Comparative proteomic analysis of cold-induced sweetening in potato (Solanum tuberosum L.) tuber

Cold-induced sweetening is one of the major factors limiting the quality of fried potato products. To understand the mechanisms of protein regulation for cold-induced sweetening in potato tubers, a comparative proteomic approach was used to analyse the differentially expressed proteins both during control (25 °C, 30 days) and cold treatment (4 °C, 30 days) using two-dimensional gel electrophoresis. Quantitative image analyses indicated that there were 25 protein spots with their intensities significantly altered more than twofold. Of these proteins, 9 were up-regulated, 13 were down-regulated, 2 were absent, and 1 was induced in the cold-stored tubers. The MALDI-TOF/TOF MS analyses led to the identification of differentially expressed proteins that are involved in several processes and might work cooperatively to maintain metabolic homeostasis in tubers during low-temperature storage. The preponderance of metabolic proteins reflects the inhibition of starch re-synthesis and the accumulation of sugars in carbon fluxes, linking starch–sugar conversion. The respiration-related proteins suggest the transfer of respiratory activity from aerobic respiration to anaerobic respiration in the cold-stored tubers. The proteins associated with defence appear to protect the tuber cells from low-temperature stress. Some heat shock proteins that act as chaperones also displayed a differential expression pattern, suggesting a potentially important role in cold-stored tubers, although their exact contribution remains to be investigated. The proposed hypothetical model might explain the interaction of these differentially expressed proteins that are associated with cold-induced sweetening in tubers.     

Proteomic profiling of Rhizobium tropici PRF 81: Identification of conserved and specific responses to heat stress

ABSTRACT: BACKGROUND: Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28degreesC and 35degreesC. RESULTS: Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses. CONCLUSIONS: Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species.     

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.     

Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)

Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response in wheat (Triticum aestivum L.), a proteomic approach was used to identify the salt stress-responsive proteins in an elite Chinese wheat cultivar, Zhengmai 9023, which exhibits a high yield, superior gluten quality and better biotic resistance. Three-week-old seedlings were treated with NaCl of four different concentrations (1.0%, 1.5%, 2.0%, and 2.5%). The total proteins from the leaves of untreated and NaCl-treated plants were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2358 protein spots were detected on the gels, among which 125 spots showed a significant change in protein abundance, and 83 differentially expressed spots were localised on preparative gels. Using Q-TOF mass spectrometry, 52 salt-responsive spots were identified, which were classified into six functional categories that included transport-associated proteins, detoxifying enzymes, ATP synthase, carbon metabolism, protein folding, and proteins with unknown biological functions. Of the 52 differentially expressed proteins, 26 were up-regulated, 21 were down-regulated, and five spots showed multi-expression patterns. In particular, some important proteins for salt tolerance were found to be up-regulated in Zhengmai 9023 under salt stress, such as H⁺-ATPases, glutathione S-transferase, ferritin and triosephosphate isomerase.     

Proteomic study for the cellular responses to Cd2+ in Schizosaccharomyces pombe through amino acid-coded mass tagging and liquid chromatography tandem mass spectrometry

Cadmium (Cd(2+)) is one of well-known toxic heavy metal ions. To gain a global understanding how Cd(2+) affects cells at the molecular level, we systematically studied the cellular response of the fission yeast Schizosaccharomyces pombe to Cd(2+) using our integrated proteomic strategy of amino acid-coded mass tagging (AACT) and liquid chromatography-tandem mass spectrometry. Our proteome-wide investigation unequivocally identified 1133 S. pombe proteins. Of which, the AACT-based quantitative analysis revealed 106 up-regulated and 55 down-regulated proteins on the Cd(2+) exposure. The most prevalent functional class in the up-regulated proteins, approximately 28% of our profile, was the proteins involved in protein biosynthesis, showing a time-dependent biphasic expression pattern characteristic with rapid initial induction and later repression. Most significantly, 27 proteins functionally classified as cell rescue and defense were up-regulated for oxygen and radical detoxification, heat shock response, and other stress response. Furthermore, the large precursor sequence coverage of our AACT approach allowed us to unequivocally identify and quantitate different isozymes for glutathione S-transferase, which have close similarity in their amino acid sequence. Our quantitative dataset also showed that 80% of the up-regulated proteins found in the S. pombe response were different from those in the Saccharomyces cerevisiae response. The function of some of the key identifications was validated through biochemical assays. It is very interesting that the induction of cysteine synthase expression was not observed in our study, although it has been proven as a critical enzyme to supply free cysteines for the enhancing synthesis of Cd(2+)-sequestering molecules such as glutathione and phytochelatins in plants and some yeasts. Our quantitative proteomic result instead suggested that, as an alternative mechanism for the detoxification of Cd(2+), S. pombe produced significantly higher level of inorganic sulfide to immobilize cellular Cd(2+) as a form of CdS nanocrystallites capped with glutathione and/or phytochelatins.     

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.     

Leaf proteome characterization in the context of physiological and morphological changes in response to copper stress in sorghum

Copper (Cu) is an essential micronutrient required for normal growth and development of plants; however, at elevated concentrations in soil, copper is also generally considered to be one of the most toxic metals to plant cells due to its inhibitory effects against many physiological and biochemical processes. In spite of its potential physiological and economical significance, molecular mechanisms under Cu stress has so far been grossly overlooked in sorghum. To explore the molecular alterations that occur in response to copper stress, the present study was performed in ten-day-old Cu-exposed leaves of sorghum seedlings. The growth characteristics were markedly inhibited, and ionic alterations were prominently observed in the leaves when the seedlings were exposed to different concentrations (0, 100, and 150 µM) of CuSO₄. Using two-dimensional gels with silver staining, 643 differentially expressed protein spots (≥1.5-fold) were identified as either significantly increased or reduced in abundance. Of these spots, a total of 24 protein spots (≥1.5-fold) from Cu-exposed sorghum leaves were successfully analyzed by MALDI-TOF-TOF mass spectrometry. Of the 24 differentially expressed proteins from Cu-exposed sorghum leaves, 13 proteins were up-regulated, and 11 proteins were down-regulated. The abundance of most identified protein species, which function in carbohydrate metabolism, stress defense and protein translation, was significantly enhanced, while that of another protein species involved in energy metabolism, photosynthesis and growth and development were severely reduced. The resulting differences in protein expression patterns together with related morpho-physiological processes suggested that these results could help to elucidate plant adaptation to Cu stress and provide insights into the molecular mechanisms of Cu responses in C₄ plants.     

Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p 相似文献   

Proteomic study of Carissa spinarum in response to combined heat and drought stress

Carissa spinarum is one of the secondary advantage plants grown in dry‐hot valleys in China, which can survive under stress conditions of high temperature and extreme low humidity. Here, we studied the physiological and proteomic changes of C. spinarum in response to 42°C heat stress treatment in combination with drought stress. Dynamic changes in the leaf proteome were analyzed at four time points during the stress treatment and recovery stages. Approximately, 650 protein spots were reproducibly detected in each gel. Forty‐nine spots changed their expression levels upon heat and drought treatment, and 30 proteins were identified by MS and 2‐D Western blot. These proteins were classified into several categories including HSP, photosynthesis‐related protein, RNA‐processing protein and proteins involved in metabolism and energy production. The potential roles of these stress‐responsive proteins are discussed.     

强休眠玉米种子休眠前后的蛋白差异表达

以强休眠玉米自交系08-641为试验材料,分别对处于休眠状态下的新鲜收获种子和经过10 d后熟作用破除休眠的种子进行了蛋白质组学差异表达分析。结果表明,通过双向电泳技术在3次重复试验下休眠状态的08-641鲜种子蛋白2-DE图谱上共检测到约600个蛋白质点,在经过10 d后熟作用破除休眠的08-641种子蛋白2-DE图谱上共检测到约620个蛋白质点,其中下调表达蛋白质点4个,上调表达蛋白质点4个,新增蛋白质点8个,缺失表达蛋白质点7个。经过质谱鉴定的差异表达蛋白质主要涉及球蛋白、胚胎晚期丰富蛋白、豆球蛋白等贮藏物蛋白质;蛋白酶体、山梨醇脱氢酶等参与物质代谢的蛋白质;热激蛋白等参与蛋白质结构、细胞功能调控的蛋白质。推测08-641种子休眠是由于种子内休眠相关蛋白的过量表达或缺失抑制了种子的正常萌发。     

Proteomic analysis of rice seedlings during cold stress

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold stress.     

达坂喜盐芽孢杆菌D-8^T在低渗冲击下的双向凝胶电泳分析

为了解革兰氏阳性中度嗜盐菌适应低渗冲击的机制,采用双向凝胶电泳技术,研究达坂喜盐芽孢杆菌(Halobacillus dabanensis)D-8T在低渗冲击下的差异蛋白表达谱。利用ImagemasterTM2D Platinum软件分析到大约650个蛋白点,大多数蛋白分子量分布在17.5~66kDa,等电点为4.0~5.9,偏酸性。在20%盐浓度中生长的D-8菌株受到0%盐浓度的低渗冲击5min及50min后34个蛋白点的表达发生改变,包括20个表达上调的点和14个点表达下调。用MALDI-TOF/MS及MASCOT软件鉴定了4个与低渗胁迫有关的蛋白,分别为热激蛋白DanK、柱状决定蛋白、青霉素结合蛋白和5-莽草酸烯醇式丙酮酸-3-磷酸合成酶。其中,热激蛋白适应低渗胁迫时表达上调为首次报道。     

Proteomic analysis of seed viability in maize

To identify specific proteins related to maize seed viability, seeds of Zhengdan 958 (one of the high-yield maize hybrids in China) were sorted based on viability evaluation with triphenyltetrazolium chloride (TTC) assay and used for comparative proteomic analysis. After TTC staining, embryos of high-viability seeds were deep red (R type), while embryos of dead seeds were white (W type). Proteomic analysis revealed that 28 protein spots identified were differently expressed significantly between R and W embryos, of which 20 were up-regulated and 8 down-regulated in R embryos. Among them were proteins involved in stress response, protein folding, and stabilization, as wells as proteins related to nutrient reservoir and metabolism. Prominently, small heat shock proteins, late embryogenesis abundant (LEA) proteins, and antioxidant enzymes were highly up-regulated, while two proteases were highly down-regulated in R embryos compared to W embryos. One of LEA proteins was EMB564, which declined in abundance during artificial aging of seeds. Our results suggested an association of EMB564 with maize seed viability. It would be of interest to use these small proteins to develop quick tests for seed quality.     

Differential expression profile of membrane proteins in zebrafish (Danio rerio) brain exposed to methyl parathion

Methyl parathion (MP) is a widely used organophosphorus pesticide, which has been related to a broad spectrum of toxic effects on environmental organisms. The present study investigated the changes in the protein profile of enriched membrane fraction from zebrafish (Danio rerio) brain exposed to three concentrations (0.5, 1 and 2 mg/L) of MP. 2-DE revealed that the abundance of 21 protein spots was significantly changed by MP stress. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database search, 16 protein spots were identified as membrane proteins, among which 8 were down-regulated, while 8 were up-regulated. These proteins are mainly involved in oxidative stress response, signal transduction, metabolism, protein synthesis and degradation, neuroplasticity and regeneration as well as synaptic transmission. These results may aid our understanding of the mechanism of MP-induced neurotoxicity and provide the possibility of the establishment of candidate biomarkers of MP.