Nathan W. Oehrle

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 Ca2+ to precipitate soybean (Glycine max) seed storage globulins, glycinin and β‐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 ≥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.

Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms

Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.

Proteomic analysis of soybean nodule cytosol

An isolation procedure for soybean (Glycine max L. cv Williams 82) nodule cytosol proteins was developed which greatly improved protein resolution by two-dimensional polyacrylamide gel electrophoresis. The most abundant proteins were selected and analyzed by mass spectrometry. The identified proteins were categorized by function (% of total proteins analyzed): carbon metabolism (28%), nitrogen metabolism (12%), reactive oxygen metabolism (12%) and vesicular trafficking (11%). The first three categories were expected based on the known physiological functions of the symbiotic nitrogen fixation process. The number of proteins involved in vesicular trafficking suggests a very active exchange of macromolecules and membrane components. Among the 69 identified proteins were the enzymes of the three carbon portion of glycolysis, which were further characterized to support their roles in the sucrose synthase pathway to provide malate for the bacteroids. Proteomic analysis provides a functional tool by which to understand and further investigate nodule function.

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 degrees C, it was carried out for 2 to 3h at -80 degrees C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 degrees C. The applicability of the method was demonstrated using several soybean tissues.

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.

Introgression of Leginsulin, a Cysteine-Rich Protein, and High-Protein Trait from an Asian Soybean Plant Introduction Genotype into a North American Experimental Soybean Line

Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur-containing amino acids could enhance the nutritive value of soybean meal. Leginsulin, a cysteine-rich peptide, predominantly accumulates in Asian soybean accessions but not in most North American cultivars. By screening diverse soybean accessions from the USDA Soybean Germplasm Collection, we were able to identify one plant introduction, PI 427138, as a high-protein line with relatively high amounts of both elemental sulfur and leginsulin. We introgressed these desirable traits from PI 427138 into an experimental line with the aim of improving the overall protein content and quality of seed proteins. Biochemical characterization of inbred progenies from the cross of LD00-3309 with PI 427138 grown at six locations revealed stable ingression of high protein, high elemental sulfur, and high leginsulin accumulation. Comparison of soybean seed proteins resolved by high-resolution 2-D gel electrophoresis in combination with Delta2D image analysis software revealed preferential accumulation of a few glycinin subunits contributed to the increased protein content in the introgressed lines. Amino acid analysis revealed that even though the leginsulin introgressed lines had higher protein, leginsulin, and elemental sulfur, the overall concentration of sulfur-containing amino acids was not significantly altered when compared with the parental lines. The experimental soybean lines developed during this study (Leg-3, Leg-7, and Leg-8) lack A5, A4, and B3 glycinin subunits and could be utilized in breeding programs to develop high-quality tofu cultivars.

Metabolic Intricacies of the Symbiotic Association between Soybean and Bradyrhizobium japonicum:A Proteomic Outlook

Symbiotic nitrogen fixation, the primary pathway by which inorganic nitrogen is made available for living organisms, requires complex communication between the bacterial microsymbiont and the host plant, beginning in the soil and ending at nodule senescence. The symbiosis takes the form of a highly complex structure referred to as a nodule, which appears as a tumor-like growth on the roots of certain leguminous plants. Both partners exchange signals and change metabolically and morphologically in response to their fellow symbiont. These changes by necessity must be coordinated and complementary. Proteomic analysis has revealed extensive changes in the proteomes of each organism during symbiosis.

Impact of overexpression of cytosolic isoform of O -acetylserine sulfhydrylase on soybean nodulation and nodule metabolome

Nitrogen-fixing nodules, which are also major sites of sulfur assimilation, contribute significantly to the sulfur needs of whole soybean plants. Nodules are the predominant sites for cysteine accumulation and the activity of O-acetylserine(thiol)lyase (OASS) is central to the sulfur assimilation process in plants. Here, we examined the impact of overexpressing OASS on soybean nodulation and nodule metabolome. Overexpression of OASS did not affect the nodule number, but negatively impacted plant growth. HPLC measurement of antioxidant metabolites demonstrated that levels of cysteine, glutathione, and homoglutathione nearly doubled in OASS overexpressing nodules when compared to control nodules. Metabolite profiling by LC-MS and GC-MS demonstrated that several metabolites related to serine, aspartate, glutamate, and branched-chain amino acid pathways were significantly elevated in OASS overexpressing nodules. Striking differences were also observed in the flavonoid levels between the OASS overexpressing and control soybean nodules. Our results suggest that OASS overexpressing plants compensate for the increase in carbon requirement for sulfur assimilation by reducing the biosynthesis of some amino acids, and by replenishing the TCA cycle through fatty acid hydrolysis. These data may indicate that in OASS overexpressing soybean nodules there is a moderate decease in the supply of energy metabolites to the nodule, which is then compensated by the degradation of cellular components to meet the needs of the nodule energy metabolism.

Whole Genome Resequencing Identifies the Molecular Genetic Cause for the Absence of a Gy5 Glycinin Protein in Soybean PI 603408

During ongoing proteomic analysis of the soybean (Glycine max (L.) Merr) germplasm collection, PI 603408 was identified as a landrace whose seeds lack accumulation of one of the major seed storage glycinin protein subunits. Whole genomic resequencing was used to identify a two-base deletion affecting glycinin 5. The newly discovered deletion was confirmed to be causative through immunological, genetic, and proteomic analysis, and no significant differences in total seed protein content were found to be due to the glycinin 5 loss-of-function mutation per se. In addition to focused studies on this one specific glycinin subunit-encoding gene, a total of 1,858,185 nucleotide variants were identified, of which 39,344 were predicted to affect protein coding regions. In order to semiautomate analysis of a large number of soybean gene variants, a new SIFT 4G (Sorting Intolerant From Tolerated 4 Genomes) database was designed to predict the impact of nonsynonymous single nucleotide soybean gene variants, potentially enabling more rapid analysis of soybean resequencing data in the future.

Recovery of nitrogenase from aerobically isolated soybean nodule bacteroids

Bacteroids, the symbiotic forms of rhizobia, express nitrogenase, which is the enzyme that catalyzes the reduction of atmospheric dinitrogen to ammonium. The extreme oxygen lability of nitrogenase requires that bacteroids be isolated under anaerobic conditions to preserve their ability to reduce atmospheric dinitrogen. Aerobically isolated bacteroids were found to exhibit nitrogenase activity as measured via the acetylene reduction method but only after incubation of the bacteroids for about 1 h under low partial pressures of oxygen. The recovery of nitrogenase activity was dependent upon the partial pressure of oxygen to which the bacteroids were exposed during the one-hour incubation. The recovery of acetylene reduction activity was prevented by addition of inhibitors of protein synthesis, but not by inhibitors of transcription. Acetylene reduction could be expressed from aerobically isolated bacteroids stored for up to three days after isolation. The recovery of nitrogenase demonstrates that both the plant and the bacteroids possess mechanisms to protect the enzyme against oxygen. The recovery of nitrogen fixation activity from aerobically isolated bacteroids may provide a facile way to obtain bacteroids for studies on symbiotic functioning.