Search

Depletion of abundant proteins is one of the effective ways to improve detection and identification of low-abundance proteins. Our previous study showed that protamine sulfate precipitation (PSP) method can deplete abundant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from leaf proteins and is suitable for their in-depth proteome investigation. In this study, we provide evidence that the PSP method can also be effectively used for depletion of abundant seed-storage proteins (SSPs) from the total seed proteins of diverse legume plants including soybean, broad bean, pea, wild soybean, and peanut. The 0.05% protamine sulfate (PS) was sufficient to deplete major SSPs from all legumes tested except for peanut where 0.1% PS was required. SDS-PAGE, Western blotting and 2DE analyses of PS-treated soybean and peanut seed proteins showed enriched spots in PS-supernatant than total proteins. Coefficient of variation percentage (%CV) and principal component analysis of 2DE spots support the reproducibility, suitability, and efficacy of the PSP method for quantitative and comparative seed proteome analysis. MALDI-TOF-TOF successfully identified some protein spots from soybean and peanut. Hence, this simple, reproducible, economical PSP method has a broader application in depleting plant abundant proteins including SSPs in addition to RuBisCO, allowing discussion for comprehensive proteome establishment and parallel comparative studies in plants., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Secreted proteins were investigated in rice suspension-cultured cells treated with rice blast fungus Magnaporthe grisea and its elicitor using biochemical and 2-DE coupled with MS analyses followed by their in planta mRNA expression analysis. M. grisea and elicitor successfully interacted with suspension-cultured cells and prepared secreted proteins from these cultures were essentially intracellular proteins free. Comparative 2-D gel analyses identified 21 differential protein spots due to M. grisea and/or elicitor over control. MALDI-TOF-MS and microLC-ESI-MS/MS analyses of these protein spots revealed that most of assigned proteins were involved in defense such as nine chitinases, two germin A/oxalate oxidases, five domain unknown function 26 (DUF 26) secretory proteins, and beta-expansin. One chitin binding chitinase protein was isolated using chitin binding beads and strong enzymatic activity was identified in an in-gel assay. Interestingly, their protein abundance correlated well at transcript levels in elicitor-treated cultures as judged by semi-quantitative RT-PCR. Each identified differentially expressed protein group was compared at transcript levels in rice leaves inoculated with incompatible (KJ401) and compatible (KJ301) races of M. grisea. Time-course profiling revealed their inductions were stronger and earlier in incompatible than compatible interactions. Identified secreted proteins and their expression correlation at transcript level in suspension-cultured cells and also in planta suggest that suspension-cultured cells can be useful to investigate the secretome of rice blast-pathogen interactions.

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) play essential and often antagonistic roles in regulating plant growth, development, and stress responses. Using a proteomics-based approach, we examined the role of GA and ABA in the modulation of protein expression levels during seed germination. Rice seeds were treated with GA (200 microM), ABA (10 microM), ABA followed by GA, GA followed by ABA, and water as a control and then incubated for 3 days. The embryo was dissected from germinated seeds, and proteins were subjected to 2-DE. Approximately, 665 total protein spots were resolved in the 2-D gels. Among them, 16 proteins notably modulated by either GA or ABA were identified by MALDI-TOF MS. Northern analyses demonstrated that expression patterns of 13 of these 16 genes were consistent with those of the proteome analysis. Further examination of two proteins, rice isoflavone resuctase (OsIFR) and rice PR10 (OsPR10), using Western blot and immunolocalization, revealed that both are specifically expressed in the embryo but not in the endosperm and are dramatically downregulated by ABA.

The present study investigated rice leaf proteome in response to heat stress. Rice seedlings were subjected to a temperature of 42 degrees C and samples were collected 12 and 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress frequently was generated in rice leaves exposed to high temperature. 2-DE, coupled with MS, was used to investigate and identify heat-responsive proteins in rice leaves. In order to identify the low-abundant proteins in leaves, samples were prefractionated by 15% PEG. The PEG supernatant and the pellet fraction samples were separated by 2-DE, and visualized by silver or CBB staining. Approximately 1000 protein spots were reproducibly detected on each gel, wherein 73 protein spots were differentially expressed at least at one time point. Of these differentially expressed proteins, a total of 34 and 39 protein spots were found in the PEG supernatant and pellet fractions, respectively. Using MALDI-TOF MS, a total of 48 proteins were identified. These proteins were categorized into classes related to heat shock proteins, energy and metabolism, redox homeostasis, and regulatory proteins. The results of the present study show that a group of low molecular small heat shock proteins (sHSPs) were newly induced by heat stress. Among these sHSPs, a low molecular weight mitochondrial (Mt) sHSP was validated further by Western blot analysis. Furthermore, four differentially accumulated proteins that correspond to antioxidant enzymes were analyzed at the mRNA level, which confirmed the differential gene expression levels, and revealed that transcription levels were not completely concomitant with translation. The identification of some novel proteins in the heat stress response provides new insights that can lead to a better understanding of the molecular basis of heat-sensitivity in plants.

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.