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.

Analysis of Differences in Biochemical Components Between Yunnan and Kenya Tea Tree Varieties

With 16 Yunnan tea tree varieties and 5 Kenya tea tree varieties as test materials,the differences in biochemical components between Yunnan and Kenya tea tree varieties were compared and analyzed.The results showed that the coefficients of variation of tea polyphenols,amino acids,caffeine,water extract,gallic acid(GA),catechin(C),epicatechin(EC),epicatechin gallate(ECG),epigallocatechin(EGC),epigallocatechin gallate(EGCG)and total catechins in Yunnan tea tree varieties were greater than those in Kenyan tea trees.The contents of tea polyphenols,amino acids,caffeine,water extract,C,EC,EGC,EGCG and total catechins in Yunnan tea tree varieties had no significant differences from those in Kenyan tea trees varieties(P>0.05),while there were significant differences in the contents of GA and ECG between Yunnan tea tree varieties and Kenya tea tree varieties(P<0.05).Therefore,it could be predicted that GA and ECG might be one of the main characteristics of the differences in biochemical components between Yunnan tea tree varieties and Kenyan tea tree varieties.The cluster analysis results showed that when the genetic distance was 15,the 21 tested tea varieties could be divided into three groups with obvious biochemical differences.