Alfalfa Root Flavonoid Production Is Nitrogen Regulated

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.     

Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue

The exchange of chemical signals between soil bacteria (rhizobia) and legumes has been termed a molecular dialogue. As initially conceived in the early 1990s, it involved two main groups of molecules: nod gene-inducing flavonoids from plants and the mitogenic lipochito-oligosaccharide Nod factors of rhizobia. This review considers how subsequent research revealed the existence of a more complex set of interactions, featuring expanded roles for the original participants and contributions from additional plant and bacterial metabolites. Rhizobia respond to chemoattractants and growth-enhancing compounds in root exudates, and several plant nonflavonoids possess nod gene-inducing properties. Expression of non-nod genes is induced by flavonoids; these include encoders of a type I secreted protein and the entire type III, and possibly also type IV, secretion systems. Many other genes and proteins in rhizobia are flavonoid-inducible but their functions are largely unknown. Rhizobia produce far more Nod factor variants than was previously envisaged and their structures can be influenced by the pH of the environment. Other symbiotically active compounds or systems of rhizobia, some of them universally present, are: the surface polysaccharides, quorum-sensing N-acyl homoserine lactones, plant growth-promoting lumichrome and two-component regulatory systems.     

Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in beta-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can "hijack" cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.     

The reaction of flavonoid metabolites with peroxynitrite

There is much interest in the bioactivity of in vivo flavonoid metabolites. We report for the first time the hierarchy of reactivity of flavonoid metabolites with peroxynitrite and characterise novel reaction products. O-Methylation of the B-ring catechol containing flavonoids epicatechin and quercetin, and O-glucuronidation of all flavonoids reduced their reactivity with peroxynitrite. The reaction of the flavanones hesperetin and naringenin and their glucuronides resulted in the formation of multiple mono-nitrated and nitrosated products. In contrast, the catechol-containing flavonoids epicatechin and quercetin yielded oxidation products which when trapped with glutathione led to the production of glutathionyl-conjugates. However, the O-methylated metabolites of epicatechin yielded both mono- and di-nitrated products and nitrosated metabolites. The 3'-O-methyl metabolite of quercetin also yielded a nitrosated species, although its counterpart 4'-O-methyl quercetin yielded only oxidation products. Such products may represent novel metabolic products in vivo and may also express cellular activity.     

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.     

Flavonoid methylation: a novel 4'-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.     

斜茎黄芪根瘤菌结瘤基因nodA PCR扩增及PCR-RFLP分析

对采自我国北方地区的16株斜茎黄芪根瘤菌代表菌株的共同结瘤基因nodA进行了PCR扩增及PCR-RFLP分析研究。来自Mesorhizobium和Rhizobium系统发育分支的代表菌株都得到了nodA PCR扩增产物;而来自Agrobacterium系统发育分支的代表菌株都没有得到nodA PCR扩增产物。进一步的nodAPCR-RFLP分析结果表明斜茎黄芪根瘤菌具有很大的nodA基因遗传多样性,具有4种不同的16S rDNAPCR-RFLP遗传图谱类型的12株斜茎黄芪根瘤菌具有8种不同的nodA PCR-RFLP遗传图谱类型。但是斜茎黄芪根瘤菌nodA基因遗传多样性随种群而变化,来自M.septentrionale的具有相同的16S rDNA PCR-RFLP遗传图谱类型的4个代表菌株具有4种不同的nodA PCR-RFLP遗传图谱类型;而来自M.tempera-tum的具有相同的16S rDNA PCR-RFLP遗传图谱类型3个代表菌株则具有相同的nodA PCR-RFLP遗传图谱类型。此外,来自不同种的具有不同16S rDNA PCR-RFLP遗传图谱类型的菌株却具有相同的nodA PCR-RFLP遗传图谱类型,说明nodA基因可能在根瘤菌的不同种间发生了水平转移。     

Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti

Flavonoids play critical roles in legume–rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti , flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non- nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula .     

Reactivity of flavonoids with 1-hydroxyethyl radical: a gamma-radiolysis study

We have investigated the reactivity between 11 flavonoids and 1-hydroxyethyl radical (HER). HER was recently implicated in many liver injuries induced by ethanol intoxication. In this study, HER was generated by radiolysis; due to its reaction rate, HER is well known to be responsible for solute degradation in irradiated ethanol. Flavonoid ethanol solutions were irradiated with gamma-rays and the flavonoid degradation was followed by HLPC. We observed the degradation of flavonols while all other flavonoids (flavones, flavanones, dihydroflavonols, catechins) were not degraded after irradiation. The major radiolysis products were identified by NMR and LC-MS and we concluded that flavonols were essentially transformed into depsides. We proposed a reactivity mechanism between flavonols and HER. In a first step, H-transfer occurred from the 3-OH group to HER. Afterwards, C-ring opening occurred due to the presence of the 2,3-double bond in flavonols. Finally, we calculated the reaction constants in order to evaluate the antioxidant activity of flavonols against HER and to compare it with reference compounds.     

5- and 4'-Hydroxylated flavonoids affect voltage gating of single alpha-hemolysin pore

Molecular mechanisms of the influence of flavonoids on the voltage gating of a single alpha-hemolysin channel in planar lipid membranes are studied. It is shown that the addition of flavonoids hydroxylated in position 5 of the A-ring and in position 4' of the B-ring into bilayer bathing solution shifts the voltage dependence of channel switching from high- to low-conductance states to voltages nearer zero. It is concluded that the effect is likely to be attributed to a specific interaction of at least three flavonoid molecules with the voltage sensor of an alpha-hemolysin pore. Possible flavonoid binding sites and identification of amino acid residues included into the voltage sensor domain of the alpha-hemolysin channel are discussed.     

Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.)

Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution. At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.     

Structure–Activity Relationships for Antioxidant Activities of a Series of Flavonoids in a Liposomal System

Structurally diverse plant phenolics were examined for their abilities to inhibit lipid peroxidation induced either by Fe(II) and Fe(III) metal ions or by azo-derived peroxyl radicals in a liposomal membrane system. The antioxidant abilities of flavonoids were compared with those of coumarin and tert-butylhydroquinone (TBHQ). The antioxidant efficacies of these compounds were evaluated on the basis of their abilities to inhibit the fluorescence intensity decay of an extrinsic probe, 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA), caused by the free radicals generated during lipid peroxidation. All the flavonoids tested exhibited higher antioxidant efficacies against metal-ion-induced peroxidations than peroxyl-radical-induced peroxidation, suggesting that metal chelation may play a larger role in determining the antioxidant activities of these compounds than has previously been believed. Distinct structure–activity relationships were also revealed for the antioxidant abilities of the flavonoids. Presence of hydroxyl substituents on the flavonoid nucleus enhanced activity, whereas substitution by methoxy groups diminished antioxidant activity. Substitution patterns on the B-ring especially affected antioxidant potencies of the flavonoids. In cases where the B-ring could not contribute to the antioxidant activities of flavonoids, hydroxyl substituents in an catechol structure on the A-ring were able to compensate and become a larger determinant of flavonoid antioxidant activity.     

Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.     

Dietary flavonoids attenuate tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells. Structure-function relationships and activity after first pass metabolism

Flavonoids have been suggested to exert human health benefits by anti-oxidant and anti-inflammatory mechanisms. In this study, we investigated whether and by what mechanisms dietary flavonoids inhibit expression of cellular adhesion molecules, which is relevant to inflammation and atherosclerosis. We found that the capacity of flavonoids to inhibit tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells was dependent on specific structural features of the flavonoids. The 5,7-dihydroxyl substitution of a flavonoid A-ring and 2,3-double bond and 4-keto group of the C-ring were the main structural requirements for inhibition of adhesion molecule expression. In striking contrast, hydroxyl substitutions of the B- and C-rings but not the A-ring were essential for antioxidant activity. Hence, only hydroxyl flavones, such as apigenin and chrysin, and flavonols, such as galangin, kaempferol, and quercetin, were able to inhibit endothelial adhesion molecule expression, whereas flavone, chromone, the flavanone, naringenin, and the flavanol, (-)-epicatechin, were ineffectual. At low concentrations, the active flavonoids significantly attenuated expression of E-selectin and intercellular adhesion molecule 1 but not vascular cell adhesion molecule 1. In addition, exposure of apigenin and kaempferol to cultured hepatocytes, mimicking first pass metabolism, greatly diminished the inhibitory effect of flavonoids on endothelial intercellular adhesion molecule 1 expression. We conclude that the effect of dietary flavonoids on endothelial adhesion molecule expression depends on their molecular structure, concentration, and metabolic transformation but not their antioxidant activity.     

Rhizobium common nod genes are required for biofilm formation

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC , whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti . Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti 's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.