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

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.

The chromosome-scale genome reveals the evolution and diversification after the recent tetraploidization event in tea plant

Tea is one of the most popular nonalcoholic beverages due to its characteristic secondary metabolites with numerous health benefits.Although two draft genomes of tea plant(Camellia sinensis)have been published recently,the lack of chromosome-scale assembly hampers the understanding of the fundamental genomic architecture of tea plant and potential improvement.Here,we performed a genome-wide chromosome conformation capture technique(Hi-C)to obtain a chromosome-scale assembly based on the draft genome of C.sinensis var.sinensis and successfully ordered 2984.7 Mb(94.7%)scaffolds into 15 chromosomes.The scaffold N50 of the improved genome was 218.1 Mb,~157-fold higher than that of the draft genome.Collinearity comparison of genome sequences and two genetic maps validated the high contiguity and accuracy of the chromosome-scale assembly.We clarified that only one Camellia recent tetraploidization event(CRT,58.9–61.7 million years ago(Mya))occurred after the core-eudicot common hexaploidization event(146.6–152.7 Mya).Meanwhile,9243 genes(28.6%)occurred in tandem duplication,and most of these expanded after the CRT event.These gene duplicates increased functionally divergent genes that play important roles in tea-specific biosynthesis or stress response.Sixty-four catechin-and caffeine-related quantitative trait loci(QTLs)were anchored to chromosome assembly.Of these,two catechin-related QTL hotspots were derived from the CRT event,which illustrated that polyploidy has played a dramatic role in the diversification of tea germplasms.The availability of a chromosome-scale genome of tea plant holds great promise for the understanding of genome evolution and the discovery of novel genes contributing to agronomically beneficial traits in future breeding programs.

METABOLIC AND TRANSCRIPTOME ANALYSIS REVEALS METABOLITE VARIATION AND FLAVONOID REGULATORY NETWORKS IN FRESH SHOOTS OF TEA(CAMELLIA SINENSIS)OVER THREE SEASONS

Metabolites,especially secondary metabolites,are very important in the adaption of tea plants and the quality of tea products.Here,we focus on the seasonal variation in metabolites of fresh tea shoots and their regulatory mechanism at the transcriptional level.The metabolic profiles of fresh tea shoots of 10 tea accessions collected in spring,summer,and autumn were analyzed using ultra-performance liquid chromatography coupled with quadrupole-obitrap mass spectrometry.We focused on the metabolites and key genes in the phenylpropanoid/flavonoid pathway integrated with transcriptome analysis.Multivariate statistical analysis indicates that metabolites were distinctly different with seasonal alternation.Flavonoids,amino acids,organic acids and alkaloids were the predominant metabolites.Levels of most key genes and downstream compounds in the flavonoid pathway were lowest in spring but the catechin quality index was highest in spring.The regulatory pathway was explored by constructing a metabolite correlation network and a weighted gene co-expression network.