Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants

Abstract

Caffeine is an important functional component in tea, which has the effect of excitement and nerve stimulation, but excessive intake can cause insomnia and dysphoria. Therefore, the production of tea with low-caffeine content can meet the consumption needs of certain people. Here, in addition to the previous alleles of the tea caffeine synthase (TCS1) gene, a new allele (TCS1h) from tea germplasms was identified. Results of in vitro activity analysis showed that TCS1h had both theobromine synthase (TS) and caffeine synthase (CS) activities. Site-directed mutagenesis experiments of TCS1a, TCS1c, and TCS1h demonstrated that apart from the 225th amino acid residue, the 269th amino acid also determined the CS activity. GUS histochemical analysis and dual-luciferase assay indicated the low promoter activity of TCS1e and TCS1f. In parallel, insertion and deletion mutations in large fragments of alleles and experiments of site-directed mutagenesis identified a key cis-acting element (G-box). Furthermore, it was found that the contents of purine alkaloids were related to the expression of corresponding functional genes and alleles, and the absence or presence and level of gene expression determined the content of purine alkaloids in tea plants to a certain extent. In summary, we concluded TCS1 alleles into three types with different functions and proposed a strategy to effectively enhance low-caffeine tea germplasms in breeding practices. This research provided an applicable technical avenue for accelerating the cultivation of specific low-caffeine tea plants.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31670685), Zhejiang Provincial Natural Science Foundation of China (LZ22C160008), Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties (2021C02067) to J.-Q.J., the Chinese Academy of Agricultural Sciences through the Agricultural Science and Technology Innovation Program (CAASASTIP-2021-TRICAAS), the China Agricultural Research System of MOF and MARA (CARS-19) to L.C., the Zhejiang Provincial Natural Science Foundation of China (LQ20C160010) to J.-D.C. The authors thank Dr Penghui Li of Anhui Agricultural University, Professor Kang Wei of TRICAAS, and three anonymous reviewers for their valuable suggestions and critical peer review.

Author contributions

J.-Q.J. and L.C. conceived and designed the research. J.-Q.J., Y.-F.L., M.-Y.W., Y.W., and C.-Y.Z. performed the experiments. Y.W. and J.-D.C. analysed the data. Y.W., J.-Q.J. and C.-Y.Z. wrote the manuscript, M.-Z.Y. and L.C. edited and contributed to the content.

Data availability

All data supporting the findings of this study are available within the paper and within its supplementary materials published online.

Conflict of interest

All authors declare that they have no conflict of interest.

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