Isoflavonoid-inducible resistance to the phytoalexin glyceollin in soybean rhizobia.

The antibacterial effect of the soybean phytoalexin glyceollin was assayed using a liquid microculture technique. Log-phase cells of Bradyrhizobium japonicum and Sinorhizobium fredii were sensitive to glyceollin. As revealed by growth rates and survival tests, these species were able to tolerate glyceollin after adaptation. Incubation in low concentrations of the isoflavones genistein and daidzein induced resistance to potentially bactericidal concentrations of glyceollin. This inducible resistance is not due to degradation or detoxification of the phytoalexin. The inducible resistance could be detected in B. japonicum 110spc4 and 61A101, representing the two taxonomically divergent groups of this species, as well as in S. fredii HH103, suggesting that this trait is a feature of all soybean-nodulating rhizobia. Glyceollin resistance was also inducible in a nodD1D2YABC deletion mutant of B. japonicum 110spc4, suggesting that there exists another recognition site for flavonoids besides the nodD genes identified so far. Exudate preparations from roots infected with Phytophthora megasperma f. sp. glycinea exhibited a strong bactericidal effect toward glyceollin-sensitive cells of B. japonicum. This killing effect was not solely due to glyceollin since purified glyceollin at concentrations similar to those present in exudate preparations had a much lower toxicity. However, glyceollin-resistant cells were also more resistant to exudate preparations than glyceollin-sensitive cells. Isoflavonoid-inducible resistance must therefore be ascribed an important role for survival of rhizobia in the rhizosphere of soybean roots.     

Glyceollin, a soybean phytoalexin with medicinal properties

This review covers the biosynthesis of glyceollin and its biological activities including antiproliferative/antitumor action (toward B16 melanoma cells, LNCaP prostate cancer cells, and BG-1 ovarian cancer cells), anti-estrogenic action (through estrogen receptors ??- and ??-), antibacterial action (toward Erwinia carotovora, Escherichia coli, Bradyrhizobium japonicum, Sinorhizobium fredii ), antinematode activity, and antifungal activity (toward Fusarium solani, Phakospora pachyrhizi, Diaporthe phaseolorum, Macrophomina phaseolina, Sclerotina sclerotiorum, Phytophthora sojae, Cercospora sojina, Phialophora gregata, and Rhizoctonia solani). Other activities include insulinotropic action and attenuation of vascular contractions in rat aorta.     

A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation

Soybean membrane preparations specifically bound [¹⁴C]mycolaminaran, a branched β-1,3-glucan produced by Phytophthora sp. which elicits production of the phytoalexin glyceollin in soybean tissues. A Scatchard plot of the binding data disclosed the presence of a single affinity class of binding sites with a K_d value of 11.5 micromolar for the glucan. To assess the physiologic importance of mycolaminaran binding in phytoalexin elicitation, several derivatives of mycolaminaran were prepared. Reduced mycolaminaran had slightly greater elicitor activity and binding affinity than the native substance, while periodinated mycolaminaran was virtually devoid of either elicitor activity orbinding capability. Phosphorylated mycolaminaran, on the other hand, gave values for both elicitor activity and membrane binding which were intermediate between the native and periodinated preparations. No other tested carbohydrates competed with the binding of [¹⁴C]mycolaminaran. Soybean membrane preparations contained β-1,3-endoglucanase activity that degraded mycolaminaran and reduced both its efficiency as a phytoalexin elicitor and its membrane binding at temperatures above 0°C. Once [¹⁴C]mycolaminaran bound to membranes, however, it was not appreciably susceptible to glucanase attack and could not be displaced with excess unlabeled ligand. Taken collectively, the observations suggest that the membrane binding sites are mycolaminaran-specific receptors which are physiologically involved in the initiation of phytoalexin production in soybean cotyledons. Because the binding of mycolaminaran to membranes was abolished by heat and proteolytic enzymes, the receptor is probably a protein(s) or glycoprotein(s).     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

Solubilization of soybean membrane binding sites for fungal beta-glucans that elicit phytoalexin accumulation

Soybean membranes contain high-affinity binding sites for fungal beta-glucans. These sites may play a role in the recognition by soybean tissues of fungal phytoalexin elicitors. We have solubilized beta-glucan-binding activity from microsomal membranes using two C12-alkyl zwitterionic detergents, Zwittergent 3-12 (ZW 3-12) and the lysolecithin analog 1-dodecanoyl propanediol-3-phosphorylcholine [corrected] (ES12H). The solubilized binding sites displayed identical affinity for beta-glucans as that found in membranes (KD = 11-34 nM). Detergent-protein micelles with glucan binding activity eluted with approximate Mr values of 300,000 in ZW 3-12 and 380,000 in ES12H in gel permeation chromatography. Maximal binding activity eluted from a chromatofocusing column in the pH range between 6.2 and 6.6 with both ES12H and ZW 3-12, suggesting an apparent pI close to neutral.     

Development and application of a radioimmunoassay for the juvenile hormones

A radioimmunochemical method for the quantification of juvenile hormones from hemolymph and whole body extracts is described. Rabbit polyclonal antiserum developed against a JH III-bovine thyroglobulin conjugate displayed minimal cross-reactivity with juvenile hormone metabolites including juvenile hormone acids, juvenile hormone diols and analogs but substantial cross-reactivity between juvenile hormone homologs. Minimum sensitivity of the assay toward racemic juvenile hormone III was 65 pg. The degree and relative order of cross-reactivities for juvenile hormones I, II and III varied according to the identity of the radioligand used. A method for isolating juvenile hormones from whole body and hemolymph for radioimmunoassay was developed utilizing organic solvent extraction followed by thin-layer chromatography. Noninterfering dyes were used to bracket the position of the hormone on thin-layer chromatography plates. Hemolymph extracts known to contain no JH did not interfere with the assay when this procedure was employed. Radioimmunoassay analysis of hemolymph samples containing known amounts of JH and corrected for recovery yielded the expected results. Quantification of total juvenile hormone in whole body and hemolymph extracts of Manduca sexta was in good agreement with total mass of JH determined by a GC/MS method.     

Development of radioimmunoassay for motilin

A highly specific and sensitive radioimmunoassay for motilin has been developed. High titers of motilin antisera were produced in rabbits by repeated immunization with synthetic porcine motilin coupled to bovine serum albumin. Motilin was radioiodinated with Na[¹²⁵I] by the chloramine-T method and was purified using a CM-Sephadex C-25 column eluted with ammonium acetate gradient at pH 5.9. A specific radioactivity of 300 to 400 μCl/μg was usually obtained. Untreated plasma showed significant nonspecific interference in the assay; however, the interference could be minimized by extracting the plasma with methanol. All of the known gastrointestinal hormones were tested and showed no cross-reactivity at 10 ng/ml. Plasma motilin levels in dogs were significantly increased within 3 min, after an intraduodenal bolus injection of 50 ml of 0,1 n HCl. This increase coincided with a marked increase in myoelectrical activities of the duodenum.     

Structure-activity relationships of oligo-beta-glucoside elicitors of phytoalexin accumulation in soybean.

The abilities of a family of chemically synthesized oligo-beta-glucosides, ranging in size from hexamer to decamer, to induce phytoalexin accumulation in soybean cotyledons were investigated to determine which structural elements of the oligoglucosides are important for their biological activity. The results of the biological assays established that the following structural motif is necessary for the oligo-beta-glucosides to have high elicitor activity: [formula; see text] The branched trisaccharide at the nonreducing end of the oligoglucosides was found to be essential for maximum elicitor activity. Substitution of either the nonreducing terminal backbone glucosyl residue or the side-chain glucosyl residue closest to the nonreducing end with glucosaminyl or N-acetylglucosaminyl residues reduced the elicitor activity of the oligoglucosides between 10-fold and 10,000-fold. Elicitor activity was also reduced 1000-fold if the two side-chain glucosyl residues were attached to adjacent backbone glucosyl residues rather than to glucosyl residues separated by an unbranched residue. In contrast, modifications of the reducing terminal glucosyl residue of an elicitor-active hepta-beta-glucoside by conjugation with tyramine and subsequent iodination had no significant effect on the elicitor activity of the hepta-beta-glucoside. These results demonstrate that oligo-beta-glucosides must have a specific structure to trigger the signal transduction pathway, which ultimately leads to the de novo synthesis of phytoalexins in soybean.     

Ethylene: indicator but not inducer of phytoalexin synthesis in soybean

Cell wall preparations (elicitors) from Phytophthora megasperma var. sojae increase C₂H₄ formation, phenylalanine ammonia lyase activity, and glyceollin accumulation in soybean cotyledons within about 1.5, 3, and 6 hours after treatment, respectively. The immediate precursor of C₂H₄, 1-aminocyclopropane-1-carboxylic acid, stimulates C₂H₄ formation like the elicitor within 1.5 hours after administration, whereas phenylalanine ammonia lyase activity and glyceollin concentration remain unchanged. Aminoethoxyvinylglycine, a specific inhibitor of C₂H₄ formation in higher plants, inhibits elicitor-induced C₂H₄ formation by about 95% but has no effects on phenylalanine ammonia lyase or glyceollin accumulation. It was concluded that C₂H₄ is a signal accompanying the specific recognition process which finally leads to the induction of phytoalexin formation, but it is not functioning as a link or messenger in the induction sequence of glyceollin accumulation.     

Development of a radioimmunoassay for colony stimulating factor.

Purified L-cell colony stimulating factor (CSF) and rabbit anti-CSF serum were used to devise a radioimmunoassay for this factor. The CSF was radiolabelled with the aid of lactoperoxidase and precipitated by a double antibody technique. Addition of unlabelled CSF caused a dose-related displacement of the labelled tracer. Similar results were noted with conditioned media and murine serum. The assay required only 4 days for completion as compared with 7 days for the conventional agar gel bioassay. Moreover, the radioimmunoassay proved more sensitive and accurate than the bioassay. This technique should allow further exploration of the role of CSF in granulopoiesis.     

Development of a homologous radioimmunoassay for secreted hamster prolactin

A specific and sensitive homologous radioimmunoassay has been developed for secreted hamster prolactin. Hamster serum and pituitary extracts showed parallel dilution-response curves with hamster prolactin. The sensitivity of the assay ranged from 0.5 to 1.0 ng/ml, and the intra- and inter-assay coefficients of variation were 6 and 10%, respectively. Additionally we have demonstrated that the rat prolactin radioimmunoassay kit distributed by the National Institute of Arthritis, Metabolism, and Digestive Diseases is an inadequate method for the measurement of hamster prolactin.     

Investigation of the mechanism of glyceollin accumulation in soybean infected by Phytophthora megasperma f. sp. glycinea

Soybean seedlings (Glycine max, cv. Harosoy 63) which had been inoculated in the hypocotyls with mycelium from either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma f. sp. glycinea were pulse labeled with ¹⁴CO₂. The time course of accumulation of glyceollin and daidzein and of ¹⁴C incorporation into these compounds was determined. Metabolic rates of glyceollin were measured by pulse-chase experiments. Differences in glyceollin accumulation between the incompatible and compatible interaction were not apparent before about 14 h after inoculation. Subsequently glyceollin accumulated to a higher level in the incompatible interaction. This difference is also reflected in the rate of ¹⁴C incorporation, which declines more rapidly in the compatible interaction. The apparent half-life of glyceollin metabolism was 28 ± 7 h for inoculation with race 1, while no metabolism was observed with race 3. In contrast to a previous report (M. Yoshikawa, K. Yamauchi, and H. Masago (1979)Physiol. Plant Pathol.14, 157–169), our data prove that the higher accumulation of glyceollin in the incompatible interaction is due to a longer duration of synthetic activity and that the level of glyceollin in both the incompatible and compatible interaction is determined predominantly by its rate of synthesis.     

Application of growth substances and mineral nutrition affecting disease development and glyceollin production of soybean

The effects of foliar application of growth substances and mineral nutrition of the host on the development of charcoal rot disease of soybean caused byMacrophomina phaseolina was tested. Among the eight growth substances examined, gibberellic acid was most successful in reducing the disease severity, followed by 3-indoleacetic acid and 2,3,5-triiodobenzoic acid. Low concentrations of these compounds stimulated while high concentrations inhibited the mycelial growth ofM. phaseolina in vitro. Substrate supplementation with different doses of N, P, K and Ca had varying effects on disease development. Disease increased considerably by both excess and defficient N and also by deficient Ca, while excess Ca conferred partial resistance. Glyceollin contents of host roots before and after excess Ca and gibberellic acid (10 mg/L) treatments were estimated. Both compounds significantly increased glyceollin production in infected roots. However, gibberellic acid induced glyceollin synthesis even in uninoculated roots. Changes in the host reaction towards increased resistance was correlated with increased phytoalexin production.     

Intracellular localization of prenyltransferases of isoflavonoid phytoalexin biosynthesis in bean and soybean

The intracellular localization of prenyltransferases involved in the biosynthesis of the phytoalexins glyceollin in soybean (Glycine max L.) and phaseollin in French bean (Phaseolus vulgaris L.) has been investigated. By sucrose- and Percoll-gradient centrifugation of microsomes of an elicitor-challenged soybean cell culture, the membranes containing prenyltransferase were separated from the endoplasmic reticulum and shown to be lighter in density. In a continuous Percoll gradient the peak of prenyltransferase activity coincided with the peak of galactolipid synthesis, as determined by incorporation of uridine 5′-diphospho-[¹⁴C]galactose (UDP-[¹⁴C]galactose). Intact chloroplasts isolated from cupricchloride-treated bean leaves contained both prenyltransferase and UDP-galactose transferase activity. Both activities increased during chloroplast isolation. Fractionation of swollen chloroplasts on a discontinuous sucrose gradient showed prenyltransferase and UDP-galactose transferase activity in the envelope membrane subfraction. It is concluded that in both plants prenyltransferase is located in the envelope membrane of plastids. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID₅₀ concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.     

Induction of phytoalexin synthesis in soybean. Stereospecific 3,9-dihydroxypterocarpan 6a-hydroxylase from elicitor-induced soybean cell cultures

A microsomal preparation from elicitor-challenged soybean cell suspension cultures catalyzes an NADPH-dependent and dioxygen-dependent 6a-hydroxylation of 3,9-dihydroxypterocarpan to 3,6a,9-trihydroxypterocarpan. The latter is a precursor for the soybean phytoalexin glyceollin. No reaction is observed with NADH. The 6a-hydroxylase is inhibited by cytochrome c. Optical rotatory dispersion spectra of the enzymatic product formed from racemic dihydroxypterocarpan and of the remaining unreacted substrate proved that the product has the natural (6aS, 11aS)-configuration and that hydroxylation proceeds with retention of configuration. The 6a-hydroxylase was also found in elicitor-challenged soybean seedlings. The results indicate that the 6a-hydroxylase is specifically involved in the biosynthesis of glyceollin.