Developing Soybean Seeds Research Articles

A seed-specific AP2-domain transcription factor from soybean plays a certain role in regulation of seed germination

Plant seed development and germination are under strict temporal and spatial regulation, and transcription factors play important roles in this regulation. In the present study we identified an EST expressed specifically in the developing soybean seeds. The full length of the gene was obtained through further RACE analysis and the gene was named GmSGR. Sequence analysis revealed that this gene belonged to the AP2/ERF transcription factor family. Its AP2 domain had the highest similarity with that of the A-3 member AtABI4 of DREB subgroup in the AP2/ERF family in Arabidopsis. GmSGR did not exhibit transcriptional activation activity in the yeast assay system. GmSGR was overexpressed in Arabidopsis and the germination rates of the transgenic seeds were significantly higher than that of the wild type seeds under higher concentrations of ABA and glucose respectively. However, the germination rates of the transgenic seeds were lower than that of control under salt stress. The expression of AtEm6 and AtRD29B was higher in the seedlings of the transgenic plants than that in the wild-type seedlings. These results suggest that GmSGR may confer reduced ABA sensitivity and enhanced salt sensitivity to the transgenic seeds through regulating the expression of AtEm6 and AtRD29B genes.

Effect of Temperature on Microsomal Omega‐3 Linoleate Desaturase Gene Expression and Linolenic Acid Content in Developing Soybean Seeds

The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post‐translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean [Glycine max (L.) Merr.] microsomal omega‐3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega‐3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic (18:2Δ9, 12) acid to produce linolenic (18:3Δ9, 12, 15) acid. At 35 d after flowering, transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5‐ to 15‐fold, GmFAD3B by 2‐ to 9‐fold, and GmFAD3C by 2‐ to 3‐fold in seeds that developed in a warm (day/night [D/N] = 30/26°C) versus a normal (D/N = 26/22°C) or a cool (D/N = 22/18°C) environment. At this stage of seed development, decreased omega‐3 desaturase gene expression levels were positively associated with reductions of 39 to 50% in the linolenic acid content of seeds of three soybean varieties examined. Thus, transcriptional regulation of the microsomal omega‐3 fatty acid desaturase gene family likely accounts, at least in part, for the reduced linolenic acid levels in soybean seeds grown at elevated temperature.

Effect of Temperature on Delta‐9 Stearoyl‐ACP and Microsomal Omega‐6 Desaturase Gene Expression and Fatty Acid Content in Developing Soybean Seeds

Delta‐9 stearoyl‐ACP (SAD) and microsomal omega‐6 oleate (FAD2) desaturases contribute to the maintenance of lipid fluidity in membranes and the fatty acid composition of storage lipids in seed. Since these enzymes must operate at varying environmental temperatures, they are under constitutive control, but they may also be subject to fine regulation both transcriptionally and posttranscriptionally. We measured transcript accumulation of the seed‐expressed SAD‐A and SAD‐B and FAD2‐1A and FAD2‐1B genes in the seeds of three soybean varieties grown at cool (22/18°C), normal (26/22°C), or warm (30/26°C) temperatures during pod fill. At the cool temperature, transcript accumulation of both the SAD and FAD2‐1 genes was significantly elevated, with FAD2‐1B 2‐ to 10‐fold or greater than FAD2‐1A at 35 d after flowering. Expression of both SAD and FAD2‐1 were significantly decreased in seed that developed at the warm temperature. Decreased FAD2‐1 transcript accumulation at the warm temperature was positively associated with significantly increased oleic and decreased linoleic acid content in the three varieties examined. Decreased SAD transcript accumulation at the warm temperature was positively associated with a significantly increased level of stearic acid but only in the high‐stearate mutant line, A6. We conclude that environmental temperature modulates oleic and linoleic acid in developing seed through regulated FAD2‐1 gene expression, but temperature modulation of stearic acid content in wild‐type soybean may be more complex, involving in addition to SAD‐A and ‐B, plastid thioesterase genes FATA and FATB.

Evaluation of Soyasaponin, Isoflavone, Protein, Lipid, and Free Sugar Accumulation in Developing Soybean Seeds

A combination of analytical techniques was used to examine and quantify seed compositional components such as protein, lipid, free sugars, isoflavones, and soyasaponins during soybean development and maturation in two Korean soybean cultivars. Protein accumulation was rapid during reproductive stages, while lipid content was only relatively moderately increased. The major carbohydrate saccarides sucrose and stachyose constantly increased during the reproductive stage. Previously published results suggest that the free sugar and lipid content reached their maximal concentrations at a relatively early stage of seed development and remain constant in comparison to other chemical components. The malonylglucosides were the predominant isoflavone form followed by the glucosides, acetyl glucosides, and aglycone forms. As soybean seed matures, total soyasaponin concentration was constantly decreased until the R8 stage. Soyasaponin beta(g) was the major soyasaponin in DDMP-conjugated group B soyasaponins, followed by the non-DDMP counterpart soyasaponin I and soyasaponin A1. The ratio of total isoflavone to total soyasaponin in the developing soybean increased from 0.06 to 1.31. Protein, lipid, and free sugar contents in the developing soybean seeds showed significant positive correlations with conjugated isoflavones and total isoflavone concentration, while the lipid contents showed a negative correlation with the isoflavone aglycone. Protein, lipid, and free sugar contents showed a negative correlation with total group A and B soyasaponins and total soyasaponins; however, only the soyasaponin A content was significantly negatively correlated with free sugar content. Total soyasaponin content was negatively correlated with isoflavone content (r = -0.828 at p < 0.01).

Molecular cloning and expression patterns of Cu/Zn-superoxide dismutases in developing soybean seeds.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

Lipoxygense Activity and C6‐Aldehyde Formation in Comparison to Germination and Vigor during Soybean Seed Development

Developmental changes in lipoxygenase (LOX) activity have been observed during embryo maturation, which suggests that the LOX pathway may be involved in seed development. A study was designed to measure changes in LOX activity and C6‐aldehyde (hexanal and hexenal) formation during seed development and relate these changes to the attainment of maximum germination and vigor. Field grown soybean [Glycine max L. (Merr.) cv. Century] seeds were sampled at regular intervals starting at 31 d after flowering and continuing until maximum dry seed weight [physiological maturity (PM)] in 1990 and 1991. Axes and cotyledons from freshly harvested seeds were evaluated for LOX activity and C6‐aldehyde formation. The remaining seeds were air dried and tested for germination and vigor. Maximum germination occurred at 46 and 43 d after flowering in 1990 and 1991, respectively. Maximum vigor, as measured by accelerated aging, occurred at 54 and 48 d after flowering in 1990 and 1991, respectively, which was 18 d before PM. During seed development, maximum LOX activity occurred in both the axes and cotyledons during the linear phase of seed growth, but declined before PM. In the axes and cotyledons, maximum hexanal and trans‐2‐hexenal formation occurred at approximately 30% of final dry weight, which was before maximum LOX activity, except for axes in 1990. Maximum hexenal and hexenal formation coincided with the first maximum expression of germination, while maximum vigor was more closely related to maximum LOX activity in the axes (1991) and cotyledons. Maximum expression of seed quality (germination and vigor) and maximum LOX and C6‐aldehyde formation were correlated during soybean seed development.

Maturation Proteins and Sugars in Desiccation Tolerance of Developing Soybean Seeds

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 mug/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 mug/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.

Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35 degrees C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20 degrees C as those cultured at 25 degrees C. Both desaturases had negligible activity in seeds cultured at 35 degrees C compared to seeds cultured at 20 degrees C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35 degrees C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20 degrees C cultures. Activities were twofold lower at 25 degrees C and a further threefold lower in 35 degrees C cultures. Cultures from 25 and 35 degrees C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20 degrees C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not.

Oligosaccharide Metabolism and Accumulation in Developing Soybean Seeds

Soluble sugar composition of developing soybean seed (Glycine max L. Merr.) vary with cultivar. This study was conducted to determine the relation between soluble sugar accumulation and associated enzyme activities in developing seed of two soybean cultivars, Williams 82 and Wolverine. Activities of sucrose synthase, invertase, galactinol synthase, and α‐galactosidase were compared to soluble sugar content (hexoses, galactinol, myo‐inositol, oligosaccharides) at seven different reproductive growth stages. A twofold higher dry matter content in mature seeds of Wolverine corresponded to a larger seed size and longer linear seed fill. Increases in sucrose synthase and alkaline invertase activities in both cultivars corresponded to accumulation of 90% total dry matter. Acid invertase activity remained below 45 nmol glucose seed−1 min−1 during seed development. Initial raffinose and stachyose formation occurred at beginning maturity in Williams 82 and full seed in Wolverine. This change in sugar partitioning during late seed development was accompanied by twofold and threefold increases in galactinol synthase activity and galactinol content, respectively, and a substantial decrease in the substrate for this enzyme, myo‐inositol. Highest levels of galactinol synthase activity at the end of linear seed fill in both cultivars, however, preceded the accumulation of the highest stachyose levels by two growth stages. Activity of α‐galactosidase increased linearly in both varieties with time. Relatively high activities of sucrose synthase and alkaline invertase during the accumulation of the majority of dry matter in both cultivars suggest that these enzymes may be associated with this process in soybean seeds. Regulation of raffinose oligosaccharide accumulation in developing soybean seeds also may depend on galactosyl transferase activity.

Phosphoenolpyruvate Carboxylase and Pyruvate Kinase Involvement in Protein and Oil Biosynthesis during Soybean Seed Development

During soybean [Glycine max (L.) Merr.] seed development, sucrose translocated to the seed is metabolized to precursors for protein and oil, the major components of mature soybean seed. while breeders have had success in selecting for increased seed protein or oil concentration, there is little understanding the metabolic events that regulate the partitioning of carbon into these seed components. Enzyme reactions that may determine carbon partitioning to seed protein and oil were examined in six soybean genotypes exhibiting genotypic differences in concentration of seed protein and oil. Developing seed were assayed for the enzymes phosphoenolpyruvate (PEP) carboxylase, pyruvate kinase, malate dehydrogenase, NADP‐malic enzyme, and aspartate and alanine aminotransferases. Correlations between these data and the protein and oil concentrations of each sample were determined. There was no significant difference between the correlation of pyruvate kinase activity with the oil and that with the protein concentration of the seed over development. The correlation of PEP carboxylase activity with protein was generally higher than with oil, suggesting that much of the oxaloacetate (OAA) produced may be used for synthesis of protein precursors. However, the high r values between PEP carboxylase and oil suggested that OAA may also be converted to malate and then to pyruvate (a precursor for oil) via a transhydrogenase system. Therefore, PEP carboxylase and pyruvate kinase activities appeared to contribute to a complex interaction that regulates the metabolic flow of glycolytic carbon into precursors for both protein and oil biosynthesis during soybean seed development.

The Timing and Rate of Phytic Acid Accumulation in Developing Soybean Seeds

The time-course of phosphorus (P) accumulation in the phytic acid fraction of developing soybean (Glycine max [L.] Merr. cv ;Williams 79') seeds as well as the relation of phytic acid P to total P content were determined. Phytic acid was detected early in embryogenesis in field-grown soybeans and accumulated in a linear fashion throughout most of seed development. Although the observed rates of accumulation ranged from 18.7 micrograms phytic acid P per seed per day in pods positioned low on the plant to 33.6 micrograms in pods positioned high on the plant, the final concentrations were the same in all cases. Nearly all of the P translocated to developing seeds was incorporated into phytic acid from the third week after flowering until physiological maturity, with the sum of nonphytic acid P compounds remaining constant. Phytic acid accumulation was also linear throughout development when soybean plants were grown in solutions having nutrient P levels that ranged from severely limiting (2.0 milligrams P per liter) to excess (50 milligrams P per liter). However, there was a pronounced effect on rate of accumulation, which ranged from 7.2 micrograms phytic acid per seed per day with limiting nutrient P to 44.7 micrograms with excess P. The change in level of phytic acid accounted for most of the alteration in total seed P that was caused by altering the P status of the plants. These results support the view that phytic acid synthesis is involved in P homeostasis of the developing soybean seed.

Relationship of Tissue Water Relations to Asparagine Uptake in Developing Soybean Seeds1

Amino acids play an important role in the N nutrition of developing soybean [Glycine max (L.) Merr.] seeds. Factors affecting cotyledonary amino acid uptake within those seeds are not well understood. The objectives of this study were to investigate the effects of altered soybean cotyledon water relations on in vitro asparagine uptake by those cotyledons. Two experimental plant introduction (PI) lines with genotypic differences in seed growth rate and final seed weight were grown in the field. They were PI 416.845 (heavy seeded), and PI 361.058 (light seeded). Cotyledons were sampled at 32 days after anthesis for the light seeded line, and 32 and 45 days after anthesis for the heavy seeded line. Cultivar Evans was used for the studies involving plants grown in the growth chamber. Cotyledons were incubated in uptake buffer solutions containing 10 mmol kg‐1 14C‐asparagine and adjusted to various water potential levels with mannitol (a slowly penetrating osmoticum) or ethylene glycol (a rapidly penetrating osmoticum). Solution water potential levels were approximately 0, −0.5, −1.0, −1.5, and −2.0 MPa. The rate of asparagine uptake for the heavy seeded genotype was greater than that for the light seeded genotype. Uptake was greater at −0.5 and −1.0 MPa than at 0 and −2.0 MPa in the experiments using plants grown in the growth chamber. In the study using field‐grown plants, asparagine uptake was similar in mannitol solutions of 0, −0.5, and −1.0 MPa, but declined in mannitol solutions of −1.5 and −2.0 MPa. Results for ethylene glycol as osmoticum were similar at 32 days after anthesis although reductions of uptake at −1.5 and −2.0 MPa were not as great. Our results indicate that asparagine uptake by excised cotyledons is influenced by the water potential components of their cells, specifically the cell turgor.

Effect of Abscisic Acid on Amino Acid Uptake and Effiux in Developing Soybean Seeds1

The role of abscisic acid (ABA) in regulating growth of developing soybean [Glycine max (L.) Merr.] seeds is not fully understood. The objectives of this study were to characterize the effect of ABA on the in vitro uptake of asparagine and glutamine by isolated immature cotyledons in three soybean plant introduction (PI) lines with genotypic differences in seed growth rate and final seed weight. Cotyledons were incubated in uptake buffer solutions plus 14C‐asparagine or 14C‐glutamine and treatment concentrations of ABA. The ABA levels in the uptake solutions were 0, 10‐7, 10‐6, and 10‐5 M. The uptake rate of glutamine was approximately three times that of asparagine. Among PI lines, the heavy seeded line had a greater rate of asparagine uptake while the light seeded line had a greater rate of glutamine uptake. For asparagine, 10‐6 M ABA depressed uptake compared to the control. For glutamine, ABA enhanced uptake compared to the control at both 10‐6 and 10‐5 M. In an additional experiment, we observed no effect of ABA and K+ on the release of labeled asparagine from excised soybean seed coats. These data indicate that amino acid uptake rates are genotypically dependent and may be influenced by ABA concentration.

The Lipoxygenases in Developing Soybean Seeds, Their Characterization and Synthesis in Vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).

Mass and Composition of Developing Soybean Seeds at Five Reproductive Growth Stages1

Historically, days after flowering (DAF) has been the criterion for sampling developing soybean [Glycine max (L.) Merr.] seeds. Seed development rate, however, differs according to genotype and environmental conditions, leading to variable DAF values for the same seed development stage. Morphologically described growth stages (GS), in contrast, allow a rapid and convenient sampling method for seed development and composition studies. The purpose of this study was to categorize the changes in mass and composition of developing ‘Williams 79’ soybean seeds at five reproductive GS. Seed mass and composition studies demonstrated that R5 and R7 were accurate indicators of seedfill initiation and physiological maturity (PM), respectively. At R5, diy weight, protein, oil, and sugar quantity were low, while moisture content was high. Moisture content decreased slowly between R5 and R7, while seed dry weight, protein, oil, and sugar content increased. Between R5 and R7, 97% of the seed dry weight at maturity had accumulated. Monosaccharides represented 82% of the seed soluble carbohydrates at RS, but decreased to 8% by R6. Sucrose increased two‐fold between R5 and R6, then remained at a high level until R8. Raffinose and stachyose were undetectable until R7, when they represented 11 and 34% of the total soluble sugar, respectively. The largest increase in fatty acid quantity occurred between R5 and R6. Palmitic, stearic, and linolenic acid accounted for 68% of the total fatty acid at R4, but only 21% at R6. Oleic and linoleic acid accumulated most rapidly between R5 and R6, representing 79% of the fatty acid content at R6. Four acetate buffer soluble proteins were resolved by electrophoresis at R5 and eight at R6. Banding patterns remained constant between R6 and R8, while protein quantity of Bands 2 through 8 decreased. The quantity (mg seed−1) of each macro‐ and micro‐element increased at varying rates between R5 and R7. After R7, the seed moisture content decreased rapidly while the quantity and composition of stored components changed little. At harvest maturity (R8), seeds possessed 380 g protein, 230 g oil, and 160 g sugar kg−1 dry weight. These data serve as a useful characterization of changes in seed composition for future studies employing GS.

Changes in Storage Protein Components of Developing Soybean Seeds (Glycine max var. Enrei).

In a study of the relationship between the dynamic changes in cellular organelles and biosynthesis of soybean storage proteins, we found that as the protein components accumulated, the α and α′-subunits of 7S globulin appeared first, followed successively by the acidic and basic subunits of 11S globulin and finally by the β-subunit of 7S globulin. Labeling experiments using tissue slices showed that the radioactivities of (3H)leucine and (14C)N-acetyl-d-glycosamine were incorporated into the trichloroacetic acid insoluble fraction and that (14C)N-acetyl-d-glycosamine was specifically incorporated into 7S.

Sucrose Concentration at the Apoplastic Interface between Seed Coat and Cotyledons of Developing Soybean Seeds

The apoplastic sucrose concentration at the interface between cotyledons and surrounding seed coats of developing soybeans (Glycine max L. Merr. cv Wye) was found by three indirect methods to be in the range of 150 to 200 millimolar. This is an order of magnitude higher than has been reported elsewhere for soybean. It was also higher than the overall sucrose concentrations in the cotyledons and seed coats, each of which was approximately 90 millimolar. By defoliating plants 24 hours before measurement, both the overall sucrose concentration in the cotyledons and the interfacial apoplastic sucrose concentration were reduced by three-fourths. However, there was no day/night difference in overall tissue sucrose concentration of cotyledons or seed coats from intact plants suggesting the existence of a homeostatic mechanism compensating for the diurnal photosynthetic cycle. About 7 hours were required for a tritiated polyethylene glycol-900 solution to fully permeate developing cotyledons (from approximately 220 milligram fresh weight embryos), implying high diffusion resistance through the tissue.These results indicate that a high interfacial sucrose concentration may exist in vivo. They suggest that the saturable carrier-mediated component of sucrose uptake may be of little physiological significance in the outermost cell layers of the cotyledons.

Production of Multiple Vacuoles as an Early Event in the Ontogeny of Protein Bodies in Developing Soybean Seeds1

We have made an ultrastructural study of developing soybean seeds, cultivar Wells [Glycine max. (L.) Merr.], using light and electron microscopy in an attempt to resolve the question of protein body ontogeny. Formation of numerous small vacuoles from the central vacuole was observed as early as 17 days after flowering (DAF). The process continued until each cell contained many small vacuoles. There was a close association between development of rough endoplasmic reticulum (RER) and Golgi bodies and the appearance of protein bodies. Proliferation of RER was observed by 34 DAF, and the vacuoles filled with reserve protein to form distinct protein bodies. Many small vesicles were derived from RER and Golgi bodies but these did not appear to develop into protein bodies. These vesicles may be concerned with transport of storage protein to multiple vacuolar sites for deposition of reserve protein into protein bodies. Protein bodies increased both in size and number as the seed developed. At later stages of seed development, around 60 DAF, RER disappeared and many large protein bodies and numerous lipid bodies were visible in cotyledon cells. The results of our study demonstrates the following: (i) early subdivision of the central vacuoles (17 DAF) appears to be the source of protein bodies; (ii) there is no apparent physical connection between the RER and protein bodies; (iii) accumulation of starch and lipid reserves in soybean seed clearly precedes that of storage protein; (iv) appearance of membrane‐bound polyribosomes (not free ribosomes) is consistent with the onset of storage protein; and (v) at physiological maturity, most cell organelles had disappeared.

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [(3)H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.

Role of Amides, Amino Acids, and Ureides in the Nutrition of Developing Soybean Seeds

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.