Catalytic Function, Tempo-spatial Expression And Tea Aroma Quality Impact Of CsLIS/NES In Camellia Sinensis

Aroma quality of Chinese teas(Camellia sinensis) is largely dependent on the spectrum and abundance of volatile terpenoids and their glycosides produced and accumulated in tea leaves. Linalool and nerolidol, possessing pleasant floral scent, are crucial for aromatic quality of oolong and black teas. In this study, the full length cDNA of CsLIS/NES was cloned, and bioinformatics analysis was performed to study the protein structure and specialized domains. Prokaryotically and eukaryotically functional analyses of CsLIS/NES, and its subcellular localization were also conducted. Besides, quantitative analysis of the main free and glycosidically bound terpenoids in leaves and flowers of tea plants, and the transcription levels of CsLIS/NES in fresh leaves and flowers at different developmental stages were carried out. The correlation of gene expression level and the free and glycosidically bound terpenoids indicates that CsLIS/NES plays a key role in linalool biosynthesis and accumulation in the tea plant. The main results as follows:1, CsLIS/NES cloning and bioinformatics analysisThe partial sequence with306bp derived from the RNA-seq transcriptome sequence data was preliminarily annotated as CsLIS/NES based on BLASTX analysis. Cloning of its full cDNA using5’and3’RACE resulted in a fragment with2050bp, containing1728bp coding sequence, encoding a protein of575amino acids and66.0kDa, pI5.93. Sequence analysis showed that the predicted protein exhibited sequence similarities to both monoterpene and sesquiterpene synthase from other plant species, and showing the greatest sequence homology71%at the amino acid level to the described linalool synthase(AaLIS) from Actinidia arguta, and69%to nerolidol synthase(AcNES) from Actinidia chinensis. The three protein structures exhibited great similarities, the N-terminal structures of CsLIS/NES is closely to AaLIS who localized in plastid, however the active site of CsLIS/NES is more similar to AcNES who is a bifunctional synthase can catalyze synthesis linalool and nerolidol in vitro. CsLIS/NES was likely targeted to the chloroplast according to the TargetP1.1(0.839) prediction program. Phylogenetic analysis showed that CsLIS/NES clustered in a well-supported manner with the TPS enzymes in the TPS-g subgroup.2, Functional analyses and subcellular localizationFunctional analysis revealed that recombinant CsLIS/NES produced in Escherichia coli catalyzed the conversion of GPP and FPP to the respective (R)-linalool and (E)-nerolidol in vitro. In transgenic tobacco expressing CsLIS/NES, substantial amount of linalool was released while control plants carrying the empty vector did not emit any detectable linalool. Besides, CsLIS/NES can also catalyzed the synthesis of much less pinene and ocimene in the transgenic tobaccos and in vitro. Fusion of CsLIS/NES with GFP reporter protein was localized in chloroplasts, suggesting that CsLIS/NES acts on plastidal terpenoid pathway.3, The tempo-spatial expression of CsLIS/NES in ’Te-xiangzao’CsLIS/NES expression levels varied spatially and temporally. The top second young leaves had the highest CsLIS/NES transcript level than other tested leaves. CsLIS/NES was expressed much higher in young leaves than that in old leaves and flowers. In flowers, the transcription of CsLIS/NES changed in the different developmental stages. Its transcript level was the highest in stamens and pistils at the flower full-opening stages.4, Main terpenoids in ’Te-xiangzao’The amount of free linalool (fresh weight) in young leaves and flowers are0.1094μg/g and0.1270μg/g, respectively, both higher than those in old leaves of0.0607μg/g. However, the abundance of glycosidic linalool in young leaves was up to0.3849μg/g·fresh weight, much higher than those in old leaves (0.1215μg/g·FW) and flowers (0.1056μg/g·FW).In summary, the catalytic function and the transcription level of CsLIS/NES are tightly correlated with the linalool biosynthesis and accumulation in tea plant. Thus, CsLIS/NES expression in young leaves is responsible for linalool synthesis and accumulation in C. sinensis, and thus is crucial for tea aroma quality.