Proteomic Analysis Of Flue-Cured Tobacco Leaves During The Yellowing Stage Reveals The Formation Mechanism Of High Quality

Tobacco(Nicotiana tabacum L.)is not only one of the most important economic crop but also a model plant organism to study fundamental biological processes.At present,the problem of flue-cured tobacco curing and the contradiction between the supply of tobacco raw materials and the industrial demand are still more prominent.The yellowing stage is the first and one of key steps associated with physiological and biochemical changes in tobacco leaves during curing,which plays an important role in the formation of tobacco quality.Therefore,we used proteomics technologies to explore the key biological processes and the molecular mechanisms associated with the formation of high quality in tobacco leaf during the yellowing stage.The main results are as follows:1.Here,molecular mechanism of senescence in tobacco leaves exposed to high temperature during curing(Fresh,38°C,42°C)were revealed by isobaric tags for relative and absolute quantitation(iTRAQ)-based quantitative proteomic analysis.In total,8903 proteins were identified,and 2034(1150 up-regulated and 1074 down-regulated)differentially accumulated/abundant proteins(DAPs)were obtained from different comparisons in tobacco leaves exposed to high temperature during senescence.These DAPs were mainly involved in posttranslational modification,protein turnover,chaperones,general function prediction only,carbohydrate transport and metabolism,and energy production and conversion,translation/ribosomal structure and biogenesis,amino acid transport and metabolism.Photosynthesis,ribosome,metabolic pathways,carbon metabolism,photosynthesis-antenna proteins,and secondary metabolite biosynthesis were the most significantly enriched(p-value < 0.05)major pathways.High temperature stress accelerated tobacco leaves senescence mainly by down-regulating the photosynthetic pathways and degrading cellular constituents to maintain cell viability and nutrient recycling.Sugar-and energy-related metabolic biological processes and pathways might be critical regulators of tobacco leaves exposed to high temperature during senescence.2.In total,5931 proteins were identified in tobacco leaves during the yellowing stage(0 h,48 h and 72 h),of which 674,724 and 95 DAPs were obtained from different comparisons(48 h vs 0 h,72 h vs 0 h and 72 h vs 48 h)using iTRAQ technology.To elucidate the molecular mechanisms of pigment metabolism and color change,nineteen DAPs involved in carotenoid metabolism and twelve DAPs related to chlorophyll metabolism were screened.For chemometric analysis,the relative concentrations of the 82 volatile components were analyzed using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry(GC×GC-TOF-MS)system,including 19 carotenoid-related metabolites and one chlorophyll-related catabolite.The total concentration of carotenoid and chlorophyll degradation products in tobacco leaves decreased during 0-48 h and increased during 48-72 h.The results showed the complex regulation of DAPs in carotenoid metabolism,a negative regulation in chlorophyll biosynthesis and a positive regulation in chlorophyll breakdown,which delayed the degradation of carotenoid/xanthophylls and accelerated the breakdown of chlorophylls and promoted the formation of yellow color during curing.Particularly,the up-regulation of the chlorophyllase-1-like isoform X2 was the key protein regulatory mechanism responsible for chlorophyll metabolism and color change.3.The withering(softening)mechanism of tobacco leaves during curing was studied by tandem mass tag(TMT)-based quantitative proteomic technology and mass-spectrometric method.The results showed that the water content,softness value and cell wall substance content of tobacco leaves decreased in the process of flue-curing.In addition,there were significant differences in the water content and softness value of tobacco leaves,but a relatively small difference in the cell wall substance content in tobacco leaves under different humid conditions(H1,75% relative humidity;H2,85% relative humidity;and H3,95% relative humidity).The softness value of tobacco leaves under medium humidity was moderate,and the degradation of cell wall substance was relatively higher.Correlation analysis showed that the water content of flue-cured tobacco was more closely related to the value of softness compared with cell wall substances.In total,11556 proteins were detected,among which 496 DAPs were identified.To elucidate the withering mechanism of tobacco leaves,27 DAPs associated with cell wall metabolism were screened.In particular,pectin acetylesterases,glucan endo-1,3-beta-glucosidases,xyloglucan endotransglucosylase/hydrolase,alpha-xylosidase 1-like,probable galactinol-sucrose galactosyltransferases,endochitinase A,chitotriosidase-1-like and expansin were the key proteins responsible for the withering of postharvest tobacco leaves.These DAPs were mainly involved in pectin metabolism,cellulose and hemicellulose metabolism,galactose metabolism,amino sugar and nucleotide sugar metabolism,and cell wall expansion.Thus,dehydration and cell wall metabolism were crucial for tobacco leaf withering under different conditions.4.Change of metabonomics of tobacco leaves under different curing conditions(H1,H2 and H3)during the yellowing stage was studied by quasi-targeted metabolomics method.A total of 480 metabolites were identified and quantified,and most metabolites were significantly down-regulated in different comparison groups(except the comparison of H3-72 h vs H2-72h).The enrichment analysis of differential metabolites showed that cyanide amino acid metabolism,aminoacyl tRNA biosynthesis,phenylalanine metabolism and amino acid biosynthesis metabolism were significantly enriched pathways of differential metabolites in several comparisons.Among them,the six key amino acids-related differential metabolites were significantly enriched in multiple metabolic pathways,namely,L-isoleucine,L-serine,L-phenylalanine,L-asparagine,L-tyrosine and valine.In addition,19 kinds of amino acids and 45 kinds of their derivatives were identified and quantified.Under different curing conditions,the average contents of amino acids and their derivatives in H2 treatment were the lowest.In addition,2-hydroxy-D-glutamic acid and L-homocysteine could be used as molecular markers of metabolic state in tobacco leaves during the yellowing stage.In a word,the curing process and amino acid metabolism play important roles in the quality formation of tobacco leaves during the yellowing stage.5.The correlations between differential proteins and metabolites in different comparisons of tobacco leaves under different humid conditions(H1,H2 and H3)were analyzed.The results showed that most of the differential proteins were positively correlated with the differential metabolites in the comparisons of H1-72 h vs H0,H2-72 h vs H0,H3-72 h vs H0,H2-72 h vs H1-72 h and H3-72 h vs H1-72 h.Namely,most of the DAPs and differential metabolites showed consistency in biological regulation in these comparisons.However,there was a negative correlation between most of the DAPs and differential metabolites in the comparison of H3-72 h vs H2-72 h,suggesting that changes in expression levels of the most DAPs were not consistent with the levels of differential metabolites in the comparison of H3-72 h vs H2-72 h.The metabolic pathways,thiamine metabolism pathway,and valine,leucine and isoleucine degradation pathway,were all enriched in the comparisons of H1-72 h vs H0,H2-72 h vs H0 and H3-72 h vs H0.However,metabolic pathways was only one enriched pathway in the comparisons of H2-72 h vs H1-72 h,H3-72 h vs H1-72 h and H3-72 h vs H2-72 h.The complex regulation of DAPs and differential metabolites in metabolic pathways was an important way to improve the quality of tobacco leaves during the yellowing stage.In conclusion,the yellowing stage is the key step for the formation of high tobacco quality during curing.Therefore,we used proteomics technologies to explore the basic biological process in tobacco leaves during senescence,the molecular mechanisms of color regulation and pigment metabolism,withering(softening)mechanism and amino acid metabolism of tobacco leaves associated with the formation of high tobacco quality during the yellowing stage.These results laid a solid foundation for improving the yellowness,softness and aroma quality of tobacco leaves during curing.