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 approach to identify wound-response related proteins from rice leaf sheath

In order to avoid the complex conditions of the intact plant for simple analysis of proteins in wound-response stress, we used the detached rice leaf sheath which is a very active part of the rice seedling. Proteins were extracted from rice leaf sheath at 0, 12, 24, 48 h after cutting and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Changes in differentially displayed proteins were found in leaf sheaths after cutting in the 0-48 h time course. Ten proteins were up-regulated, while 19 proteins were down-regulated compared with those on the four 2-D gels. Among them, 14 proteins were analyzed by N-terminal, or internal amino acid sequence. The clear functions of nine proteins could be identified. Six proteins did not yield amino acid sequence information due to their blocked N-termini. Furthermore, 11 proteins were determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and identified protein database matching. It was shown that the down-regulated proteins were calreticulin (nos. 5, 6), histone H1 (no. 15) and hemoglobin (no. 17), putative peroxidase (no. 19); the up-regulated proteins were Bowman-Birk trypsin inhibitor (no. 23), putative receptor-like protein kinase (nos. 24, 25), calmodulin-related protein (no. 26), small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (no. 27), mannose-binding rice lectin (nos. 28, 29). Among all the above proteins, four (nos. 23, 24, 25, 26) have been confirmed to be wound-response proteins. The others cannot be excluded as also being related to wound-responses, such as the signal transduction-related proteins (nos. 5, 6), photosynthesis-related protein (no. 27), and stress-response proteins (nos. 19, 28, 29). This is the first time protein changes in response to wounding in rice leaf sheath have been shown.     

Proteomics analysis reveals multiple regulatory mechanisms in response to selenium in rice

Selenium (Se) shows both beneficial and toxic effects on plant growth. Rice (Oryza sativa L.) seedlings cultivated under lower concentrations of sodium selenite showed enhanced growth, whereas higher concentrations of sodium selenite repressed seedling growth. To acquire detailed regulatory mechanisms underlying these effects, a comparative proteomics study using 2-dimensional gel electrophoresis and MALDI-TOF/TOF MS was performed. By comparison of gel images between Se treatments and control, 66 and 97 differentially expressed proteins were identified in shoot and root, respectively under at least one of the Se treatment concentrations. Gene Ontology and Clustering analysis reveal primary metabolism, photosynthesis and redox homeostasis are the most highly affected biological processes by Se treatments. Lower Se treatments (2 and 6 mg/L sodium selenite) activated antioxidative system, enhanced photosynthesis and primary metabolism. However, higher Se treatment (10 mg/L sodium selenite) damaged photosynthesis apparatus, inhibited photosynthesis and primary metabolism. Protein ubiquitination and phosphorylation may also play important roles in Se response in rice. In conclusion, our study provided novel insights into Se response in rice at the proteome level, which are expected to be highly useful for dissecting the Se response pathways in higher plants and for producing Se enriched rice cultivars in the future.     

Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

PLASTOCHRON2 regulates leaf initiation and maturation in rice

In higher plants, leaves initiate in constant spatial and temporal patterns. Although the pattern of leaf initiation is a key element of plant shoot architecture, little is known about how the time interval between initiation events, termed plastochron, is regulated. Here, we present a detailed analysis of plastochron2 (pla2), a rice (Oryza sativa) mutant that exhibits shortened plastochron and precocious maturation of leaves during the vegetative phase and ectopic shoot formation during the reproductive phase. The corresponding PLA2 gene is revealed to be an orthologue of terminal ear1, a maize (Zea mays) gene that encodes a MEI2-like RNA binding protein. PLA2 is expressed predominantly in young leaf primordia. We show that PLA2 normally acts to retard the rate of leaf maturation but does so independently of PLA1, which encodes a member of the P450 family. Based on these analyses, we propose a model in which plastochron is determined by signals from immature leaves that act non-cell-autonomously in the shoot apical meristem to inhibit the initiation of new leaves.     

Proteomic analysis of drought-responsive proteins in rice reveals photosynthesis-related adaptations to drought stress

Drought stress inhibits rice growth and biomass accumulation. To identify novel regulators of drought-stress responses in rice, we conducted a proteome-level study of the stress-susceptible (SS) Oryza sativa L. cv. ‘Leung Pratew 123’ and its stress-resistant (SR) somaclonal mutant line. In response to osmotic-stress treatments, 117 proteins were differentially accumulated, with 62 and 49 of these proteins detected in the SS and SR rice lines, respectively. There were six proteins that accumulated in both lines. The proteins in the SS line were mainly related to metabolic processes, whereas the proteins identified in the SR line were primarily related to retrotransposons. These observations suggest that retrotransposons may influence the epigenetic regulation of gene expression in response to osmotic stress. To identify the biological processes associated with drought tolerance in rice, we conducted a co-expression network analysis of 55 proteins that were differentially accumulated in the SR line under osmotic-stress conditions. We identified a major hub gene; LOC_Os04g38600 (encoding a glyceraldehyde-3-phosphate dehydrogenase), suggesting that photosynthetic adaptation via NADP(H) homeostasis contributes to drought tolerance in rice.     

Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating

Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction.     

Proteomics to identify pathogenesis-related proteins in rice roots under water deficit

Upland and lowland rice (Oryza sativa L.) showed different mechanisms of water stress resistance. Hydroponically grown 3-week-old seedlings of a lowland variety IR64 and an upland variety were exposed to 15% polyethylene glycol (PEG-6000). After 7 d of treatment, IR64 maintained high relative water content and developed a well-branched root. Therefore, IR64 had better water-deficit tolerance than Azucena under PEG treatment. To identify water-deficit-responsive proteins associated with the tolerance differences between two ecotypes, a comparative proteomic analysis of roots was conducted. Out of 700 proteins reproducibly detected on two-dimensional electrophoresis gels, 65 proteins exhibited significant changes in at least one ecotype at 48 h of water deficit. Only 15 proteins showed different responses to water deficit between the two ecotypes. Twelve proteins were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry, which involved in energy and metabolism, protein processing and degradation, detoxification and pathogenrelated (PR) proteins, i.e. PR-1a, RSOsPR10 and JIOsPR10. All three PR proteins were induced more strongly in IR64 than in Azucena by water deficit at both protein and mRNA level. The results suggested that PR-1a, RSOsPR10 and JIOsPR10 may play important roles in protecting root cells against water deficit in rice.     

Comparative analysis of JmjC domain-containing proteins reveals the potential histone demethylases in Arabidopsis and rice

Histone methylation homeostasis is achieved by controlling the balance between methylation and demethylation to maintain chromatin function and developmental regulation. In animals, a conserved Jumonji C (JmjC) domain was found In a large group of histone demethylases. However, it is still unclear whether plants also contain the JmjC domaincontaining active histone demethylases. Here we performed genome-wide screen and phylogenetic analysis of JmjC domaincontaining proteins in the dicot plant, Arabidopsis, and monocot plant rice, and found 21 and 20 JmjC domain-containing, respectively. We also examined the expression of JmjC domain-containing proteins and compared them to human JmjC counterparts for potential enzymatic activity. The spatial expression patterns of the Arabidopsis JmjC domaincontaining genes revealed that they are all actively transcribed genes. These active plant JmjC domain-containing genes could possibly function in epigenetic regulation to antagonize the activity of the large number of putative SET domaincontaining histone methyltransferase activity to dynamically regulate histone methylation homeostasis.     

应激心肌损伤的蛋白质组学研究

目的:寻找应激心肌损伤相关蛋白.方法:建立束缚应激心肌损伤模型,制备心室肌2DE蛋白样品和心肌2DE图谱,图像分析软件分析应激后蛋白表达差异点,MALDI-TOF-MS-数据库搜索鉴定蛋白质.结果:应激前后10个蛋白表达量发生改变,其中8个应激后表达显著升高,经质谱鉴定为心肌肌球蛋白、白蛋白、脂蛋白A-I前体等;2个显著降低,经质谱鉴定为线粒体能量代谢酶类和UCP3.结论:这些差异蛋白可能参与应激机体心肌损伤的发生.     

Proteomics analysis of human serum of patients with non-small-cell lung cancer reveals proteins as diagnostic biomarker candidates

Non-small-cell lung carcinomas (NSCLC) is the most common type of lung cancer and it has a poor prognosis, because overall survival after 5 years is 20–25% for all stages. Thus, it is extremely important to increase the survival rate in the early stages NSCLC by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging and patient prognosis. In this study, we focused our attention on quantitative proteomics of three heavily glycosylated serum proteins: AMBP, α2 macroglobulin, and SERPINA1. In particular, we analyzed serum samples from 20 NSCLC lung adenocarcinoma cancer patients in early and advanced stages, and 10 healthy donors to obtain a relative quantification through the MRM analysis of these proteins that have shown to be markers of cancer development and progression. AMBP, α2 macroglobulin, and SERPINA1 were chosen because all of them possess endopeptidase inhibitor activity and play key roles in cancer. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like α2 macroglobulin, αmicroglobulin/bikunin, and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.     

Proteomics analysis of the peritoneal dialysate effluent reveals the presence of calcium-regulation proteins and acute inflammatory response

Background

Peritoneal dialysis (PD) is a form of renal replacement used for advanced chronic kidney disease. PD effluent holds a great potential for biomarker discovery for diagnosis and prognosis. In this study a novel approach to unravelling the proteome of PD effluent based-on dithiothreitol depletion followed by 2D-SDS-PAGE and protein identification using tandem mass spectrometry is proposed.

Results

A total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.

Conclusions

Remarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury. Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

An approach to identify cold-induced low-abundant proteins in rice leaf

A proteomic approach has been adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposed to different temperatures, such as 5 or 10 degrees C, and samples were collected after different time course. To eliminate the high-abundant proteins in leaf tissues such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), proteins were fractionated by polyethylene glycol (PEG). The elimination of Rubisco from the protein samples was confirmed by Western blot analysis. The PEG fractionated protein samples were separated by 2-DE and visualized by silver or CBB staining. A total 12 up-regulated protein spots were identified using the analysis of MALDI-TOF mass spectrometry or ESI MS/MS. We identified some novel proteins such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like, drought-inducible late embryogenesis abundant, and a fibrillin-like protein that had not yet been reported in the earlier reports on cold proteomic analysis. The identification of some novel low-abundant proteins in response to cold stress may provide a new homeostasis to develop enhanced cold tolerance transgenic plants. Thus, we propose that a PEG fractionation system can be used as an influential protein extraction method from the leaf samples, which can lead to knowledge of the expression pattern of low-abundant proteins in response to various biotic or abiotic stresses.     

Proteomics analysis of two heat shock proteins in insects

Heat shock proteins (HSPs) are found in all living organisms, from bacteria to humans, are expressed under stress. In this study, characterization of two families of HSP including HSP60 and HSP70 protein was compared in different insect species from different orders. According to the conserved motifs analysis, none of the motifs were shared by all insects of two protein families but each family had their own common motifs. Functional and structural analyses were carried out on seven different insect species from each protein family as the representative samples. These analyses were performed via ExPASy database tools. The tertiary structure of Drosophila melanogater as the sample of each protein family were predicted by the Phyre2 and TM-score servers then their qualities were verified by SuperPose and PROCHECK. The tertiary structures were predicted through the “c4pj1E” model (PDB Accession Code: 4pj1) in HSP60 family and “c3d2fC” model (PDB Accession Code: 3d2f) in HSP70 family. The protein phylogenetic tree was constructed using the Neighbor-joining (NJ) method by Molecular Evolutionary Genetic Analysis (MEGA) 6.06. According to the results, there was a high identity of HSP60 and HSP70 families so that they should be derived from a common ancestor however they belonged to separate groups. In protein–protein interaction analysis by STRING 10.0, 10 common enriched pathways of biological process, molecular function and Kyoto Encyclopedia of Genes and Genomes (KEGG) were identified in D. melanogaster in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of insects and other organisms.

Communicated by Ramaswamy H. Sarma     

Indel marker analysis of putative stress-related genes reveals genetic diversity and differentiation of rice landraces in peninsular Thailand

Physiology and Molecular Biology of Plants - Genetic assessment of rice landraces is important for germplasm evaluation and genetic resource utilization. Rice landraces in peninsular Thailand have...