Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.     

Elicitor-induced accumulation of glyceollin and callose in soybean roots and localized resistance against Phytophthora megasperma f.sp. glycinea

Immersion of roots of 2-day-old soybean seedlings (Glycine max cv. Harosoy 63) into solutions of several glucan elicitors caused the accumulation to various degrees of the soybean phytoalexin glyceollin. Laminarin and polytran proved to be more effective elicitors in this system than the glucan elicitor from Phytophthora megasperma f.sp. glycinea (Pmg). Digitonin and tomatin caused, in addition to glyceollin accumulation, the deposition of callose in the rhizodermis. Pretreatment of the soybean roots with laminarin effected an increase in resistance of the seedlings against a compatible race of Pmg.     

De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin.     

Ultrastructural localization of Bowman-Birk inhibitor on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method

The soybean Bowman-Birk inhibitor (BBI), a polypeptide of MW 8,000, has a specificity directed against trypsin and chymotrypsin. BBI was localized at the ultrastructural level by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledon and embryonic axis, BBI was found in all protein bodies, the nucleus and, to a lesser extent, the cytoplasm. Contrary to the Kunitz trypsin inhibitor (Horisberger and Tacchini-Vonlanthen 1983), BBI was not present in the cell wall but was found in the intercellular space. Intensity of marking in cotyledons of four-day-old seedlings was similar with the exception of the intercellular space which was free of BBI. In two lines lacking the Kunitz inhibitor (P.I. 157440 and 196168), data indicated that marking intensity was similar to that of cv. Maple Arrow. In contrast, in varieties lacking the lectin (Norredo, T-102) marking was more intense than in cv. Maple Arrow.     

Distribution of Lectins in the Jumbo Virginia and Spanish Varieties of the Peanut, Arachis hypogaea L

Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs.     

Rapid release of protease inhibitors from soybeans: immunochemical quantitation and parallels with lectins

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.     

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.     

Ultrastructural localization of Kunitz inhibitor on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method

The soybean trypsin inhibitor (SBTI, Kunitz type) was localized by immunofluorescence and, at the ultrastructural level, by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. SBTI was localized in cell walls, protein bodies, the cytoplasm between the lipid-containing spherosomes, and the nucleus of the cotyledon and embryonic axis. In the nucleus, SBTI was present in the chromatin deposit and the nucleolus. The intensity of marking by the gold method decreased in the cell wall from the center of the cotyledon to the periphery. In four-days-old seedlings marking intensity of cell wall was much reduced. No SBTI could be detected in the hypocotyl. In two lines lacking soybean agglutinin (Norredo, T-102) the location of SBTI was similar to that observed in Maple Arrow. In P.I. 196168, a line lacking SBTI, marking intensity of the organelles was reduced to a very low level. Although this study was not designed to discern the cellular function of SBTI, if any, it may establish criteria consistent with its role in soybean.     

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.     

Distribution of Lectins in Tissues, Derived Callus, and Roots of Psophocarpus tetragonolobus (Winged Bean)

The distribution of lectin in parental tissues, roots formed de novo from parental stem tissue, and derived callus cells of Psophocarpus tetragonolobus has been measured by hemagglutinating activity and radioimmunoassay. The antisera used for the radioimmunoassay was raised in rabbits to lectin isolated from seeds by affinity chromatography using insolubilized hog gastric mucin. The distribution of lectin in buffer extracts of the tissues (or cells) and the extracellular medium favors the tissues for in vitro grown roots, regardless of the culture conditions used. The lectin content of the extracellular medium is more significant for callus, regardless of its conditions of culture. The lectin activity of extracts of in vitro grown roots was higher than that of mature roots from whole plants. Differences in relative levels of lectin activity measured by hemagglutination and by radioimmunoassay, and differences in saccharide inhibition of hemagglutination, suggest the presence of multiple lectins in extracts of different tissues.     

Development and Distribution of a Lectin from the Stems and Leaves of Dolichos biflorus

The stems and leaves of the Dolichos biflorus plant contain a lectin that cross-reacts with antiserum against the seed lectin. This cross-reactive material (CRM) was followed during early seedling growth, stem elongation, and seed development using a specific radioimmunoassay.

No CRM was detected in developing seeds, but very low levels were found in dormant and imbibed seeds. As germination proceeds, the CRM accumulates at the apex of both etiolated and green seedlings in the epicotyl and leaves. Lower amounts of CRM are found in the cotyledons and hypocotyl, but no CRM was detected in the roots.

The amount of CRM in the first and second stem internodes increases during elongation and gradually declines after the completion of elongation. Approximately 80% of the CRM in the stems of 19-day-old Dolichos biflorus plants is associated with the elongating tissues. These results are discussed with respect to the possible roles of lectins in plants.

     

Role of Lectins in Plant-Microorganism Interactions: II. Distribution of Soybean Lectin in Tissues of Glycine max (L.) Merr

Three different assay procedures have been used to quantitate the levels of soybean (Glycine max [L.] Merr.) lectin in various tissues of soybean plants. The assays used were a standard hemagglutination assay, a radioimmunoassay, and an isotope dilution assay. Most of the lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was present in all of the tissues of young seedlings, but decreased as the plants matured and was not detectable in plants older than 2 to 3 weeks. Soybean lectin isolated from seeds of several soybean varieties were identical when compared by several methods.     

Phospholipids in the developing soybean seed

The distribution of phospholipids in developing soybean seeds [Glycine max (L.) Merr., var. “Chippewa 64,” “Harosoy 63,” “Wayne,” and “Clark 63”] was followed. From 30 to 60 days after flowering expressed as mole per cent of phospholipid phosphorus phosphatidic acid decreased from 14.8 to 9.1; phosphatidylinositol increased from 0 to 9.1; phosphatidylcholine increased from 8.2 to 9.8; phosphatidylethanolamine increased from 5.3 to 8.6; phosphatidylglycerol increased from 3.2 to 4.8; diphosphatidylglycerol increased from 2.7 to 4.1; and N-acylphosphatidylethanolamine decreased from 65.8 to 54.6. However, from 60 days after flowering to maturity, phosphatidic acid decreased to 0; phosphatidylinositol increased roughly 2-fold; phosphatidylcholine increased roughly 4.7-fold; phosphatidylethanolamine increased 3-fold; N-acylphosphatidylethanolamine decreased 11-fold; whereas phosphatidylglycerol and diphosphatidylglycerol remained essentially constant. Percentages of individual phospholipid species were not statistically different between any two varieties at a given time period.     

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10⁴Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.     

Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes.Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons.     

Studies on the metabolism of lipid molecular species in immature soybean cotyledons

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

Citrate cleavage enzymes from developing soybean cotyledons: incorporation of citrate carbon into Fatty acids

Data are presented which demonstrate a citrate cleavage enzyme in the supernatant of a developing soybean (Glycine max L. Merr., var. Harosoy 63) cotyledon homogenate following a 126,000g spin for 2 hours. Activity of the enzyme was observed directly in the supernatant enzyme preparation and in a desalted supernatant preparation by measuring the formation of acetylhydroxamate. Acetylhydroxamate production was dependent on citrate and coenzyme A. The reaction increased with time, citrate, and coenzyme A concentrations.     

Expression of flavonoid 6-hydroxylase candidate genes in normal and mutant soybean genotypes for glycitein content

Soybean seeds accumulate large amounts of isoflavones (genistein, daidzein and glycitein), secondary metabolites known for their phytoestrogenic activities. Isoflavone composition depends on the seed part and glycitein is almost found exclusively in hypocotyls. Moreover, two major phenotypes are encountered in soybean cultivars, with either low (35 %) or high (55 %) levels of glycitein in their hypocotyls. This trait was under a quasi-mendelian heredity, implicating at most one or two genes. A CYP71D9 cDNA displaying a flavonoid 6-hydroxylase (F6H) activity had previously been isolated from elicitor-induced soybean (Glycine max L.) cells. This enzyme allows the synthesis of the glycitein flavanone intermediate (6,7,4′- trihydroxyflavanone) by catalyzing the A-ring hydroxylation of liquiritigenin. In this study, the CYP71D9 gene (F6H1) and two other candidates (F6H2 and F6H3) were studied using contrasted soybean cultivars for glycitein content (0, 35, 55 and 80 %). Their expression was observed in chitosan elicited leaves. They encode P450 proteins of 496, 469 and 481 amino acids respectively and were expressed in leaves with or without elicitation. The expression patterns of these three genes were performed in cotyledons and hypocotyls at different developmental stages. F6H1 and F6H2 were not expressed in the developing seed. F6H3 was only expressed in hypocotyls. Its expression levels did not correlate with hypocotyls glycitein content, but it was not expressed in the null mutant for glycitein. Thus, this F6H3 gene is a good potential candidate for glycitein biosynthesis in soybean seed.     

Development and Distribution of Dolichos biflorus Lectin as Measured by Radioimmunoassay

A radioimmunoassay, capable of detecting the Dolichos biflorus lectin at concentrations as low as 400 ng/ml, was developed and used to follow the distribution of this lectin in the plant during its life cycle.

The lectin was first detected in the seeds of the plant 27 days after flowering and rapidly attained the high level of lectin present in the mature seed. The lectin content of the plant is highest in the seeds and cotyledons and decreases as the storage materials of the cotyledons decrease.

A low but measurable amount of material that reacts with antibodies to the seed lectin was detected in the leaves, stems, and pods of the plant. This material gives a precipitin band of only partial identity to the seed lectin when tested in immunodiffusion against antiserum to the seed lectin.

No lectin was detected by the radioimmunoassay in the roots of the plant at any stage of development.