iTRAQ-based comparative proteomic analysis reveals high temperature accelerated leaf senescence of tobacco (Nicotiana tabacum L.) during flue-curing.

Tobacco (Nicotiana tabacum) is extensively cultivated all over the world for its economic value. During curing and storage, senescence occurs, which is associated with physiological and biochemical changes in postharvest plant organs. However, the molecular mechanisms involved in accelerated senescence due to high temperatures in tobacco leaves during curing need further elaboration. We studied molecular mechanisms of senescence in tobacco leaves exposed to high temperature during curing (Fresh, 38 °C and 42 °C), revealed by isobaric tags for relative and absolute quantification (iTRAQ) for the proteomic profiles of cultivar Bi'na1. In total, 8903 proteins were identified, and 2034 (1150 up-regulated and 1074 down-regulated) differentially abundant proteins (DAPs) were obtained from tobacco leaf samples. These DAPs were mainly involved in posttranslational modification, protein turnover, energy production and conversion. Sugar- and energy-related metabolic biological processes and pathways might be critical regulators of tobacco leaves exposed to high temperature during senescence. High-temperature stress accelerated tobacco leaf senescence mainly by down-regulating photosynthesis-related pathways and degrading cellular constituents to maintain cell viability and nutrient recycling. Our findings provide a valuable inventory of novel proteins involved in senescence physiology and elucidate the protein regulatory network in postharvest organs exposed to high temperatures during flue-curing.

Comparative Proteomic Analysis by iTRAQ Reveals that Plastid Pigment Metabolism Contributes to Leaf Color Changes in Tobacco (Nicotiana tabacum) during Curing.

Tobacco (Nicotiana tabacum), is a world's major non-food agricultural crop widely cultivated for its economic value. Among several color change associated biological processes, plastid pigment metabolism is of trivial importance in postharvest plant organs during curing and storage. However, the molecular mechanisms involved in carotenoid and chlorophyll metabolism, as well as color change in tobacco leaves during curing, need further elaboration. Here, proteomic analysis at different curing stages (0 h, 48 h, 72 h) was performed in tobacco cv. Bi'na1 with an aim to investigate the molecular mechanisms of pigment metabolism in tobacco leaves as revealed by the iTRAQ proteomic approach. Our results displayed significant differences in leaf color parameters and ultrastructural fingerprints that indicate an acceleration of chloroplast disintegration and promotion of pigment degradation in tobacco leaves due to curing. In total, 5931 proteins were identified, of which 923 (450 up-regulated, 452 down-regulated, and 21 common) differentially expressed proteins (DEPs) were obtained from tobacco leaves. To elucidate the molecular mechanisms of pigment metabolism and color change, 19 DEPs involved in carotenoid metabolism and 12 DEPs related to chlorophyll metabolism were screened. The results exhibited the complex regulation of DEPs in carotenoid metabolism, a negative regulation in chlorophyll biosynthesis, and a positive regulation in chlorophyll breakdown, which delayed the degradation of xanthophylls and accelerated the breakdown of chlorophylls, promoting 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. The expression pattern of 8 genes was consistent with the iTRAQ data. These results not only provide new insights into pigment metabolism and color change underlying the postharvest physiological regulatory networks in plants, but also a broader perspective, which prompts us to pay attention to further screen key proteins in tobacco leaves during curing.

Changes in water loss and cell wall metabolism during postharvest withering of tobacco (Nicotiana tabacum L.) leaves using tandem mass tag-based quantitative proteomics approach.

Withering is an important biological process accompanied by dehydration and cell wall metabolism in postharvest plant organs during curing/processing and storage. However, dynamics involved in cell wall metabolism and resultant water loss during withering in postharvest tobacco leaves is not well-documented. Here, tandem mass tag (TMT)-based quantitative proteomic analysis in postharvest tobacco leaves (cultivar K326) under different withering conditions was performed. In total, 11,556 proteins were detected, among which 496 differentially abundant proteins (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, hemicellulose and galactose metabolism, amino sugar and nucleotide sugar metabolism as well as cell wall expansion. Furthermore, relative water content and softness values were significantly and positively correlated. Thus, dehydration and cell wall metabolism were crucial for tobacco leaf withering under different conditions. Nine candidate DAPs were confirmed by parallel reaction monitoring (PRM) technique. These results provide new insights into the withering mechanism underlying postharvest physiological regulatory networks in plants/crops.

Separation and characterization of sucrose esters from Oriental tobacco leaves using accelerated solvent extraction followed by SPE coupled to HPLC with ion-trap MS detection.

Sucrose esters (SEs) were successfully extracted from Oriental tobacco leaves using a new methodology based on accelerated solvent extraction followed by hydrophilic-lipophilic balanced cartridge cleanup step. The SEs were detected by HPLC with ion-trap MS detection using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the highly sensitive characterization offered by ion-trap MS. Under the optimized conditions, 14 SEs were first identified among a total of 23 SEs found in Oriental tobacco leaves. Under the same conditions, only four new SEs were extracted by using traditional ultrasound-assisted extraction and liquid-solid extraction methods. The present method might be potentially useful in high-efficiency extraction and sensitive characterization of SEs from complex matrices such as tobacco leaves.