The Response Mechanism Of Tea Leaves To Yaoqing Mechanical Force Stimuli: A Multi-omics

Yaoqing is the key procedure of quality formation in Oolong tea manufacturing process.Through the mechanical force to stimulate it,combining with airing procedure,eventually formed the unique flavor quality of Olong tea by mechanical forces,water loss and other adversity stress.In particular the mechanical force Yaoqing is also widely used in the process of floral black tea,white tea and green tea production to improve quality.Under stress conditions,self-protection and adaptation could be activated rapidly in plants to adapt and enhance the ability to tolerate stress,and to regulate responses at physiological,transcriptional,proteomic and metabolic levels,thus leading to the accumulation of abundant primary and specialized metabolites.In order to systematically reveal the response mechanism(s)of tea leaves to mechanical force stimuli,Tieguanyin tea leaves were used as materials in this study and the vibrating Yaoqing was chosen as the model of mechanical force stimulus.The effects of mechanical force on leaves were analyzed from physiological changes,transcriptome and proteome.Coupled with metabolomics,the metabolic regulation mechanism of tea leaves in response to mechanical force stimuli was analyzed combining with the data of transcriptome and proteome.The main research results were as follows:1 physiological changes in tea leaves during mechanical force stimulationCompared with the naturally spreading leaves and withering leaves,the water content of leaves had no significant difference within the first 30 min.The water content within leaves subject to 60 mins’ mechanical force stimulation was significantly lower than the respective control and withering leaves.The electric conductivity of stimulated leaves was significantly higher than the naturally spreading leaves at 10 min(V10).Y(Ⅱ)and ETR significantly reduced in V5 and V10.The observation of subcellular microstructure showed that with the increased duration of mechanical force stimulation,the close and clear lamellar structures of chloroplast(CH)and thylakoid(Mt)gradually loosened,deformed,contracted,disordered and disintegrated,and the starch grains increased significantly in V5.Meanwhile,the osmiophilic granules increased significantly in V5 and V15,and aggregated in the latter sample.The activities of antioxidant enzymes APX,DHAR,GR and MDAR in leaves exposed to 5-10 min of stimulation were significantly higher than those in natural spreading leaves.This indicated that the effect of water content in leaves was not significantly affected by mechanical force stimulus within a certain period of time(30 min).The mechanical force stimulus resulted in the damage of the membrane systems in cells,increased cell permeability,facilitated the release of intracellular contents,increased cell conductivity,promoted thylakoid degradation,severely damaged chloroplasts,destroyed PSⅡand elevated the activities of defense enzymes to cope with leaf damages imposed by ROS.2.Transcriptional responses of tea leaves to mechanical force stimulationAccording to the above physiological results,mechanical force stimulated leaves(V5 and V15)and controls(C0 and C15)were chosen for transcriptome analysis.The results showed that more than 84% of the sequences obtained f could matchthose of tea genomes.Compared with C15,the up-regulated GO function in V15 was enriched in the metabolic process,protein modification,aromatic compound synthesis,transferase activity and ion binding,etc.The KEGG metabolic pathways were enriched in plant-pathogen interaction,glutathione metabolism,α-linolenic acid metabolism and plant hormone signal transduction.Moderate mechanical force stimulated leaves(15 min)based on the defense response genes and stress resistance gene,such as MAPK,CDPK and Ca2+ signaling pathways of CNGC,MPK6,MPK4,WRKY22,Ca CML and HSP90 up-regulated expression,to improve the defense reaction of leaves.At the same time,promote the NO and ROS accumulation to induce the gens of three REDOX cycle in glutathione metabolism,such as 6PGDP,GPX,APX,GSTU,GSH1 and GSH2 up-regulated expression;The genes AOS,AOC,ACX and JMT were used as synthesize JAs,as well as COI1,JAZ and MYC2 in the JA signaling pathway were up-regulated,to clear the ROS produced by intracellular mechanical stress,cause the expression of defension-related genes and the production of signaling substances,and improve the resistance and adaptability of leaves to mechanical stress.Tea leaves promoted the accumulation of volatiles by up-regulating the expression of key genes such as SDP1,DAD1,LOX and HPL1 in the metabolic pathways of linolenic acid,which may be the reason for the heavy grassy smells in shaking leaves.3.Proteomic responses of tea leaves to mechanical force stimulationTMT-based proteomics profiling of V5,V15,V30,C0 and C15 samples obtained a total of 868 differential proteins,which are mainly located in chloroplast,nucleus and cytoplasm.Compared with C0,differentially expressed proteins in V were mostly up-regulated.But with the increased duration of stimulus time,the number of up-regulated proteins gradually decreased.Differential GO terms wereenriched in biological stimulation,defense response,host cell virus transport,hydrolase activity and cytoskeleton structure composition,etc.The KEGG metabolic pathways were enriched in plant-pathogen interaction,glutathione metabolism and phenylalanine metabolism,etc.,which was consistent with the transcriptomic results.Chloroplast is the main biochemical reaction site of leaves in response to mechanical force stimulation.Tea eaves demonstrate a more intense response to short time mechanical force stimulation(5-15min),while a longer stimulation time(30min)could inhibit the change of protein number in tea leaves,which provides a theoretical explanation why it is necessary to perform rotation/Yao Qing for multiple times and to control timing at each step..Moderate mechanical force stimulated leaves(V15)depended on upregulating proteins in CDPK and Ca2+ signaling pathways,such as CDPK,Rboh,CNGCs and CML,to mediate the accumulation of NO and ROS respectively,induced proteins of oxidative decomposition in GSH metabolism pathway,such as GGT1-5,GPX,GST,APX,etc,upregulated to promote the oxidation of GSH and the combination with electrophilic material,improve the ability of metabolism of other different source material’s to achieve the role of antioxidant stress and the detoxification in leaves.However,GSH synthetic proteins GSHA,GSS and reducing proteins GSR were down-regulated.The results showed that the protective mechanism of REDOX reaction of GSH was activated at the protein level after the sensory stimulation of mechanical force in tea leaves,and GSH consumption was dominant.The defense reactive proteins RIN4,RPM1,HSP90,etc.,were also up-regulated in the mechanical force stimulated leaves to resist and adapt to the stress formed by mechanical force stimulus.Mechanical force promoted the synthesis of phenylacetaldehyde,phenylacetic acid and phenylpyruvate by increasing the expression of enzymes such as DDC,AOC3,PAT and GOT2.Prolonged mechanical force stimulation of leaves(V30)promoted the synthesis of catechin by up-regulating theexpression of proteins such as PAL,C4 H,4CL,CHS,CHI,FLS,etc.Just the strong stress caused by mechanical force stimulation could promote the synthesis of flavonoids and affecte the quality of Yaoqing.4.Metabolomic conjoint analysis of the response of tea leaves to mechanical force stimulusNon-volatiles and volatiles of MV5,MV15,MV30,MC0 and MC15 were profiled by widely targeted metabolomics.A total of 772 nonvolatile compounds and 36 volatile compounds were obtained.Compared with MC0,the number of up-regulated metabolites increased first and then decreased(153<161>150)with the extension of the stimulus time,while the number of down-regulated metabolites increased over time(55<83<105).KEGG enrichment analysis of differential metabolites showed that they were enriched in the metabolic pathways of α-linolenic acid,glutathione,phenylalanine and flavonoid biosynthesis.It is shown that only a moderate degree of mechanical force stimulation could increase the number of metabolites whereas a longer stimulation leads to the increase in the number of down-regulated metabolites.By regulating the number of metabolites in these metabolic pathways,tea leaves enhance their toleration and resistance towards mechanical force stimulation.Combined with the results of proteome studies,mechanical force stimulation regulated the decrease of GSH content in leaves and the increase of GSSG by down-regulating the expression reducing proteins of GSH synthesis and up-regulating the expression of GSH oxidationdecomposed proteins.By upregulating the expression of APX protein,ASA content was reduced,and the expression of PGD and G6 PD were upregulated,NADP content was reduced,so that ROS produced by leaves to remove mechanical force stimulus stress caused the consumption of reducing substances(GSH,ASA)and the generation of oxidized substances(GSSG),and NADP+-NADPH redox was upregulated to maintain GSH balance.By improving the content of substrate of GSH,reduce the metabolite content of GSH catabolism downstream,adjust the leaves for a long period of time(30 min)mechanical force stimulation make GSH excessive consumption and to break its GSSG oxidation process of steady state,to make it to improve the content of substrate of GSH to strengthen synthesis,so as to maintain the REDOX steady continuous removal of ROS in leaves.Metabolism and protein levels together indicate the regulatory mechanism by which mechanical force stimulates leaves to produce GSSG by consuming GSH and ASA,and to remove ROS produced by mechanical stress to resist the stress effect of mechanical force stimulation.Mechanical force stimulated the up-expression of the key gene LOX6 in tea leaves,which led to the formation of the intermediate metabolite 9(S)–Hp OTr E in the branch metabolism of the low-boiling aromatic compounds.The content of 9(S)-Hp OTr E increased in MV5,and the expression of ADH1 was up-regulated to regulate the content of 3-hexenol.The content of 9(S)-HOTr E 9 and 9,10-EOTr E increased in MV15 and MV30 through the up-regulated expression regulation of AOS and HPL1.Both transcriptional and metabolic data indicated that short time mechanical force stimulation promotes the reaction of this metabolic branch,and produces volatiles with grass smells,which provides a scientific basis for producing strong green leaf odor in the early stage of rotation/Yao Qing.It is shown by transcriptional and metabolic data that a long time(15 min,30 min)mechanical force could direct α-linolenic acid metabolic pathways to the formation of JA branch and raise JA responsivegenes in response to mechanical force stimulation.In reality,with the increase of Yaoqing time,more JAs could be accumulated to provide a prerequisite for the formation of more aroma substances in the later stage,and provide a theoretical basis for the gradual extension of Yaoqing time.The increase of phenylethylamine,phenylethanol and phenylacetic acid in MV30 was facilitated by ethe elevated expression of the rate-limiting enzymes DDC,AOC3,PAT,GOT1 and GOT2 in tea leaves under mechanical force stimulation.The results indicated that the long time(15min and 30min)mechanical force stimulation was beneficial to the formation of fruit-scented aromatic compounds.Another metabolic branch by up-regulating the expression of key enzymes including PAL,C4 H,4CL,CHS,CHI,F3 ’H,F3 H,DFR,FLS and LAR increased the production of intermediates such as trans-cinnamic acid,p-coumaric acid,5,7’,3’,4-tetrahydroxy flavanone,5,7,3’,4’,5’-pentahydroxy flavanone,2,3-dihydrokaempferol,kaempferol,quercetin,myricetin,naringenin chalcone,naringenin and 2,3-dihydroquercetin content is higher in MV30,metabolic end products in addition to the EGC,the remaining 5 kinds of catechin monomer content is higher in MV15.From protein and metabolic data,itis suggested that mechanical force stimulation could promote phenylalanine metabolism and the synthesis of aromatic compounds,and only moderate mechanical force stimulation(15-30min)could promote the synthesis of most catechins.Twenty-two amino acids were detected,except for threonine,aspartic acid and citrulline,which were present in low concentrationos.Contents of other free amino acids were higher in mechanically stimulated leaves,which suggests that moderate mechanical stimulation could effectively promote the accumulation of amino acids.It provides the basis for improving the quality of other tea products using Yaoqing procedure.Aromatic compounds including 3,5-Dimethyl benzaldehyde,methyl salicylate,benzyl alcohol and jasmine lactone,aliphatic compounds with short chains such as cis-3-Hexenyl butyrate,(Z)-3-Hexenyl hexanoate and 2-Heptanol were present in higher concentrations in the mechanical force stimulated leaves,and as the stimulus duration increased,their concentrations increased suggesting that the mechanical force stimulation is beneficial for their accumulation.Only certain mechanical force stimulation can promote the accumulation of(Z)-3-hexenol,α-farnescene and trans-Nerolidol.The long time stimulation was beneficial for the accumulation of phytoland its derivatives as well as phenylacetaldehyde.Mechanical force stimulation inhibited the accumulation of 2-hexenaldehyde,β-Ocimene and indole.To sum up,the response of tea leaves to mechanical force stimulation could be divided into the primary response stage with up-regulated expression of genes and proteins,activation of leaf cell defense and antioxidant reactions,generation of grassy gas,and tension of leaves.The secondary reaction stage is characterized by damage of chloroplasts,increase and aggregation of starch granules and osmiophilic granules,and the accumulation of defense signaling substances,the accumulation of metabolites such as aromatic compounds,catechins,some amino acids and volatile substances,and down-regulated expression patterns of genes and proteins.