Developing of a simple method for screening soybean seedling cadmium accumulation to select soybean genotypes with low seed cadmium

Significant inter-cultivar differences of soybean seed cadmium (Cd) concentrations arise from the inter-cultivar differences in root Cd accumulation ability. The Cd concentration in the shoots of plants at the vegetative stage is already controlled by the roots Cd concentration in the same way that it determines seed Cd concentration. Based on these results we conjectured that there is no need to wait until the full maturity stage because the inter-cultivar difference in seed Cd concentration can be predicted from the Cd concentration in the shoots of seedlings. To test this theory, we cultivated 150 cultivars/lines to the harvest stage in a field not contaminated with Cd and measured seed Cd concentration. We also planted seeds in pots filled with contaminated soil, cultivated them for 3 weeks, and measured the Cd concentration of the seedling obtained at the 5th-node (V5) stage when the 4th trifoliolate leaf had expanded. The 150 cultivars/lines were roughly divided into 2 groups based on the relationship between these 2 Cd concentrations. One group was cultivars in which seedlings and seeds both had low Cd concentrations (low Cd accumulation group, n?=?129), and the other group was the opposite (high Cd accumulation group, n?=?21). Further, when we predicted seed Cd concentration using the ratio of Cd and Zn concentrations in seedlings, we were able to clearly divide the 2 groups with no overlap. Measuring Cd/Zn in seedlings therefore makes it possible to select cultivars with low Cd accumulation tendency readily, without waiting to harvest the seeds. Additionally, by investigating genealogies we found that varieties in the high-Cd accumulation group were descended from certain cultivars such as Harosoy.     

Relationships between soybean shoot nitrogen components and soybean aphid populations

Defining the relationships between soybean (Glycine max [L.] merr.) shoot nitrogen (N) components and soybean aphid (Aphis glycines Matsumura) populations will increase understanding of the biology of this important insect pest. In this 2-year field study, caged soybean plants were infested with soybean aphids (initial infestation of 0, 10, 50, or 100 aphids plant^?1) at the fifth node developmental stage. Soybean aphid populations, soybean shoot dry weight, and shoot concentrations of nitrate-N, ureide-N, and total N were measured starting at full bloom through full seed soybean development stages. Soybean aphid population as well as shoot concentration of ureide-N increased rapidly starting at full bloom, peaked at beginning seed, and dramatically decreased by full seed soybean reproductive stages. Regression analysis indicated significant relationships (P = 0.01; r = 0.71) between soybean aphid populations and shoot ureide-N concentration. Thus, soybean aphid population levels appear to coincide with shoot ureide-N concentrations in the soybean plant.     

Two subtilisin-like proteases from soybean

Two subtilisin-like proteases (SLP) were identified in soybean ( Glycine max [L.] Merr.). The first, SLP-1, was localized in seed coats early in seed development, but became undetectable with anti-SLP-1 antibodies as seed fill progressed. A partial purification of SLP-1 was achieved using a two step chromatographic procedure. NH₂-terminal sequence analysis of the partially purified enzyme permitted primers to be designed that were used to amplify cDNA encoding SLP-1. A genomic clone encoding SLP-1 was also obtained. Characterization of the cDNA and partially purified SLP-1 revealed the initial translation product was an 82 694 MW precursor. After removal of a signal peptide, the mature protein was formed by removal of an NH₂-terminal propeptide. A COOH-terminal peptide also appeared to be removed from some of the protease molecules. DNA blot analysis suggested that at least one additional SLP gene was present in soybean. The second gene, SLP-2, was subsequently cloned and characterized. Although the coding regions for SLP-1 and SLP-2 were homologous, their promoters were quite divergent. RT-PCR revealed that SLP-2 message was found in the mature plant and in cotyledons of germinating seeds. Although SLP-2 mRNA could be identified in developing seeds, the message was at least an order of magnitude less abundant than that for SLP-1, and it was mis-spliced such that a chain termination event would preclude obtaining a product. As with SLPs from other organisms, the functions of the soybean proteases are unknown. However, SLP-1 is one of only a few proteins from soybean seed coats that have been described.     

Delayed luminescence of air-dry soybean seeds as a measure of their viability

Delayed luminescence (DL) excited by visible light has been observed in air-dry seeds of various plants. Changes in seeds of soybeans ( Glycine max L. W.) which occur during natural and accelerated aging were reflected in subsequent loss of vigor and DL increase. The increase of the luminescence is caused by the changes in seed biopolymers, leading to a decrease in their ability to take up water. The method makes it possible to measure DL from a single seed. With aging, the DL was shifted toward higher intensities and there was greater variability. By monitoring the DL of seeds it may be possible to evaluate their quality and storage capability.     

Membrane organization in soybean seeds during hydration

The ability of seeds to withstand dehydration indicates that their membranes may maintain structural integrity even when dry. Analysis of polar lipids (the principal lipidic constituents of the membranes) from soybean seeds (Glycine-max (L.) Merr.) by X-ray diffraction indicated that even in the dehydrated state the lipids retained a lamellar (bilayer) configuration. As the degree of hydration was raised, evidence of some structural alteration (apparent as an abrupt increase in bilayer spacing) was obtained from diffraction patterns of both the extracted lipid and particles of seed tissue. In seed tissue this increase in bilayer spacing occurred at a hydration level just above that at which free water could be detected by nuclear-magnetic-resonance analysis. The water content at which the increase in bilayer spacing occurred was higher in the seed tissue than in the extracted polar lipids, probably because other cell components restricted the availability of free water in the seed.Abbreviation NMR nuclear-magnetic resonance     

Expression of polyhydroxybutyric acid as a model for metabolic engineering of soybean seed coats

The feasibility of genetically engineering soybean seed coats to divert metabolism towards the production of novel biochemicals was tested. The genes phbA, phbB, phbC from Ralstonia eutropha each under the control of the seed coat peroxidase promoter were introduced into soybean and the production of polyhydroxybutyrate (PHB) was assayed. The analysis of seed coats arising from 4 independent transformation events demonstrated that PHB was produced at a mean of 0.12% seed coat dried weight with individual values up to 0.36%. These values demonstrate that it is possible to metabolically engineer soybean seed coats.     

Soy polyol formulations as novel seed treatments for the management of soil-borne diseases of soybean

Polyurethanes prepared from vegetable oils display a number of desirable properties useful for many commercial and industrial applications. One unique application is that of an agricultural seed treatment. Seed treatments are used to incorporate pesticides onto the seed coat and to decrease the disease susceptibility of the seed during its germination in the soil. In addition, by altering the movement of water across the seed coat and by incorporating protective pesticides in the coating, seed coating polymers can enhance the germination and survival of the seed under adverse environmental conditions. Soy polyols alone, and in combination with glycerin, polymerized with 4,4'-diphenylmethane diisocyanate (MDI) were studied for their seed treating properties and impact on soybean seed germination. The cross-linking density and properties of these polyurethane compounds were varied by changing the isocyanate/hydroxyl molar ratios. In order to optimize the coating qualities and to increase the efficiency of the coating, acetone was also studied as a diluting solvent to reduce the viscosity of the polyurethane mixture prior to polymerization on the seed coat. Optimal polymerization and resulting germination (95%) were obtained using a 1:1 isocyanate/hydroxyl molar ratio consisting of a mixture of soy polyol 180 and glycerin, and the use of an equal volume of acetone as a dilution solvent. This optimal polyurethane seed treatment had several desirable qualities including: reduced viscosity, decreased seed coating thickness, increased seed coating uniformity and permitted larger volumes of seed to be treated with the same volume of polymer. This optimal seed treatment increased the soybean seed germination by 15%, as compared with untreated seed. In addition, preliminary studies of the compatibility of these unique formulations with commercial and experimental fungicides also support the use of these polymers as seed treatments due to their enhanced stability, longevity and slow active ingredient water teaching characteristics. Compatibility of these seed coating polymers as formulations with captan, metalaxyl, thiabendazole and novel antimicrobial lipids and triterpenoid compounds display that the active ingredients can readily provide a zone of fungal inhibition around the seed as it germinates in the presence of Macrophomina phaseolino, causal agent of charcoal rot of soybeans. However, the release of the active ingredient from the polyol seed treatments is less affected by water leaching as compared to commercially available water-soluble seed treating polymer formulations. This is most likely due to the polyols unique polymer cross-linking characteristics. These results support the continued exploration of soy polyol derived polymers as seed coating compounds.     

Lipoxygenases affect protease inhibitor levels in soybean seeds

No physiological role has been assigned to lipoxygenases (LOX) (linoleate:oxygen oxidoreductase, EC 1.13.11.12) present in soybean seeds. The development of LOX null lines has provided an ideal genetic material to determine possible physiological roles for seed LOX. Genetic elimination of seed LOX was followed by a corresponding decrease in the levels of protease inhibitors in soybean seeds. Participation of LOX in the biosynthetic pathway leading to jasmonates, which activate protease inhibitor genes, has been demonstrated in leaves of several plant species. It is conceivable that LOX has a similar role in the developing seed.     

Differential responses to K deficiency among soybean cultivars

The seed yield per unit of potassium applied differed for five soybean cultivars which were grown to maturity under different K regimes in a glasshouse. Whereas Dodds was the most responsive cultivar to moderate increases in K supply, the cultivar Bragg was the most efficient in its ability to produce seed with low levels of available K; Lee and Forest were the least efficient cultivars while Bossier and Dodds were of intermediate efficiency. The basis for the efficiency of cv. Bragg was that the growth of its tops, as indicated by mature stem weights and its roots, were less affected by reduced K supply than those of other cultivars. This enabled it to produce more pods under K-deficient regimes, resulting in a greater seed yield per plant. The percentage reduction in oil/protein ratios in the seed of the five cultivars under moderate K deficiency correlated closely with reductions in seed yield. However, changes in this ratio were poorly related to the K percentages in the seed. All cultivars experienced an impairment of plant senescence under K deficiency as evidenced by a reduction in leaf abcission and a delay in pod maturity. The existence of genetic diversity in K-use efficiency means that breeding programmes could utilize K-efficient germplasm in developing new cultivars for soils not naturally high in potassium.     

SCB1, a BURP-domain protein gene,from developing soybean seed coats

We describe a gene, SCB1 (Seed Coat BURP-domain protein 1), that is expressed specifically within the soybean (Glycine max [L.] Merrill) seed coat early in its development. Northern blot analysis and mRNA in situ hybridization revealed novel patterns of gene expression during seed development. SCB1 mRNA accumulated first within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurred prior to the period of seed coat maturation and seed filling and before either of the layers started to degrade. SCB1 may therefore play a role in the differentiation of the seed coat parenchyma cells. In addition, the protein product appears to be located within cell walls. The SCB1 gene codes for a new member of a class of modular proteins that possess a carboxy-terminal BURP domain and a variety of different repeated sequences. The sequence of the genomic clone revealed the insertion of a Tgm transposable element in the upstream promoter region but it is not certain whether it contributes to the tissue-specific pattern of SCB1 expression.     

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.     

The outermost cuticle of soybean seeds: chemical composition and function during imbibition

Seeds of different cultivars of Glycine max (L.) Merr. (soybean) have strikingly different rates of water imbibition. Seeds that readily imbibe water are termed 'soft', while those that remain non-permeable, even after several days in water, are referred to as 'hard', 'stone', or 'impermeable' seeds. What prevents soybean hard seeds from taking up water? Previous work established that the initial imbibition of soft soybean seeds correlates with the presence of small cracks in the outermost cuticle that covers the seed coat, prompting a detailed analysis of soybean seed coat cutin. In this paper, it is shown that the outermost cuticle of the seed coat has an unusual chemical composition, lacking typical mid-chain-hydroxylated fatty acids but being relatively rich in other types of hydroxylated fatty acids. The cuticle of the impermeable cultivar studied contained a disproportionately high amount of hydroxylated fatty acids relative to that of the permeable ones. Moreover, a brief treatment with hot alkali released the omega-hydroxy fatty acid component of the outermost cuticle and created holes in it that caused the seeds to become permeable. This demonstrates that the outermost cuticle of the seed is the critical structure that prevents water uptake by hard seeds.     

Agronomic evaluation of tissue-culture-derived soybean plants

Summary Genetic alterations of regenerated plants based on the tissue culture process (somaclonal variation) have become common for many plant species including soybean [Glycine max (L.) Merr.]. The objective of this study was to test for the presence of tissue-culture-derived genetic variation in eight agronomic traits in homozygous progeny regenerated by organogenesis using the commercially important cultivar Asgrow A3127. A total of 86 lines derived by repeated self-pollination of nine regenerated plants was grown in two locations for 2 years. When compared to the unregenerated parent, statistically significant variation (P<0.05) was found for maturity, lodging, height, seed protein and oil, but not for seed quality, seed weight, or seed yield. All of the variation noted was beneficial and did not involve decreased yield. Since the differences were not large, the results indicate that the tissue culture process is not necessarily detrimental to plant performance, which is an important consideration since tissue culture techniques are used in many genetic engineering methods.     

Reversal of the effects of aging in soybean seeds

Accelerated aging predisposed seeds to imbibition injury. Slowing the rate of hydration prevented the loss of germinability due to imbibition injury. Germinability of accelerated aged seeds (50 hours) was increased from 10 to 90% by controlling the rate of imbibition. Slow hydration also prevented seed electrolyte leakage. This may indicate that cell membrane permeability or rupture was a major factor contributing to the loss of germinability after aging.

Reversal of the effects of aging (repair) was accomplished by slowly inbibing and then redrying seeds (priming). This treatment lowered steep water conductivity by a factor of 2 to 5. Priming also increased the per cent germination of low vigor seeds. The mechanism of this reversal was probably metabolic because it depended on temperature, seed moisture, and treatment duration.

Priming doubled the survival of seeds in the accelerated aging vigor test. The `rejuvenation' was accepted as evidence for metabolic repair. Since the `vigor' of seeds was increased by priming, metabolic repair probably included other subcellular components as well as the plasma membrane.

     

Patterns and kinetics of water uptake by soybean seeds

Soybean [Glycine max (L.) Merr.] plants produce some seeds (called stone or impermeable seeds) that do not take up water for long periods of time. The present investigation confirmed that the stone seed trait is a feature of the seed coat: isolated embryos from both stone and permeable seeds took up water equally quickly. A whole, permeable seed typically imbibed water initially through its dorsal side, forming wrinkles in the seed coat and delivering water to the underlying cotyledons. Later, some lateral movement of water through the coat occurred, presumably through the air spaces of the osteosclereid layer. Imbibition by seeds was a two-phase process, the first dominated by hydration of the seed coat and the second by hydration of the cotyledons, which was rate-limited by the coat. When hydrated, coats of stone seeds were permeable to water but their hydraulic conductivity, as measured with a pressure probe, was smaller than that of coats from permeable seeds by a factor of five. Hydrated coats of both permeable and stone seeds showed weak osmometer properties.     

Preparation of soybean seed samples for FT-IR microspectroscopy.

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4 degrees C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Structure and possible ureide degrading function of the ubiquitous urease of soybean

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.

The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection (“Western Blot”). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.

The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.