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.     

Polyacetylenes accumulation in <Emphasis Type="Italic">Ambrosia maritima</Emphasis> hairy root and cell cultures after elicitation with methyl jasmonate

Three lines of hairy root culture of Ambrosia maritima induced by Agrobacterium rhizogenes ATCC15834 were established. Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol and their precursor pentayneene were produced by the hairy roots after elicitation with methyl jasmonate, the common signal molecule in the plant defense and development. Thiarubrine A diol was the main form detected in the medium. Maximum yield was achieved when the 13-day-old hairy root cultures were exposed to 40 M methyl jasmonate for 72 h. Callus and cell suspension cultures were established and maintained on Murashige and Skoog medium supplied with -naphthylacetic acid (NAA) and kinetin. When the cell suspension cultures were elicited with methyl jasmonate, pentayneene was the only polyacetylene produced. The yield of pentayneene in hairy root cultures was much higher (9.6 times) than that of cell suspension cultures.     

High isoflavone content and estrogenic activity of 25 year-old Glycine max tissue cultures

Soy isoflavones are phytoestrogens which have been associated with several health benefits. In the present study, we report the production of isoflavones in a collection of 40 strains of soya cell cultures established in 1975. A large variability in the isoflavone composition was observed and high-producing strains, with an isoflavone content of up to 46.3 mg g(-1) dry wt., were found. In comparison with soybeans, many callus strains had a higher isoflavone concentration (10-40 times) and a different ratio of genistin to daidzin forms. The highest producing strain was transferred to liquid medium in an Erlenmeyer flask and in a 10 l stirred-tank bioreactor where high isoflavone content (7% dry wt.), concentration (880 mg l(-1)) and a maximum productivity estimated to 60 mg l(-1) d(-1) were obtained. We further studied the estrogenic activity of pure compounds compared to plant cell culture extracts in the estrogen-responsive human endometrial Ishikawa cell line. Estrogen was confirmed to be 1000-10,000 times more active than isoflavones. The estrogenic activity of the extracts correlated to their isoflavone content. The activity of the malonyl isoflavones, assessed here for the first time, was lower than the aglycones. Taken together, these results suggest that soya cell cultures can be used as an alternative source to soybeans to provide high concentrations of bioactive isoflavones.     

茉莉酸甲酯、水杨酸和一氧化氮诱导怀槐悬浮细胞合成异黄酮及细胞结构变化

本文对比研究了茉莉酸甲酯(MeJA)、水杨酸(SA)和一氧化氮(NO)三种激发子对怀槐悬浮培养物异黄酮合成及细胞结构变化的影响。结果表明,在三种激发子的作用下怀槐细胞异黄酮合成量显著提高:200μmol/L MeJA、100μmol/L SA及50μmol／L SNP处理培养细胞9d后,异黄酮含量分别为同期对照的417．18％、185．45％和222．45％。同时细胞内发现染色很深的电子致密小体(EDB),其数量随着异黄酮含量的升高而增加,亦在第九天达到最多,与异黄酮积累呈现正相关性。推测激发子可能诱导植物细胞结构变化来响应次生代谢产物的合成。     

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.     

Relationship of viability and apoptosis to taxol production in Taxus sp. suspension cultures elicited with methyl jasmonate

Taxus cuspidata P991 in plant cell suspension culture is capable of producing the important anticancer agent Taxol (paclitaxel) and related taxanes. High-level production is obtained by elicitation with methyl jasmonate, but successful elicitation leads to loss of cell viability that cannot be recovered by subculture. Here, we test whether the loss of viability is due to a direct effect of methyl jasmonate. Upon subculture, the reduced viability continued in methyl jasmonate elicited cultures, but not in nonelicited control cultures. The growth reduction in elicited T. cuspidata P991 suspension cultures was evaluated by viability reduction measurements using phenosafranin and fluorescein diacetate. The viability reduction does not appear to be related to apoptosis based on DNA laddering analysis because it occurred very late (at day 35) in the culture period. DNA laddering was also found only after day 28 in T. canadensis C93AD (a Taxol-producing cell line) elicited with methyl jasmonate, implying that apoptosis is not the major death mechanism after elicitation. As compared to Taxol-producing cell lines, the viability of a nonproducing cell line, T. canadensis CO93D, was not severely affected by methyl jasmonate, indicating that methyl jasmonate itself is not the primary factor for viability reduction. Based on Northern analysis of taxadiene synthase mRNA from both elicited and nonelicited T. cuspidata P991, methyl jasmonate directly induces the production of this enzyme, which is the first committed step in the biosynthetic pathway for Taxol. As a result, both viability reduction and growth reduction appear related to a high production level of Taxol (and related taxanes) upon methyl jasmonate elicitation, rather than to the direct effect of methyl jasmonate.     

Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis

Taxus chinensis suspension cells were cultured in the modified Gamborg's B5 medium. Addition of 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ resulted in the greatest paclitaxel production, at 25 mg l^–1 in the cultures, being almost 40 times higher than that of the control culture, 10 times higher than that of the culture exposed to Ag⁺, 6 times higher than that of the culture elicited by chitosan and almost double that of the culture elicited by methyl jasmonate.     

Elicitation of rosmarinic acid by <Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension culture with abiotic elicitors

The growth of Lavandula vera MM plant cell suspension culture and rosmarinic acid biosynthesis under elicitation with benzothiadiazole and methyl jasmonate were investigated. Upon elicitation with 50 μM methyl jasmonate, the production of rosmarinic acid was enhanced 2.4-fold (3348 mg/l) compared to the non-elicited cells. The influence of benzothiadiazole on rosmarinic acid biosynthesis was weaker and 12 h after its addition the achieved yields were 20–30% higher compared to the control variant at this time. The influence of both elicitors on rosmarinic acid secretion into the culture medium was also discussed.     

Isoflavone production in Cyclopia subternata Vogel (honeybush) suspension cultures grown in shake flasks and stirred-tank bioreactor

Suspension cultures of the endemic South-African plant Cyclopia subternata were established for the first time and evaluated for the presence of isoflavones. The influence of light, as well as medium supplementation strategies with phenylalanine, casein hydrolysate and coconut water on biomass growth and isoflavone production were examined. The highest levels of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (275.57, 125.37 and 147.28 mg/100 g DW, respectively) were recorded for cultures grown in the absence of light, whereas coconut water substantially promoted biomass growth. Cell suspensions were subsequently grown in the 2-l stirred-tank bioreactor. Maximum productivity of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (0.96, 0.44 and 0.22 mg l^?1 day^?1, respectively) in bioreactor-cultivated cells was obtained for biomass grown in the dark and supplemented with coconut water. The results indicate that C. subternata suspension cultures can be utilised for the production of the specified isoflavone derivatives absent in the intact plant.     

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％。     

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.     

Biotic elicitors as a means of increasing isoflavone concentration of soybean seeds

Soybean (Glycine max) seeds contain isoflavones that have positive impacts on human health. Four greenhouse experiments were conducted to determine if isoflavone concentration of mature soybean seeds could be increased using elicitor compounds. The effects on soybean seed isoflavone concentrations following foliar applications of two lipo‐chitooligosaccharides (LCO) [Bj V (C_18:1 MeFuc) and Bj V (Ac, C¹⁶, MeFuc)], chitosan, actinomycetes spores (Streptomyces melanosporofaciens strain EF‐76) and yeast extract at different concentrations and growth stages were evaluated. Combined chitosan seed treatment and foliar applications were also evaluated. Concentrations of daidzein, genistein, glycitein, and total isoflavones were determined by HPLC. Foliar applications of LCOs, chitosan, and actinomycetes caused a marked increase in individual and total isoflavone concentration (ranging between 21% and 84%) of mature seeds when compared to untreated control plants. There were limited differences between the different concentrations and stages of application tested for chitosan and actinomycetes; however, response to LCOs was greatest at higher concentrations (i.e. 10^‐6 M) when applied at the early podding stage. Compared to untreated plants, combined seed treatment and foliar applications of chitosan increased individual and total isoflavone concentration of mature soybean seeds by 16% to 93%. Trends were similar for different cultivars, however, the magnitude of the response varied. Finally, response to foliar applications of yeast extract was highly concentration dependent with increases of up to 56% in total isoflavone observed with 2 mg mL^‐1. Results indicate that elicitors hold promise as a way of increasing isoflavone concentration of mature soybean seeds.     

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

Cell cycle-dependent disruption of microtubules by methyl jasmonate in tobacco BY-2 cells

Summary Methyl jasmonate, a growth-regulating substance that is ubiquitous in the plant kingdom, was found to disrupt cortical microtubules in tobacco cultured cells. It exerted a microtubule-disrupting effect only in cells at the S phase of the cell cycle. Neither microtubules in preprophase bands, spindles and phragmoplasts nor cortical microtubules at stages of the cell cycle other than the S phase were disrupted by methyl jasmonate. Jasmonic acid was as effective as methyl jasmonate in disrupting cortical microtubules.Abbreviations BUdR 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EGTA ethylene glycol bis(2-aminoethyl ether)-tetraacetic acid - FITC fluorescein isothiocyanate - FUdR 5-fluoro-2-deoxyuridine - JA jasmonic acid - JA-Me methyl jasmonate - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride     

Bioavailability of isoflavones

Isoflavones are disease protective components of soybeans. Isoflavone metabolism and bioavailability are key to understanding their biological effects. Isoflavone glucuronides, dominant biotransformation products in humans that are more hydrophilic than isoflavone aglycones, activate human natural killer cells in vitro but are less toxic to NK cells than the parent aglycones. Gut microbial isoflavone metabolites have also been identified, but remain to be well characterized. Gut transit time (GTT) seems to be a significant determinant of isoflavone bioavailability because women with more rapid GTT (<40 h) experienced 2-3-fold greater absorption of isoflavones than did women with longer GTT (>65 h). Isoflavone metabolism varies a great deal among individuals, thus limiting the quantitative value of urine or plasma isoflavones as biomarkers of soy ingestion. Defining and lessening interindividual variation in isoflavone bioavailability, and characterizing health-related effects of key isoflavone metabolites are likely to be crucial to further understanding of the health benefits of isoflavones.     

Differential metabolic response of narrow leafed lupine (Lupinus angustifolius) leaves to infection with Colletotrichum lupini

Flavones and isoflavones are a major group of phenolic secondary metabolites which occur in leaves of narrow leafed lupine (Lupinus angustifolius) either as free aglycones or in a form of glycosides and malonyl-glycosides. Profiles of phenolic compounds in leaves of seedlings infected with anthracnose causing fungus Colletotrichum lupini were compared to those of healthy plants. A HPLC with diode array UV detector was used as the analytical method and identification of these secondary metabolites was confirmed with a HPLC/MSⁿ instrument. Isomers of several target compounds differing in the glycosilation and/or malonylation pattern were detected in the studied samples. However, the application of standard HPLC with C18 columns resulted in the co-elution of several glyconjugates in single chromatographic peaks whereas for isoflavonoid aglycones complete resolution was achieved. Lupine plants grown in a greenhouse were either sprayed with the C. lupini spore suspension or the suspension was spotted on to wounded leaves. Profiles of the isoflavones were altered in result to infection with both methods. In particular, the concentration of isoflavone free aglycones detected in extracts from diseased plants was substantially increased in all of the studied samples. However, the pattern of these compounds depended on the age of lupine leaves as well as on the method of infection. Synthesis of luteone and 2′-hydroxygenistein was enhanced in the youngest leaves of plants sprayed with spores as well as in wound-infected leaves. Wighteone synthesis was induced mainly in older leaves of plants sprayed with the spore suspension.     

Effects of fermentation temperature on the content and composition of isoflavones and beta-glucosidase activity in sufu

Sufu is a popular fermented tofu product in China. The low quality of sufu produced in the hot summer is a big problem in sufu manufacture, so we prepared sufu at two different temperatures, 26 degrees C as normal and 32 degrees C as high temperature, and the effects of temperature on isoflavones and beta-glucosidase activity were investigated. Fermentation temperature did not cause significant differences in the recovery of isoflavones, but resulted in a different redistribution of isoflavone isomers in sufu. Sufu fermented at 26 degrees C was richer in isoflavone aglycones than at 32 degrees C; the enrichment of isoflavone aglycones might have the advantage of enhancing the physiological function. No 6'-O-malonyl-glucosides were detected in sufu fermented at 26 degrees C, whereas some 6'-O-malonyl-glucosides were found at 32 degrees C. A fermentation temperature of 26 degrees C benefited the beta-glucosidase production by fungi, which contributed to valid conversion from beta-glucosides to aglycones. It was also found that beta-glucosidase converted beta-glucosides more effectively than 6'-O-malonyl-glucosides and 6'-O-acetyl-glucosides into aglycones.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

Effects of elicitation on the production of saponin in cell culture of Panax ginseng

This study was initiated to investigate the impacts of elicitor concentration and elicitor-adding time on the saponin synthesis and the cell growth of Panax ginseng cell suspensions. Both of the elicitors tested, yeast extract and methyl jasmonate, significantly improved saponin production. The highest additive level of the seven ginsenosides tested was 2.07% (dry weight basis), which was 28-fold higher than that in the control. The optimum time to add either elicitor was found to be on the day of inoculation. The addition of either elicitor did not show as significant an influence on cell growth as on saponin production. It was advisable to remove 2,4-dichlorophenoxyacetic acid (2,4-D) from the medium when methyl jasmonate was used as the elicitor as methyl jasmonate interacts antagonistically with 2,4-D. These results suggest that the addition of an elicitor to ginseng cell suspension cultures could stimulate saponin production.     

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.