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.

Assessment of indigenous Nepalese soybean as a potential germplasm resource for improvement of protein in North American cultivars.

Soybeans contain approximately 40% protein and 20% oil and represents an important source of protein in animal rations and human diets. Attempts are being made to increase further the overall protein content of soybeans by utilization of exotic germplasms. In this study, soybean cultivars from Nepal have been characterized and their potential as a germplasm resource for improvement of the protein content and quality of North American cultivars assessed. Soybean cultivars 'Sathia', 'Seti', 'Kavre', and 'Soida Chiny', indigenous to various regions of Nepal, contained 42-45% protein, which is significantly higher in comparison to that of the North American cultivar 'Williams 82' (39%). Fractionation of seed protein by high-resolution two-dimensional gel electrophoresis revealed differences in the protein profiles of these cultivars. Various isoelectric forms of glycinin and beta-conglycinin were identified by comparing the matrix-assisted laser desorption ionization time-of-flight mass fingerprinting data against the National Center for Biotechnology Information nonredundant database. Nepalese cultivar Sathia was distinct, lacking some isoelectric forms of acidic and basic glycinin subunits while expressing other unique forms. The contribution of these unique protein spots present in either Sathia or Williams 82 to the total protein content was quantified using scanning laser densitometry. Distinct restriction fragment length polymorphisms (RFLP) for group 1 glycinin genes were observed among the tested Nepalese genotypes, indicating sequence variation among the cultivars. Conversely, evaluation of RFLP for the genes encoding group 2 glycinins, beta-conglycinin, and Bowman-Birk proteinase inhibitors indicated a high degree of conservation in these genes. Determination of amino acid composition, a reflection of protein quality, indicated that the arginine content of the Nepalese soybeans ranged from 7.7 to 8.1%, which was 5-10% higher than the 7.4% expressed in Williams 82. Additionally, Karve and Seti contained significantly more cysteine than Williams 82. Nepalese high-protein soybeans having a desirable amino acid composition hold potential to increase the protein quality and diversity of North American cultivars.

Effect of six decades of selective breeding on soybean protein composition and quality: a biochemical and molecular analysis.

To evaluate the extent of the genetic change and its effects on the seed protein composition of soybean cultivars released during the past 60 years, representative ancestral cultivars and those derived from selective breeding were grown in a side-by-side comparison. Total seed protein content, determined by combustion analysis of nitrogen, revealed a decline in the protein content after decades of selection and breeding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis comparison of protein profiles of the soybean cultivars indicated that relative expression of most of the seed storage proteins had not varied substantially from the ancestral lines to the present commercial cultivars. There was noticeably less beta-subunit of beta-conglycinin, a protein devoid of sulfur amino acids, in the modern cultivars represented by Mustang, Pioneer 93B09, and Asgrow 3602. Comparison of the amino acid profiles of soybean seed, a benchmark of the protein's nutritional quality, revealed that the ancestral progenitor, G. soja, was significantly higher in cysteine, glutamic acid, histidine, and arginine than either the ancestral or the modern cultivars. Selective breeding over the past 60 years minimally affected the overall amino acid composition. The degree of divergence in the DNA sequence of the genes encoding glycinin and beta-conglycinin in the ancestral and modern cultivars was investigated using Southern hybridization and the polymerase chain reaction. Even though some restriction fragment polymorphisms could be detected, overall, the banding patterns were remarkably similar among the ancestral cultivars and those derived from them, suggesting a high degree of conservation of seed-storage protein genes. The results of our study suggest that selection and breeding for yield during the past 60 years had no major influence on the protein composition, ostensibly because of limited genetic diversity among the parental lines.

Nitrogen lowers the sulfur amino acid content of soybean (Glycine max [L.] Merr.) by regulating the accumulation of Bowman-Birk protease inhibitor.

Soybeans in general contain 35-40% protein. Efforts are underway to increase further this protein content, thus enhancing their nutritive value. Even though higher protein is a desirable characteristic, whether such an increase will be accompanied by enhanced protein quality is not known. Soybean protein quality could be significantly improved by increasing the concentration of the sulfur-containing amino acids, cysteine and methionine. To ascertain if a correlation existed between protein quantity and quality, a comparison of the amino acids of soybeans differing in protein content was made. Soybeans with higher protein content had a significantly lower percentage of sulfur amino acids, while those with lower protein exhibited a higher content of cysteine and methionine. Nitrogen application elevated the protein content but lowered that of the sulfur amino acids. Transmission electron microscopy examination of thin sections of low protein soybean seeds revealed several protein storage vacuoles that were partially filled with storage proteins. Fluorescence two-dimensional difference gel electrophoresis of soybean seed proteins revealed that nitrogen application favored the accumulation of the beta-subunit of beta-conglycinin while decreasing the accumulation of Bowman-Birk protease inhibitor (BBI), a protein rich in cysteine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 60% 2-propanol-extracted proteins showed a drastic reduction in the accumulation of BBI with increasing protein content. Northern blot analysis indicated that nitrogen had a negative influence on the expression of the BBI gene. Our results indicate that the negative correlation between total protein and sulfur amino acid content is mostly mediated by the differential accumulation of BBI.

Accumulation of genistein and daidzein, soybean isoflavones implicated in promoting human health, is significantly elevated by irrigation.

To circumvent drought conditions persisting during seed fill in the mid-south U.S. soybean production region, researchers have developed the early soybean (Glycine max [L.] Merr.) production system (ESPS), which entails early planting of short-season varieties. Because soybean supplies a preponderance of the world's protein and oil and consumption of soy-based foods has been associated with multiple health benefits, the effects of this agronomic practice on seed quality traits such as protein, oil, and isoflavones should be investigated. Four cultivars of soybean, two from maturity group IV and two from maturity group V, were planted in April (ESPS) and May (traditional) in a two-year study at Stoneville, MS. Near-infrared analysis of soybean seed was utilized to determine the percentages of protein and oil. Dependent upon variety, the oil content of the early-planted crop was increased by 3-8%, whereas protein was not significantly changed. Visualization of protein extracts fractionated by sodium dodecyl sulfate-polyacrylamide electrophoresis and fluorescence two-dimensional difference gel electrophoresis revealed that early planting did not affect the relative accumulation of the major seed-storage proteins; thus, protein composition was equal to that of traditionally cultivated soybeans. Maturity group IV cultivars contained a higher percentage of oil and a lower percentage of protein than did the maturity group V cultivars, regardless of planting date. Gas chromatographic separation of fatty acids revealed that the percentages of saturated and unsaturated fatty acids were not significantly altered by planting date. Methanol extracts of seed harvested from different planting dates when analyzed by high-performance liquid chromatography showed striking differences in isoflavone content. Dependent upon the variety, total isoflavone content was increased as much as 1.3-fold in early-planted soybeans. Irrigation enhanced the isoflavone content of both early- and late-planted soybeans as much as 2.5-fold. Accumulation of individual isoflavones, daidzein and genistein, was also elevated by irrigation. Because this cultural practice improves the quality traits of seeds, ESPS provides an opportunity for enhancing the quality of soybean.

Positional effect on protein and oil content and composition of soybeans.

Soybean (Glycine max [L.] Merr.) protein and oil qualities, with respect to monogastric nutrition, have been linked to the relative abundance of specific protein subunits and fatty acids, respectively. An analysis of field-grown soybean seeds by near-infrared spectroscopy revealed significant differences in their protein and oil contents as a function of nodal position. Seed proteins from the plant apex were high in protein and low in oil content, while those from the basal region exhibited an opposite pattern of accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total seed proteins revealed that the beta-subunit of beta-conglycinin content was 4-fold higher in seeds from the apical nodes than in seeds from basal nodes. The glycinin A3 polypeptide content gradually increased in successively lower nodes from the top of the plant. Its accumulation was drastically reduced when nitrogen was applied at specific growth stages. Exogenous nitrogen did not alter the pattern of beta-subunit accumulation, but accrual of the acidic and basic polypeptides of glycinin was diminished. The remaining seed storage protein components were not influenced by nodal position or nitrogen application. Gas chromatographic analysis of fatty acids indicated that only oleic (18:0) and linoleic (18:2) acids showed variability in accumulation at different nodes. Neither the abundance nor the distribution of the fatty acids was altered by nitrogen application.