【作者】 郝志龙；

【导师】 金心怡； 杨贞标； 俞晓敏；

【作者基本信息】 福建农林大学 ， 茶学， 2020， 博士

【摘要】 摇青是乌龙茶品质形成的关键工序,通过机械力刺激青叶,结合晾青工序,利用机械力、失水等环境胁迫最终形成乌龙独特的风味品质,特别是机械力摇青工序还广泛应用于花香红茶、花香白茶、花香绿茶等其他茶类的加工改善品质。植物在环境胁迫下可以迅速启动自我保护与适应机制,在生理、转录、蛋白、代谢水平产生一系列应答调控,从而导致丰富的初、次生代谢物积累。为系统研究青叶对摇青机械力的响应机制,本研究以铁观音青叶为材料,选用易于控制的振动摇青为机械力刺激模型,从生理变化、转录和蛋白水平分析机械力作用对青叶的影响,并结合代谢组学联合转录组和蛋白组数据分析青叶响应机械力刺激的代谢调控机制。研究结果如下:1青叶响应机械力刺激的生理变化机械力刺激30 min内青叶水分含量与自然摊放叶和萎凋叶比无显著差异,60 min时显著低于自然摊放叶和萎凋叶。处理5 min青叶叶态变化不明显,15 min后叶色变深,叶缘红变,30 min时顶叶叶缘略背卷,随着机械力作用时间延长,青臭气变浓,清香减退。10 min时电导率显著升高,5 min和10 min时光系统Y(Ⅱ)和ETR显著降低。随着机械力作用时间的延长,叶绿体(CH)和类囊体(Thy)紧密而清晰的片层结构逐渐松散、变形、收缩、紊乱、瓦解,5 min时淀粉粒明显增加,15 min时嗜锇颗粒明显增多,出现聚集。青叶抗氧化酶APX、DHAR、GR和MDAR活性在刺激5-10min显著高于自然摊放叶。说明一定时间(30 min以内)机械力刺激对青叶水分含量影响不显著,青气变浓,叶色变深,加剧青叶细胞膜系统损伤,提高其透性,促进膜内可溶物渗出,电导率升高,类囊体降解,叶绿体严重受损,PSⅡ被破坏,并通过提高活性氧代谢酶活性抵御机械力胁迫产生ROS对叶细胞的损伤。2青叶对机械力刺激的转录响应模式根据机械力刺激青叶生理变化结果,取刺激5 min(V5)、15min(V15)青叶与萎凋叶(C0)、自然摊放15 min(C15)青叶进行有参转录分析。结果表明,所获高质量转录文库中84%以上序列能与已有茶树基因匹配。与C15相比,V15中差异基因以上调表达为主,GO功能富集在代谢过程、蛋白修饰、芳香族化合物合成、转移酶活性和离子结合等;KEGG代谢通路富集在植物-病原互作、谷胱甘肽代谢、α-亚麻酸代谢和植物激素信号转导等途径。适度的机械力刺激青叶(15 min)通过防御应答和抗逆基因,如MAPK、CDPK和Ca2+信号途径的CNGC、MPK6、MPK4、WRKY22、Ca CML、HSP90等上调表达,增强青叶细胞的防御反应,同时促进NO和ROS积累诱导谷胱甘肽代谢中3个氧化还原对关键基因6PGDp、GPX、APX、GSTU、GSH1和GSH2上调表达;合成JAs的基因AOS、AOC、ACX和JMT等以及JA信号途径的COI1、JAZ和MYC2上调表达,从而清除胞内因机械力胁迫产生的ROS,引起细胞防御相关基因表达和信号物质的产生,提高青叶对机械力胁迫的抵抗力和适应性。青叶通过调控α-亚麻酸代谢途径SDP1、DAD1、LOX和HPL1等基因上调表达,促进绿叶挥发物积累,可能是刺激5 min产生青气的原因。3青叶对机械力刺激的蛋白响应模式对V5、V15、V30和C0、C15进行蛋白质TMT检测分析,共获得868个差异蛋白,主要定位在叶绿体、细胞核和细胞质上。与C0相比,机械力刺激青叶差异蛋白以上调表达为主,随着作用时间延长,上调表达蛋白逐渐减少(389(?)301(?)174)。差异蛋白GO功能富集在生物刺激、防御应答、宿主细胞病毒转运、水解酶活性和细胞骨架结构组成等,KEGG代谢通路富集在植物-病原互作、谷胱甘肽代谢和苯丙氨酸代谢等途径,与转录结果较吻合。说明叶绿体是青叶响应机械力刺激的主要生化反应场所,青叶对短时间(5-15min)机械力刺激应答反应明显,长时间(30 min)刺激反而抑制蛋白质数量的变化,为生产上乌龙茶做青须分次摇青,并控制摇青时间从蛋白水平提供理论依据。适度机械力刺激青叶(15 min)通过调控CDPK和Ca2+信号途径CDPK和Rboh蛋白与CNGCs、CALM和CML上调表达,分别介导细胞内NO和ROS积累,诱导GSH代谢途径氧化分解蛋白GGT1-5、GPX、GST、APX等上调表达,促进GSH氧化以及与亲电物质的结合,提高代谢其他异源物质能力,达到青叶抗氧化胁迫和解毒的作用;但GSH合成蛋白GSHA、GSS和还原蛋白GSR下调表达。说明青叶感知机械力刺激后,在蛋白水平启动GSH氧化还原反应保护机制,以GSH消耗占优势。防御反应蛋白RIN4、RPM1、HSP90等在机械力刺激青叶中也上调表达,抵抗和适应机械力刺激产生的胁迫。机械力刺激青叶通过上调合成苯乙醛、苯乙酸等芳香族物质的调控蛋白DDC、AOC3、PAT、GOT2等,促进苯乙醛、苯乙酸和苯丙酮酸的合成。较长时间机械力刺激青叶(30 min)通过调控蛋白PAL、C4H、4CL、CHS、CHI等上调表达,促进儿茶素的合成。16个候选基因q RT-PCR检测结果与转录组RNA-seq和蛋白组TMT分析的表达趋势基本一致,证实青叶在分子水平调控CDPK、Ca2+信号途径和GSH、苯丙氨酸代谢途径,提高其抵抗和适应机械力胁迫的能力,影响代谢的方向和结果,进而影响摇青品质。4青叶对机械力刺激响应的代谢组学联合分析对相同的样品MV5、MV15、MV30与MC0、MC15进行广泛靶向代谢物和挥发性物质分析,共获得772个代谢物和36种挥发性物质,与MC0相比,随着刺激时间延长,上调代谢物先增后降(153<161>150),下调代谢物呈增加趋势(55<83<105)。差异代谢物KEGG富集在α-亚麻酸代谢、谷胱甘肽代谢、苯丙氨酸代谢和黄酮类生物合成等途径上。说明只有适度的机械力刺激,才能促进青叶上调代谢物增多,刺激时间越长,下调代谢物越多,为生产上控制摇青时间,改善乌龙茶品质提供代谢物依据。结合蛋白组研究结果,机械力刺激通过下调还原型谷胱甘肽GSH合成蛋白和还原蛋白,上调GSH氧化分解蛋白调控青叶中GSH含量下降,GSSG升高;通过上调APX蛋白调控ASA含量降低,上调蛋白PGD和G6PD调控NADP含量降低,使青叶为清除机械力胁迫产生的ROS促使还原型物质(GSH、ASA)的消耗,生成氧化型物质(GSSG),并上调NADP+-NADPH氧化还原对维持GSH平衡。通过提高合成GSH的底物含量,降低GSH分解代谢下游的代谢物含量,调节青叶因长时间(30 min)机械力刺激使GSH过量消耗而打破其向GSSG氧化的稳态过程,使其重新提高GSH合成底物含量加强合成。代谢和蛋白水平共同揭示机械力刺激青叶通过消耗GSH、ASA,生成GSSG,清除机械胁迫产生的ROS,抵抗机械力胁迫的调控机制。机械力刺激青叶通过关键基因LOX6上调表达调控α-亚麻酸代谢形成低沸点芳香族化合物的分支代谢中间代谢物9(S)-Hp OTr E在MV5中含量升高,ADH1上调表达调控3-己烯醇含量升高;通过AOS、HPL1上调表达调控9(S)-HOTr E和9,10-EOTr E在MV15中含量升高。转录和代谢水平说明短时间机械力刺激促进该代谢分支反应,形成具有青草气的挥发物,为生产上摇青前期产生强烈的青臭气提供科学依据。转录和代谢水平揭示长时间(15-30 min)机械力作用促进α-亚麻酸代谢途径向JAs分支进行,通过上调JA途径的关键基因应答机械力胁迫,实际生产上随着摇青次数的增加而逐渐延长摇青时间,可能积累更多的JAs物质为后期形成丰富的香气物质提供前提条件,为摇青时间逐渐延长提供理论依据。蛋白和代谢水平揭示机械力刺激能促进苯丙氨酸代谢合成芳香族化合物,只有适度的机械力刺激(15-30 min)才能促进大部分儿茶素的合成。检测到的22种氨基酸中除了苏氨酸、天冬氨酸和瓜氨酸含量较低外,其他游离氨基酸含量均有在机械力刺激不同时间点青叶中含量较高,说明适度的机械力刺激能有效促进青叶氨基酸的积累,为其他茶类应用摇青工序改善品质提供依据。芳香族化合物3,5-二甲基苯甲醛、苯甲醇、水杨酸甲酯、苯乙醇和茉莉内酯含量和短链脂肪族化合物顺-3-己烯醇丁酯、顺-3-己烯醇己酯和2-庚醇在机械力刺激青叶中含量较高,并随着刺激的时间延长呈增加趋势,说明机械力刺激有利于这些与茶叶香气密切相关的挥发性物质的积累。只有一定的机械力刺激才能促进顺-3己烯醇、α-法尼烯和反式橙花叔醇的积累。较长时间的刺激作用有利于叶绿醇及其同分异构体和苯乙醛的积累。30 min以内的机械力作用并不能直接促进β-罗勒烯和吲哚的积累,但能从转录水平上调JA合成途径,促进其积累为摇青之后吲哚的合成提供前提条件。综上所述,青叶对机械力刺激的响应可分为以基因和蛋白质上调表达,启动叶细胞防御和抗氧化反应,产生青草气,叶张呈紧张状态的初级响应阶段;以叶绿体受损,淀粉粒和嗜锇颗粒增多并聚集,促进防御信号物质积累,芳香族化合物、儿茶素类、部分氨基酸和挥发性物质等代谢物的积累,基因和蛋白质呈下调表达模式为特征的次级反应阶段。更多还原

【Abstract】 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.更多还原