Proteomics of Physcomitrella patens protonemata subjected to treatment with 12-oxo-phytodienoic acid.

12-Oxo-phytodienoic acid (OPDA) is induced by mechanical wounding and suppresses the growth of Physcomitrella patens; OPDA is considered as a signal compound in this moss species. In this study, a proteomic analysis of P. patens protonemata treated with OPDA was performed. The abundance levels of 41 proteins were significantly altered by OPDA, with decreased levels for 40 proteins. The proteins for which abundance decreased in response to OPDA at the protonema developmental stage were mainly involved in the metabolism of proteins and carbohydrates. The effects of inhibition on protein abundance are likely a major physiological function of OPDA in P. patens. OPDA also suppressed the expression of histones at the protein level and gene transcription level. Suppression of histone expression might be an OPDA-specific function in P. patens protonemata. In P. patens, a subset of the physiological responses caused by OPDA is shown to differ between protonema and gametophore developmental stages.

Proteomic Techniques and Management of Flooding Tolerance in Soybean.

Climate change is considered a major threat to world agriculture and food security. To improve the agricultural productivity and sustainability, the development of high-yielding stress-tolerant, and climate-resilient crops is essential. Of the abiotic stresses, flooding stress is a very serious hazard because it markedly reduces plant growth and grain yield. Proteomic analyses indicate that the effects of flooding stress are not limited to oxygen deprivation but include many other factors. Although many flooding response mechanisms have been reported, flooding tolerance mechanisms have not been fully clarified for soybean. There were limitations in soybean materials, such as mutants and varieties, while they were abundant in rice and Arabidopsis. In this review, plant proteomic technologies are introduced and flooding tolerance mechanisms of soybeans are summarized to assist in the improvement of flooding tolerance in soybeans. This work will expedite transgenic or marker-assisted genetic enhancement studies in crops for developing high-yielding stress-tolerant lines or varieties under abiotic stress.

Quantitative proteomic and transcriptomic analyses of molecular mechanisms associated with low silk production in silkworm Bombyx mori.

To investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain, we used both SDS-PAGE-based and gel-free-based proteomic techniques and transcriptomic sequencing technique. Combining the data from two different proteomic techniques was preferable in the characterization of the differences between the ZB silkworm strain and the original Lan10 silkworm strain. The correlation analysis showed that the individual protein and transcript were not corresponded well, however, the differentially changed proteins and transcripts showed similar regulated direction in function at the pathway level. In the ZB strain, numerous ribosomal proteins and transcripts were down-regulated, along with the transcripts of translational related elongation factors and genes of important components of fibroin. The proteasome pathway was significantly enhanced in the ZB strain, indicating that protein degradation began on the third day of fifth instar when fibroin would have been produced in the Lan10 strain normally and plentifully. From proteome and transcriptome levels of the ZB strain, the energy-metabolism-related pathways, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle were enhanced, suggesting that the energy metabolism was vigorous in the ZB strain, while the silk production was low. This may due to the inefficient energy employment in fibroin synthesis in the ZB strain. These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis, the degradation of proteins, and the inefficiency of the energy exploiting.

Proteomic analysis of Physcomitrella patens treated with 12-oxo-phytodienoic acid, an important oxylipin in plants.

12-Oxo-phytodienoic acid (OPDA) is biosynthesized in the octadecanoid pathway and is considered to be a signaling molecule in plants. In Physcomitrella patens, OPDA is induced by bacterial infection and mechanical stress and is known to suppress growth; however, the functional mechanism of OPDA signaling remains elusive. In this study, we performed a proteomic analysis of P. patens treated with OPDA and found that the expression of 82 proteins was significantly altered, with approximately 80% of these proteins being downregulated by OPDA. The identified proteins were mainly categorized as being involved in photosynthesis, metabolism, and protein synthesis, and most of the proteins that were upregulated by OPDA are involved in light-dependent reactions, suggesting that OPDA regulates a function in chloroplasts. Additionally, OPDA induced the expression of an allene oxide cyclase (PpAOC1) in the octadecanoid pathway, demonstrating positive feedback regulation by OPDA in P. patens.

Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean.

Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere.

Label-free quantitative proteomic analysis of abscisic acid effect in early-stage soybean under flooding.

Flooding is a serious problem for soybean cultivation because it markedly reduces growth. To investigate the role of phytohormones in soybean under flooding stress, gel-free proteomic technique was used. When 2-day-old soybeans were flooded, the content of abscisic acid (ABA) did not decrease in the root, though its content decreased in untreated plant. When ABA was added during flooding treatment, survival ratio was improved compared with that of soybeans flooded without ABA. When 2-day-old soybeans were flooded with ABA, the abundance of proteins related to cell organization, vesicle transport and glycolysis decreased compared with those in root of soybeans flooded without ABA. Furthermore, the nuclear proteins were analyzed to identify the transcriptional regulation. The abundance of 34 nuclear proteins such as histone deacetylase and U2 small nuclear ribonucleoprotein increased by ABA supplementation under flooding; however, 35 nuclear proteins such as importin alpha, chromatin remodeling factor, zinc finger protein, transducin, and cell division 5 protein decreased. Of them, the mRNA expression levels of cell division cycle 5 protein, C2H2 zinc finger protein SERRATE, CCCH type zinc finger family protein, and transducin were significantly down-regulated under the ABA treatment. These results suggest that ABA might be involved in the enhancement of flooding tolerance of soybean through the control of energy conservation via glycolytic system and the regulation on zinc finger proteins, cell division cycle 5 protein and transducin.

The synthesis of cytosolic ascorbate peroxidases in germinating seeds and seedlings of soybean and their behavior under flooding stress.

Cytosolic ascorbate peroxidases (cAPXs) of soybean have been found by proteome analysis to be downregulated in submerged seedlings. To elucidate the physiological meaning of this downregulation, soybean cAPXs were characterized in this study. Vigorous synthesis was detected in germinating seeds and seedlings. Expression of the corresponding genes was detected clearly in tissues that actively underwent cell division. The gene expression was suppressed by flooding stress, but not by salinity, cold or drought stress. The expression recovered 1 d after release from flooding stress, accompanied by growth resurgence.

Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings.

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.

The rice alpha-amylase glycoprotein is targeted from the Golgi apparatus through the secretory pathway to the plastids.

The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner.

Comprehensive analysis of mitochondria in roots and hypocotyls of soybean under flooding stress using proteomics and metabolomics techniques.

Flooding is a serious problem for soybeans because it reduces growth and grain yield. Proteomic and metabolomic techniques were used to examine whether mitochondrial function is altered in soybeans by flooding stress. Mitochondrial fractions were purified from the roots and hypocotyls of 4-day-old soybean seedlings that had been flooded for 2 days. Mitochondrial matrix and membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis, respectively. Differentially expressed proteins and metabolites were identified using mass spectrometry. Proteins and metabolites related to the tricarboxylic acid cycle and γ-amino butyrate shunt were up-regulated by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were down-regulated. The amounts of NADH and NAD were increased; however, ATP was significantly decreased by flooding stress. These results suggest that flooding directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle.

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature.

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 degrees C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 degrees C to 38 degrees C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.

Transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings.

To understand the transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings, genome-wide changes in gene expression were analyzed using a soybean microarray chip containing 42,034 60-mer oligonucleotide probes. More than 6,000 of flooding-responsive genes in the roots including hypocotyl of soybean seedlings were identified. The transcriptional analysis showed that genes related to photosynthesis, glycolysis, Ser-Gly-Cys group amino acid synthesis, regulation of transcription, ubiquitin-mediated protein degradation and cell death were significantly up-regulated by flooding. Meanwhile, genes related to cell wall synthesis, secondary metabolism, metabolite transport, cell organization, chromatin structure synthesis, and degradation of aspartate family amino acid were significantly down-regulated. Comparison of the responses with other plants showed that genes encoding pyrophosphate dependent phosphofructokinase were down-regulated in flooded soybean seedlings, however, those in tolerant plants were up-regulated. Additionally, genes related to RNA processing and initiation of protein synthesis were not up-regulated in soybean, however, those in tolerant plants were up-regulated. Furthermore, we found that flooding-specific up-regulation of genes encoding small proteins which might have roles in acclimation to flooding. These results suggest that functional disorder of acclimative responses to flooding through transcriptional and post-transcriptional regulations is involved in occurring flooding injury to soybean seedlings.

Ozone stress-induced proteomic changes in leaf total soluble and chloroplast proteins of soybean reveal that carbon allocation is involved in adaptation in the early developmental stage.

Considerable soybean yield losses caused by ozone (O3) stress have been demonstrated by large-scale meta-analyses of free-gas concentration enrichment systems. In this study, comparative proteomic approach was employed to explore the differential changes of proteins in O3 target structures such as leaf and chloroplasts of soybean seedlings. Acute O3 exposure (120 parts-per-billion) for 3 days did not cause any visible symptoms in developing leaves. However, higher amounts of ROS and lipid peroxidation indicated that severe oxidative burst occurred. Immunoblot analysis of O3-induced known proteins revealed that proteins were modulated before symptoms became visible. Proteomic analysis identified a total of 20 and 32 differentially expressed proteins from O3-treated leaf and chloroplast, respectively. Proteins associated with photosynthesis, including photosystem I/II and carbon assimilation decreased following exposure to O3. In contrast, proteins involved in antioxidant defense and carbon metabolism increased. The activity of enzymes involved in carbohydrate metabolism increased following exposure to O3, which is consistent with the decrease in starch and increase in sucrose concentrations. Taken together, these results suggest that carbon allocation is tightly programmed, and starch degradation probably feeds the tricarboxylic acid cycle while the photosynthesis pathway is severely affected during O3 stress.

Comparative proteomic analysis of early-stage soybean seedlings responses to flooding by using gel and gel-free techniques.

Gel-based and gel-free proteomics techniques were used to investigate early responses to flooding stress in the roots and hypocotyls of soybean seedlings. Proteins from 2-day-old soybean seedlings flooded for 12 h were extracted and analyzed. Two mass-spectroscopy-based proteomics analyses, two-dimensional fluorescence difference gel electrophoresis, and nanoliquid chromatography identified 32 from 17 spots and 81 proteins, respectively, as responsive to flooding stress. On the basis of the number and function of proteins identified, glycolysis and fermentation enzymes and inducers of heat shock proteins were key elements in the early responses to flooding stress. Analysis of enzyme activities and carbohydrate contents in flooded seedlings showed that glucose degradation and sucrose accumulation accelerated during flooding due to activation of glycolysis and down-regulation of sucrose degrading enzymes. Additionally, the methylglyoxal pathway, which is detoxification system linked to glycolysis, was up-regulated. Furthermore, two-dimensional polyacrylamide gel electrophoresis-based phosphoproteomics analysis showed that proteins involved in protein folding and synthesis were dephosphorylated under flooding conditions. These results suggest that translational and post-translational control during flooding possibly induces an imbalance in the expression of proteins involved in several metabolic pathways including carbohydrate metabolism that might cause flooding injury of soybean seedlings.

Proteome analysis of soybean leaves, hypocotyls and roots under salt stress.

BACKGROUND: Salinity is one of the most widespread agricultural problems in arid and semi-arid regions that makes fields unproductive, and soil salinization is a serious problem in the entire world. To determine the effects of salt stress on soybean seedlings, a proteomic technique was used. RESULTS: Soybean plants were exposed to 0, 20, 40, or 80 mM NaCl for one week. The effect of treatment at 20 mM NaCl on plant growth was not severe, at 80 mM NaCl was lethal, and at 40 mM NaCl was significant but not lethal. Based on these results, proteins were extracted from the leaves, hypocotyls and roots of soybean treated with 40 mM NaCl. Nineteen, 22 and 14 proteins out of 340, 330 and 235 proteins in the leaves, hypocotyls and roots, respectively, were up- and down-regulated by NaCl treatment. In leaves, hypocotyls and roots, metabolism related proteins were mainly down-regulated with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was down-regulated in the leaf/hypocotyls, and fructokinase 2 was down-regulated in the hypocotyls/root with NaCl treatment. Stem 31 kDa glycoprotein precursor was up-regulated in all three organs with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was specifically down-regulated at the RNA and protein levels by salt stress. CONCLUSION: These results suggest that metabolism related proteins play a role in each organ in the adaptation to saline conditions.

Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress.

Flooding is a major problem for soybean crop as it reduces the growth and grain yield. To investigate the function of the soybean cell wall in the response to flooding stress, cell wall proteins were analyzed. Cell wall proteins from roots and hypocotyls of soybeans, which were germinated for 2 days and subjected to 2 days of flooding, were purified, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Sixteen out of 204 cell wall proteins showed responses to flooding stress. Of these, two lipoxygenases, four germin-like protein precursors, three stem 28/31 kDa glycoprotein precursors, and one superoxide dismutase [Cu-Zn] were downregulated. A copper amine oxidase was found to have shifted from the basic to acidic zone following flooding stress. Based on these results, it was confirmed by the lignin staining that the lignification was suppressed in the root of soybean under the flooding stress. These results suggest that the roots and hypocotyls of soybean caused the suppression of lignification through decrease of these proteins by downregulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.

Identification of flooding stress responsible cascades in root and hypocotyl of soybean using proteome analysis.

Flooding inducible proteins were analyzed using a proteomic technique to understand the mechanism of soybean response to immersion in water. Soybeans were germinated for 2 days, and then subjected to flooding for 2 days. Proteins were extracted from root and hypocotyl, separated by two-dimensional polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue, and analyzed by protein sequencing and mass spectrometry. Out of 803 proteins, 21 proteins were significantly up-regulated, and seven proteins were down-regulated by flooding stress. Of the total, 11 up-regulated proteins were classified as related to protein destination/storage and three proteins to energy, while four down-regulated proteins were related to protein destination/storage and three proteins to disease/defense. The expression of 22 proteins significantly changed within 1 day after flooding stress. The effects of flooding, nitrogen substitution without flooding, or flooding with aeration were analyzed for 1-4 days. The expression of alcohol dehydrogenase increased remarkably by nitrogen substitution compared to flooding. The expression of many proteins that changed due to flooding showed the same tendencies observed for nitrogen substitution; however, the expression of proteins classified into protein destination/storage did not.

A comprehensive analysis of the soybean genes and proteins expressed under flooding stress using transcriptome and proteome techniques.

The inducible genes and proteins were analyzed using transcriptome and proteome techniques to explore the mechanisms underlying soybean response to flooding stress. Soybean seedlings were germinated for 2 days and subjected to flooding for 12 h, and the total RNAs and proteins were extracted from the root and hypocotyl. High-coverage gene expression profiling analysis as transcriptome technique was performed. Ninety-seven out of the 29,388 peaks observed demonstrated a greater than 25-fold change following 12 h of flood-induced stress. Furthermore, 34 proteins out of 799 proteins were changed by 12 h stress. Genes associated with alcohol fermentation, ethylene biosynthesis, pathogen defense, and cell wall loosening were significantly up-regulated. Hemoglobin, acid phosphatase, and Kunitz trypsin protease inhibitor were altered at both transcriptional and translational levels. Reactive oxygen species scavengers and chaperons were changed only at the translational level. It is suggested that the early response of soybean under flooding might be important stress adaptation to ensure survival against not only hypoxia but also the direct damage of cell by water.

Analysis of plasma membrane proteome in soybean and application to flooding stress response.

The plasma membrane acts as the primary interface between the cellular cytoplasm and the extracellular environment. To investigate the function of the plasma membrane in response to flooding stress, plasma membrane was purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method. Purified plasma membrane proteins with 81% purity were analyzed using either two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry and protein sequencing (2-DE MS/sequencer)-based proteomics or nanoliquid chromatography followed by mass spectrometry (nanoLC-MS/MS)-based proteomics. The number of hydrophobic proteins identified by nanoLC-MS/MS-based proteomics was compared with those identified by 2-DE MS/sequencer-based proteomics. These techniques were applied to identify the proteins in soybean that are responsive to flooding stress. Results indicate insights of plasma membrane into the response of soybean to flooding stress: (i) the proteins located in the cell wall are up-regulated in plasma membrane; (ii) the proteins related to antioxidative system play a crucial role in protecting cells from oxidative damage; (iii) the heat shock cognate protein plays a role in protecting proteins from denaturation and degradation during flooding stress; and (iv) the signaling related proteins might regulate ion homeostasis.