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Article|21 Dec 2022|OPEN
Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants
Yi Wang1 ,† , Yu-Fei Liu1,2 ,† , Meng-Yuan Wei1 , Chen-Yu Zhang1 , Jie-Dan Chen1 , Ming-Zhe Yao1 and Liang Chen1 , , Ji-Qiang Jin,1 ,
1Key Laboratory of Biology, Genetics and Breeding of Special Economic Animals and Plants, Ministry of Agriculture and Rural Affairs; Tea Research Institute of the Chinese Academy of Agricultural Sciences, Hangzhou 310008, China
2Yunnan Provincial Key Laboratory of Tea Science, Tea Research Institute, Yunnan Academy of Agricultural Sciences, 2 Jingnan Road, Menghai, Yunnan 666201, China
*Corresponding author. E-mail:,
Both authors contributed equally to the study.

Horticulture Research 10,
Article number: uhac279 (2023)
Views: 75

Received: 19 Sep 2022
Accepted: 12 Dec 2022
Published online: 21 Dec 2022


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 TCS1aTCS1c, 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.