Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani.

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.374') to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [(14)C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus.     

Development of a radioimmunoassay for the soybean phytoalexin glyceollin I

A radioimmunoassay for glyceollin I, the major phytoalexin produced by soybean (Glycine max [L.] Merr.), has been developed. Antibodies were raised in rabbits against a glyceollin I-bovine serum albumin conjugate. The antisera were used to establish a radioimmunoassay for glyceollin I using [¹²⁵I]glyceollin I as the tracer. A logit plot of a standard concentration series yielded a straight line in the range of 1 to 100 picomoles (0.34-34 nanograms) of glyceollin I. The structurally related pterocarpan phytoalexins, glyceollins II and III, glyceollidin II and glycinol, which also accumulate in infected soybean tissue, show a low cross-reactivity in the radioimmunoassay (0.5-5% at 50% displacement of the tracer). Two related isoflavones present constitutively in soybean tissue, daidzein and genistein, have cross-reactivities of less than 0.84% and 1.1%, respectively. The radioimmunoassay permitted the quantitative determination of glyceollin I in 15-micrometer microtome sections of soybean hypocotyl tissue infected with zoospores of Phytophthora megasperma f. sp. glycinea.     

Phytoalexins and their Role in the Resistance of Plants to Nematodes

Phytoalexins are antibiotic compounds synthesized in an infected plant in response to infection. Nematodes are capable of eliciting phytoalexins in resistant plants. Resistant lima bean (Phaseolus lunatus) infected by Pratylenchus penetrans produces the phytoalexin coumestrol; soybean (Glycine max) infected by Meloidogyne incognita produces glyceollin; cotton (Gossypium hirsuturn) infected by M. incognita produces terpenoid aldehydes.     

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.     

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.     

Rhizosphere salinity and phytoalexin accumulation in soybean

Summary Rhizosphere salinity decreased the capacity of soybean to accumulate a pterocarpanoid phytoalexin (glyceollin) in the stem in response toPhytophthora megasperma var.sojae. Rapid (48h) accumulation was depressed by NaCl, Na₂SO₄, CaCl₂ and MgSO₄ applications. Time-course accumulations was slowed by applications. Time-course accumulation was slowed by application of 0.131M NaCl. Glyceollin accumulation was also reduced in plants subjected to a period of high salinity stress (0.177M NaCl, 72 h) after a period of nonsalinized growth. Calcium chloride completely suppressed glyceollin accumulation in normally-resistant plants but no susceptibility to the fungus was observed.     

Involvement of Oxidative Processes in the Signaling Mechanisms Leading to the Activation of Glyceollin Synthesis in Soybean (Glycine max)

The efficiency of hydroperoxides (tert-butyl hydroperoxide, hydrogen peroxide) and sulfhydryl reagents (iodoacetamide, p-chloromercuribenzene sulfonic acid) as glyceollin elicitors was examined in relation to sulfhydryl oxidation, or alteration, and to lipid peroxidation, in 3-d-old soybean hypocotyl/radicle, Glycine max. These oxidative events were investigated as possible early steps in the transduction mechanisms leading to phytoalexin synthesis. Free protein sulfhydryl groups were not modified after any of the eliciting treatments, thus indicating that immediate massive protein oxidation or modification cannot be considered a signal transduction step. Unlike sulfhydryl reagents, which led to a decrease of the free nonprotein sulfhydryl group (free np-SH) pool under all of the eliciting conditions, the results obtained with hydroperoxides indicated that immediate oxidation of the np-SH is not required for the signal transduction. Moreover, elicitation with 10 mM tertbutyl hydroperoxide did not lead to further oxidation or to changes in np-SH level during the critical phase of phenylalanine ammonialyase activation (the first 20 h), suggesting that np-SH modifications are probably not involved in hydroperoxide-induced elicitation. On the other hand, all treatments leading to significant glyceollin accumulation were able to trigger a rapid (within 2 h) lipid peroxidation process, whereas noneliciting treatments did not. In addition, transition metals, such as Fe2+ and Cu+, were shown to stimulate both hydrogen peroxide-induced lipid peroxidation and glyceollin accumulation, again emphasizing that the two processes are at least closely linked in soybean. Among the oxidative processes triggered by activated oxygen species, oxidation of sulfhydryl compounds, or lipid peroxidation, our results suggest that lipid peroxidation is sufficient to initiate glyceollin accumulation in soybean. This further supports the hypothesis that lipid peroxidation could be involved as a step in the signal cascade that leads to induction of plant defenses.     

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.     

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.     

Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: structures, syntheses and antifungal activity

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-methoxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.     

Quantification of glyceollins in non-elicited seedlings of Glycine max by gas chromatography-mass spectrometry

A sensitive gas chromatography-mass spectrometry method is described for detection and quantification of small amounts of phytoalexins, especially glyceollins. This method allowed identification of canescacarpin (glyceollin V) besides appreciable amounts of glyceollin isomers I–III and glyceofuran in unchallenged parts of soybean seedlings. Based on this quantification method the glyceollin content of soybean roots, either untreated, wounded or incubated with a glucan elicitor from Phytophthora sojae, was compared. The proportions of glyceollin isomers in the mixture of compounds were determined in different soybean tissues. Single-ion monitoring revealed the presence of four additional glyceollin isomers with benzofuranoid structure.     

信号传导拮抗物对大豆细胞植保素和异黄酮积累的影响

磷酸酶的抑制剂花萼海绵诱癌素A、芫菁素和冈田酸都能诱导大豆植保素(大豆素)的积累。相反,激酶的抑制剂K252a几乎完全阻止它们诱导大豆素的合成。与磷酸酶抑制剂相比较,酵母细胞壁激发子(YE)有利于诱导黄苷元、染料木苷等异黄酮中间体的积累,而磷酸酶的抑制剂有利于大豆素的合成。YE和芫菁素还呈现出协同诱导大豆素积累的效果。通过磷脂酶A2的抑制剂与阻止线粒体ATP合酶活性的化合物的分别处理,发现茉莉酸信号传导途径是参与调控大豆植保素合成的重要途径之一,而植保素的合成需要线粒体提供能量。     

Effect of three resistant soybean genotypes on the fecundity, mortality, and maturation of soybean aphid (Homoptera: Aphididae)

The fecundity, longevity, mortality, and maturation of the soybean aphid, Aphis glycines Matsumura (Homoptera: Aphididae), were characterized using three resistant soybean, Glycine max (L.) Merrill, genotypes ('Dowling', 'Jackson', and PI200538 'Sugao Zarai') and two susceptible genotypes ('Pana' and 'Loda'). Antibiosis in the resistant genotypes was demonstrated by a significant decrease in fecundity and longevity and increased mortality of A. glycines. Aphid fecundity, measured as number of offspring produced in the first 10 d by each viviparous aptera, was higher on Pana than on the resistant genotypes. Aphid longevity, the mean number of days a 1-d-old adult lived, was 7 d longer on Pana than on Dowling and Jackson. The mortality of both viviparous apterae and nymphs on resistant genotypes was significantly higher than on susceptible genotypes. A greater number of first instars survived to maturation stage (date of first reproduction) on susceptible plants than on resistant plants. None of the first instars placed on Dowling and PI200538 leaves survived to maturation. Observations of aphid behavior on leaves indicated that aphids departed from the leaves of resistant plants 8-24 h after being placed on them, whereas they remained indefinitely on leaves of susceptible cultivars and developed colonies. Reduced feeding due to ingestion of potentially toxic compounds in soybean may explain the possible mechanism of resistance to the soybean aphid.     

Analysis of isoflavones and coumestrol in soybean sprouts

High-performance liquid chromatography analysis is used to examine the distribution of isoflavones in different parts of soybean sprouts. Between the seed leaf and hypocotyl, the ratio of isoflavones differs depending on the aglycone type. Glycitein exists predominantly in the hypocotyl. Three compounds isolated from 4-day-old seed coats were identified as coumestrol and its glycosides.     

The diphenylether herbicide lactofen induces cell death and expression of defense-related genes in soybean

Lactofen belongs to the diphenylether class of herbicides, which targets protoporphyrinogen oxidase, which in turn causes singlet oxygen generation. In tolerant plants like soybean (Glycine max), the chemical nonetheless causes necrotic patches called "bronzing" in contact areas. Here it is shown that such bronzing is accompanied by cell death, which was quantified from digital microscopic images using Assess Software. Cellular autofluorescence accompanied cell death, and a homolog of the cell death marker gene, Hsr203j, was induced by lactofen in treated soybean tissues. Thus, this form of chemically induced cell death shares some hallmarks of certain types of programmed cell death. In addition to the cell death phenotype, lactofen caused enhanced expressions of chalcone synthase and chalcone reductase genes, mainly in the exposed and immediately adjacent (proximal) cells. Furthermore, isoflavone synthase genes, which are wound inducible in soybean, were up-regulated by lactofen in both proximal and distal cell zones in minimally wounded cotyledons and further enhanced in wounded tissues. Moreover, if the wall glucan elicitor from Phytophthora sojae was present during lactofen treatment, the induction of isoflavone synthase was even more rapid. These results are consistent with the fact that lactofen triggers massive isoflavone accumulations and activates the capacity for glyceollin elicitation competency. In addition, lactofen induces late expression of a selective set of pathogenesis-related (PR) protein genes, including PR-1a, PR-5, and PR-10, mainly in treated proximal tissues. These various results are discussed in the context of singlet oxygen-induced responses and lactofen's potential as a disease resistance-inducing agent.     

Investigation of the mechanism of phytoalexin accumulation in soybean induced by glucan or mercuric chloride

Soybean cotyledons which had been treated with glucan from Phytophthora megasperma f.sp. glycinea or with mercuric chloride were pulse-labeled with ¹⁴CO₂ and then the ¹⁴C-incorporation into the phytoalexins was determined. The kinetics of ¹⁴C-incorporation into phytoalexins (glyceollin isomers and 3,6α,9-trihydroxypterocarpan) was very similar with the two types of elicitors. Metabolic rates of phytoalexins were determined by pulse-chase experiments. The apparent half-life of metabolism was about 100 h for glyceollin with either glucan or HgCl₂. The half-lives for trihydroxypterocarpan were 39 h with glucan and 14 h with HgCl₂. According to our results levels of glyceollins in soybean cotyledons are mainly controlled by their rates of synthesis. Biotic (glucan) and abiotic (HgCl₂) elicitors have similar induction effects. Both types of elicitors could act by effecting the release of endogenous elicitors.     

Effects of Ca2+ on phytoalexin induction by fungal elicitor in soybean cells

A glucan elicitor from the cell walls of the fungus Phytophthora megasperma f.sp. glycinea caused increases in the activities of the phytoalexin biosynthetic enzymes, phenylalanine ammonia-lyase and chalcone synthase, and induced the production of the phytoalexin, glyceollin, in soybean (Glycine max) cell suspension cultures when tested in culture medium containing 1.2 mmol/liter Ca2+. Removal of extracellular Ca2+ by treatment with ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid followed by washing the cells with Ca2+-free culture medium abolished the elicitor-mediated phytoalexin response. This suppression was largely reversed on readdition of Ca2+. Elicitor-mediated enhancement of biosynthetic enzyme activities and accumulation of glyceollin was strongly inhibited by La3+; effective concentrations for 50% inhibition were (mumol/liter) 40 for phenylalanine ammonia-lyase, 100 for chalcone synthase, and 30 for glyceollin. Verapamil caused similar effects only at concentrations higher than 0.1 mmol/liter, whereas trifluoperazine and 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate did not affect enzyme induction by the elicitor in the concentration range tested. Uptake of alpha-amino isobutyric acid into soybean cells, which was rapidly inhibited in the presence of the glucan elicitor, was not affected by La3+ nor was uptake inhibition by the elicitor relieved by La3+. The Ca2+ ionophore, A23187, enhanced phytoalexin biosynthetic enzyme activities and glyceollin accumulation in a dose-dependent manner, with 50% stimulation (relative to the elicitor) occurring at about 5 mumol/liter. The results suggest that the glucan elicitor causes changes in metabolite fluxes across the plasma membrane of soybean cells, among which changes in Ca2+ fluxes appear to be important for the stimulation of the phytoalexin response.     

Protocol for assessing soybean antixenosis to Heliothis virescens

Larvae of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) often infest soybean crops, Glycine max (L.) (Fabaceae), causing significant yield losses in important soybean-producing regions. The use of soybean varieties resistant to lepidopteran larvae is a major approach in soybean integrated pest management. However, standardization and optimization of bioassays that are used to screen genotypes for insect resistance are essential for high-throughput phenotyping. Methodologies for screening were assessed to determine the most effective method for discriminating levels of antixenosis to H. virescens in soybean plants. Feeding and oviposition preference assays were performed to determine optimal densities of larvae and adults, and optimal plant structures and growth stages for conducting assays. In addition, trichome densities, and fiber and lignin contents were quantified in plant structures of soybean cultivars differing in resistance. Resistance levels of cultivars were best differentiated using nine neonate larvae and two 6-day-old larvae, and by using young leaves of plants at the vegetative stage. This was likely due to the more pronounced differences in lignin and fiber contents in young leaves of vegetative-stage plants. Density of adult pairs, plant structure, and growth stage did not affect ability to distinguish differences in oviposition preference by H. virescens. Higher numbers of eggs were found on the leaves, which were the plant structures that exhibited the lowest trichome densities. The protocol developed in this work will benefit future evaluations of soybean genotypes for antixenosis against H. virescens.     

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.     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.