Study On The Dynamic Regulation Of Volatiles In Leaves Responding To Wounding Stress During Oolong Tea “Zuoqing” Process

The “Zuoqing” process in oolong tea is a typical example of inducing metabolic accumulation through external stress,involving complex metabolic pathways.Understanding the transcriptional level changes or functions of a single gene in the oolong tea processing process is not conducive to our overall understanding of the transcriptional/metabolic reprogramming events.Therefore,in this study,we used representative varieties of oolong tea,Tieguanyin and Jinguanyin,as experimental materials,combined with metabolomics,and transcriptomics methods,to clarify the important role of the Zuoqing process in the formation of oolong tea aroma quality.A shaking mechanical wounding stress experiment was designed,and a high-resolution transcriptome was used to construct a transcriptional regulatory temporal model mediated by wounding stress.Furthermore,by combining epigenomics methods,the study elucidated the involvement of chromatin accessibility and DNA methylation epigenetic regulatory mechanisms in the formation of aroma quality during the “Zuoqing” stage,aiming to deepen our understanding of the molecular mechanisms of aroma formation during the making green process of oolong tea.The main conclusions obtained are as follows:(1)Study on the dynamic changes of gene transcription levels and volatile metabolite contents during the processing of oolong teaIn this study,processing samples of two representative varieties were analyzed using a combination of sensory analysis,metabolomics and transcriptomics.The metabolomics results showed that a total of 190 volatile metabolites were identified in the two varieties.The total content of volatile metabolites during the processing of oolong tea reached the highest level at the end of the last “Liangqing” stage.The same volatile substance classes in both varieties had similar dynamic patterns of change during processing,with the highest levels of esters and terpenes volatiles at the end of the last “Liangqing” stage.The contents of key volatiles in oolong tea,such as nerolidol,α-farnesene,β-farnesene,limonene,indole,cis-caryophyllene,hexyl caproate,linalool and phenylethylaldehyde,reached the peak at the end of “Liangqing” stage.Combined with sensory analysis,linalool(OAV=50.6)and geraniol(OAV=1.9)were found to be key components in the formation of clear floral aroma characteristics of Tieguanyin and sweet floral aroma of Jinguanyin.At the transcriptional level,based on the dynamic expression of genes during the processing of oolong tea,it could be clearly clustered into 10 Clusters with specific change trends,and most of the gene expression levels increased significantly during the “Zuoqing” stage.Some conserved differential genes induced by specific processing processes were identified,and the number and binding motifs of ERF and bHLH transcription factors were significantly enriched in this conserved gene set,implying that the ERF and bHLH transcription factor families play an important role in the processing of oolong tea.In conclusion,we comprehensively analyzed the dynamic changes of volatiles and gene transcription levels during the processing of oolong tea,and emphasized the important role of the“Zuoqing” stage of oolong tea on the formation of aroma quality.(2)Analysis of transcriptional regulation-mediated regulation of volatiles in response to mechanical wounding stress during“Zuoqing”stageIn order to further explore the transcriptional and metabolic reprogramming events mediated by mechanical wounding stress during oolong tea “making green” stage,High-density sampling of tea leaves after a single mechanical wonding treatment(including a total of 63 samples)was performed for transcriptome sequencing and volatile determination.The results showed that wounding stress during oolong tea processing induced a rapid burst of transcriptional activity.Transcription kinetics results showed that the expression gene pattern in response to injury stress was divided into 18 gene sets with specific biological processes,and transcription factors(TFs)were involved in regulating specific biological processes of different gene sets.Among them,CsbHLH,ERF,NAC,and WRKY transcription factor family may be the main regulators that positively regulate wounidng-stress-induced genes.wounding-mediated time-series models reveal a specific sequence of transcriptional reprogramming events,and tea exhibits a complex and rapid pattern of transcriptional regulation in response to wounding stress,that is,following a fast-slow-fast pattern of transcriptional regulation within 200 min.Most of the genes and TFs that showed significant up-regulation early(up-phase I and II)were involved in signaling pathways in response to wounding stress,suggesting that this process is one of the primary targets of wounding-mediated transcriptional upregulation events;in genes and TFs in up-phase III and IV were involved in the regulation of primary metabolism of tea plant;in subsequent up-phase stages(up-phase stages V and VI),structural defense pathways of tea plant were activated.These results suggest that wounding-mediated transcriptional reprogramming events display a distinct temporal profile of up-regulated genes that begins with activation of wounding-stress-related pathways followed by activation of defense-related secondary and primary metabolic pathways.The weighted gene co-expression network analysis showed that transcription factors such as C2H2,ERF,and bHLH positively regulated the expression of CsTSB(CsTGY08G0000242),CsOCS(CsTGY08G0000372),and CsNES(CsTGY08G0000357,CsTGY18G0000361)genes,leading to accumulation of key volatiles such as indole and nerolidol during the “Zuoqing” process.(3)Analysis of chromatin accessibility-mediated regulation of volatiles in response to mechanical wounding stress during“Zuoqing”stageThe ATAC-seq technique was used to investigate the dynamics of chromatin opening in tea plants after shaking green damage stress.A total of 1406 shared THS(transposase hypersensitive sites)were identified in fresh leaves(YL,same material as used in Chapter 3),10 min after wouding stress(Y10,same material as used in Chapter 3)and 40 min after wounding stress(Y40,same material as used in Chapter 3),respectively.624 and 1838 shared THSs were identified at 40 min(Y10,same material as in Chapter 3)and 40 min(Y40,same material as in Chapter 3),respectively,of which 462 THSs were shared by three samples.The majority of THSs were distributed in the distal region of the gene,followed by the promoter region 2000 bp upstream of the gene.The openness of the distal regulatory region in the treated group was smaller than that in the control group,while the openness of the proximal promoter region increased significantly after wounding treatment,indicating that dynamic changes in chromatin accessibility are an effective means for tea plants to cope with wounding stress.Combined with the transcriptome data,a transcriptional regulatory network was constructed with MYB and with bHLH,ERF and Dof transcription factors as the cores in response to wounding stress.Further,the yeast effector expression vector was constructed and the DNA library was randomly screened using the TF-centered Y1 H method yeast single hybridization system,and it was found that tea CsMYC2 a could bind specifically to the known binding element G-box(-CACGTG-)and four new binding elements.Combined with ATAC-seq data,the MYC regulator network under wounding stress was constructed,and it was found that tea tree MYC transcription factors could positively regulate tea tree ethanol dehydrogenase CsADH and glycosyltransferase and thus indirectly participate in the quality formation during the greening process of oolong tea.In addition,the chromatin accessibility and transcript levels of the promoter regions of three structural genes(CsLOX,CsTPS,and CsTSB),which are involved in the synthesis of key aroma of oolong tea,were significantly increased after the mechanical damage stress of shaking green,indicating that the epistatic regulatory mechanism by means of chromatin opening changes played an important role in the response to the mechanical damage stress of shaking green and the formation of key aroma substances in oolong tea.4.Study on DNA methylation regulation of tea leaves in response to wounding stress during the oolong tea manufacturing processTo further clarify the epigenetic regulation mechanism of Oolong tea responding to mechanical stress,the single-base resolution DNA methylation landscape of tea tree in fresh leaf stage(YL),10 min(Y10)and 40 min(Y40)after shaking green damage stress was detected by Whole Genome Bisulfite Sequencing(WGBS)technology.The results showed that the methylation rate of C sites in the whole genome was 15.08% to 18.1%;the methylation ratio of Cp G and CHG sequence environment was relatively high,among which the methylation rate of m Cp G was 37.08% to 41.95%,and the methylation rate of m CHG was 34.15% to 40.53%;the methylation rate of m CHH sites supported was relatively low,only 7.93% to 10.31%.At the chromosomal scale,there was no significant difference in the methylation locus density between the treatment group and the control group in the three sequence environments.The distribution of CG and CHG methylation loci before and after treatment was basically the same in each chromosome of tea tree,and it showed a complementary relationship with CHH methylation loci.There were significant differences in the methylation levels of genes with different functions,and the CG methylation degree in the gene intron region was the highest,followed by the promoter region.In the CHG and CHH sequence background,the methylation degree of the gene promoter region was the highest.After mechanical damage stress treatment of tea tree,the methylation levels of the three different sequence environments in the gene promoter region were increased to different degrees,indicating that DNA methylation may participate in the regulation of target gene expression as an important regulatory mechanism in the response of tea tree to mechanical stress.The difference methylation regions mainly concentrated in gene promoter region,exon region and intron region between the treatment group and the control group,suggesting that in the process of tea tree responding to mechanical stress,the methylation and demethylation of the above three functional regions are mainly used to regulate the transcription of target genes.After wounding treatment at 10 min and 40 min,more than 1000 high methylation regions were annotated in the gene promoter region of the tea tree genome,mainly in CHH methylation.In the YL vs Y10 and YL vs Y40 comparison groups,2042 and 3287 differential methylation genes were respectively anchored,and KEGG enrichment analysis results showed that these differential methylation genes involved a wide range of secondary metabolic pathways.Combined with ATAC-seq,RNA-seq and WGBS data,it was found that the demethylation process of the promoter region of the key gene CsTSB(tryptophan synthase β-subunit)was the main reason for the change of its promoter chromatin accessibility and transcription level.