Study On Aroma Formation In Tieguanyin Processing Mediated By Jasmonic Acid Synthesis And Signal Transduction Pathway

Using the Chinese Oolong tea variety Tieguanyin leaves as test materials,Headspace Solid Phase Micro-extraction followed by Gas Chromatography-Mass Spectrometric detection（HS-SPME-GC-MS）method was used to detect the dynamic changes of volatile substances during Tieguanyin processing（withering and Zuo-qing）,to analyze the different volatiles among samples,and to explore the main aroma substances and main contributing aroma substances in the processing.The genes related to jasmonic acid synthesis and signal transduction pathway,and the CsGro/Tup1 family numbers which are coregulater of this pathway,were identified and analyzed by bioinformatics.According to the prediction of cis-acting elements,real-time fluorescence quantitative polynucleotide chain reaction（q RT-PCR）was used to analyze the expression patterns of genes related to jasmonic acid synthesis and signal transduction pathway in Tieguanyin processing（withering,Zuo-qing）,and the expression patterns of Gro/Tup1 family genes under Me JA,GA₃,ABA,drought and low temperature treatments.The correlation between the change of aroma content and the expression level of genes related to jasmonic acid synthesis and signal transduction pathway in tea plant was analyzed.The main results are as follows:1.A total of 330 kinds of volatiles were detected in Tieguanyin processing samples,including 42 alcohols,22 aldehydes,8 acids,60esters,73 terpenes,4 phenols,5 amines,1 ethers,29 ketones,30 aromatic hydrocarbons,Hydrocarbons,34 heterocyclic compounds and 8 sulfur compounds.Terpenoids account for the highest proportion of volatile compounds in all process samples.The total peak area of volatiles reached the peak,indicating that the aroma abundance of tea leaves increased significantly after Zuo-qing.The volatile substances with floral characteristics,including 2-phenylethanol,phenylacetaldehyde,nerol,α-farnesene,geranyl acetone,β-farnesene,hexyl caproate,cedarene,methyl jasmonate,γ-juniperene,ionone,α-curcumin,and lauryl aldehyde,are the main aroma substances.Their relative contents increased significantly after Zuo-qing.Based on the analysis of distinct volatile matter and ACI value,it was found that（Z）-6-nonanal,p-methylmercaptan,β-ionone,linalool,geraniol,citral,（E）-2-nonenal had low threshold value and high ACI value,which made an important contribution to the aroma composition.2.72 jasmonic acid biosynthesis pathway genes and 61 jasmonic acid signal transduction genes were identified in tea genome.According to their tissue expression profiles,19 genes highly expressed in old and tender leaves were analyzed.It was found that there were more plant hormone response elements and stress response elements in the prediction of cis-acting elements in the promoter region.All of them have one or more of three related elements:jasmonic acid response,damage response and anaerobic induction.The content of jasmonic acid in Tieguanyin gradually increased to stable and at a high level,and the genes on jasmonic acid synthesis and signal transduction pathway could respond to a variety of stresses during Tieguanyin processing and induce defense response,and jasmonic acid signal transduction pathway was more responsive to mechanical damage caused by rotating.During Tieguanyin processing,genes related to JA synthesis pathway such as CsLOX11,CsLOX12,CsAOS2,CsAOC1,CsACX4,CsACX8,CsJAR9,CsJAR12,and key signal transduction factors CsMYC2＿4 and CsMYC2＿21 were positively correlated with ester volatiles such as hexenyl acetate,linalyl acetate and ethyl heptate,as well as terpene volatiles such asβ-pinene,β-basilene,geraniol,beta-ionone,myrcene,β-hydrocelene and（+）-limonene.The expression levels of negative feedback regulator structural gene JAZ and corepressor structural gene TPL in JA signal transduction pathway were negatively correlated with them.The jasmonic acid content of postharvest fresh leaves increased significantly after withering,and maintained a high level in the process of Zuo-qing.This showed that multiple stresses in the withering and Zuo-qing of Tieguanyin stimulated jasmonic acid synthesis and signal transduction pathway genes expression,increased jasmonic acid content and promoted the formation and accumulation of tea aroma substances,especially flower and fruit aroma substances.3.The CsGro/Tup1 family had 13 members,which were divided into two subfamilies:TOPLESS/TOPLESS-related（TPL/TPR）and LEUNIG/LEUNIG HOMOLOG（LUG/LUH）.The prediction results of cis-acting elements showed that Gro/Tupls’promoter region had a large number of cis-acting elements in response to GA,ABA,Me JA,drought and low temperature.RT-q PCR results demonstrated that Gro/Tup1s presented different expression patterns under different treatments.In severe drought,the expressions of CsLUG3 and CsLUG4 genes were significantly up-regulated,the expressions of CsTPL2 were inhibited at light drought,CsTPL8 and CsTPL9 were not regulated by drought.The expressions of CsTPL2 and CsTPL3 were significantly up-regulated under low temperature treatment.Under GA₃treatment,most gene expressions were up-regulated.Under ABA treatment,the expressions of most genes significantly increased in the beginning and significantly decreased later.Most of the gene expressions were significantly inhibited under Me JA treatment.In Tieguanyin processing,after withering,jasmonic acid content increased significantly,CsLUG4 expression increased significantly,while CsTPL1,CsTPL2,CsTPL3,CsTPL5 and CsTPL8 content did not change significantly;jasmonic acid content accumulated slowly during the green process,and decreased significantly after reaching the peak after four shaking,while the expression of CsLUG1,CsLUG4,CsTPL3 and CsTPL8 decreased gradually,and decreased most sharply before green treatment.There was a certain correlation between the content of different aroma substances and the expression level of most members of CsGro/Tup1 family.To sum up,13members of the CsGro/Tup1 family are highly conserved in structural evolution;can respond to exogenous hormones and low temperature and drought stress,and their gene responses show different expressions;they can participate in jasmonic acid signal transduction pathways to respond to stresses involved in the withering and green processes of Tieguanyin,thus affecting the formation of Tieguanyin aroma.