Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. BIOLOGICAL SIGNIFICANCE: The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from genome sequences, though there are over lapped proteins. Based on the demonstrated application of data stored in the database for functional analyses, it is suggested that these data will be useful for analyses of biological mechanisms in soybean. Furthermore, coupled with recent advances in information and communication technology, the usefulness of this database would increase in the analyses of biological mechanisms.

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.

Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions. To better understand the flooding-responsive mechanisms of soybean, the effect of exogenous calcium on flooding-stressed soybeans was analyzed using proteomic technique. An increase in exogenous calcium levels enhanced soybean root elongation and suppressed the cell death of root tip under flooding stress. Proteins were extracted from the roots of 4-day-old soybean seedlings exposed to flooding stress without or with calcium for 2 days and analyzed using gel-free proteomic technique. Proteins involved in protein degradation/synthesis/posttranslational modification, hormone/cell wall metabolisms, and DNA synthesis were decreased by flooding stress; however, their reductions were recovered by calcium treatment. Development, lipid metabolism, and signaling-related proteins were increased in soybean roots when calcium was supplied under flooding stress. Fermentation and glycolysis-related proteins were increased in response to flooding; however, these proteins were not affected by calcium supplementation. Furthermore, urease and copper chaperone proteins exhibited similar profiles in 4-day-old untreated soybeans and 4-day-old soybeans exposed to flooding for 2 days in the presence of calcium. These results suggest that calcium might affect the cell wall/hormone metabolisms, protein degradation/synthesis, and DNA synthesis in soybean roots under flooding stress.

Analyses of flooding tolerance of soybean varieties at emergence and varietal differences in their proteomes.

Flooding of fields due to heavy and/or continuous rainfall influences soybean production. To identify soybean varieties with flooding tolerance at the seedling emergence stage, 128 soybean varieties were evaluated using a flooding tolerance index, which is based on plant survival rates, the lack of apparent damage and lateral root development, and post-flooding radicle elongation rate. The soybean varieties were ranked according to their flooding tolerance index, and it was found that the tolerance levels of soybean varieties exhibit a continuum of differences between varieties. Subsequently, tolerant, moderately tolerant and sensitive varieties were selected and subjected to comparative proteomic analysis to clarify the tolerance mechanism. Proteomic analysis of the radicles, combined with correlation analysis, showed that the ratios of RNA binding/processing related proteins and flooding stress indicator proteins were significantly correlated with flooding tolerance index. The RNA binding/processing related proteins were positively correlated in untreated soybeans, whereas flooding stress indicator proteins were negatively correlated in flooded soybeans. These results suggest that flooding tolerance is regulated by mechanisms through multiple factors and is associated with abundance levels of the identified proteins.

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.

Proteomic analyses of soybean root tips during germination.

Plant root systems form complex networks with the surrounding soil environment and are controlled by both internal and external factors. To better understand the function of root tips of soybean during germination, three proteomic techniques were used to analyze the protein profiles of root tip cells. Proteins were extracted from the root tips of 4-day-old soybean seedlings and analyzed using two-dimensional (2D) gel electrophoresis-based proteomics, SDS-gel based proteomics, and gel-free proteomics techniques. A total of 121, 862, and 341 proteins were identified in root tips using the 2D gel-based, SDS gel-based, and gel-free proteomic techniques, respectively. The proteins identified by 2D gel-based proteomic analysis were predominantly localized in the cytoplasm, whereas nuclear-localized proteins were most commonly identified by the SDS gel-based and gel-free proteomics techniques. Of the 862 proteins identified in the SDS gel-based proteomic analysis, 190 were protein synthesis-related proteins. Furthermore, 24 proteins identified using the 2D-gel based proteomic technique shifted between acidic and basic isoelectric points, and 2 proteins, heat shock protein 70.2 and AAA-type ATPase, displayed two different molecular weights at the same isoelectric point. Taken together, these results suggest that a number of proteins related to protein synthesis and modification are activated in the root tips of soybean seedlings during germination.

Analysis of soybean root proteins affected by gibberellic acid treatment under flooding stress.

Flooding is a serious abiotic stress for soybean because it restricts growth and reduces grain yields. To investigate the effect of gibberellic acid (GA) on soybean under flooding stress, root proteins were analyzed using a gel-free proteomic technique. Proteins were extracted from the roots of 4-days-old soybean seedlings exposed to flooding stress in the presence and absence of exogenous GA3 for 2 days. A total of 307, 324, and 250 proteins were identified from untreated, and flooding-treated soybean seedlings without or with GA3, respectively. Secondary metabolism- and cell-related proteins, and proteins involved in protein degradation/synthesis were decreased by flooding stress; however, the levels of these proteins were restored by GA3 supplementation under flooding. Fermentation- and cell wall-related proteins were not affected by GA3 supplementation. Furthermore, putative GA-responsive proteins, which were identified by the presence of a GA-responsive element in the promoter region, were less abundant by flooding stress; however, these proteins were more abundant by GA3 supplementation under flooding. Taken together, these results suggest that GA3 affects the abundance of proteins involved in secondary metabolism, cell cycle, and protein degradation/synthesis in soybeans under flooding stress.

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

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.

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.

Identification of nuclear proteins in soybean under flooding stress using proteomic technique.

Flooding stress restricts soybean growth, it results in decrease the production. In this report, to understand how nuclear proteins in soybean affected by flooding, abundance changes of those proteins was analyzed. Nuclear proteins were extracted from the root tips of soybean treated with or without flooding stress. The extracted proteins were analyzed using a label-free quantitative proteomic technique. Of a total of 94 nuclear proteins that were found to be responsive to flooding, the 19 and 75 proteins were increased and decreased, respectively. The identified flooding-responsive proteins were functionally classified, revealing that 8 increased proteins changed in protein synthesis, posttranslational modification, and protein degradation, while 34 decreased proteins were involved in transcription, RNA processing, DNA synthesis, and chromatin structure maintenance. Among these proteins, those whose levels changed more than 10 fold included two poly ADP-ribose polymerases and a novel G-domain-containing protein that might be involved in RNA binding. The mRNA expression levels of these three proteins indicated a similar tendency to their protein abundance changes. These results suggest that acceleration of protein poly-ADP-ribosylation and suppression of RNA metabolism may be involved in root tip of soybean under flooding stress.

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging.

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 (TaBWPR-1.2), TaBWPR-1.2#2 and TaBWPR-1.2#13. Using microprojectile bombardment, both cDNAs were introduced into "Bobwhite". Despite low transformation efficiency, four independent T2 homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi:TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.

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.

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.

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.

Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

Flooding stress of soybean is a serious problem because it reduces growth; however, flooding-tolerant cultivars have not been identified. To analyze the flooding tolerance mechanism of soybean, the flooding-tolerant mutant was isolated and analyzed using a proteomic technique. Flooding-tolerance tests were repeated five times using gamma-ray irradiated soybeans, whose root growth (M6 stage) was not suppressed even under flooding stress. Two-day-old wild-type and mutant plants were subjected to flooding stress for 2days, and proteins were identified using a gel-based proteomic technique. In wild-type under flooding stress, levels of proteins related to development, protein synthesis/degradation, secondary metabolism, and the cell wall changed; however, these proteins did not markedly differ in the mutant. In contrast, an increased number of fermentation-related proteins were identified in the mutant under flooding stress. The root tips of mutant plants were not affected by flooding stress, even though the wild-type plants had damaged root. Alcohol dehydrogenase activity in the mutant increased at an early stage of flooding stress compared with that of the wild-type. Taken together, these results suggest that activation of the fermentation system in the early stages of flooding may be an important factor for the acquisition of flooding tolerance in soybean.

Comprehensive analysis of endoplasmic reticulum-enriched fraction in root tips of soybean under flooding stress using proteomics techniques.

Flooding is a serious problem for soybean cultivation because it markedly reduces growth and grain yields. Here, 2 proteomics techniques were used to evaluate whether endoplasmic reticulum (ER)-enriched fraction is altered in soybean under flooding stress. Two-day-old soybeans were treated with flooding for 2 days, and rough ER-enriched fraction was then purified from root tips. Flooding-responsive protein of ER-enriched fraction was identified using gel-free and 1D-gel based proteomics techniques, and 117 proteins were increased and 212 proteins were decreased in soybean root tips in response to flooding stress. Among the identified proteins, 111 were functionally categorized as being involved in protein synthesis, post-translational modification, protein folding, protein degradation, and protein activation. Among differentially regulated proteins, the mRNA expression levels of 14 proteins that were predicted to be localized in the ER were analyzed. Notably, 3-ketoacyl-CoA reductase 1 was up-regulated and eight genes related to stress, hormone metabolism, cell wall and DNA repair were down-regulated within 1 day under flooding conditions. In addition, the expression of luminal-binding protein 5 was specifically induced in flood-stressed roots, whereas arabinogalactan protein 2 and methyltransferase PMT2 were down-regulated. Taken together, these results suggest that flooding mainly affects the function of protein synthesis and glycosylation in the ER in root tips of soybean.

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding.

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.

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.