Differential Regulation Of Catechin Metabolism In Albino And Normal Tea(camellia Sinensis) Leaves And Tea Linalool Biosynthesis

Tea,produced from fresh tea(Camellia sinensis)leaves,is an important,widely popular non-alcohol beverage due to two aspects:(i)tea has many healthy benefit functions;and(ii)tea has pleasant flavor.Characteristic compounds those determined tea flavor and healthy benefit functions are catechins,theanine,caffeine and terpenes.In addition,these compounds also vital for growth,development and stress defense of tea plant.Though some results have been gained on catechin and theanine biosynthesis and many studies focused on the identification of tea volatiles and their conversion pathways,however,the biosynthesis and regulation of these compounds need further studies.In this study,transcriptome,metabolome,reverse genetics and other daily-used molecular biological methods were used for studying the mechanisms of “Yujinxiang” pale tea and the possible biosynthetic pathways and their regulations of catechins and theanine as well as functional characterization of CsLIS/NESandCsNES.The main findings are as follows: Pale “Yujinxiang” leaves contained lower content of catechins(p<0.05)and higher content of theanine(p<0.05)compared to re-greening leaves and control leaves.No significant difference on caffeine content between pale “Yujinxiang” leaves and control leaves(p>0.05).Processed “Yujinxiang” tea had significant lower terpenoid contents(p<0.05).TEM observations on chloroplast size,thylakoid membrane and number of starch granuals revealed deficiency and malfunction of chloroplast in pale “Yujinxiang” leaves.Shading regreening and DEGs analysis showed pale “Yujinxiang” leaves was suffering high light stress.QPCR confirmed that the expression of ClpP5 in pale leaves was almost 18 folds higher than regreening leaves,suggesting a role of ClpP5 for pale mechanism.The transcript level of FLS was significantly higher(p<0.05)than control leaves while DFR was significantly lower(p<0.05),which consistent with the content of catechins and quercetin.These results indicated that metabolic re-direction of catechins to flavanol quercetin,which can effectively defense photo-damage,existed in pale “Yujinxiang” leaves.Consistently,transcript levels of upstream flavonoid biosynthetic pathway genes were higher(p<0.05)in pale “Yujinxiang” leaves,e.g.CHS,CHI,F3 H and F3'5'H,whereas downstream genes LAR1 and-2,ANS,ANR-1 and-2 were lower(p<0.05).Together with metabolic re-direction findings,it was likely that CHS,CHI,F3 H,F3'5'H,DFR and FLS are the turning-points in flavonoid branch pathways.SAMDC was lower(p<0.05)expressed in pale “Yujinxiang” leavescompared to re-greening leaves and control leaves,which was consistent with theanine contents in these leaf samples,while the expressions of other gene were opposite to theanine contents,strongly suggesting a role of SAMDC in theanine biosynthesis.GC-MS showed that linalool and nerolidol are abundant in tea leaves and flowers,and that tea leaves had more bound volatile than their free forms,which was consistent with previous reports.RACE-PCR,sequencing,qRT-PCR and Western blot confirmed the first exon skipping alternative splicing isoform at both mRNA and protein level.In silico analysis showed that may have same catalytic function.In vitro assay confirmed their similar catalytic functions: both isoforms catalyze GPP to produce linalool and minor products and FPP to produce(E)-nerolidol.The product linalool had roughly same percent of 3R-linalool and 3S-linalool,which was never reported before.CsLIS/NES-1,with a signal peptide,localized in plastid.Significantly more linalool(p<0.05)was produced in CsLIS/NES-1 overexpressed tobacco leaves was found and AsODN experiment resulted in lower expression(p<0.05)of CsLIS/NES-1 and lower content of linalool and linalool oxides(p<0.05).These indicated CsLIS/NES-1 is a linalool synthase gene.While CsLIS/NES-2,without this signal peptide located to cytoplasm.In vivo assay using CsLIS/NES-2 overexpressed tobacco leaf discs produced more(E)-nerolidol(p<0.05);AsODN experiment resulted in lower expression of CsLIS/NES-2(p<0.05)and lower content of(E)-nerolidol,indicated a role of CsLIS/NES-2 as nerolidol synthase gene.Distinct expression pattern of CsLIS/NES-1 and-2 were revealed in this study.MeJA induced CsLIS/NES-1 expression(p<0.05)but had no effect on CsLIS/NES-2.Diurnal expression peak of CsLIS/NES-1 was about 8 o'clock while CsLIS/NES-2 was about 12 o'clock.These results suggesting a natural role of CsLIS/NES-1 and alternative splicing controlled role of CsLIS/NES-2.Tea nerolidol synthase gene CsNES has 551 amino acid residues.Phylogenetic tree analysis showed that CsNES clustered to TPS-g sub-family.Multiple protein analysis showed CsNES had TPS conserved ion binding sites “DDXXD” and “DDxxSxxxE”.In vitro assay showed CsNES catalyzed GPP to form linalool at a low efficiency and(E)-nerolidol using FPP.Product linalool was predominantly 3S-linalool.Online prediction showed CsNES located to cytoplasm.Transient expression of GFP fusion in N.benthamiana confirmed this prediction and suggesting its role as nerolidol synthase gene.Above all,this study focused on and uncovered the biosynthesis and regulation of catechins,theanine,linalool and nerolidol.This study revealed that albino tea lacks aroma,MeJA treatment for inducing terpene biosynthesis can be a way to improve its aroma.Shade treatment resulted in roughly the same levels of flavor compounds and can improve growth of tea plants and the yields of fresh tea leaves,thus shade treatment should be an important step in tea industry.This study revealed that CHS,CHI,F3 H,F3'5'H,DFRand FLS were most probably to be the turning point of flavonoid flux to either flavan-3-ols or flavonols,these results fundamentally supported for further regulation verification of catechins.The role of SAMDC on theanine biosynthesis is interesting and needs further studies.This study showed the biosynthesis and regulation of linalool in tea.Moreover,CsLIS/NES was revealed to be bifunctional in planta due to alternative splicing,the two isoforms showed distinct spatiotemporal expression pattern,these results are important for future studies on functional characterization of terpene synthase genes and other genes.Differences on I323,I326,D474,S478 and K487 of CsLIS/NES compared to other linalool synthase gene might be the key aspects of selective production of linalool enantiomers,this result was important for uncovering the mechanisms of linalool enantiomers biosynthesis.Thus,this study will be important for further investigations on the biosynthesis and regulation of these compounds as well as the improvement of processed tea flavor.