Multi-omics Approach Revealed The Quality Formation Mechanism Of Fresh Tea Leaves During Withering Treatment

Following harvest,the fresh tea leaves will naturally undergo a withering treatment,in which they are moved to a proper environment and spread evenly on a flat surface before being subjected to the enzyme-deactivating pan-firing process.In traditional tea manufacturing process,the fresh tea leaves were supposed to be pan-fired as soon as possible after harvest,while withering was treated as a “unnecessary” process,and its significance was neglected.During withering,the water content of fresh tea leaves will drop,and their texture will become pliable.This will make the following fixation and shaping processes easier to conduct.In the meanwhile,since the enzyme is still active,dynamic metabolic changes will occur during this process.There are several researches that investigate the metabolic changes of tea leaves during withering process.However,despite these changes being well documented and a general notion that a proper withering treatment before pan firing was beneficial to green tea's quality,the underlying mechanisms of these metabolic changes still remains unknown.To understand the quality formation of withering tea leaves and its underlying mechanism more comprehensively and systematically,UHPLC-Orbitrap-MS metabolomics,as well as tandem mass tag(TMT)-based proteomics analysis were applied to samples of tea leaves that were collected at different times of withering(up to 15 h).Global metabolic changes in tea leaves undergoing withering treatment and the underlying proteinic mechanisms were explored,and an integrated multi-omics analysis were conducted using both the metabolomic and proteomic results.The results are as follows:(1)A total of 69 metabolites were identified in metabolomic analysis.During withering(up to 15 h),The differentiated metabolites in fresh tea leaves were showing a monotonic shift.Among them,the levels of 12 amino acids significantly increased.The levels of catechins and several glycosidically bound volatiles decreased.The level of caffeine remained relatively unchanged.And the levels of flavonoids and their glycosides did not demonstrate specific patterns of change.(2)Up to 136 differentially expressed proteins(13h / 0h comparison)were obtained through pair-wise comparisons in proteomic analysis.The number of differentially expressed proteins increased with the time of withering,and most differentially expressed proteins showed monotonic changes of expression.The differentially expressed proteins mainly participated in dehydration response,starch synthesis and catalysis,plant hormone and signal regulation,flavonoid biosynthesis,the synthesis and catalysis of glycosidically bound volatiles,and so on.In general,the proteome of withering tea leaves was changed ascribing to the tea leaves' response to multiple stresses in the withering process.These changes will in turn lead to the metabolic shift of tea leaves.(3)During the withering process,the levels of metabolites in fresh leaves changed synchronically with the expression levels of related proteins.Among them,the increase of amino acids' level could be ascribed to the overall up-regulation of proteases and peptidases;the biosynthesis of catechins and flavonoids were inhibited,thus cause the drop of catechins' level;the decrease of glycosidically bound volatiles' level could be attributed to the downregulation of UDP-glycosyltransferase and up-regulation of glycosyl hydrolase.Overall,the withering process could help lower the phenol/amino ratio of fresh tea leaves,but would also cause the loss of aroma substances by turning them from glycosidically-binding state to free state.This suggests that the withering process is beneficial to the quality of fresh tea leaves in some respects.A prolonged withering process,however,may cause negative effect to the aroma of made tea.