Katsumi Sakata

Proteomic study on the effects of silver nanoparticles on soybean under flooding stress.

UNLABELLED Flooding negatively affects the soybean growth; however, silver nanoparticles (AgNPs) enhanced the growth under stress. To study the effects of AgNPs on soybean under flooding, a gel-free proteomic technique was used. The morphological analysis of early-stage soybean exposed to flooding with AgNPs of various sizes and concentrations revealed enhanced seedling growth by treatment with 15n m AgNPs at 2 ppm. Differentially changed 107 root proteins were predominantly associated with stress, signaling, and cell metabolism. Hierarchical clustering divided these proteins into 3 clusters. Based on cluster analysis, the abundances of glyoxalase II 3 and fermentation related proteins were time-dependently increased under flooding stress, but decreased in response to AgNPs. Six enzymes involved in metabolic pathways were analyzed at the transcriptional level. The alcohol dehydrogenase 1 and pyruvate decarboxylase 2 genes were up-regulated under flooding stress while down-regulated in response to AgNPs. Moreover, comparatively low transcript level of glyoxalase II 3 under AgNPs treatment implies that less cytotoxic by-products of glycolysis are produced in AgNPs exposed soybeans as compared to flooded soybean. These results suggest that the AgNPs treated soybeans might have experienced less oxygen-deprivation stress, which might be the key factor for better growth performance of AgNPs treated soybeans under flooding stress. BIOLOGICAL SIGNIFICANCE This study highlighted the effect of silver nanoparticles (AgNPs) on the soybean under flooding stress. Silver nanoparticles (2 ppm AgNPs, 15 nm in size) treatment facilitate the soybean under flooding stress enhancing seedling growth. A time-course comparative gel-free proteomic study was performed to analyze the changes inproteome profiles in response to AgNPs treatment under flooding. The 107 differentially changed root proteins were predominantly associated with stress, signaling, cell metabolism. The abundances of the glyoxalase II 3 and fermentation related proteins were significantly increased on exposure to flooding; however, decreased by AgNPs treatment. Comparatively low transcript level of glyoxalase II 3 under AgNPs treatment implies that less cytotoxic by-products of glycolysis are produced in AgNPs exposed soybeans as compared to flooded soybean. Moreover, the observed up-regulation of the alcohol dehydrogenase 1 and pyruvate decarboxylase 2 genes under flooding stress condition and its down-regulation in response to AgNPs treatment might be related to a metabolic shift towards normal cellular processes.

Soybean Proteome Database 2012: update on the comprehensive data repository for soybean proteomics

The Soybean Proteome Database (SPD) was created to provide a data repository for functional analyses of soybean responses to flooding stress, thought to be a major constraint for establishment and production of this plant. Since the last publication of the SPD, we thoroughly enhanced the contents of database, particularly protein samples and their annotations from several organelles. The current release contains 23 reference maps of soybean (Glycine max cv. Enrei) proteins collected from several organs, tissues, and organelles including the maps for plasma membrane, cell wall, chloroplast, and mitochondrion, which were analyzed by two-dimensional polyacrylamide gels. Furthermore, the proteins analyzed with gel-free proteomics technique have been added and are available online. In addition to protein fluctuations under flooding, those of salt and drought stress have been included in the current release. A case analysis employing a portion of those newly released data was conducted, and the results will be shown. An ‘omics table has also been provided to reveal relationships among mRNAs, proteins, and metabolites with a unified temporal-profile tag in order to facilitate retrieval of the data based on the temporal profiles. An intuitive user interface based on dynamic HTML enables users to browse the network as well as the profiles of the multiple “omes” in an integrated fashion. The SPD is available at: http://proteome.dc.affrc.go.jp/Soybean/

Insights into the proteomic response of soybean towards Al₂O₃, ZnO, and Ag nanoparticles stress.

Understanding the complex mechanisms involved in plant response to nanoparticles is indispensable in assessing the impact of nano-pollutants on environment. The present study compares the phytotoxicity of three different metal-based nanoparticles (Al2O3, ZnO, and Ag) in soybean seedling at proteome level. Plant growth, rigidity of roots, and root cell viability were markedly affected by ZnO- and Ag-NPs stress; while, Al2O3-NPs challenged soybean maintained normal seedling growth like control. Moreover, severe oxidative burst was evident in ZnO-NPs and Ag-NPs treatments. Gel-free proteomic analysis of NPs stressed soybean roots revealed 104 commonly changed proteins primarily associated with secondary metabolism, cell organization, and hormone metabolism. Oxidation-reduction cascade related genes, such as GDSL motif lipase 5, SKU5 similar 4, galactose oxidase, and quinone reductase were up-regulated in Al2O3-NPs challenged roots and down-regulated in ZnO- and Ag-NPs treatments. In comparison to root, 16 common proteins were found to be significantly changed in leaves of NPs exposed soybean that were predominantly associated to photosystem and protein degradation. The proteomic findings suggest that high abundance of proteins involved in oxidation-reduction, stress signaling, hormonal pathways related to growth and development might be the principal key for optimum growth of soybean under Al2O3-NPs stress.

Gel-free quantitative proteomic approach to identify cotyledon proteins in soybean under flooding stress

UNLABELLED Flooding stress causes growth inhibition and ultimately death in most crop species by limiting of energy production. To better understand plant responses to flooding stress, here, flooding-responsive proteins in the cotyledons of soybean were identified using a gel-free quantitative proteomic approach. One hundred forty six proteins were commonly observed in both control and flooding-stressed plants, and 19 were identified under only flooding stress conditions. The main functional categories were protein and development-related proteins. Protein-protein interaction analysis revealed that zincin-like metalloprotease and cupin family proteins were found to highly interact with other proteins under flooding stress. Plant stearoyl acyl-carrier protein, ascorbate peroxidase 1, and secretion-associated RAS superfamily 2 were down-regulated, whereas ferretin 1 was up-regulated at the transcription level. Notably, the levels of all corresponding proteins were decreased, indicating that mRNA translation to proteins is impaired under flooding conditions. Decreased levels of ferritin may lead to a strong deregulation of the expression of several metal transporter genes and over-accumulation of iron, which led to increased levels of reactive oxygen species, resulting to detoxification of these reactive species. Taken together, these results suggest that ferritin might have an essential role in protecting plant cells against oxidative damage under flooding conditions. BIOLOGICAL SIGNIFICANCE This study reported the comparative proteomic analysis of cotyledon of soybean plants between non-flooding and flooding conditions using the gel-free quantitative techniques. Mass spectrometry analysis of the proteins from cotyledon resulted in the identification of a total of 165 proteins under flooding stress. These proteins were assigned to different functional categories, such as protein, development, stress, redox, and glycolysis. Therefore, this study provides not only the comparative proteomic analysis but also the molecular mechanism underlying the flooding responsive protein functions in the cotyledon.

Proteomic analysis of flooded soybean root exposed to aluminum oxide nanoparticles.

Aluminum oxide (Al2O3) nanoparticles are used in agricultural products and cause various adverse growth effects on different plant species. To study the effects of Al2O3 nanoparticles on soybean under flooding stress, a gel-free proteomic technique was used. Morphological analysis revealed that treatment with 50 ppm Al2O3 nanoparticles under flooding stress enhanced soybean growth compared to ZnO and Ag nanoparticles. A total of 172 common proteins that significantly changed in abundance among control, flooding-stressed, and flooding-stressed soybean treated with Al2O3 nanoparticles were mainly related to energy metabolism. Under Al2O3 nanoparticles the energy metabolism was decreased compared to flooding stress. Hierarchical clustering divided identified proteins into four clusters, with proteins related to glycolysis exhibiting the greatest changes in abundance. Al2O3 nanoparticle-responsive proteins were predominantly related to protein synthesis/degradation, glycolysis, and lipid metabolism. mRNA expression analysis of Al2O3 nanoparticle-responsive proteins that displayed a 5-fold change in abundance revealed that NmrA-like negative transcriptional regulator was up-regulated, and flavodoxin-like quinone reductase was down-regulated. Moreover, cell death in root including hypocotyl was less evident in flooding-stressed with Al2O3 nanoparticles compared to flooding-treated soybean. These results suggest that Al2O3 nanoparticles might promote the growth of soybean under flooding stress by regulating energy metabolism and cell death.

Analysis of initial changes in the proteins of soybean root tip under flooding stress using gel-free and gel-based proteomic techniques.

UNLABELLED Flooding has a severe negative effect on soybean cultivation in the early stages of growth. To obtain a better understanding of the response mechanisms of soybean to flooding stress, initial changes in root tip proteins under flooding were analyzed using two proteomic techniques. Two-day-old soybeans were treated with flooding for 3, 6, 12, and 24h. The weight of soybeans increased during the first 3h of flooding, but root elongation was not observed. Using gel-based and gel-free proteomic techniques, 115 proteins were identified in root tips, of which 9 proteins were commonly detected by both methods. The 71 proteins identified by the gel-free proteomics were analyzed by a hierarchical clustering method based on induction levels during the flooding, and the proteins were divided into 5 clusters. Additional interaction analysis of the proteins revealed that ten proteins belonging to cluster I formed the center of a protein interaction network. mRNA expression analysis of these ten proteins showed that citrate lyase and heat shock protein 70 were down-regulated, whereas calreticulin was up-regulated in initial phase of flooding. These results suggest that flooding stress to soybean induces calcium-related signal transduction, which might play important roles in the early responses to flooding. BIOLOGICAL SIGNIFICANCE Flooding has a severe negative effect on soybean cultivation, particularly in the early stages of growth. To better understand the response mechanisms of soybean to the early stages of flooding stress, two proteomic techniques were used. Two-day-old soybeans were treated without or with flooding for 3, 6, 12, and 24h. The fresh weight of soybeans increased during the first 3h of flooding stress, but the growth then slowed and no root elongation was observed. Using gel-based and gel-free proteomic techniques, 115 proteins were identified in root tips, of which 9 proteins were commonly detected by both methods. The 71 proteins identified by the gel-free proteomics were analyzed by a hierarchical clustering method based on induction levels during the flooding stress, and 5 protein clusters were recognized. Protein interaction analysis revealed that ten proteins belonging to cluster I formed the center of a protein interaction network. mRNA expression analysis of these ten proteins showed that citrate lyase and heat shock protein 70 were down-regulated in response to flooding stress, whereas calreticulin was up-regulated. These results suggest that flooding stress to soybean induces calcium-related signal transduction, which might play important roles in the early responses to flooding.

Gel-free/label-free proteomic analysis of root tip of soybean over time under flooding and drought stresses.

Growth in the early stage of soybean is markedly inhibited under flooding and drought stresses. To explore the responsive mechanisms of soybean, temporal protein profiles of root tip under flooding and drought stresses were analyzed using gel-free/label-free proteomic technique. Root tip was analyzed because it was the most sensitive organ against flooding, and it was beneficial to root penetration under drought. UDP glucose: glycoprotein glucosyltransferase was decreased and increased in soybean root under flooding and drought, respectively. Temporal protein profiles indicated that fermentation and protein synthesis/degradation were essential in root tip under flooding and drought, respectively. In silico protein-protein interaction analysis revealed that the inductive and suppressive interactions between S-adenosylmethionine synthetase family protein and B-S glucosidase 44 under flooding and drought, respectively, which are related to carbohydrate metabolism. Furthermore, biotin/lipoyl attachment domain containing protein and Class II aminoacyl tRNA/biotin synthetases superfamily protein were repressed in the root tip during time-course stresses. These results suggest that biotin and biotinylation might be involved in energy management to cope with flooding and drought in early stage of soybean-root tip.

Proteomic analysis of canola root inoculated with bacteria under salt stress.

UNLABELLED Plant-growth promoting bacteria can ameliorate the negative effects of salt stress on canola. To better understand the role of bacteria in canola under salt stress, salt-sensitive (Sarigol) and salt-tolerant (Hyola308) cultivars were inoculated with Pseudomonas fluorescens and protein profiles of roots were compared. Bacterial inoculation increased the dry weight and length of canola roots under salt stress. Using a gel-free proteomic technique, 55 commonly changed proteins were identified in Sarigol and Hyola308 roots inoculated with bacteria under salt stress. In both canola cultivars, proteins related to amino acid metabolism and tricarboxylic acid cycle were affected. Hierarchical cluster analysis divided the identified proteins into three clusters. Proteins related to Clusters II and III, which were secretion-associated RAS super family 1, dynamin-like protein, and histone, were increased in roots of both Sarigol and Hyola308 inoculated with bacteria under salt stress. Based on pathway mapping, proteins related to amino acid metabolism and the tricarboxylic acid cycle significantly changed in canola cultivars inoculated with or without bacteria under salt stress. These results suggest that bacterial inoculation of canola roots increases tolerance to salt stress by proteins related to energy metabolism and cell division. BIOLOGICAL SIGNIFICANCE Plant-growth promoting bacteria as an emerging aid can ameliorate the negative effect of salt stress on canola. To understand the role of bacteria in canola under salt stress, salt sensitive Sarigol and tolerant Hyola308 cultivars were used. Dry weight and length of canola root were improved by inoculation of bacteria under salt stress. Using gel-free proteomic technique, 55 commonly changed proteins identified in Sarigol and Hyola308 inoculated with bacteria under salt stress. In both canola cultivars, the number of proteins related to amino acid metabolism and tricarboxylic acid cycle was more than other categories with higher change in protein abundance. Hierarchical cluster analysis divided into 3 clusters. Cluster II including secretion-associated RAS super family 1 and dynamin-like protein and Cluster III including histones H2A were increased by bacterial inoculation in both cultivars. Furthermore, pathway mapping highlighted the importance of S-denosylmethionine synthetase and malate dehydrogenase that decreased in canola inoculated with bacteria under salt stress. These results suggest that bacterial inoculation helps the canola to endure salt stress by modulating the proteins related to energy metabolism and cell division.

‘Omics’ techniques and their use to identify how soybean responds to flooding

Various environmental stresses reduce crop growth and productivity. Plants respond to stresses through changes in gene expression, protein abundance, and metabolite accumulation. Among functional genomics tools, ‘omics’ techniques such as transcriptomics, proteomics, and metabolomics are extensively used to reveal these responses. Altering plant phenotype by changing transcripts, proteins, and metabolites is one of the main challenges for improving crop production. The application of ‘omics’ techniques may facilitate the development of stress-tolerant crops. This review introduces ‘omics’ techniques and their use to identify how soybean responds to flooding.

Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress

UNLABELLED Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse drought tolerance characteristics were subjected to WS, and their leaf proteome was compared using two-dimensional electrophoresis complemented with MALDI-TOF/TOF mass spectrometry. Ninety-six protein spots were differentially abundant to water stress in both cultivars that corresponded to 60 non-redundant proteins. Protein interaction prediction analysis suggests that 42 unique proteins showed interactions in tolerant cultivar while 20 showed interactions in the susceptible cultivar, activating other proteins in directed system response networks. Four proteins: glutamine ammonia ligase, chitin class II, actin isoform B, and beta tubulin, involved in metabolism, defense and cellular biogenesis, are unique in tolerant cultivar and showed positive interactions with other proteins. In addition, four proteins: serine/threonine protein phosphate PP1, choline monooxygenase, peroxidase 43, and SNF1-related protein kinase regulatory subunit beta-2, that play a role as cryoprotectants through signal transduction, were induced in drought tolerant cultivar following WS. Eleven interologs of these proteins were found in Arabidopsis interacting with several proteins and it is believed that similar mechanisms/pathways exist in peanut. SIGNIFICANCE Peanuts (Arachis hypogaea L.) are a major source of plant protein grown in subtropical and tropical regions of the world. Pre-harvest aflatoxin contamination is a major problem that affects peanut crop yield and food safety. Poor understanding of molecular and cellular mechanisms associated with aflatoxin resistance is largely responsible for the lack of progress in elucidating a process/methodology for reducing aflatoxin contamination in peanuts. Drought perturbs the invasion of the aflatoxin producing fungus and thus affects the quality and yield of peanut. Therefore, more studies involving the effects of drought stress to determine the molecular changes will enhance our understanding of the key metabolic pathways involved in the combined stresses. The changes associated with the biotic and abiotic interactions within the peanut will be used to determine the metabolic pathways involved in the stress tolerance. This research would be beneficial in identifying the tolerant molecular signatures and promoting food safety and consumer health through breeding superior quality peanut cultivars.