Study On Molecular Mechanism Of Volatiles Metabolism And Quality Regulation During Light Withering Process In Oolong Tea

Light is an external signal factor,which was accepted during first manufacturing process of Oolong tea.Solar withering was regarded as an essential and indispensability process in the formation of excellent flavor quality of Oolong tea.Under stress conditions,self-protection mechanism could be activated rapidly in plants to adapt and enhance the ability of stress tolerance.Moreover,when the transcriptional level took place,primary and secondary metabolic reaction would be induced,intermediate and extremity products would be accumulated.In this study,fresh leaves of Tieguanyin were plucked as research material.We initially analyzed the physiological changes of light response and stress,and observation of subcellular structure during light withering afterpostharvest in Oolong tea.Secondary,based on transcriptome sequencing,patterns of differential gene expression and network regulation of molecular mechanism on volatiles metabolic pathways were analyzed in comparation groups,including dark withering to fresh leaves group and solar withering to dark withering group.Additionally,based on analysis of metabolic profiling,we further analyzed the spatial and temporal variation between expression profiling and metabolic profiling on volatile metabolic pathways.Finally,we combined analysis results with flavor quality to uncover regulatory mechanism on formation of flower-fruit scent during solar withering in Oolong tea.The achievements would provide a scientific basis for constructing all-weather production models in Oolong tea.The main results were as follows:1 The physiological changes during light withering in Oolong teaThe physiological indicator of light response and stress and subcellular structure were changed during light withering in Oolong tea.Compared to fresh leaves,phenotype of leaves,moisture content,Fv/Fm of photosystem II,net photosynthetic rate and relative conductivity in dark withering samples showed a non significantly difference,but leave temperature and proline content were slightly increased.However,the leaves turned to dark green and wilt quickly when exposed to solar after 15mins and 30mins,then turned to slightly and seriously reddish brown after 60mins and 120 mins,respectively.The leave temperature,relative conductivity and proline content increased rapidly,moisture content and photosystem ? Fv/Fm decreased quickly,and net photosynthetic rate was increased quickly at first,then continuous declination.The cell wall,cell membrane and vacuole of mesophyll cell were observed intact both in dark withering and solar withering after postharvest by electron microscopy.However,thylakoids lamella of chloroplast were dissociated and dispersed after solar withering,starch granule and osmiophilic granule were enlarged and increased,respectively.2 Transcriptome database construction and quality evaluation during light withering in Oolong teaTotal RNA extraction,library construction,quality evaluation,sequence assembling and annotation of ten samples were carried out,including different time of fresh leave,dark withering,solar withering and white LED withering treatments.The quality of ten samples and cDNA library were qualified.A total of 52,164,238 clean reads and 7.82G?9.95G data volume were obtained.Then,307,121 assembled transcripts and 190,914 assembled unigenes were obtained.Meanwhile,compared to seven public database,a highest sequencing similarity could be found to vitis vinifera.Among them,42,050 unigenes were assigned to 46 categories by GO classification and 17,935 unigenes were classified to 19 categories of the secondary metabolic pathways by KO database.3 The analysis of volatile metabolic pathways and differentially expressed genes during dark withering in Oolong teaBased on transcriptome,differential genes between fresh leave and three dark withering treatments were analyzed.The results showed that three dark withering treatments performed an entirely different expression pattern to fresh leaves.Differentially expressed genes of compared groups were enriched on the second categories by GO terms,including metabolic process,catalytic activity,oxidation-reduction process and oxidoreductase activity.Relative volatile metabolic pathways were significantly up-enriched including terpenoid backbone biosynthesis,alpha-Linolenic acid metabolism,monoterpenoid and sesquiterpenoid biosynthesis and phenylalanine metabolism.However,flavonoid biosynthesis,fatty acid biosynthesis,photosynthesis were down-enriched.The molecular mechanism on network regulation of relative volatile metabolic pathways in dark withering process were analyzed.The results showed that expression level of rate-limiting LOX in alpha-Linolenic acid metabolism was significantly up-regulated.Meanwhile,expression level of HPL in HPL branch and AOS,AOC,OPR,OPCL,ACX,MFP,ACAA in AOS branch were also dramatically up-regulated.For terpenoid backbone biosynthesis pathway,MEP pathway in plastid showed a remarkable up-regulated expression level than MVA pathway in cytosol.The genes up-regulated in MEP and MVA pathway including DXS,DXR,ISPG,ISPH,GPPS,LIS and HMGS,HMGR,FPPS,AFS.For phenylalanine metabolism pathway,relative genes expression in synthesis of phenyl acetaldehyde branch,including ASP?TAT?PER,were up-regulated.However,it was worth noting that there showed a dramatically down-regulate expression in flavonoid biosynthesis,including PAL?C4H?4CL?CHS,F3'H?F3H?DFR?FLS?ANR.In addition,we also discovered that accumulation of pyruvic acid were significantly positive correlated to free amino acid metabolism and formation of intermediate products in TCA cycle.Accumulation of pyruvic acid did not take part in gluconegenesis,oxidative decarboxylation for acetyl-CoA and free amino acid biosynthesis.As an importance precursor substrate,pyruvic acid directly participate in terpenoid backbone biosynthesis pathway for synthesis of monoterpenoids and sesquiterpenoids.4 The analysis of gene expression pattern in volatile metabolic pathways during light withering in Oolong teaIn order to sum up the regulation mechanism on the formation of volatiles when exposed to solar in Oolong tea.The comparation of expression pattern between dark withering and solar withering treatments were analyzed.The results showed a similarity expression pattern between solar withering after 15min and three dark withering groups.However,expression pattern in solar withering after 30min and 60min were clustered in different group.Based on GO and KEGG enrichment,we found that metabolic process and catalytic activity were significantly down-enriched in solar withering after 30min and 60min,and relative metabolic pathways of disease,senescence and death were significantly up-enriched.Compared to dark withering,the expression level of key genes in three volatile metabolic pathways were up-regulated in solar withering after 15 min.However,significantly down-regulation was showed in solar withering after 30min and 60min,especially in terpenoid backbone biosynthesis pathway.Meanwhile,a large number of relative genes in heat shock and stress tolerance showed a dramatically up-regulated expression.Therefore,the expression level of key genes in volatile metabolic pathways could be significantly up-regulated in solar withering,but could be facility inhibited by up-regulated genes of heat shock and stress tolerance.A reverse coexpression regulation effect were performed between volatile metabolic genes and heat shock relative genes.Thirteen candidate genes expression were measured by real-time fluorescence quantification PCR for three different time groups during dark and light withering after postharvest in Oolong tea.The changes of relative expression pattern were similar to those observed by RNA-seq.Influence of high temperature on gene expression level of volatiles during light withering were further verified.The results showed that compared to normal temperature treatment(30?),gene expression of heat shock protein in high temperature treatment(43?)showed dramatically up-regulated and volatiles relative genes down-regulated under similar condition of sunlight.It also showed a lower expression level of volatiles relative genes in dark withering treatment.5 The analysis of metabolic profiling and quality regulation during light withering in Oolong teaThe volatiles,carbohydrates and organic acids of leaves were measured by diethyl ether extraction combined GC-TOF-MS and GC-TOF-MS derivatization.Volatiles of raw tea were measured by SPME-GC-MS.The free amino acids and catechines were measured by UPLC-QqQ-MS.The result indicated that volatile fatty acids,terpenes,aromatic and alkane were detected as main volatile compounds during dark and solar withering in Oolong tea.Compared to fresh leaves,the relative content of volatile compounds were significantly increased during dark and solar withering,especially in solar withering after 15 min,ranked the highest.The gene expression profiling in volatile metabolic pathways and metabolic profiling showed a distinctly positive correlation in temporal and spatial variation during dark and solar withering after postharvest in Oolong tea.In addition,the content of catechins including non-gallated catechin(C?EC?GC?EGC)and gallated catechin(ECG?EGCG)ranked highest in fresh leaves,followed by dark withering treatments and solar withering treatments were lowest.Meanwhile,the content of free amino acids in solar withering treatments were exceeded to dark withering treatments.The content of aroma compounds in raw tea showed higher and the concentration of catechins showed lower in solar withering after 15 min and 30 min.Furthermore,the aroma accumulation and catechins degradation had non-significantly positive correlation from 30 min to 120 min?6 Influence of alternately irradiation by LED on flavor compounds in Oolong teaFeasibility and effect on the formation of quality characteristic in Oolong tea via irradiation repeatedly and alternately by LED were discussed.The results showed that compared to control treatment,all catechines reduced in different times irradiation treatments.The bitterness was dramatically decreased by 20?35%in S-2 and S-3 treatments accordingly.The relative content of alpha-farnesene and nerolidol in S-2 treatment were increased by 56.28%and 62.51%,respectively.The sensory evaluation of aroma in raw tea showed a similar trend to principal component analysis.