Immature Soybean Seeds Research Articles

Purification and some properties of inositol 1,3,4,5,6-Pentakisphosphate 2-kinase from immature soybean seeds.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase was purified from immature soybean seeds harvested approximately 5 weeks post-anthesis. A crude extract was clarified using polyethyleneimine and purified by chromatography on DEAE-cellulose, Cibacron Blue 3GA-agarose, Toyopearl DEAE 650M, and Toyopearl phenyl 650M columns. The enzyme had a relative molecular mass, M(r), of 52,000 as determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and retained 50% of its activity after 6 weeks at 0 degrees C. The Km values for inositol 1,3,4,5,6-pentakisphosphate and MgATP, respectively, were 2.3 microM and 8.4 microM, and the Vmax was 243 nmol/min/mg. The pH and temperature optima, respectively, were 6.8 and 42 degrees C. Maximum activity was obtained when the magnesium ion concentration was 4 mM. The kinase specifically phosphorylated the 2-position on the inositol ring and could also utilize D-inositol 1,4,5,6-tetrakisphosphate as a substrate. The K for the reaction was 14, indicating that the enzyme may be involved in both inositol hexakisphosphate formation in maturing seeds and ATP resynthesis in germinating seeds. Substrate concentrations in mature seeds were favorable for ATP formation, whereas additional factors appeared to drive the accumulation of inositol hexakisphosphate in maturing seeds.

Carotenoid Components in Soybean Seeds Varying with Seed Color and Maturation Stage

The difference in carotenoid components among various color types of soybean seeds, and the changes in carotenoid composition during seed development were examined by reverse-phase high-performance liquid chromatogrphy (HPLC). Lutein was the major carotenoid component in seed extracts from the common yellow soybean and from a variety having a black seed coat. Green soybean seeds contained several xanthophylls in addition to lutein. None of the mature soybean seeds contained β-carotene, a part from a trace amount being detected in a local variety of green soybean. The total carotenoid and lutein contents were higher in green soybeans than in the yellow types, and the estimated total amount of carotenoids correlates with that of chlorophylls. The thylakoid membrane residue in the plastids of green soybean had lost its functional lamella structure. Immature soybean seeds contained a green-vegetable type of carotenoids including α- and β-carotene. The amount of β-carotene decreased more rapidly than that of l...

A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean

The assembly of 11S globulin seed storage proteins in plants is regulated in part by the activity of a protease that cleaves between asparagine and glycine residues. Post-translational cleavage of subunit precursors into acidic and basic polypeptides is associated with the ability of subunits in trimers to aggregate into hexamers in vitro. An activity is present in extracts from immature soybean seeds that specifically cleaves immature 11S seed storage proteins of soybean and Vicia faba into the polypeptides of the mature proteins. Sequence microanalysis has been used to demonstrate that proglycinin and prolegumin are cut at the legitimate site when proteins synthesized in vitro are used as substrates. A single amino acid change in the cleavage site renders the substrate uncleavable. The protease responsible for this activity also hydrolyzes a synthetic octapeptide whose sequence reproduces four amino acids on either side of the glycinin subunit G4 cleavage site. This assay permitted the purification and characterization of the protease. It is a glycosylated enzyme with an acidic pH optimum and a molecular mass of about 45 kDa in solution.

Effects of Exogenous Auxins on Expression of Lipoxygenases in Cultured Soybean Embryos

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.

Starch breakdown in developing soybean seeds(Glycine max cv. Enrei).

Immature soybean seeds accumulate starch as a transient reserve material that declines later in seed development. Phosphorylase activity in cotyledons rapidly increased 40-50 days after flowering (DAF) corresponding to the starch breakdown. Decomposition of starch granules was intracellularly compartmented to arm loplasts which were associated with rough endoplasmic reticulum. Simultaneously with the phosphorylase activation, phytase activity appeared and increased till 45 DAF, and transitory decline of phytic acid was observed at the maximum of phytase activity. These results suggest that phosphorolysis may be involved in starch breakdown, and that phytic acid is mobilized in developing soybean seeds.

The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using Mössbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in Mössbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect Mössbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) Mössbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The Mössbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these Mössbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.

Inheritance and expression of foreign genes in transgenic soybean plants

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R(1)) and second (R(2)) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for beta-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for beta-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R(1) generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R(2) generation. R(1) plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R(2) generation, demonstrating recovery of both homozygous and heterozygous R(1) plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.

Nuclear factors interact with a soybean beta-conglycinin enhancer.

Upstream sequences of the gene encoding the alpha' subunit of beta-conglycinin were analyzed for interactions with nuclear proteins from immature soybean seeds. Two factors were identified that interact with specific sequence elements within 257 base pairs 5' of the transcription start site. One factor, SEF 3, binds exclusively to a region composed of two elements located at -183 to -169 base pairs and -153 to -134 base pairs relative to the start of transcription. Each of these sites includes the hexanucleotide sequence AACCCA, which may serve as a primary recognition sequence. During seed development, SEF 3 binding activity was found to increase in soybean embryos during the time of beta-conglycinin synthesis and to decrease as seeds neared maturity. The position of the SEF 3 binding sequence corresponds with a previously reported seed-specific enhancer region, and it seems likely that this factor may act as a positive regulator of transcription of the beta-conglycinin, alpha' subunit gene in developing soybean seeds. The second factor, SEF 4, also binds within the -257 to -77 region but also interacts with sites located further upstream.

Immunoaffinity Techniques Applied to the Purification of Gibberellins from Plant Extracts

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.

Stable Transformation of Soybean (Glycine Max) by Particle Acceleration

We have used particle acceleration by electric discharge to introduce DNA-coated gold particles into meristems of immature soybean seeds. Approximately 2 percent of shoots derived from these meristems via organogenesis were chimeric for expression of the introduced gene. Experiments using this protocol have resulted in mature RO and R1 plants expressing the introduced gene(s). We regard this work as a major step in the practical application of genetic engineering to the useful modification of food-crop species.

Changes in Viability, Germination, and Respiration of Freshly Harvested Soybean Seed During Development

As soybean [Glycine max (L.) Merr.] seeds develop and mature, they gradually desiccate to cause a switch in metabolic processes from a developmental to a germination phase. This investigation was conducted to determine when fresh (nondesiccated), immature soybean seeds acquire the ability to germinate and develop into normal seedlings. Soybean pods were detached from ‘DeSoto’ plants at four developmental stages ranging from full seed (first time the seed filled the locule) to yellow pod [physiological maturity (PM)] in 1981 to 1983. Freshly harvested seeds were immediately evaluated for seed moisture, dry seed weight, viability (radicle protrusion through the testa), germination (normal seedling development), and respiration. Seed and axis dry weight accumulation and moisture loss were nearly identical throughout development. Near maximum viability (93%) of freshly harvested seeds occurred when the seed first filled the locule (35% of final dry seed weight); however, maximum germination (development of normal seedlings) did not occur until maximum accumulation of dry seed weight (PM). Respiration during germination of fresh seed harvested before PM declined prior to radicle emergence without a concomitant decrease in seed moisture, followed by an increase in respiration typical of a germinating seed. Contrary to previous reports, freshly harvested immature soybean seeds did not need a desiccation period prior to expression of maximum viability and normal seedling development.

Biology of Megalotomus parvus (Hemiptera: Alydidae) on Selected Leguminous Food Plants

Laboratory studies with Megalotomus parvus (Hemiptera: Alydidae), feeding on seeds or pods of legumes, and on different parts of the soybean plant, Glycine max, demonstrated that nymphal survivorship, developmental time and body weight at adult emergence, and adult reproductive parameters, survivorship, longevity and body weight changes, exhibited differences depending on the food source utilized. In general, mature soybean seeds (R8 stage) were the most suitable food. The greatest nymph and adult survivorship and the greatest adult longevity were obtained from this food source. Body weight at certain ages and fecundity of females was also greater on mature soybean seeds than on the other foods. Immature soybean seeds for nymphs, and soybean pods for adults, were the second most suitable food, followed by green beans, Phaseolus vulgaris and immature lupin, Lupinus luteus seeds (for nymphs) and lupin pods (for adults); nymph and adult performance was drastically reduced on lupin seeds/pods and no nymphs survived to adult on soybean stems or leaves. On all foods tested, duration of the nymphal stages differed significantly, with the first stage being the shortest, and the fifth the longest. Females achieved significantly greater body weight than males on all foods, except on lupin pods. Results of these laboratory studies and those of field observations suggest that mature soybean seeds are the most important food source in the seasonal phenology of M. parvus

Daño de hongos e insectos a semillas inmaduras de soja en el trópico

El propósito del trabajo que informamos fue estudiar si la incidencia de plagas y sus daños cambian al cosechar semilla de soja en diferentes etapas de desarrollo en condiciones tropicales. Las cultivares 'BSR 201', del Grupo de Madurez II, y 'Cumberland', del Grupo de Madurez III, se sembraron en tres ambientes en Puerto Rico en 1981 y 1982. Para determinar la edad de las semillas en desarrollo, las flores se identificaron el día que abrieron con una etiqueta colocada inmediatamente debajo del nudo donde estaban las flores. La etiqueta decía la fecha de floración y el número de flores por nudo. Las semillas se comenzaron a cosechar a los 24 días después de la floración (DAF) y continuó semanalmente hasta que las plantas alcanzaron su madurez fisiológica a los 59 DAF. Los síntomas de cada plaga se identificaron y se contó su incidencia en una muestra de 50 semillas de cada cultivar, cada fecha de cosecha y cada ambiente. Las semillas inmaduras cosechadas a los 24 DAF presentaron síntomas de mancha púrpura [Cercospora kikucii (T. Matsu. & Tomoyasu) Gardner], añublo pulverulento [Peronospora manshurica (Naum.) Syd. ex Gaum.], antracnosis [Colletotrichum dematium (Pers. ex Fr.) Grove var. truncatum (Schw.) Arx.], tizón de la vaina (Phomopsis spp.) y daños de las larvas del barrenador de la vaina [Etiella zinckenella (Treitschke)] y de la chinche apestosa [Nezara viridula (L.)]. Los daños máximos causados por estas plagas se observaron en las semillas cosechadas 38 DAF o más edad dependiendo de la plaga y del ambiente. Nuestros resultados indican que la cosecha de semillas de soja inmaduras a los 31 DAF disminuye la incidencia del daño de las plagas indentificadas en este estudio.

Infection of immature soybean pods and seeds by Phomopsis longicolla

Phomopsis longicolla Hobbs, a causal agent of Phomopsis seed decay, colonizes the seed coats and embryos of mature soybean (Glycine max (L.) Merr.) seeds. The mode of infection of immature soybean pods and seeds by P. longicolla was examined using scanning electron microscopy. Phomopsis longicolla penetrated directly through pod walls by appressoria. Penetration through stomata in the pod surface was not observed. Immature seeds were also penetrated directly. Heavily infected immature seeds showed extensive colonization of the seed coats and embryos.

Isolation and Sustained Division of Protoplasts From Cotyledons of Seedlings and Immature Seeds of Glycine max L.

Five enzyme mixtures were evaluated for the isolation of protoplasts from seedling cotyledons of five varieties of soybean. An enzyme mixture of 0.1% w/v Pectolyase Y23, 0.2% Onozuka RS cellulase, and 1.0% Rhozyme gave the highest protoplast yield. This mixture also released protoplasts from cotyledons of immature seeds of one of the soybean varieties. A mixture of 4.0% Meicelase, 2.0% Rhozyme and 0.03% Macerozyme R10, previously used for isolating protoplasts from seedling cotyledons of several genera other than Glycine, failed to release protoplasts from cotyledons of soybean seedlings, but produced protoplasts from cotyledons of immature soybean seeds. These results suggest some differences in the composition of walls of cotyledon cells of immature seeds and seedlings of soybean, and also between soybean seedlings and those of other plants so far examined as a source of protoplasts. The protoplast culture medium of Kao (1977) sustained protoplast division. On average, in excess of 30% of the seedling cotyledon protoplasts from each of the five varieties tested had undergone division within the first eight days of culture.

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)(+) RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I-IV. Polypeptides synthesized in vitro were labeled with ((35)S)-cysteine and ((3)H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I-IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.

Soluble carbohydrates of dry and developing seeds

Sucrose was present in seeds of 31 species at all ages and stages of their development. The raffinose family of oligosaccharides is present in most mature and dry seeds; tomato and tobacco seeds contain planteose, whereas sesame seeds contain this sugar and a higher member of the planteose series. Cotton seeds contain raffinose, stachyose, verbascose and an unidentified ketose. Free monosaccharides were not detected in any of the dry seeds; although free glucose and fructose were detected in some immature seeds, these sugars decreased in amount and eventually disappeared during seed maturation. Sucrose, stachyose, raffinose and verbascose accumulated, in developing soybeans, in that sequence. Maltose, a sugar rarely found in plant tissues, is present in immature soybean and honey locust seeds but does not occur in the other seeds examined. It increases to a maximum during development, subsequently decreases in amount during maturation and ripening and eventually disappears completely. The petioles of old leaves and stems of the soybean plant contain maltose, but the petioles of young soybean leaves, empty pods, leaf blades and roots do not.

Iron Availability to Rats from Soybeans

Experiments were carried out to determine the effects of endogenous phytic acid in soybean seeds (Glycine max, var. "Amsoy") on the bioavailability of 59Fe to iron-depleted rats. Soybean plants were grown in nutrient solutions labeled with 59Fe supplied at either 0.4 or 1.0 ppm iron in solution. Immature and mature soybean seeds were harvested and fed to male rats as a single dose. The phytic acid content of the immature and mature seeds averaged 0.61 and 1.71% dry weight, respectively. Varying the nutrient solution iron concentration in the mature soybean seeds. When the 59Fe-labeled seeds were fed to iron-deficient rats, 59Fe from the mature seeds was more available than that from immature seeds even though the mature seeds contained approximately three times as much phytic acid. Autoclaving the mature seeds did not affect the absorption of 59Fe by rats from the seeds. Immature soybean seeds contained much more insoluble 59Fe than did mature soybean seeds. It was concluded that the directly correlated to the phytate content of the seeds; also immature soybean seeds apparently contain a factor or factors (other than phytic acid) that depresses iron availability.

Desaturation of palmitate and stearate by cell-free fractions from soybean cotyledons.

AbstractHomogenates of cotyledons from immature soybean seeds were fractionated by centrifugation. Cell‐free preparations actively desaturated 1‐14C‐palmitate and 1‐14C‐stearate to produce 9,10‐unsaturated acids. The 9‐desaturase activity was present mainly in supernatant fractions (22,000 and 105,000 xg). These fractions also desaturated oleate to linoleate and elongated C14, C16 and C18 acids. In view of this versatility of desaturase systems in the soybean, including the 9‐desaturase(s) for C14, C16 and C18 saturated acids, there would not seem to be further need to consider a separate plant pathway for biosynthesis of unsaturated fatty acids.

Enzymatic Synthesis of Steryl Glucoside by a Particulate Preparation from Immature Soybean Seeds<xref ref-type="fn" rid="fn1"><sup>*</sup></xref>

Enzymatic Synthesis of Steryl Glucoside by a Particulate Preparation from Immature Soybean Seeds<xref ref-type="fn" rid="fn1"><sup>*</sup></xref>