Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways.

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with ß-oxidation of α-linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value-added soybeans.

Genomic changes and biochemical alterations of seed protein and oil content in a subset of fast neutron induced soybean mutants.

BACKGROUND: Soybean is subjected to genetic manipulation by breeding, mutation, and transgenic approaches to produce value-added quality traits. Among those genetic approaches, mutagenesis through fast neutrons radiation is intriguing because it yields a variety of mutations, including single/multiple gene deletions and/or duplications. Characterizing the seed composition of the fast neutron mutants and its relationship with gene mutation is useful towards understanding oil and protein traits in soybean. RESULTS: From a large population of fast neutron mutagenized plants, we selected ten mutants based on a screening of total oil and protein content using near infra-red spectroscopy. These ten mutants were regrown, and the seeds were analyzed for oil by GC-MS, protein profiling by SDS-PAGE and gene mapping by comparative genomic hybridization. The mutant 2R29C14Cladecr233cMN15 (nicknamed in this study as L10) showed higher protein and lower oil content compared to the wild type, followed by three other lines (nicknamed in this study as L03, L05, and L06). We characterized the fatty acid methyl esters profile of the trans-esterified oil and found the presence of five major fatty acids (palmitic, stearic, oleic, linoleic, and linolenic acids) at varying proportions among the mutants. Protein profile using SDS-PAGE of the ten mutants did exhibit discernable variation between storage (glycinin and ß-conglycinin) and anti-nutritional factor (trypsin inhibitor) proteins. In addition, we physically mapped the position of the gene deletions or duplications in each mutant using comparative genomic hybridization. CONCLUSION: Characterization of oil and protein profile in soybean fast neutron mutants will assist scientist and breeders to develop new value-added soybeans with improved protein and oil quality traits.

Biochemical and Anatomical Investigation of Sesbania herbacea (Mill.) McVaugh Nodules Grown under Flooded and Non-Flooded Conditions.

Sesbania herbacea, a native North American fast-growing legume, thrives in wet and waterlogged conditions. This legume enters into symbiotic association with rhizobia, resulting in the formation of nitrogen-fixing nodules on the roots. A flooding-induced anaerobic environment imposes a challenge for the survival of rhizobia and negatively impacts nodulation. Very little information is available on how S. herbacea is able to thrive and efficiently fix N2 in flooded conditions. In this study, we found that Sesbania plants grown under flooded conditions were significantly taller, produced more biomass, and formed more nodules when compared to plants grown on dry land. Transmission electron microscopy of Sesbania nodules revealed bacteroids from flooded nodules contained prominent polyhydroxybutyrate crystals, which were absent in non-flooded nodules. Gas and ion chromatography mass spectrometry analysis of nodule metabolites revealed a marked decrease in asparagine and an increase in the levels of gamma aminobutyric acid in flooded nodules. 2-D gel electrophoresis of nodule bacteroid proteins revealed flooding-induced changes in their protein profiles. Several of the bacteroid proteins that were prominent in flooded nodules were identified by mass spectrometry to be members of the ABC transporter family. The activities of several key enzymes involved in nitrogen metabolism was altered in Sesbania flooded nodules. Aspartate aminotransferase (AspAT), an enzyme with a vital role in the assimilation of reduced nitrogen, was dramatically elevated in flooded nodules. The results of our study highlight the potential of S. herbacea as a green manure and sheds light on the morphological, structural, and biochemical adaptations that enable S. herbacea to thrive and efficiently fix N2 in flooded conditions.

Identification of a Halotolerant Mutant via In Vitro Mutagenesis in the Cyanobacterium Fremyella diplosiphon.

Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. In this study, the effect of salinity on the freshwater cyanobacterium, Fremyella diplosiphon, was investigated and mutagenesis-based efforts were undertaken to enhance salt tolerance. Salinity at a concentration of 10 g/L sodium chloride (NaCl) inhibited growth of wild type F. diplosiphon under white, red, and green light. Efforts to enhance halotolerance resulted in a mutant that could survive in 20 g/L NaCl for 15 generations with no significant reduction in phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) or chlorophyll a. Gene expression measured by quantitative reverse transcription-polymerase chain reaction revealed a three-fold increase in tripartite ATP-independent periplasmic transporters (TRAP) solute receptor transcript in the mutant compared to wild type. Our discovery of a TRAP transporter system in F. diplosiphon and its possible role in salinity response enables growth in brackish waters, which enhances its potential for biotechnological applications.

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors.

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.

Protein profiling of fast neutron soybean mutant seeds reveals differential accumulation of seed and iron storage proteins.

A fast neutron (FN) radiated mutant soybean (Glycine max (L.) Merr., Fabaceae) displaying large duplications exhibited an increase in total seed protein content. A tandem mass tag (TMT) based protein profiling of matured seeds resulted in the identification of 4338 proteins. Gene duplication resulted in a significant increase in several seed storage proteins and protease inhibitors. Among the storage proteins, basic 7 S globulin, glycinin G4, and beta-conglycinin showed higher abundance in matured FN mutant seeds in addition to protease inhibitors. A significantly higher abundance of L-ascorbate peroxidases, acid phosphatases, and iron storage proteins was also observed. A higher amount of albumin, sucrose synthase, iron storage, and ascorbate family proteins in the mutant seeds was observed at the mid-stage of seed filling. We anticipate that the duplicated genes might have a cascading effect on the genome constituents, thus, resulting in increased storage and iron-containing protein content in the mutant seeds.

Two-dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines.

2-DE reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. The results showed that proteins involved in metabolic, developmental and biological regulation processes were the most abundant.

Protein and metabolite composition of xylem sap from field-grown soybeans (Glycine max).

The xylem, in addition to transporting water, nutrients and metabolites, is also involved in long-distance signaling in response to pathogens, symbionts and environmental stresses. Xylem sap has been shown to contain a number of proteins including metabolic enzymes, stress-related proteins, signal transduction proteins and putative transcription factors. Previous studies on xylem sap have mostly utilized plants grown in controlled environmental chambers. However, plants in the field are subjected to high light and to environmental stress that is not normally found in growth chambers. In this study, we have examined the protein and metabolite composition of xylem sap from field-grown cultivated soybean plants. One-dimensional gel electrophoresis of xylem sap from determinate, indeterminate, nodulating and non-nodulating soybean cultivars revealed similar protein profiles consisting of about 8-10 prominent polypeptides. Two-dimensional gel electrophoresis of soybean xylem sap resulted in the visualization of about 60 distinct protein spots. A total of 38 protein spots were identified using MALDI-TOF MS and LC-MS/MS. The most abundant proteins present in the xylem sap were identified as 31 and 28 kDa vegetative storage proteins. In addition, several proteins that are conserved among different plant species were also identified. Diurnal changes in the metabolite profile of xylem sap collected during a 24-h cycle revealed that asparagine and aspartate were the two predominant amino acids irrespective of the time collected. Pinitol (D-3-O-methyl-chiro-inositol) was the most abundant carbohydrate present. The possible roles of xylem sap proteins and metabolites as nutrient reserves for sink tissue and as an indicator of biotic stress are also discussed.

Distinct cell surface appendages produced by Sinorhizobium fredii USDA257 and S. fredii USDA191, cultivar-specific and nonspecific symbionts of soybean.

Sinorhizobium fredii USDA257 and S. fredii USDA191 are fast-growing rhizobia that form nitrogen-fixing nodules on soybean roots. In contrast to USDA191, USDA257 exhibits cultivar specificity and can form nodules only on primitive soybean cultivars. In response to flavonoids released from soybean roots, these two rhizobia secrete nodulation outer proteins (Nop) to the extracellular milieu through a type III secretion system. In spite of the fact that Nops are known to regulate legume nodulation in a host-specific manner, very little is known about the differences in the compositions of Nops and surface appendages elaborated by USDA191 and USDA257. In this study we compared the Nop profiles of USDA191 and USDA257 by one-dimensional (1D) and 2D gel electrophoresis and identified several of these proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-tandem MS (LC-MS/MS). Examination of the surface appendages elaborated by these two strains of soybean symbionts by transmission electron microscopy revealed distinct differences in their morphologies. Even though the flagella produced by USDA191 and USDA257 were similar in their morphologies, they differed in their flagellin composition. USDA257 pili resembled long thin filaments, while USDA191 pili were short, rod shaped, and much thinner than the flagella. 2D gel electrophoresis of pilus-like appendages of USDA191 and USDA257 followed by mass spectrometry resulted in the identification of several of the Nops along with some proteins previously undetected in these strains. Some of the newly identified proteins show homology to putative zinc protease and a LabA-like protein from Bradyrhizobium sp. ORS278, fimbrial type 4 assembly proteins from Ralstonia solanacearum, and the type III effector Hrp-dependent protein from Rhizobium leguminosarum bv. trifolii.

Natural variability in abundance of prevalent soybean proteins.

Soybean is an inexpensive source of protein for humans and animals. Genetic modifications (GMO) to soybean have become inevitable on two fronts, both quality and yield will need to improve to meet increasing global demand. To ensure the safety of the crop for consumers it is important to determine the natural variation in seed protein constituents as well as any unintended changes that may occur in the GMO as a result of genetic modification. Understanding the natural variation of seed proteins in wild and cultivated soybeans that have been used in conventional soybean breeding programs is critical for determining unintended protein expression in GMO soybeans. In recent years, proteomic technologies have been used as an effective analytical tool for examining modifications of protein profiles. We have standardized and applied these technologies to determine and quantify the spectrum of proteins present in soybean seed. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of different classes of soybean seed proteins. We have observed significant variations in different classes of proteins, including storage, allergen and anti-nutritional protein profiles, between non-GMO cultivated and wild soybean varieties. This information is useful for scientists and regulatory agencies to determine whether the unintended expression of proteins found in transgenic soybean is within the range of natural variation.

A rapid and simple procedure for the depletion of abundant storage proteins from legume seeds to advance proteome analysis: a case study using Glycine max.

2-D analysis of plant proteomes containing thousands of proteins has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the non-abundant proteins within seeds is difficult when 60-80% is storage proteins. Resolution can be improved through sample fractionation using separation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) to precipitate soybean (Glycine max) seed storage globulins, glycinin and beta-conglycinin. This method removes 87+/-4% of the highly abundant seed proteins from the extract, allowing for 541 previously inconspicuous proteins present in soybean seed to be more detectable (volume increase of >or=50%) using fluorescent detection. Of those 541 enhanced spots, 197 increased more than 2.5-fold when visualized with Coomassie. The majority of those spots were isolated and identified using peptide mass fingerprinting. Fractionation also provided detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2-D electrophoretic separation and an in-gel phosphoprotein stain. Application of this methodology toward other legumes, such as peanut, bean, pea, alfalfa and others, also containing high amounts of storage proteins, was examined, and is reported here.

An efficient extraction method to enhance analysis of low abundant proteins from soybean seed.

Large amounts of the major storage proteins, beta-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.

Influence of sample preparation on the assay of isoflavones.

The complexity of sample matrices, coexistence of multiple forms of bioactive phytochemicals, and their interaction of with other cellular components pose a significant challenge for optimized extraction and accurate estimation of bioactive phytochemicals in foods and dietary supplements. This article discusses the significance of optimizing extraction procedures for accurate assay of phytochemicals from different matrices using bioactive isoflavones as model substrate because isoflavones are known to exist in nature as free aglycones or as conjugates with sugars and/or acids. The wide structural diversity and polarities of free and conjugated isoflavones makes optimum extraction and accurate quantification of isoflavones a challenging task. This paper reviews variables, extraction solvent composition (aqueous-organic solvents mixtures at different acidification levels), physical extraction techniques (Soxhlet, stirring, ultrasonic, microwave, pressurized, supercritical-fluid, high-speed counter-current chromatography), and parameters (temperature, pressure, number of cycles, solid-solvent ratio) that influence quantitative extraction of isoflavones from different matrices. In addition, this review covers a brief overview of structures, sources, bioactivities, separation, and detection used for isoflavones analysis. Optimum extraction efficiencies of isoflavones were obtained with EtOH : H (2)O : DMSO (70 : 25 : 5, v/v/v) as the extraction solvent and acidification of extraction solvent favored partial degradation of conjugated forms to their corresponding aglycones. Accurate quantification of isoflavones in foods, plants, and dietary supplements will allow researchers and regulators to provide more precise guidelines on dietary intake and safety levels necessary to achieve optimum health.

Utility of proteomics techniques for assessing protein expression.

Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential unintended (collateral) effects due to transgenic modifications in genetically modified (GMO) soybeans. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to separate, identify and quantify the different classes of soybean seed proteins. Sixteen soybean genotypes, including four wild and twelve cultivated genotypes, belonging to four different subgroups were used as models for protein profile evaluation. Significant variations of allergen and anti-nutritional protein profiles were observed between two different groups, cultivated and wild soybean genotypes. However, only minor variations in protein profiles were observed within the soybean samples from the same group (cultivated or wild). These results may be useful to scientists needing to compare GMO and non-GMO soybeans once additional data are generated on additional soybean varieties and the same varieties grown at different geographical locations.

Proteomic Comparison of Three Extraction Methods Reveals the Abundance of Protease Inhibitors in the Seeds of Grass Pea, a Unique Orphan Legume.

Grass pea is an orphan legume that is grown in many places in the world. It is a high-protein, drought-tolerant legume that is capable of surviving extreme environmental challenges and can be a sole food source during famine. However, grass pea produces the neurotoxin ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a neurological disease. This crop is promising as a food source for both animals and humans if ß-ODAP levels and other antinutritional factors such as protease inhibitors are lowered or removed. To understand more about these proteins, a proteomic analysis of grass pea was conducted using three different extraction methods to determine which was more efficient at isolating antinutritional factors. Seed proteins extracted with Tris-buffered saline (TBS), 30% ethanol, and 50% isopropanol were identified by mass spectrometry, resulting in the documentation of the most abundant proteins for each extraction method. Mass spectrometry spectral data and BLAST2GO analysis led to the identification of 1376 proteins from all extraction methods. The molecular function of the extracted proteins revealed distinctly different protein functional profiles. The majority of the TBS-extracted proteins were annotated with nutrient reservoir activity, while the isopropanol extraction yielded the highest percentage of endopeptidase proteinase inhibitors. Our results demonstrate that the 50% isopropanol extraction method was the most efficient at isolating antinutritional factors including protease inhibitors.

Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani.

Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris, T. praticola) is a basidiomycetous fungus and a major cause of root diseases of economically important plants. Various isolates of this fungus are also beneficially associated with orchids, may serve as biocontrol agents or remain as saprophytes with roles in decaying and recycling of soil organic matter. R. solani displays several hyphal anastomosis groups (AG) with distinct host and pathogenic specializations. Even though there are reports on the physiological and histological basis of Rhizoctonia-host interactions, very little is known about the molecular biology and control of gene expression early during infection by this pathogen. Proteamic technologies are powerful tools for examining alterations in protein profiles. To aid studies on its biology and host pathogen interactions, a two-dimensional (2-D) gel-based global proteomic study has been initiated. To develop an optimized protein extraction protocol for R. solani, we compared two previously reported protein extraction protocols for 2-D gel analysis of R. solani (AG-4) isolate Rs23. Both TCA-acetone precipitation and phosphate solubilization before TCA-acetone precipitation worked well for R. solani protein extraction, although selective enrichment of some proteins was noted with either method. About 450 spots could be detected with the densitiometric tracing of Coomassie blue-stained 2-D PAGE gels covering pH 4-7 and 6.5-205 kDa. Selected protein spots were subjected to mass spectrometric analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Eleven protein spots were positively identified based on peptide mass fingerprinting match with fungal proteins in public databases with the Mascot search engine. These results testify to the suitability of the two optimized protein extraction protocols for 2-D proteomic studies of R. solani.

Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines.

Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the high-protein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of beta-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of beta-conglycinin and glycinin, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing beta-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin, beta-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin, beta-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of beta-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.

Proteomic Analysis of Pigeonpea (Cajanus cajan) Seeds Reveals the Accumulation of Numerous Stress-Related Proteins.

Pigeonpea is one of the major sources of dietary protein for more than a billion people living in South Asia. This hardy legume is often grown in low-input and risk-prone marginal environments. Considerable research effort has been devoted by a global research consortium to develop genomic resources for the improvement of this legume crop. These efforts have resulted in the elucidation of the complete genome sequence of pigeonpea. Despite these developments, little is known about the seed proteome of this important crop. Here, we report the proteome of pigeonpea seed. To enable the isolation of maximum number of seed proteins, including those that are present in very low amounts, three different protein fractions were obtained by employing different extraction media. High-resolution two-dimensional (2-D) electrophoresis followed by MALDI-TOF-TOF-MS/MS analysis of these protein fractions resulted in the identification of 373 pigeonpea seed proteins. Consistent with the reported high degree of synteny between the pigeonpea and soybean genomes, a large number of pigeonpea seed proteins exhibited significant amino acid homology with soybean seed proteins. Our proteomic analysis identified a large number of stress-related proteins, presumably due to its adaptation to drought-prone environments. The availability of a pigeonpea seed proteome reference map should shed light on the roles of these identified proteins in various biological processes and facilitate the improvement of seed composition.

Overexpression of hlyB and mdh genes confers halotolerance in Fremyella diplosiphon, a freshwater cyanobacterium.

Fremyella diplosiphon is a freshwater cyanobacterium that has great potential as a biofuel agent due to its ability to grow in low light intensity and acclimation to different wavelengths. To enhance its halotolerance for growth in 35gL-1 sodium chloride (NaCl), plasmids harboring hemolysin B (hlyB) and malate dehydrogenase (mdh) genes were transformed into wild type F. diplosiphon (WT-Fd33). Electroporation-mediated overexpression of the genes resulted in two transformants, HSF33-1 and HSF33-2, with 9- and 20-fold increases in hlyB and mdh transcript levels. In addition, up-regulation of proteins at the expected size ranges of 50-60kDa for HlyB and 40-50kDa for MDH was observed. Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed a protein spot corresponding to HlyB in HSF33-1 with a significant MOWSE score of 164 and 3% sequence coverage, and a spot corresponding to MDH in HSF33-2 gave a significant MOWSE score of 124 with 10% sequence coverage. Physiological evaluation in BG11/HEPES medium and seawater adjusted to 35gL-1 NaCl confirmed that the transformants could thrive in high salinity with no loss of photosynthetic pigments. Results of the study indicate that overexpression of hlyB and mdh genes confer halotolerance in F. diplosiphon, thus maximizing its potential as a large-scale biofuel agent.

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis.

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.