Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

Genetic analysis of field germination in soybean (Glycine max. (L) merill.)

Summary Genetic analysis for germination percentage was carried out in the F₃ and F₄ generations of a diallel cross involving six promising genotypes of soybean. Results indicated a high amount of genetic variability and a moderately high heritability together with genetic advance, suggesting a possible improvement for this character through hybridization and selection. Correlations at different levels revealed a strong negative association of germination with only one seed character: seed weight. This observation was further confirmed from path coefficient analysis. These findings strongly suggest that to base selection on seed weight which may not influence the seed quality of soybean.     

Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation,biotinylated protein

A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized. DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa. GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes. The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds. It degrades rapidly at the early stage of seed germination. These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated.     

QTL analyses of seed weight during the development of soybean (Glycine max L. Merr.)

At harvest traits such as seed weight are the sum of development and responses to stresses over the growing season and particularly during the reproductive phase of growth. The aim here was to measure quantitative trait loci (QTL) underlying the seed weight from early development to drying post harvest. One hundred forty-three F(5) derived recombinant inbred lines (RILs) developed from the cross of soybean cultivars 'Charleston' and 'Dongnong 594' were used for the analysis of QTL underlying mean 100-seed weight at six different developmental stages. QTL x Environment interactions (QE) were analyzed by a mixed genetic mode based on 3 years' data. At an experiment-wise threshold of a=0.05 and by single-point analysis 94 QTL unaffected by QE underlay the mean seed weight at different developmental stages. Sixty-eight QTL affected by QE that also underlay mean seed weight were identified. From the 162 QTL 42 could be located on 12 linkage groups by composite interval mapping (LOD>2.0). The numbers, locations and types of the QTL and the genetic effects were different at each developmental stage. On linkage group C2 the distantly linked QTL swC2-1, swC2-2 and swC2-3 each affected mean seed weight throughout the different developmental stages. The DNA markers linked to the QTL possessed potential for use in marker-assisted selection for soybean seed size. The identification of QTL with genetic main effects and QE interaction effects suggested that such interactions might significantly alter seed weight during seed development.     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

Molecular-genetic identification and certification of soybean (Glycine max L.) cultivars

An approach to certification of soybean genotypes has been developed. The procedure employs three methods of DNA analysis based on polymerase chain reaction (PCR): PCR with arbitrary primers (AP PCR), simple sequence repeat polymorphism (SSRP) analysis, and inter-simple sequence repeat (ISSR) analysis. The approach to certification proposed may be used in both genetic and breeding research and seed production. A "certificate" form that reflects the unique characteristics of each cultivar studied is proposed. The results of molecular genetic analysis of allele distribution in genotypes of soybean from different ecological geographic zones permit estimation of the adaptive significance of individual alleles.     

Soluble carbohydrate content of soybean [Glycine max (L.) merr.] somatic and zygotic embryos during development

Summary Somatic and zygotic embryos of soybean cv. Jack were analyzed for soluble carbohydrate, total lipids, and protein during development. Zygotic embryos accumulated trace amounts of fructose, galactose, and galactinol., whereas somatic embryos contained only trace amounts of galactose. Somatic embryos accumulated much higher glucose levels than zygotic embryos. Both somatic and zygotic embryos contain low levels of sucrose, myoinositol, and pinitol. Raffinose and stachyose accumulated in the late developmental stages of zygotic embryos, but only stachyose was found to accumulate in the late stage somatic embryos. Zygotic embryos contained low total lipid levels up to 50 d after flowering (DAF) and then the levels increased to 16% by 55 DAF and 21% at 65 DAF. Somatic embryos had low levels of total lipids throughout development with the maximum of only 4.7%. Soybean zygotic embryos contained about 40% protein throughout development, while the protein concentration of somatic embryos decreased from 44% to 25% as maturation approached. These studies demonstrate that the composition of Jack zygotic embryos is similar to that described for other cultivars during development while the somatic embryo composition and size is markedly different. The low somatic embryo germination often noted might be due to the abnormal development as shown by a composition different from that of mature zygotic embryos. The low concentration of the raffinose series sugars might be especially important factors.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Cadmium-induced senescence in nodules of soybean (Glycine max L.) plants

The relationship between cadmium-induced oxidative stress and nodule senescence in soybean was investigated at two different concentrations of cadmium ions (50 and 200 μM), in solution culture. High cadmium concentration (200 μM) resulted in oxidative stress, which was indicated by an increase in thiobarbituric acid reactive substances content and a decrease in leghemoglobin levels. Consequently, nitrogenase activity was decreased, and increases in iron and ferritin levels were obtained. Senescent parameters such as ethylene production, increased levels of ammonium and an increase in protease activity were simultaneously observed. Glutamate dehydrogenase activity was also increased. Peroxidase activity decreased at the higher cadmium concentration while the lower cadmium treatment produced changes in peroxidase isoforms, compared to control nodules. Ultrastructural investigation of the nodules showed alterations with a reduction of both bacteroids number per symbiosome and the effective area for N₂-fixation. These results strongly suggest that, at least at the higher concentration, cadmium induces nodule senescence in soybean plants.     

Fatty acid composition of soybean (Glycine max (L.) merr.) somatic embryos

Summary Somatic embryos from four soybean cultivars were matured for 30 and 45 d. Success of embryo germination was evaluated for each length of maturation. The percentage of somatic embryos undergoing successful germination, as defined by rooting and shoot emergence, was greater for embryos matured 45 d than for embryos matured 30 d. Therefore, embryos matured for 45 d are probably physiologically more mature than embryos matured for 30 d. Relative percentages of fatty acids comprising oils and lipids of somatic embryos were determined for each length of maturation and for each cultivar. Variation in relative percentages of palmitic acid, oleic acid, and linoleic acid was affected by length of maturation. However, these changes were genotype dependent. A significant interaction between the cultivars Clark and Maple Arrow and stage of maturation was observed for levels of oleic acid. No other interactions were observed. These data suggest that if changes in relative percentages of certain fatty acids are associated with soybean somatic embryo maturation the changes are genotype dependent. This is journal paper No. J-12870 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2763. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that also may be suitable. This research was supported in part by grants from the American Soybean Association Development Foundation and the Iowa Soybean Promotion Board.     

Phytochrome-driven changes in respiratory electron transport partitioning in soybean (Glycine max. L.) cotyledons

After it was observed that light induces changes in electron partitioning between the cytochrome and the alternative pathway, the focus interest was directed to assessing what type of photoreceptors are involved and the extent of such modifications. Studies on 5-day-old soybean (Glycine max L.) cotyledons using an oxygen isotope fractionation technique showed that phytochrome is involved in changes in electron partitioning between the cytochrome and the alternative respiratory pathway. A follow-up of a previous study, showing that 5 min of white light caused changes in mitochondrial electron partitioning, demonstrated that while blue light was not involved in any such changes, red light caused a significant shift of electrons toward the alternative pathway. The major shift, observed after 24 h of light, is mainly due to both a decrease in the activity of the cytochrome pathway and an increase in the activity of the alternative pathway. The involvement of a phytochrome receptor was confirmed by demonstration of reversibility by far-red light. The implications of the possible involvement of phytochrome in the regulation of mitochondrial electron transport are discussed.     

Growth and nitrogen fixation in soybean (Glycine max (L.) Merr.) as affected by gibberellic acid treatment during reproductive development

Summary Nodulated soybean (Glycine max (L.) Merr.) plants cv. Williams were grown on a N-free medium and sprayed daily, after flowering, with a solution of 0,0.5 or 1.0 mg/l gibberellic acid (GA₃). Relative growth rate, net assimilation rate and mean N₂-fixation rate decreased in control plants from flowering to mid-pod filling. The decline of growth and N₂-fixation was attenuated by GA₃. Lesser decline of growth and N₂-fixation in GA₃-treated plants was linked to enhancement of vegetative growth, to delay of fruit development and to arrest of root decay.

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Resumen Plantas de soja (Glycine max L. Merr.) cv. Williams, noduladas, crecieron en un medio libre de N; se rociaron diariamente, después de la floración, con una solución de 0, 0.5 ó de l mg l^–1 de àcido gibberélico (GA₃). Las tasas de crecimiento relativo, de asimilación neta y de fijación de nitrógeno disminuyeron en las plantas control desde la floración hasta la mitad del periodo de crecimiento de las vainas. La declination del crecimiento y fijación, de N₂ fue atenuada por el ácido gibberélico. Esta menor disminución del crecimiento y de la fijación de N₂ en las plantas tratadas con GA₃, esta asociada a un mayor crecimiento vegetativo, a un retraso en el desarrollo del fruto y a una menor descomposición radicular.

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Résumé Des plants nodulés de soja (Glycine max L. Merr.) ont été cultivés en milieu dépourvu d'azote et aspergés quotidiennement, après la floraison, avec une solution d'acide gibbérellique (GA₃) à des concentrations de 0,0,5 et 1,0 mg/l. Dans le cas des plants témoins, les taux de croissance, d'assimilation nette et de fixation moyenne de l'azote diminuent entre la floraison et la moitié du remplissage des gousses. Chez les plants traités par GA₃, la croissance et la fixation de N₂ diminuent moins, et on constate un accroissement de la croissance végétative, un ralentissement du développement des fruits et un arrêt de la dégénérescence racinaire.

     

两种羟基苯乙酸对大豆萌发的化感效应研究

通过田间和室内试验相结合,利用高效液相色谱检测大豆根际土境和残茬腐解液中的对羟基苯乙酸和间羟基苯乙酸的含量;在培养皿内研究两种酚酸对大豆生长抑制效应;研究了二者对大豆DNA熔点(Tm)的影响,并采用SDS—PAGE方法研究二者对根系蛋白合成的影响．结果表明,在大豆根际土境和残茬腐解液中检测到对羟基苯乙酸;两种羟基苯乙酸处理后,大豆例根效和主根长均显著减少,表现出明显的化感抑制效应,间羟基苯乙酸的抑制效应更明显;两种羟基苯乙酸使大豆DNA的熔点(Tm)下降,一些小分子蛋白的合成受到影响,从而抑制大豆生长,表现出典型的化感效应。     

Identification of genomic regions determining flower and pod numbers development in soybean （Glycine max L.）

Flower and pod numbers per plant are important agronomic traits underlying soybean yield.So far quantitative trait loci (QTL) detected for flower and pod-related traits have mainly focused on the final stage,and might therefore have ignored genetic effects expressed during a specific developmental stage.Here,dynamic expressions of QTL for flower and pod numbers were identified using 152 recombinant inbred lines (RILs) and a linkage map of 306 markers.Wide genetic variation was found among RILs;17 unconditional and 18 conditional QTL were detected for the two traits at different developmental stages over two years.Some QTL were detected only at one stage and others across two or more stages,indicating that soybean flower and pod numbers development may be governed by time-dependent gene expression.Three main QTL (qfn-Chr18-2,qfn-Chr20-1,and qfn-Chr19) were detected for flower number,and two main QTL (qpn-Chr11 and qpn-Chr20) were detected for pod number.The phenotypic variation explained by them ranged from 6.1% to 34.7%.The markers linked to these QTL could be used in marker-assisted selection for increasing soybean flower and pod numbers,with the ultimate aim of increasing soybean yield.Comparison of the QTL regions for flower and pod numbers traits with the related genes reported previously showed that seven and four related genes were located in the QTL regions of qfn-Chr11 and qfn-Chr19,respectively.Tbese results provide a basis for fine mapping and cloning of flower and pod development-related genes.     

Antioxidant enzymes and isoflavonoids in chilled soybean (Glycine max (L.) Merr.) seedlings

Changes of activity antioxidant enzymes and of levels of isoflavonoids were studied in the roots and hypocotyls of the etiolated soybean (Glycine max (L.) Merr. var. Essor) seedlings, submitted to cold. Prolonged exposure to 1 degrees C inhibited hypocotyl and root elongation and limited their growth after seedlings were transferred to 25 degrees C. Roots were more sensitive to chilling than hypocotyls. At 1 degrees C a gradual increase in MDA concentration in roots but not in hypocotyls was observed. An increase in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activity in hypocotyls was observed both at 1 degrees C and after transfer of plants to 25 degrees C. In roots, CAT activity increased after 4 days of chilling, while SOD activity only after rewarming. L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity decreased in roots of chilled seedlings, but did not change in hypocotyls until activity increased after transfer to 25 degrees C. The content of genistein and daidzein increased after 24 h of treatment by low temperature and then decreased with prolonged chilling in hypocotyls and remained high in roots. However, it should be noted that genistin level (genistein glucoside) in chilled hypocotyls is 10 times higher than in roots, despite falling tendency. The role of antioxidant enzymes and isoflavonoids in preventing chilling injury in hypocotyls and roots of soybean seedlings is discussed.     

Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45–50% were obtained in liquid medium and 55–60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1–2 mm in size) were obtained in 5–6 weeks, which upon transfer to MSB medium with 0.5 mg 1^–1 each of 2,4-D, BA, Kn and 500 mg 1^–1 CH produced compact green calli in 4–6 weeks. After 3–4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1^–1 each of BA, Kn, ZT, 0.1 mg 1^–1 NAA and 500 mg 1^–1 CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA₃ enhanced shoot regeneration up to 30%. Shoots of 0.5–1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1^–1 TH and 0.5 mg 1^–1 GA₃ for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2–3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1^–1 IBA or 0.5 mg 1^–1 NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.Abbreviations BA 6-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellic acid - IBA indole-3-butyric acid - Kn kinetin - MES 2[N-morpholino] ethane sulfonic acid - NAA naphthaleneacetic acid - TH thidiazuron - ZT zeatin     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.