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.     

Isoflavone glucoside stress metabolites of soybean leaves

The isoflavone glucosides daidzin, genistin and ononin, the isoflavones daidzein and formononetin, and glyceollins I-III accumulated in soybean leaves inoculated with phytopathogenic bacteria. Treatment of leaves with sodium iodoacetate or yeast extract also led to isoflavonoid accumulation. Various other stress-inducing treatments were not effective. Bacterially-induced accumulation of isoflavone glucosides and the occurrence of ononin and formononetin in soybean are reported for the first time.     

丹贝发酵过程中大豆异黄酮组分与含量的变化

用HPLC方法检测大豆[Glycine max(Linn.)Merr.]发酵食品－丹贝的发酵过程中异黄酮各组分含量变化。在发酵的最初24h内,大部分异黄酮由糖甙转化成甙元,随着发酵时间的延长转化率逐渐降低,发酵终产物中异黄酮主要以大豆甙元和染料木素形式存在,发酵64h的丹贝中总异黄酮摩尔含量比未发酵的大豆高51．25％。     

Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan

Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells.     

Signaling in Soybean Phenylpropanoid Responses (Dissection of Primary,Secondary, and Conditioning Effects of Light,Wounding, and Elicitor Treatments)

The spatial and temporal deployment of plant defense responses involves a complex interplay of signal events, often resulting in superimposition of signaling processes. We have employed a minimal-wound protocol to clearly separate and characterize the specific contributions of light, wounding, and a wall glucan elicitor preparation (PWG) from Phytophthora sojae (Kauf. and Gerde.) to the regulation of phenylpropanoid defense responses in soybean (Glycine max L. [Merr.]) cotyledon tissues. The assay also allowed us to clearly reconstitute responses to combinations of these primary signals and to examine the effects of other pathogenesis-related molecules on the responses in a defined manner. Light specifically triggers accumulation of malonylglucosyl conjugates of the 5-hydroxy-isoflavone, genistein, which is normally found in epidermal cells. PWG selectively induces accumulation of conjugates of the 5-deoxy-isoflavone daidzein, the first committed precursor of the phytoalexin glyceollin. Wounding initiates phenolic polymer deposition, a process greatly potentiated by PWG and light. Whereas glutathione selectively enhances light induction of genistein conjugates, methyl jasmonate enhances both light and PWG-induced isoflavone conjugate accumulations. Wound exudate fully activates the cell's capacity (competency) for the phenolic polymer and glyceollin responses to PWG, whereas glutathione partially restores competency, favoring coumestrol and phenolic polymer responses to PWG. Abscisic acid inhibits all induced phenylpropanoid responses.     

光照对大豆幼苗组织中异黄酮含量和分布的影响

利用高效液相色谱（ＨＰＬＣ）测定了不同光照处理的大豆（Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒｒｉ．）幼苗不同组织的异黄酮类含量。子叶中最高,叶片和根部相对较少。子叶的异黄酮以大豆甙和染料木甙及其丙二酰基结合体为主,且在光照条件下,异黄酮含量随光照时间的增加而显著升高;相反,黑暗中的异黄酮含量随苗龄的增加呈下降趋势;当子叶由黑暗转为光照处理以后,异黄酮含量同样随光照时间的增加而升高。在叶片和根部异黄酮含量和种类也因光照条件的不同而有很大差异。光照条件下,叶片中以染料木甙及其丙二酰结合体和黄酮芦丁为主,且随时间增加呈上升趋势;黑暗中的黄化叶片,则以大豆甙和丙二酰结合体为主,但随时间的变化不明显。在幼苗根部,黑暗条件下几乎检测不出异黄酮的存在;光照条件下,则可检测到５种异黄酮,其中以大豆甙元及其衍生物占主要部分。实验证实了光照对大豆异黄酮的积累有明显的促进作用     

Light Effect on the Tissue Contents and Distribution of Isoflavones in the Developing Seedling of Soybean

The contents of isoflavones in the developing soybean seedling tissues under different light conditions were examined by high-performance liquid chromatography (HPLC). The results indicated that the total contents of isoflavones in soybean seedling tissues were remarkably different, being the highest in the cotyledons and lower in the leaves and roots. In the cotyledons, the isoflavones like daidzin, genistin and their malonyl conjugates were the major metabolites and were increased markedly with the time of light exposure. In contrast, there was a decrease in the content of isoflavones in the dark. When the dark grown seedlings were tranferred to the light, an increase of the isoflavone content was observed. In the leaves and roots, there was also a marked difference in the contents and types of isoflavones due to various light conditions. Leaves contained mainly the isoflavones such as genistin, malonyl gensistin and flavones rutin, which were increased gradually under light condition, but in the dark, daidzin and malonyl daidzin were the major metabolites and did not change markedly with time. In the roots, no isoflavone was detected in the dark, while five isoflavones were found in the light-grown seedlings, and all root sections were predominated by daidzein and its conjugates. These results provided the evidence that light strongly stimulates the accumulation of isoflavones in soybean seedling tissues.     

Effect of Circulating Forms of Soy Isoflavones on the Oxidation of Low Density Lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Developmental and nutritional regulation of isoflavone secretion from soybean roots

Isoflavones play important roles in plant–microbe interactions in rhizospheres. Soybean roots secrete daidzein and genistein to attract rhizobia. Despite the importance of isoflavones in plant–microbe interactions, little is known about the developmental and nutritional regulation of isoflavone secretion from soybean roots. In this study, soybeans were grown in hydroponic culture, and isoflavone contents in tissues, isoflavone secretion from the roots, and the expression of isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) were investigated. Isoflavone contents did not show strong growth-dependent changes, while secretion of daidzein from the roots dramatically changed, with higher secretion during vegetative stages. Coordinately, the expression of ICHG also peaked at vegetative stages. Nitrogen deficiency resulted in 8- and 15-fold increases in secretion of daidzein and genistein, respectively, with no induction of ICHG. Taken together, these results suggest that large amounts of isoflavones were secreted during vegetative stages via the hydrolysis of (malonyl)glucosides with ICHG.     

Isoflavones Daidzein and Genistein: Preparation by Acid Hydrolysis of Their Glycosides and the Effect on Phospholipid Peroxidation

A mixture of isoflavones was obtained by acid hydrolysis of isoflavone glycosides isolated from the products of soybean processing by successive extraction with aqueous acetone and methanol. The homogeneous isoflavones daidzein and genistein were isolated from the aglycone mixture by adsorption chromatography and identified by spectral and chromatographic methods. The effect of both isoflavones on lipid peroxidation of soy phospholipids in multilamellar vesicles was studied at various concentrations. These aglycones were found to inhibit the formation of lipid hydroperoxides and malonic dialdehyde at concentrations as low as 1 mM.     

Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates

The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event.     

Profiling changes in metabolism of isoflavonoids and their conjugates in Lupinus albus treated with biotic elicitor

Liquid chromatography with ultraviolet and mass spectrometric detection was applied to monitor changes in profiles of isoflavonoid glycosides and free isoflavonoid aglycones in Lupinus albus L. Four isoflavonoid aglycones, fourteen isoflavonoid glycosides, four flavonol glycosides and flavone glycoside were identified in lupin tissue after LC/ESI/MS analyses. An elicitor preparation from purified yeast cell wall was used to inject the shoots of 3-week old seedlings or to infiltrate the cut lupin leaves. Qualitative and quantitative changes of isoflavonoids were measured at different time points after elicitation. In elicited lupin seedlings increased amounts of prenylated isoflavone aglycones were identified. The concentrations of glycosidic conjugates of isoflavones present in plant tissue were less affected.     

Wound-Associated Competency Factors Are Required for the Proximal Cell Responses of Soybean to the Phytophthora sojae Wall Glucan Elicitor

Intact soybean (Glycine max L. [Merr.]) tissues show distinct proximal and distal cell responses to the Phytophthora sojae (Kauf. and Gerde.) wall glucan elicitor. Proximal cells respond with accumulations of glyceollin and phenolic polymers, whereas distal cells respond with an increase of isoflavone conjugates. Comparison of the activities of the P. sojae glucan in the classical cut cotyledon and a cotyledon infiltration assay suggests that the proximal, but not the distal, responses to elicitor require tissue wounding. Washing the surface of cut cotyledons prior to elicitor treatment also greatly diminishes the proximal responses, which can be restored in a dose-dependent manner by prior treatment of the washed cells with wound exudate from cut "donor" cotyledons. Thus, discrete wound-associated factors, which we term elicitation competency factors, are required for the proximal cell response to the glucan elicitor. The wound factors induce a competent state that is transient in nature. Maximal elicitor response is seen 2 to 3 h after wounding, and cells become elicitor nonresponsive after 4 h. Competency is markedly affected by the age of tissues; cotyledons become more inherently competent as they approach senescence. The time course of attainment of the competent state and its duration are strongly affected by light and temperature. Since the wound-associated competency factors can also be obtained from washings of hypersensitive lesions, we hypothesize that similar competency factors may be released from hypersensitively dying cells in incompatible infections. This event may program the immediately surrounding cells to make them competent for the proximal defense responses.     

Anti-obesity activities of fermented soygerm isoflavones by Bifidobacterium breve

Soygerm isoflavones were subjected to fermentation by Bifidobacterium breve. Most of isoflavone glycosides (daidzin, glycitin and genistin) in soygerms were deglycosylated to their corresponding isoflavone aglycones (daidzein, glycitein and genistein) within 24 h fermentation. Fermented isoflavones significantly inhibited pancreatic lipase activity in fermentation-time and dosage dependant manner. When fermented isoflavones were orally administered with olive oil to SD rats, the triglyceride (TG) level in plasma after 2 h of ingestion was significantly lower than the control of only olive oil administered group whereas no such significant decrease in plasma TG was observed in unfermented isoflavone administered group. This result indicates that oral administration of fermented isoflavones effectively suppressed absorption of excessive lipid into a body. Addition of either unfermented or fermented soygerm isoflavones effectively inhibited adipocyte differentiation from 3T3-L1 in a dose dependent manner. In conclusion, B. breve successfully converted soygerm isoflavones into their aglycones, and these aglycones were more effective in suppressing lipid absorption as well as adipocytes differentiation than their glycosides.     

Tissue distribution of isoflavones in ewes after consumption of red clover silage

When discovered in the 50’s, isoflavones were suspected to provoke infertility syndrome in sheep grazing on clover. Many others effects of these phytoestrogens have been documented afterwards. To determine the distribution of isoflavone metabolites in ewe tissues and look for a link with their physiological impact, two ewes were fed a diet containing 50% red clover silage (variety Pawera) for one month with a daily intake of 157.6 mg/kg bw of total isoflavones. Only aglycones were fed due to the fermentation stage of the silage. At the sacrifice, isoflavone metabolites and aglycones were analyzed in blood, liver, kidney, lung, heart, muscle, ovaries, uterus, mammary glands, suprarenal glands, thymus, aorta, thyroid, pituitary gland, cerebellum, olfactory lobes, and brain hemispheres using HPLC-Coularray and LC-MS-MS. The major compounds recovered in tissues were equol and daidzein, present as glucuronides. Kidney concentrations were 10-fold higher than in other tissues. Penetration in brain was very limited. Reproductive organs contained higher concentrations of isoflavones than heart, muscle, or thymus. Distribution of isoflavones in ewe tissues is unequal and may reflect specific impact in some target tissues.     

Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations

The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein, and daidzein together make up 0.1–0.2 % of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by X-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of Saccharopolyspora erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations, the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance also found isoflavone coproduct potential.