Isoflavonoids in non-leguminous taxa: a rarity or a rule?

Isoflavonoids are characteristic metabolites in legumes and an overwhelming number of reports concerning them come from the Leguminosae. Nevertheless, the spectrum of isoflavonoid producing taxa includes the representatives of four classes of multicellular plants, namely the Bryopsida, the Pinopsida, the Magnoliopsida and the Liliopsida. At least 59 non-leguminous families have been reported to produce isoflavones sensu lato; coumestans have been reported in 3 families, coumaronochromones in 3, pterocarpans in 9 and rotenoids in 8 families. Prenylated isoflavones have been found in 15 non-leguminous families and isoflavone dimers, heterodimers or oligomers in three families. More than two hundred different isoflavonoid aglycones have been reported in non-legumes altogether. The number of individual structures is even greater if the variety of glycosides are considered. Enzymology and genetics of isoflavonoid biosynthesis have been studied almost exclusively in legumes, with the exception of a few model plants (i.e. Beta vulgaris, Arabidopsis thaliana, Nicotiana tabacum and Zea mays). The key step at the very beginning of the isoflavonoid metabolic pathway is the oxidation of flavanone connected with the migration of aryl moiety from C2 to C3 mediated by a CYP450 enzyme isoflavone synthase (IFS), which has been identified and cloned in multiple legumes and in sugar beet (Beta vulgaris, Chenopodiaceae). No information is available about the enzyme(s) responsible for the biosynthesis of isoflavonoid core in other taxa. Experimental data demonstrates the capability of numerous enzymes of non-legume origin to metabolize isoflavones as alternative substrates to other phenolics.     

Genetic and metabolic engineering of isoflavonoid biosynthesis

Isoflavonoids are a diverse group of secondary metabolites derived from the phenylpropanoid pathway. These compounds are distributed predominantly in leguminous plants and play important roles in plant–environment interactions and human health. Consequently, the biosynthetic pathway of isoflavonoid compounds has been widely elucidated in the past decades. Up to now, most of the structural genes and some of the regulatory genes involved in this pathway have been isolated and well characterized. Nowadays, the protective effects of the legume isoflavonoids against hormone dependent cancers, cardiovascular disease, osteoporosis, and menopausal symptoms have generated considerable interest within the genetic and metabolic engineering fields to enhance the dietary intake of these compounds for disease prevention. Subsequently, there are some great progresses in genetic and metabolic engineering to improve their yields in leguminous and non-leguminous plants and/or microorganisms. Because of the field of flavonoid biosynthesis has been reviewed fairly extensively in the past, this review concentrates on the more recent development in the isoflavonoid branch of phenylpropanoid pathway, including gene isolation and characterization. In addition, we describe the state-of-the-art research with respect to genetic and metabolic engineering of isoflavonoid biosynthesis.     

In vitro isoflavonoid production in callus from different organs of Pueraria lobata (Wild.) Ohwi

Pueraria lobata (Wild.) Ohwi is a medicinal plant producing large amounts of isoflavonoid glycosides. Here, the ability of in vitro callus cultures to synthesize isoflavonoids was tested. Callus cultures have been initiated from different explants of in vitro germinated plants using modified MS medium. Roots, leaves and stem segments were the best sources of callus tissue. The isoflavonoid profile and content was determined by means of chromatographic methods. Callus from all organs contained isoflavonoid aglycones: genistein and daidzein and daidzein glycosides: daidzin, puerarin and 3'-methoxypuerarin. The differences between each kind of explant were observed in both the total amount of isoflavonoids and in the proportion of individual compounds. The highest content was in root callus, followed by leaf- and stem callus.     

Isoflavonoids in the Rutaceae family: 1. Fortunella obovata, Murraya paniculata and four Citrus species

Several types of compounds with immunoreactivity similar to isoflavonoids were detected in water: ethanol extracts of leaves of Fortunella obovata Hort. ex Tanaka, Murraya paniculata Jack. and four Citrus species, namely C. aurantium L, C. grandis Osbeck, C. limonia Osbeck., and C. sinensis Osbeck (Rutaceae). The chromatographic mobilities of the immunoreactive substances were compared with those of authentic standards, revealing a spectrum of isoflavonoid metabolites in all plants studied. Aglycones as well as glycosides were recognized, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavonoids. A subsequent HPLC-MS study verified the identities of the main immunoreactive isoflavonoids and established the identities of several others, viz. glycitein, glycitin, ononin and sissotrin, including the malonylated and acetylated isoflavonoid glucosides. The estimated content of the individual immunoreactive entities ranged from a few microg to about 2 mg/kg (dry weight). It is concluded that the isoflavonoid metabolic pathway is present throughout the Rutaceae family.     

Major isoflavonoid contents of the 1-year-cultivated phytoestrogen-rich herb, Pueraria mirifica

Pueraria mirifica is a tuberous plant enriched with active phytoestrogens. There is no established information about the factors influencing isoflavonoid storage in the tubers. We investigated the tuberous storage of the major isoflavonoids of 1-year-old plants. Four cultivars of P. mirifica were cultivated in the same field trial during the same period to establish a unique plant age and differentiation under the same environment and soil conditions. The tubers collected from the 1-year-old plants in the summer, rainy season and winter were submitted to an HPLC analysis with a gradient system comprising 0.1% acetic acid and acetonitrile. Five major isoflavonoids, puerarin, daidzin, genistin, daidzein and genistein, were adopted as standards. P. mirifica tubers of different cultivars collected in the same season exhibited significant differences in individual and total isoflavonoid contents, showing chemovariety. P. mirifica tubers of the same cultivar collected from different seasons also exhibited significant differences in individual and total isoflavonoid contents, showing the influence of season. In conclusion, the tuberous storage of major isoflavonoids in 1-year-cultivated plants was greatly diverse and was strongly influenced by the season and plant genetics.     

Isoflavonoid derivatives from Lophira alata stem heartwood

Six isoflavonoid derivatives among which three are new have been isolated from the stem heartwood of Lophira alata. The structures were elucidated from spectroscopic and chemical evidences. Two have unusual carbon skeletons, possibly resulting from a variant of isoflavonoid biogenesis. The two compounds form the first members of a new subclass of flavonoid compounds which we call "isobiflavonoids". The presence of these isoflavonoid compounds in this plant of the Ochnaceae family has important chemotaxonomic implications since it modifies the botanic distribution of isoflavonoid compounds in non-leguminous plants.     

Production of isoflavone genistein in transgenic IFS tobacco roots and its role in stimulating the development of arbuscular mycorrhiza

Flavonoids and isoflavonoids are secondary metabolites in plants. With the goal of obtaining isoflavonoids from a wide range of plants, a few key studies have proven that isoflavonoids can be produced in non-leguminous plants by transgenic engineering. Many earlier studies investigate genistein biosynthesis in leaves and petals of isoflavone synthase (IFS) transgenic tobacco. However, most reports do not attempt to analyze quantification of genistein or do not check the presence of genistein in transgenic plant roots. In addition, little is known about the influence of genistein on arbuscular mycorrhiza (AM). In this paper, we reported that genistein was obtained from transgenic IFS tobacco roots. In addition, we revealed that endogenous genistein and 10???g?g^?1 exogenous genistein enhanced the development of AM symbiosis. We also revealed the relative expression levels of pertinent genes during the development of AM symbiosis. Our results suggest that genistein plays a positive role in the development of AM symbiosis in tobacco roots.     

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.     

Affinity of isoflavonoids for lipid bilayers evaluated with liposomal systems

Three parameters, i.e., the proportion of the amount incorporated into the liposomes, the partition coefficient in a system of n-octanol/phosphate buffered saline, and the retention times by HPLC, were measured to determine the lipophilicity of isoflavonoids. The presence of a hydroxyl group at 5-position of the A-ring and a methoxyl group at 4'-position of the B-ring in the isoflavonoid structure increased the three parameters. The localization of isoflavonoids in lipid bilayers was investigated by a liposome system with fluorescent probes. The location of the isoflavonoid depended on its structure. The cytotoxicity of isoflavonoids was investigated by a colony-formation assay with Chinese hamster lung fibroblast V79 cells. The structure-activity relationship of the cytotoxic activity partly reflected those of the three parameters. This suggests that the biological activities of isoflavonoids in vitro could be attributable to their affinity for lipid components in the cases where the estrogen receptors have no role.     

崖豆藤属植物中异黄酮类化学成分及其药理活性的研究进展

本文对豆科崖豆藤属植物中的异黄酮类化学成分及此类成分药理活性的国内外研究进行综述。从崖豆藤属植物中分离得到异黄酮类化合物159个,包括异黄酮及其苷类、鱼藤酮类、异黄烷类、紫檀烷类、二氢异黄酮类,它们具有抗肿瘤、抗炎、抗雌激素等作用。     

Effect of localized nitrate application on isoflavonoid concentration and nodulation in split-root systems of wild-type and nodulation-mutant soybean plants

Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypernodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized NO(3) (-) application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypernodulating mutant (NOD1-3). Nitrate application markedly decreased isoflavonoid concentrations in non-inoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar NO(3) (-) compared with the other root-half receiving 0 millimolar NO(3) (-). High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar NO(3) (-) was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of NO(3) (-) on root growth. The inoculated NOD1-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of NO(3) (-). These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of NO(3) (-) on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO(3) (-) also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that internal levels of root isoflavonoids may affect nodule development.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Pterocarpans from Bituminaria morisiana and Bituminaria bituminosa

The aerial parts of Mediterranean papilionaceous plants Bituminaria morisiana and B. bituminosa afforded, along with known phenolics, the prenylated pterocarpans bitucarpin A and B, whose structure was elucidated by spectroscopic techniques. A known isoflavonoid (8-prenyldaidzein) was also obtained for the first time as a genuine plant constituent. The accumulation of pterocarpans at the expense of biogenetically more primitive shikimate metabolites like furanocoumarins or isoflavonoids supports the inclusion of this plant, once part of the genus Psoralea, into the distinct genus Bituminaria.     

Growth and isoflavonoid accumulation of Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

We established cell suspension cultures derived from leaf, stem, and root calli of Pueraria candollei var. candollei and P. candollei var. mirifica using liquid Murashige and Skoog (MS) medium supplemented with 0.56 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Growth of the cell suspension cultures progressed to the stationary phase within 15–24 days. Methanolic extracts of cell suspension cultures of both varieties of P. candollei were analyzed using a validated HPLC protocol. All cell lines derived from leaf, stem, and root explants produced four major isoflavonoids: daidzein, daidzin, genistein, and genistin; these isoflavonoids were detected only in the roots of intact plants. Furthermore, the isoflavonoid contents of the cell suspension cultures were higher than those of intact plants. Thus, cell suspension culture of both varieties of P. candollei may be an effective tool for isoflavonoid production.     

Elicitor-Induced Association of Isoflavone O-Methyltransferase with Endomembranes Prevents the Formation and 7-O-Methylation of Daidzein during Isoflavonoid Phytoalexin Biosynthesis

The bioactive isoflavonoids of the Leguminosae often are methylated on the 4'-position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O-methyltransferase (IOMT) in the biosynthesis of 4'-O-methylated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O-methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT-green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to endomembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum-associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4',7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and subsequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoalexin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion.     

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.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.