Comparative proteomics of dehydration response in the rice nucleus: New insights into the molecular basis of genotype‐specific adaptation

Dehydration is the most crucial environmental factor that considerably reduces the crop harvest index, and thus has become a concern for global agriculture. To better understand the role of nuclear proteins in water‐deficit condition, a nuclear proteome was developed from a dehydration‐sensitive rice cultivar IR‐64 followed by its comparison with that of a dehydration‐tolerant c.v. Rasi. The 2DE protein profiling of c.v. IR‐64 coupled with MS/MS analysis led to the identification of 93 dehydration‐responsive proteins (DRPs). Among those identified proteins, 78 were predicted to be destined to the nucleus, accounting for more than 80% of the dataset. While the detected number of protein spots in c.v. IR‐64 was higher when compared with that of Rasi, the number of DRPs was found to be less. Fifty‐seven percent of the DRPs were found to be common to both sensitive and tolerant cultivars, indicating significant differences between the two nuclear proteomes. Further, we constructed a functional association network of the DRPs of c.v. IR‐64, which suggests that a significant number of the proteins are capable of interacting with each other. The combination of nuclear proteome and interactome analyses would elucidate stress‐responsive signaling and the molecular basis of dehydration tolerance in plants.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Dehydration-responsive reversible and irreversible changes in the extracellular matrix: comparative proteomics of chickpea genotypes with contrasting tolerance

Dehydration is the most crucial environmental factor that limits plant growth, development, and productivity affecting agriculture throughout the world. Studies on genetic variations for dehydration tolerance in plants is crucial because divergent cultivars with contrasting traits aid the identification of key cellular components that confer better adaptability. The extracellular matrix (ECM) is a dynamic structure that serves as the repository for important signaling components and acts as a front-line defense. To better understand dehydration adaptation, a proteomic study was performed on the extracellular matrix of ICCV-2, a dehydration-susceptible genotype of chickpea. The proteome was generated with ECM-enriched fractions using two-dimensional gel electrophoresis. The LC-ESI-MS/MS analysis led to the identification of 81 dehydration-responsive proteins. The proteome was then compared with that of JG-62, a tolerant genotype. Comparative proteomics revealed genotype-specific expression of many proteins involved in a variety of cellular functions. Further, the reversible and irreversible changes in the proteomes revealed their differing ability to recover from dehydration-induced damage. We propose that cell wall restructuring and superior homeostasis, particularly the management of reactive oxygen species, may render better dehydration-adaptation. To our knowledge, this is the first report on the comprehensive comparison of dehydration-responsive organellar proteome of two genotypes with contrasting tolerance.     

Proteomics Reveals New Salt Responsive Proteins Associated with Rice Plasma Membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

Proteomics reveals new salt responsive proteins associated with rice plasma membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

Modification of the mitochondrial proteome in response to the stress of ethanol-dependent hepatotoxicity

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.     

Proteomic comparison of Gibberella moniliformis in limited-nitrogen (fumonisin-inducing) and excess-nitrogen (fumonisin-repressing) conditions

The maize pathogen Gibberella moniliformis produces fumonisins, a group of mycotoxins associated with several disorders in animals and humans, including cancer. The current focus of our research is to understand the regulatory mechanisms involved in fumonisin biosynthesis. In this study, we employed a proteomics approach to identify novel genes involved in the fumonisin biosynthesis under nitrogen stress. The combination of genome sequence, mutant strains, EST database, microarrays, and proteomics offers an opportunity to advance our understanding of this process. We investigated the response of the G. moniliformis proteome in limited nitrogen (N0, fumonisininducing) and excess nitrogen (N+, fumonisin-repressing) conditions by one- and two-dimensional electrophoresis. We selected 11 differentially expressed proteins, six from limited nitrogen conditions and five from excess nitrogen conditions, and determined the sequences by peptide mass fingerprinting and MS/MS spectrophotometry. Subsequently, we identified the EST sequences corresponding to the proteins and studied their expression profiles in different culture conditions. Through the comparative analysis of gene and protein expression data, we identified three candidate genes for functional analysis and our results provided valuable clues regarding the regulatory mechanisms of fumonisin biosynthesis.     

Proteomics analysis of BHK‐21 cells infected with a fixed strain of rabies virus

Rabies is a neurotropic virus that causes a life threatening acute viral encephalitis. The complex relationship of rabies virus (RV) with the host leads to its replication and spreading toward the neural network, where viral pathogenic effects appeared as neuronal dysfunction. In order to better understand the molecular basis of this relationship, a proteomics study on baby hamster kidney cells infected with challenge virus standard strain of RV was performed. This cell line is an in vitro model for rabies infection and is commonly used for viral seed preparation. The direct effect of the virus on cellular protein machinery was investigated by 2‐DE proteome mapping of infected versus control cells followed by LC‐MS/MS identification. This analysis revealed significant changes in expression of 14 proteins, seven of these proteins were viral and the remaining were host proteins with different known functions: cytoskeletal (capping protein, vimentin), anti‐oxidative stress (superoxide dismutase), regulatory (Stathmin), and protein synthesis (P0). Despite of limited changes appeared upon rabies infection, they present a set of interesting biochemical pathways for further investigation on viral‐host interaction.     

Shotgun proteome analysis of Bordetella pertussis reveals a distinct influence of iron availability on the bacterial metabolism,virulence, and defense response

One of the mechanisms involved in host immunity is the limitation of iron accessibility to pathogens, which in turn provokes the corresponding physiological adaptation of pathogens. This study reports a gel‐free nanoLC‐MS/MS‐based comparative proteome analysis of Bordetella pertussis grown under iron‐excess and iron‐depleted conditions. Out of the 926 proteins covered 98 displayed a shift in their abundance in response to low iron availability. Forty‐seven of them were found to be increased in level while 58 were found with decreased protein levels under iron starvation. In addition to proteins previously reported to be influenced by iron in B. pertussis, we observed changes in metabolic proteins involved in fatty acid utilization and poly‐hydroxybutyrate production. Additionally, many bacterial virulence factors regulated by the BvgAS two‐component system were found at decreased levels in response to iron limitation. These results, together with the increased production of proteins potentially involved in oxidative stress resistance, seem to indicate that iron starvation provokes changes in B. pertussis phenotype that might shape host–pathogen interaction.     

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.     

Label‐free quantitative proteome analysis of skeletal tissues under mechanical load

Skeletal tissue has the capability to adapt its mass and structure in response to mechanical stress. However, the molecular mechanism of bone and cartilage to respond to mechanical stress are not fully understood. A label‐free quantitative proteome approach was used for the first time to obtain a global perspective of the response of skeletal tissue to mechanical stress. Label‐free quantitative analysis of 1D‐PAGE‐LC/MS/MS based proteomics was applied to identify differentially expressed proteins. Differential expression analysis in the experimental groups and control group showed significant changes for 248 proteins including proteins related to proliferation, differentiation, regulation of signal transduction and energy metabolic pathways. Fluorescence labeling by incorporation of alizarin/calcein in newly formed bone minerals qualitatively demonstrated new bone formation. Skeletal tissues under mechanical load evoked marked new bone formation in comparison with the control group. Bone material apposition was evident. Our data suggest that 39 proteins were assigned a role in anabolic process. Comparisons of anabolic versus catabolic features of the proteomes show that 42 proteins were related to catabolic. In addition, some proteins were related to regulation of signal transduction and energy pathways, such as tropomyosin 4, fibronectin 1, and laminin, might be new molecular targets that are responsive to mechanical force. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the effects of mechanical force on skeletal tissue. J. Cell. Biochem. 108: 600–611, 2009. © 2009 Wiley‐Liss, Inc.