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Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [(14)C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35 degrees C, saturating illumination (pH 6), and 21% oxygen, the apparent K(m) for sucrose is approximately 8 millimolar and V(max) is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone.

Metabolism of lipid molecular species in soybean cotyledons (Glycine max [L.] Merr. var. "Harosoy 63") was determined from incorporation studies with radioactive acetate and glycerol. Lipid synthetic activity was highest in immature cotyledons at 30 days after flowering. Distinct differences in labeling patterns of molecular species within lipid classes demonstrated that selective utilization of diglyceride intermediates occurred in complex lipid biosynthesis in soybean. The phospholipid molecular species in this tissue that displayed the highest turnover rates had the following acyl combinations: saturate-linoleic and dioleic in phosphatidic acid; saturate-oleic in phosphatidylinositol and phosphatidylethanolamine; dioleic in phosphatidylcholine; oleic-dilinoleic in N-acylphosphatidylethanolamine. Saturate-dilinoleic, oleic-dilinoleic, trioleic, and trilinoleic structures were rapidly synthesized species of triglyceride in immature soybean cotyledons.

There is a limited understanding of the carbon assimilation capacity of nonfoliar green tissues and its impact on yield and seed quality since most photosynthesis research focuses on leaf photosynthesis. In this study, we investigate the photosynthetic efficiency of soybean (Glycine max) pods and seeds in a field setting and evaluate its effect on mature seed weight and composition. We demonstrate that soybean pod and seed photosynthesis contributes 13% to 14% of the mature seed weight. Carbon assimilation by soybean pod and seed photosynthesis can compensate for 81% of carbon loss through the respiration of the same tissues, and our model predicts that soybean pod and seed photosynthesis contributes up to 9% of the total daily carbon gain of the canopy. Chlorophyll fluorescence (CF) shows that the operating efficiency of photosystem II in immature soybean seeds peaks at the 10 to 100 mg seed weight stage, while that of immature pods peaks at the 75 to 100 mg stage. This study provides quantitative information about the efficiency of soybean pod and seed photosynthesis during tissue development and its impact on yield. [ABSTRACT FROM AUTHOR]

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a9 domain among the a, a9, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a9 domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a9 domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants. [ABSTRACT FROM AUTHOR]

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α' - and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α;' /α-subunits were complexes of more than 720 kD. The α' - and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α' /α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α' - and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions. [ABSTRACT FROM AUTHOR]

The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either alpha-naphthaleneacetic acid or indoleacetic acid but not by 2,4-dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by alpha-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination-associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos.

Immature soybean (Glycine max L.) embryos from commercially important cultivars were the targets of rapidly accelerated, DNA-coated, gold particles. Protoplasts were prepared from these tissues and propagated in culture under selection conditions for the introduced neomycin phosphotransferase II gene. Kanamycin-resistant calli were obtained at a rate of approximately 10(-5). Enzyme assays and Southern blot hybridization confirmed the expression of the foreign gene and its stable integration into the soybean genome. Our results show that particle acceleration can be used for the introduction of foreign DNA into the soybean genome and indicate the technique may be useful in the recovery of engineered plants by transformation of regenerable tissues.

Immature soybean (Glycine max L. Merrill cv Provar) cotyledons were grown aseptically for 6 days in complete culture medium with zero, deficient (17 micromolar), sufficient (1.5 millimolar), or supraoptimal (7.5 millimolar) levels of sulfate. Some cotyledons at each sulfate concentration were supplemented with l-methionine. No sulfate or 17 micromolar sulfate were inadequate for growth and protein accumulation, but all the major subunits of the 7S and 11S storage protein fractions were detected. The ratio of 11S to 7S proteins was <1.0. Addition of 8.4 millimolar methionine overcame the restriction of cotyledon growth and protein accumulation in the sulfate-deficient media, and the ratio of 11S to 7S proteins was significantly increased. The amino acid compositions of the 7S and 11S fractions from sulfate-sufficient cotyledons and from sulfate-deficient cotyledons were not significantly different. There was no difference in fresh weight or total protein accumulation in cotyledons grown in 1.5 millimolar or 7.5 millimolar sulfate. At 7.5 millimolars sulfate, the 11S to 7S ratio was significantly increased, and the amount of beta-subunit in the 7S fraction decreased. At all sulfate levels supplemented with methionine, the 11S to 7S ratio was greater than 1, and no beta-subunit was detected in the 7S fraction. Supplemental methionine in media of any sulfate concentration increased growth and protein methionine content to a greater extent than high (7.5 millimolar) sulfate only. Adding supraoptimal sulfate is not equivalent to supplementing with methionine. Results of this study of in vitro growth are compared to results of studies of seed development on intact plants supplied with various sulfur concentrations.

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. "Harosoy 63" were frozen with liquid N(2), dry ice, or stored in a freezer (-20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at -20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with (3)H, and fatty acids with (14)C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.

Messenger RNAs (mRNAs), isolated from immature soybean (Glycine max L., Merr.) seeds, that bound to oligo(dT)-cellulose were fractionated by centrifugation in sucrose density gradients containing dimethyl sulfoxide. mRNAs with sedimentation values between 21S and 25S coded for the in vitro translation of polypeptides with electrophoretic mobilities similar to those of the alpha' and alpha subunits of the 7S seed storage protein. High pressure liquid chromatographic analyses of the trypsin-induced fragments ("column fingerprinting") verified that the polypeptides produced in vitro were closely related to authentic alpha' and alpha subunits.The fractions of RNA that coded for the in vitro synthesis of these subunits contained three major species of nonribosomal RNA (molecular weights of 1.1, 0.84, and 0.75 x 10(6)) sufficiently large to code for the synthesis of the alpha' and alpha subunits.

The various nitrogenous solutes important to embryo development in symbiotic soybean plants were determined during the midpodfilling stage. Glutamine was the principal form of nitrogen, contributing 55% of the embryo nitrogen requirement. Asparagine was the second most important, contributing 20%. The ureides allantoin and allantoic acid directly contributed only insignificantly to the total nitrogen requirement of the embryo. These conclusions were based upon analyses of tissue extracts, translocation studies of radiolabeled solutes, analysis of in vivo seed coat exudate collected from the freespace of attached, surgically altered seeds, and the in vitro culture of isolated immature soybean embryos.

The maturation of soybean (Glycine max L. Merr.) somatic embryos was characterized. Maturation was assayed by evaluating the ability of somatic embryos to make the transition to a plantlet through a germination-like process. Somatic embryos were organized from cotyledons of immature soybean embryos. Maturation of somatic embryos occurred on a Murashige-Skoog basal medium supplemented with activated charcoal and 0.28 molar sucrose. After 8 weeks on this medium, somatic embryos exhibited vigorous, high frequency development to plantlets. The "germination" frequency (conversion) of somatic embryos, and plantlet recovery frequency varied concurrently with maturation period. Conversion and plant recovery required no exogenous growth regulators. Desiccation of immature somatic embryos under controlled humidity regimes resulted in increased frequency of conversion of immature somatic embryos. Morphological abnormalities appeared in the somatic embryos, but few were detrimental to conversion velocity. There was little effect of genotype on conversion velocity or frequency.

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA(1), GA(3), GA(4), GA(5), GA(7), and GA(9) conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA(7) was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA(20) and GA(29) were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA(9) were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA(9) was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA(20) and GA(51) were also observed in testae.