Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

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 Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

The Comparatively Proteomic Analysis in Response to Cold Stress in Cassava Plantlets

Cassava (Manihot esculenta Crantz) is a tropical root crop and sensitive to low temperature. However, it is poorly to know how cassava can modify its metabolism and growth to adapt to cold stress. An investigation aimed at a better understanding of cold-tolerant mechanism of cassava plantlets was carried out with the approaches of physiology and proteomics in the present study. The principal component analysis of seven physiological characteristics showed that electrolyte leakage (EL), chlorophyll content, and malondialdehyde (MDA) may be the most important physiological indexes for determining cold-resistant abilities of cassava. The genome-wide proteomic analysis showed that 20 differential proteins had the same patterns in the apical expanded leaves of cassava SC8 and Col1046. They were mainly related to photosynthesis, carbon metabolism and energy metabolism, defense, protein synthesis, amino acid metabolism, signal transduction, structure, detoxifying and antioxidant, chaperones, and DNA-binding proteins, in which 40 % were related with photosynthesis. The remarkable variation in photosynthetic activity and expression level of peroxiredoxin is closely linked with expression levels of proteomic profiles. Moreover, analysis of differentially expressed proteins under cold stress is an important step toward further elucidation of mechanisms of cold stress resistance.     

Comparative Physiological and Proteomic Analysis Reveals the Leaf Response to Cadmium-Induced Stress in Poplar (Populus yunnanensis)

Excess amounts of heavy metals are important environmental pollutants with significant ecological and nutritional effects. Cdmium (Cd) is of particular concern because of its widespread occurrence and high toxicity. We conducted physiological and proteomic analyses to improve our understanding of the responses of Populus yunnanensis to Cd stress. The plantlets experienced two apparent stages in their response to Cd stress. During the first stage, transiently induced defense-response molecules, photosynthesis- and energy-associated proteins, antioxidant enzymes and heat shock proteins (HSPs) accumulated to enhance protein stability and establish a new cellular homeostasis. This activity explains why plant photosynthetic capability during this period barely changed. During the second stage, a decline of ribulose-1, 5-bisphosphate carboxylase (RuBisCO) and HSP levels led to imbalance of the plant photosynthetic system. Additionally, the expression of Mitogen-activated protein kinase 3 (MPK3), Mitogen-activated protein kinase 6 (MPK6) and a homeobox-leucine zipper protein was higher in the second stage. Higher expression of caffeoyl-CoA O-methyltransferase (CCoAOMT) may regulate plant cell wall synthesis for greater Cd storage. These genes may be candidates for further research and use in genetic manipulation of poplar tolerance to Cd stress.     

Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a non-ethylene pathway for induction

Antisera to acidic isoforms of pathogenesis-related proteins were used to measure the induction of these proteins in tobacco (Nicotiana tabacum) leaves. Endo-(1-4)-β-xylanase purified from culture filtrates of Trichoderma viride was a strong elicitor of pathogenesis-related protein synthesis in tobacco leaves. The synthesis of these proteins was localized to tissue at the area of enzyme application. The inhibitors of ethylene biosynthesis and ethylene action, 1-aminoethoxyvinylglycine and silver thiosulfate, inhibited accumulation of pathogenesis-related proteins induced by tobacco mosaic virus and α-aminobutyric acid, but did not inhibit elicitation by xylanase. Likewise, the induction of these proteins by the tobacco pathogen Pseudomonas syringae pv. tabaci was not affected by the inhibitors of ethylene biosynthesis and action. The leaf response to tobacco mosaic virus and α-aminobutyric acid was dependent on light in normal and photosynthetically incompetent leaves. In contrast, the response of leaves to xylanase was independent of light. Tobacco mosaic virus and α-aminobutyric acid induced concerted accumulation of pathogenesis-related proteins. However, xylanase elicited the accumulation of only a subset of these proteins. Specifically, the plant (1-3)-β-glucanases, which are normally a part of the concerted response, were underrepresented. These experiments have revealed the presence of a novel ethylene-independent pathway for pathogenesis-related protein induction that is activated by xylanase.     

Two poplar methyl salicylate esterases display comparable biochemical properties but divergent expression patterns

Two genes encoding proteins of 98% sequence identity that are highly homologous to tobacco methyl salicylate (MeSA) esterase (SABP2) were identified and cloned from poplar. Proteins encoded by these two genes displayed specific esterase activities towards MeSA to produce salicylic acid, and are named PtSABP2-1 and PtSABP2-2, respectively. Recombinant PtSABP2-1 and PtSABP2-2 exhibited apparent Km values of 68.2 ± 3.8 μM and 24.6 ± 1 μM with MeSA, respectively. Structural modeling using the three-dimensional structure of tobacco SABP2 as a template indicated that the active sites of PtSABP2-1 and PtSABP2-2 were highly similar to that of tobacco SABP2. Under normal growing conditions, PtSABP2-1 showed the highest level of expression in leaves and PtSABP2-2 was most highly expressed in roots. In leaf tissues of poplar plants under stress conditions, the expression of PtSABP2-1 was significantly down-regulated by two stress factors, whereas the expression of PtSABP2-2 was significantly up-regulated by four stress factors. The plausible mechanisms leading to these two highly homologous MeSA esterase genes involved in divergent biological processes in poplar are discussed.     

Differential proteomic analysis of cadmium-responsive proteins in wheat leaves

To gain a comprehensive understanding of plant response to Cd, physiological and proteomic changes in wheat (Triticum aestivum L.) leaves exposed to a range of Cd concentrations (10, 100 and 200 μM) were investigated. Leaf elongation was decreased, whereas the H₂O₂ and malondialdehyde content increased significantly at higher Cd concentrations. Changes in protein profiles were analyzed by two-dimensional electrophoresis. Twenty-one proteins which showed 1.5-fold change in protein abundance in response to Cd were identified. These proteins can be functionally grouped into three groups: 1) oxidative stress response, 2) photosynthesis and sugar metabolism and 3) protein metabolism and others. These results provide a new insight into our understanding of the molecular basis of heavy metal response in plants.     

Differential Expression of Proteins in Response to Molybdenum Deficiency in Winter Wheat Leaves Under Low-Temperature Stress

Molybdenum (Mo) is an essential micronutrient for plants. To obtain a better understanding of the molecular mechanisms of cold resistance enhanced by molybdenum application in winter wheat, we applied a proteomic approach to investigate the differential expression of proteins in response to molybdenum deficiency in winter wheat leaves under low-temperature stress. Of 13 protein spots that were identified, five spots were involved in the light reaction of photosynthesis, five were involved in the dark reaction of photosynthesis, and three were highly involved in RNA binding and protein synthesis. Before the application of cold stress, four differentially expressed proteins between the Mo deficiency (?Mo) vs. Mo application (+Mo) comparison are involved in carbon metabolism and photosynthetic electron transport. After 48 h of cold stress, nine differentially expressed proteins between the ?Mo vs. +Mo comparison are involved in carbon metabolism, photosynthetic electron transport, RNA binding, and protein synthesis. Under ?Mo condition, cold stress induced a more than twofold decrease in the accumulation of six differential proteins including ribulose bisphosphate carboxylase large-chain precursor, phosphoglycerate kinase, cp31BHv, chlorophyll a/b-binding protein, ribulose bisphosphate carboxylase small subunit, and ribosomal protein P1, whereas under +Mo condition cold stress only decreased the expression of RuBisCO large subunit, suggesting that Mo application might contribute to the balance or stability of these proteins especially under low-temperature stress and that Mo deficiency has greater influence on differential protein expression in winter wheat after low-temperature stress. Further investigations showed that Mo deficiency decreased the concentrations of chlorophyll a, chlorophyll b, and carotenoids; the maximum net photosynthetic rate; the apparent quantum yield; and carboxylation efficiency, even before the application of the cold stress, although the decrease rates were greater after 48 h of cold treatment, which is consistent with changes in the expressions of differential proteins in winter wheat under low-temperature stress. These findings provide some new evidence that Mo might be involved in the light and dark reaction of photosynthesis and protein synthesis.     

Proteome analysis of tobacco leaves under salt stress

The mechanisms responsible for the effects of salt stress on tobacco plants were examined by means of proteomic analysis. Tobacco plants were exposed to 0, 150, 250, 300, or 400 mM NaCl. At 150 mM NaCl or above, the plants showed a reduction in fresh weight and an increase in proline levels. Proteins extracted from the leaves of tobacco plants exposed to 150 mM NaCl were separated by 2-DE. Of 205 protein spots that were detected reproducibly in each gel, 18 were differentially expressed under NaCl treatment. Up-regulated proteins belonged to the photosynthesis category, whereas down-regulated proteins correspond to defense-related functions. Dose- and time-dependent studies showed that a stromal 70-kDa heat shock-related protein was markedly down-regulated by NaCl. Thus, down-regulation of the stromal 70-kDa heat shock protein in response to salt stress is likely the cause of failure to protect cells against salt stress of tobacco plants.     

Comparative proteomic analysis of <Emphasis Type="Italic">Phalaenopsis</Emphasis> leaves in the vegetative and flowering phase

Phalaenopsis, an epiphytic crassulacean acid metabolism (CAM) plant, requires moderate variations of day/night temperatures for flowering. In this study, changes in chlorophyll content, chlorophyll fluorescence, sugar components, titratable acidity and soluble protein content in Phalaenopsis leaves during flowering were observed. Comparative proteomic analysis of Phalaenopsis leaves in the vegetative and flowering phase was performed for the first time using iTRAQ (isobaric tags for relative and absolute quantification). A total of 126 proteins were differentially expressed in Phalaenopsis leaves. Analysis of potential functions revealed that the major categories of predicted function of the up-regulated proteins were protein destination (27 %), photosynthesis (15.9 %), primary metabolism (14.3 %) and defense (12.7 %) in the flowering phase, while the major categories of predicted function of the down-regulated proteins were protein destination (33.3 %), primary metabolism (20.6 %), transportation (14.3 %) and signal transduction (11.1 %). Proteome profile analysis indicated that the proteome changes were consistent with changes in sugar and protein metabolites. Some novel proteins were differentially expressed, most of which were identified as signaling proteins, including 14-3-3 proteins, fibrillin, rapid alkalinization factors (RALF), the Ras-related protein RABB1c, calreticulin and calmodulin. Histone, importin alpha, multidrug resistance proteins and the ABC transporters were also differentially expressed. These results provide insights into the mechanisms that regulate flowering in complex flowering plants.     

Low nitrogen-induced expression of cyclophilin in Nicotiana tabacum

Leaf morphology and the leaf protein expression profiles of flue-cured tobacco grown in central Henan province of China under low nitrogen (low-N) and normal nitrogen (normal-N) nutrition were examined. The leaf length and width were measured at 50, 60, and 70 days after transplanting. Leaves grown under low-N conditions were shorter and more narrow than those grown under normal-N conditions. The protein expression profiles of tobacco leaves harvested at 70 days after transplanting were analyzed by 2-dimensional electrophoresis, and five differentially expressed proteins including a putative protein were identified. Except for the MCM protein-like protein, the other three differentially expressed proteins of cyclophilin-like protein, vacuolar invertase INV2, MAR-binding protein and the one putative protein showed increased expression in the low-N nutrition group. Among these proteins, the cyclophilin-like protein, which is a stress-responsive signal protein, may play pivotal roles in regulating leaf development under stress conditions. Real-time quantitative PCR analysis showed that the mRNA expression level of the cyclophilin-like protein at day 50, 60 and 70 under low-N conditions was 0.90, 1.43 and 6.9-fold higher than that under normal-N conditions, indicating that the gene expression of cyclophilin-like protein was strongly induced by low-N conditions.