烟草叶片衰老期过程中的蛋白质组学分析

大田烟叶生产过程中因打顶打叉的处理,改变了烟叶正常的衰老模式。为研究这一特殊的衰老机制,我们自旺长期开始,对‘云烟87’不同发育阶段烟株的中部叶片,进行形态观测、生理生化分析及蛋白质组学检测。结果显示：随着烟叶的逐渐成熟和衰老,烟草的叶色逐渐变黄,叶片逐渐变短、变窄,厚度减少;解剖结构清晰看到栅栏组织和海绵组织从最初的整齐排列到逐渐排列紊乱,组织细胞间轮廓不明显,细胞间隙明显增大;亚显微观测表明,淀粉粒在叶绿体中逐渐积累,类囊体片层结构被挤散,叶绿体膜被撑破。生理与生化分析表明衰老过程伴随着光合作用速率下降,光合色素降解加速,呼吸代谢的增加,这可能与衰老叶片中叶绿体逐渐崩塌和细胞膜透性增加相一致。iTRAQ标记方法共检测到不同发育阶段432个差异表达蛋白质,其中注释到308个与多种生命过程相关。蛋白差异富集分析表明,烟草叶片衰老过程中与光合作用等合成代谢相关蛋白多下调表达,而逆境反应及呼吸作用等分解代谢相关蛋白多上调表达。     

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.     

Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus

Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non‐senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.     

砂仁不同叶位叶片的光合作用和氧化胁迫

衰老时砂仁叶片Pmax降低,这与叶片Gs、Chi含量和可溶性蛋白质含量的降低有关.随着叶片的衰老,NPQ、AQY、F/Fm、φPsIl和qp均降低,热耗散减少,光抑制加剧,衰老后期出现光破坏.但这些参数下降的幅度均小于Pmax下降幅度.光暗反应失衡,活性氧生成增加.衰老初期(老化)叶片MDA含量没有升高,衰老中后期叶片MDA含量显著升高,表明老化叶片能有效地耗散或清除活性氧,衰老叶片则不能,尽管其sOD、APX和POD等抗氧化酶活力显著升高.上述结果表明砂仁叶片老化与氧化胁迫关系不大,衰老与氧化胁迫密切相关.     

Quantitative proteomics analysis of proteins involved in leaf senescence of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Delaying leaf senescence and prolonging the available time for photosynthesis is one of the important approaches to increase grain yield of rice (Oryza sativa L.). Here, iTRAQ-based quantitative proteomics approach was used to comparative analyze the expression profiles of proteins in rice leaves in response to senescence. Totally 5067 proteins were identified. Compared with the proteins in the flag leaves at early stage of grain-filling in rice Liang-You-Pei 9 (LYP9), 240 and 188 proteins were up-regulated and down-regulated in the flag leaves at middle stage of grain-filling, and 387 and 202 proteins were up-regulated and down-regulated in the flag leaves at late stage of grain-filling, respectively. In addition, 39 and 18 identified proteins were constantly up-regulated and down-regulated in the leaves from early to middle and late stages of grain-filling, respectively. Among them, chloroplast chaperonin 10, geranylgeranyl diphosphate reductase, Mg chelatase subunit ChLD, porphobilinogen deaminase, protochlorophyllide reductase B and thioredoxin-like protein CITRX might have involved in the senescence of leaves. This study provided important information for understanding the age-sensitive mechanism of LYP9, and offered a foundation for future studying and improving it.     

Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings

Key message

Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings.

Abstract

Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

     

RING-box proteins regulate leaf senescence and stomatal closure via repression of ABA transporter gene ABCG40

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.     

Analysis of differentially expressed genes in response to endogenous cytokinins during cotton leaf senescence

Cytokinins have been implicated in delaying leaf senescence. We previously generated transgenic cotton (Gossypium hirsutum L.) plants that harbor the Agrobacterium isopentenyl transferase gene (ipt) directed by a proteinase gene promoter. Here, we report that mRNAs were isolated from ipt cotton leaves and azygous leaves and were subsequently sequenced using Illumina Solexa technology. The sequence tags were searched against the TIGR database and the related gene expression profiles were compared resulting in the identification of 1 218 differentially expressed genes (DEGs): 719 up-regulated and 499 down-regulated. Analyzing the DEGs in the ipt cotton leaves showed that these genes belonged to four pathways: flavone biosynthesis, arginine and proline metabolism, glyoxylate and dicarboxylate metabolism, and RNA degradation. These pathways increased the activities of antioxidants, inhibited the effect of ethylene, and prevented degradation of macromolecules during senescence. The expression patterns of 17 genes were evaluated by real-time PCR and results were in agreement with the patterns of sequencing analysis. The identification of the DEGs may help us to understand a role of cytokinins in leaf senescence.     

不同生态区烟草叶片蛋白质组学的比较

为探讨不同生态区烟叶香气风格形成的机理,应用蛋白质双向电泳联用质谱技术,对河南平顶山（浓香型烟叶的典型生态区）和福建龙岩（清香型烟叶的典型生态区）的烟草（Nicotina tobaccum L．cv．K326）叶片蛋白质组成进行了比较研究。结果发现,51个蛋白质在两个生态区发生了差异表达,其中在河南表达量上升的有15个,在福建表达量上升的有25个。另外,还分别有2个和9个蛋白点为在河南和在福建样品中特异表达。采用MALDI-TOF/MS进行肽质量指纹图谱分析,经MSDB、NCBInr和SwissPort数据库查询,共鉴定出25种蛋白质,其中参与叶绿体发育、色素代谢、光合作用相关的蛋白在福建烟区高表达,与糖酵解途径相关的蛋白质在河南烟区中高表达。另外,在两生态区烟叶中都发现有相当数量的特异表达的抗逆和防御蛋白。首次在蛋白质组学水平对不同香气风格烟叶的形成机理进行了探讨。     

Differentially expressed proteins during an incompatible interaction between common bean and the fungus Pseudocercospora griseola

The common bean (Phaseolus vulgaris L.) is the main source of protein and an important source of minerals in several countries around the world. Angular leaf spot, caused by the fungus Pseudocercospora griseola, is one of the major diseases of the common bean. In this work, we used two-dimensional gel electrophoresis and mass spectrometry to analyze alterations in the proteome of common bean leaves challenged with an incompatible race of P. griseola. Twenty-three differentially expressed proteins were detected in leaves of cultivar AND 277 collected at 12, 24 and 48 h after inoculation. The proteins were digested with trypsin and submitted to MALDI-TOF/TOF and MicrOTOF-Q electrospray mass spectrometry. Nineteen of them were identified upon MS/MS fragmentation. Most of these proteins are involved with amino acid metabolism, terpenoid metabolism, phenylpropanoid biosynthesis, antioxidant systems, vitamin and cofactor metabolism, plant–pathogen interaction, carbohydrate metabolism, photosynthesis, or genetic information processing, showing that the interaction in this pathosystem affects different genes from various metabolic pathways and processes.     

Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related proteins

Genes that are expressed during leaf senescence in Brassica napus were identified by the isolation of representative cDNA clones. DNA sequence and deduced protein sequence from two senescence-related cDNAs, LSC94 and LSC222, representing genes that are expressed early in leaf senescence before any yellowing of the leaves is visible, showed similarities to genes for pathogenesis-related (PR) proteins: a PR-1a-like protein and a class IV chitinase, respectively. The LSC94 and LSC222 genes showed differential regulation with respect to each other; an increase in expression was detected at different times during development of healthy leaves. Expression of both genes was induced by salicylic acid treatment. These findings suggest that some PR genes, as well as being induced by pathogen infection, may have alternative functions during plant development, for example in the process of leaf senescence.     

Regulation of photosynthesis in developing leaves of soybean chlorophyll-deficient mutants

In this report we examine the factors that regulate photosynthesis during leaf ontogeny in y3y3 and Y11y11, two chlorophyll-deficient mutants of soybean. Photosynthetic rates were similar during wild type and Y11y11 leaf development, but the senescence decline in photosynthesis was accelerated in y3y3. Photosynthetic rates fell more rapidly than chlorophyll concentrations during senescence in wild type leaves, indicating that light harvesting is not strongly limiting for photosynthesis during this phase of leaf development. Chlorophyll concentrations in Y11y11, though significantly lower than normal, were able to support normal photosynthetic rates throughout leaf ontogeny. Chlorophyll a/b ratios were constant during leaf development in the wild type, but in the mutants they progressively increased (y3y3) or decreased (Y11y11). In all three sets of plants, photosynthetic rates were directly proportional to Rubisco contents and activities, suggesting that Rubisco plays a dominant role in regulating photosynthesis throughout leaf ontogeny in these plants. The expression of some photosynthetic proteins, such as Rubisco activase, was coordinately regulated with that of Rubisco in all three genotypes, i.e. an early increase, coincident with leaf expansion, followed by a senescence decline in the fully-expanded leaf. On the other hand, the light harvesting chlorophyll a/b-binding proteins of PS II (the CAB proteins), while they showed a profile similar to that of Rubisco in the wild type and y3y3, progressively increased in amount during Y11y11 leaf development. We conclude that Y11y11 may be defective in the accumulation of a component required for LHC II assembly or function, while y3y3 has more global effects and may be a regulatory factor that controls the duration of senescence.