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.

Figure 1

Comparison of TCS1 allele promoter sequences and amino acid sequences. A Comparison of promoter sequences between TCS1 alleles. The yellow box marks the initiation codon (ATG). B Comparison of amino acid sequences of TCS1h and other TCS1s. The proposed SAM-binding motifs (A, B′, and C) and conserved region that are nominated as the ‘YFFF-region’ are shown in red empty boxes. The amino acid residues are marked in a blue empty box, and they played a critical role in substrate recognition. The nominated amino acids in substrate binding are indicated by purple (SAM) and red (methyl acceptor) boxes. The TCS1b, TCS1c, TCS1g, and TCS1i variant sites compared with TCS1a, TCS1d, TCS1e, TCS1f, and TCS1h are indicated by a brownish yellow triangle. The important mutation sites are marked with red triangles. The TCS1b, TCS1c, TCS1g, and TCS1i deletion motif ‘ELATA’ is marked with a yellow empty box.

Figure 2

Functional studies of proteins encoded by different TCS1 alleles and comparison of phylogenetic trees of N-methyltransferases. A Activity assay results of TCS1 recombinant proteins (n = 3). B Phylogenetic tree of N-methyltransferase coding region sequences related to purine alkaloids in Camellia (red), Coffea (yellow), and Theobroma (blue) is performed by the MEGA Program. Green, blue, and red groups indicate TS, CS, and TcS (theacrine synthase gene) classes.

Figure 3

Expression level analysis of TCS1 and TcS in 27 accession tea plants with diverse genetic background. A Purine alkaloid contents in 27 accessions. B–D Determination of TCS1 allele expression levels in 27 accessions of tea plants by qRT-PCR. Primer pairs TCS1-TS, TCS1-CS, and TcS were used to detect gene whose encoded protein had only theobromine synthase (TS), both TS and caffeine synthase (CS), and TcS (theacrine synthase) activities, respectively.

Figure 4

Cis-acting elements and promoter activity analysis of different TCS1 alleles. A Distribution of cis-acting elements across the promoter sequences of different TCS1 alleles. Predictive analysis with online software PlantCARE. B GUS histochemical staining of T₃ generation Arabidopsis thaliana transduced with different promoters of TCS1 alleles. Red circles indicate important variations in the light-response element. C Determination of the promoter activity of different TCS1 alleles by dual-luciferase assay (n = 6); the difference in promoter activity was determined by detecting the ratio of firefly luciferase and renilla luciferase activities with an empty vector as the control; ***P < 0.001. EV, empty vector.

Figure 5

Promoter activity analysis of the key regions and cis-elements of truncate promoters of TCS1a. A Distribution of cis-acting elements on the TCS1a promoter sequence with deletions of different lengths. B GUS histochemical staining of T₃ generation of Arabidopsis thaliana transfected with 5′ end fragment deletion of TCS1a promoter. C Determination of the promoter activity of different deleted fragments of the TCS1a promoter (n ≥ 6); ***P < 0.001, **P < 0.01. D Schematic representation of large indel mutations in TCS1e and TCS1f and the absence of a G-Box cis-element. E Comparison of GUS histochemical staining between TaP6 and mutant promoter. TaP6 represents the 331 bp of the TCS1a promoter, TeP6 and TfP6 represent the corresponding promoter sequences of TCS1e and TCS1f, and TaP6m represents a G-Box element near the 3′ end mutated with one base (G-214A). EV indicates empty vector.

Figure 6

The strategy for enhancing high-theobromine and low-caffeine tea germplasms.