Comparative chloroplast proteome analysis of exogenously supplied trehalose to wheat seedlings under heat stress

The aim of our study was to investigate the underlying molecular mechanisms of exogenously supplied trehalose affecting wheat photosynthesis under heat stress. The amount of ATP synthase (ATPase), oxygen-evolving enhancer protein (OEE), PsbP, Rubisco, chloroplast fructose-bisphosphate aldolase (FBPA), and ferredoxin-NADP(H) oxidoreductase (FNR) were downregulated, while PSI reaction center subunits were upregulated under heat stress. However, in the trehalose-pretreated groups, the amount of FNR, cytochrome b₆f complex, PSI reaction center subunits, ATPase, FBPA, and Rubisco were upregulated under normal growth conditions and heat stress. Besides, during the recovery period, the upregulation in CAB, PsbP, OEE2, and ATPase suggested that trehalose pretreatment might help to the recovery of PSII and PSI. These results indicate that trehalose pretreatment effectively regulates the levels of the photosynthesis-related proteins and relieves the damage of heat stress to wheat chloroplast.     

镉胁迫后旱柳转录组变化分析

随着重金属镉(Cd)应用范围的扩大,由此引发的土壤镉污染问题日益严重。以具有植物恢复潜力的旱柳Salix matsudana作为研究对象,探究不同浓度的Cd (2.5 mg/L, 50 mg/L)胁迫后旱柳无性系1 d、7 d和30 d后基因表达与代谢通路的变化。转录组测序结果表明:共获得102 595个非冗余基因(Unigenes),相同浓度不同时间的差异基因总数为26 623个和32 154个;相同时间不同浓度的差异基因总数为8 550个、3 444个和11 428个。从中筛选得到与Cd胁迫响应密切相关的基因25个,其中金属硫蛋白、ABC转运蛋白、锌和锰转运蛋白等基因的表达不仅会随着Cd胁迫浓度变化而且同时受到胁迫时间的改变而发生改变;油菜素内酯合成通路的ROT3和黄酮类化合物合成通路的FLS、F3H均明显上调。此外Cd胁迫引起旱柳在代谢过程、细胞过程、膜、细胞器、细胞、细胞部分、催化活化和结合蛋白这8个方面发生改变,参与这些GO条目的差异表达基因数随着Cd浓度和胁迫时间的增加而增加。并对转录组信息的可靠性用RT-PCR和酶活性生理实验数据进行了验证。文中通过转录组测序分析旱柳Cd胁迫后的响应机制,从而为旱柳修复土壤Cd污染提供理论指导。     

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H⁺ ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.     

Comparative proteome analysis of Helicobacter pylori

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.     

镉胁迫下金银花的生长反应及积累特性

采用水培试验方法,研究了藤本植物金银花在不同浓度镉（Cd）胁迫下的生长反应和积累特 性,结果表明,Cd对金银花生长未造成显著影响,甚至低浓度(5和10 mg·L^-1) Cd具有一定的生长促进作用,表现为植物的株高和叶绿素含量有所增加。当Cd处理浓度达到50 mg·L^-1时,植物的株高和根长虽有所降低,但与对照相比并无显著差异,且耐性系数均＞0.8,表明金银花对Cd具有较强的耐性。当Cd处理浓度为25 mg·L^-1时,金银花地上部中Cd含量达到286.12 μg·g^-1,而富集系数均＞1,说明金银花具备Cd超富集植物的特征,对Cd污染土壤的修复具有很大的应用潜力。     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.     

Comparative proteomic analysis of canola leaves under salinity stress

Although canola is a moderately salt‐tolerant species, its growth, seed yield, and oil production are markedly reduced under salt stress, particularly during the early vegetative growth stage. To identify the mechanisms of salt responsiveness in canola, the proteins expressed in the second and third newly developed leaves of salt‐tolerant, Hyola 308, and salt‐sensitive, Sarigol, cultivars were analyzed. Plants were exposed to 0, 175, and 350 mM NaCl during the vegetative stage. An increase in the Na content and a reduction in growth were observed in the third leaves compared to the second leaves. The accumulation of Na was more pronounced in the salt‐sensitive compared with the salt‐tolerant genotype. Out of 900 protein spots detected on 2‐DE gels, 44 and 31 proteins were differentially expressed in the tolerant and susceptible genotypes, respectively. Cluster analysis based on the expression level of total and responsive proteins indicated that the second leaves had a discriminator role between the two genotypes at both salinity levels. Using MS analysis, 46 proteins could be identified including proteins involved in responses to oxidative stress, energy production, electron transport, translation, and photosynthesis. Our results suggest that these proteins might play roles in canola adaptation to salt stress.     

Physiological responses of peanut seedlings to exposure to low or high cadmium concentration and the alleviating effect of exogenous nitric oxide to high cadmium concentration stress

Abstract

The physiological responses of peanut seedlings exposed to low (5 µM) or high (200 µM) cadmium (Cd) concentration and the ability of sodium nitroprusside (SNP, a donor of NO) to reverse the harmful effects of Cd on peanut (Arachis hypogaea L.) were studied. Changes in plant growth parameters, chlorophyll content, antioxidant system, nutrient contents and Cd accumulation were investigated. The results showed that SNP and 5 µM Cd improved plant growth and chlorophyll content. Furthermore, antioxidative system was up-regulated, and as a result, the production rate of superoxide radical (O₂^??) was reduced. Moreover, the absorption of nutrient elements was not impacted, and Cd toxicity was not observed. However, 200 µM Cd had negative effects on the above measured parameters and dramatically increased the accumulation of Cd in all the plant organs. In the 200 µM Cd treatment, addition of 250 µM SNP stimulated plant growth and increased chlorophyll content. It also enhanced the regulation of antioxidative system and reduced the production rate of O₂^?? and malondialdehyde (MDA) content. Besides, SNP supply enhanced the absorption of nutrient elements and restrained the absorption and transport of Cd.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

Comparative proteome analysis of human epithelial ovarian cancer

Background  

Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer.     

Altered proteome biology of cardiac mitochondria under stress conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.     

Comparative membrane proteome analysis of three Borrelia species

The versatility of the surface of Borrelia, the causative agent of Lyme borreliosis, is very important in host-pathogen interactions allowing bacteria to survive in ticks and to persist in a mammalian environment. To identify the surface proteome of Borrelia, we have performed a large comparative proteomic analysis on the three most important pathogenic Borrelia species, namely B. burgdorferi (strain B31), B. afzelii (strain K78), and B. garinii (strain PBi). Isolation of membrane proteins was performed by using three different approaches: (i) a detergent-based fractionation of outer membrane proteins; (ii) a trypsin-based partial shedding of outer cell surface proteins; (iii) biotinylation of membrane proteins and preparation of the biotin-labelled fraction using streptavidin. Proteins derived from the detergent-based fractionation were further sub-fractionated by heparin affinity chromatography since heparin-like molecules play an important role for microbial entry into human cells. All isolated proteins were analysed using either a gel-based liquid chromatography (LC)-MS/MS technique or by two-dimensional (2D)-LC-MS/MS resulting in the identification of 286 unique proteins. Ninety seven of these were found in all three Borrelia species, representing potential targets for a broad coverage vaccine for the prevention of Lyme borreliosis caused by the different Borrelia species.     

Comparative plasma proteome analysis of lymphoma-bearing SJL mice

In SJL mice, growth of RcsX lymphoma cells induces an inflammatory response by stimulating V(beta)16+ T cells. During inflammation, various serum protein levels can increase (e.g., acute phase reactants) or decrease (e.g., albumin), and most of these altered proteins are thus potential biomarkers. Although blood plasma is a valuable and promising sample for biomarker discovery for diseases or for novel drug targets, its proteome is complex. To address this, we have focused on a comprehensive comparison of the plasma proteomes from normal and RcsX-tumor-bearing SJL mice using the 1D-Gel-LC-MS/MS method after removing albumin and immunoglobulins. This analysis resulted in the identification of a total of 1079 nonredundant mouse plasma proteins; more than 480 in normal and 790 in RcsX-tumor-bearing SJL mouse plasma. Of these, only 191 proteins were found in common. The molecular weights ranged from 2 to 876 kDa, covering the pI values between 4.22 and 12.09, and included proteins with predicted transmembrane domains. By comparing the plasma proteomic profile of normal and RcsX-tumor-bearing SJL mice, we found significant changes in the levels of many proteins in RcsX-tumor-bearing mouse plasma. Most of the up-regulated proteins were identified as acute-phase proteins (APPs). Also, several unique proteins i.e., haptoglobin, proteosome subunits, fetuin-B, 14-3-3 zeta, MAGE-B4 antigen, etc, were found only in the tumor-bearing mouse plasma; either secreted, shed by membrane vesicles, or externalized due to cell death. These results affirm the effectiveness of this approach for protein identification from small samples, and for comparative proteomics in potential animal models of human disorders.