with the control, while theanine and glutamate contents increased by about 23.19% and 34.53%, respectively, accompanied by a significant increase in GS/GOGAT activities. Conclusion Exogenous TDIF treatment significantly inhibites GS/GOGAT activity and the accumulation of theanine and glutamate, whereas transient silencing of CsTDIFL enhances amino acid levels and key nitrogen metabolism enzyme activities. This supports the conclusion that the TDIF signal negatively regulates nitrogen metabolism in tea plants, likely through suppression of GS and GOGAT activities, thereby limiting glutamate supply and theanine biosynthesis.

Key words: tea plant, CsTDIFL, gene cloning, amino acids synthesis, nitrogen metabolism, tissue expression analysis, bioinformatic analysis

JIN Xuan, XIANG Fen, DAI Cui-ting, YANG Hui, LI Sai-jun, LIU Hong-yan, QIAN Si-wei, LIN Wan-huang, LI Wei. Cloning and Expression Analysis of CsTDIFL and Its Regulation on Amino Acid Synthesis in Tea Plants （Camellia sinensis）[J]. Biotechnology Bulletin, 2026, 42(5): 323-331.

Figures/Tables 6

Fig. 1 Root, stem, and leaf of the tea plant (Camellia sinensis L.)

Fig. 2 Cloning, sequence homology and phylogenetic analysis of the CsTDIFL geneA:Amino acid sequence alignment analysis of CsTDIFL protein with the Arabidopsis CLE family. B:Phylogenetic tree and motif analysis of CsTDIFL protein and the Arabidopsis CLE family proteins

Fig. 3 Expressions of CsTDIFL and CsTDR in different tissues of tea plant** indicates extremely significant difference (P<0.01)

Fig. 4 Responses of CsTDIFL and CsTDR to exogenous TDIF peptide in tea plantA‒C refer to the relative expressions of CsTDIFL and CsTDR in tea tree leaf, stem, and root samples, respectively, under treatments of 0 μmol/L (MOCK), 1 μmol/L, and 10 μmol/L TDIF polypeptide hormone. The horizontal axis (1‒24 h) indicates the sampling time, with the pre-treatment time denoted as 0 h. The data in the figure (Panel A‒C) are the mean±SD of three biological replicates. * indicates a significant difference (P<0.05), and ** indicates a highly significant difference (P<0.01). The same below

Fig. 5 Dynamics of amino acid contents and key nitrogen metabolic enzyme activities under TDIF treatmentA: Accumulation of major free amino acids after 24 h of TDIF treatment. B: Dynamic changes in key nitrogen metabolism enzyme activities under TDIF treatment at 0, 1, 4, 12, and 24 h

Fig. 6 Effects of antisense oligonucleotide inhibiting CsTDIFL on theanine contentA: CsTDIFL expressions after 24 h of asODN treatment. B: Theanine and glutamate contents after 24 h of asODN treatment. C: GOGAT/GS enzyme activity after 24 h of asODN treatment

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