Influence of Flavonoids on Nodulation and Carbon Partitioning in Pea-Rhizobium Symbiosis

Rhizospheric application of nod regulators influenced the nodulation status, nodule efficiency and partitioning of assimilated CO₂ in Pisum sativum-Rhizobium leguminosarum symbiosis. Depending upon the plant growth stage, naringenin and flavone enhanced nodule number (22.8–87.6%), nodule weight (19.2–35.2%) and nitrogenase activity (17.8–108.6%). Syringaldehyde had negative effect on various parameters of symbiosis. Proportion of ¹⁴C-assimilates was higher in nodules of plants from naringenin and flavone treatments as compared to the control or syringaldehyde treatments. The enhanced nodule efficiency in naringenin and flavone treatments was reflected in the increased N content (12.7–50.4%) and biomass (4.3–35.1%) of plants.     

Effect of Low Temperature and Rh izospheric Application of Naringenin on Pea-Rhizobium leguminosarum biovar viciae Symbiosis

The production of Tsr factor by Rhizobium leguminosarum bv. viciae was influenced by low temperature (10°C) In the presence of seed exudate collected at 10°C and 25°C or naringenin (10fuM). Root exudate collected at 25°C and naringenin induced Tsr factor in R. leguminosarum causing thick and short root phenotype and root hair curling and deformation of host root. Root exudate collected at 10°C also induced root hair curling but Tsr activity was low. low temperature grown plants had poor nodulation, nitrogen fixation, nitrogen content and total blomass as compared to plants grown at 25°C. Rhizospheric application of naringenin partially alleviated the deleterious effect of low temperature on nodulation status and nodule efficiency.     

Preincubation of Mesorhizobium ciceri with flavonoids improves its nodule occupancy

Strains ofM. ciceri, symbionts of chickpea (Cicer arietinum) were incubated with the flavonoids naringenin, daidzein and quercetin which have earlier been reported as inducers and inhibitors ofnodABC-lacZ fusion ofM. ciceri. Preincubation ofM. ciceri with naringenin and daidzein (100 nmol/L) for 1 d improved the competitive ability of the inoculated strain while preincubation with quercetin decreased the nodule occupancy of inoculated strain under sterile conditions. Under nonsterile conditions induced strains of Rcd 301 and HT-6 formed by 23 and 18% more nodules, respectively, than untreated control. Quercetin-treated strains showed by 13–20% fewer nodules than untreated controls. Therefore, it is possible to regulate the competitive ability of inoculated strains by flavonoid treatment.     

Enantiomers of naringenin as pleiotropic,stereoselective inhibitors of cytochrome P450 isoforms

Interactions between naringenin and the cytochrome P450 (CYP) system have been of interest since the first demonstration that grapefruit juice reduced CYP3A activity. The effects of naringenin on other CYP isoforms have been less investigated. In addition, it is well known that interactions with enzymes are often stereospecific, but due to the lack of readily available pure naringenin enantiomers, the enantioselectivity of its effects has not been characterized. We isolated pure naringenin enantiomers by chiral high‐performance liquid chromatography and tested the ability of (R)‐,(S)‐ and rac‐naringenin to inhibit several important drug‐metabolizing CYP isoforms using recombinant enzymes and pooled human liver microsomes. Naringenin was able to inhibit CYP19, CYP2C9, and CYP2C19 with IC₅₀ values below 5 μM. No appreciable inhibition of CYP2B6 or CYP2D6 was observed at concentrations up to 10 μM. Whereas (S)‐naringenin was 2‐fold more potent as an inhibitor of CYP19 and CYP2C19 than (R)‐naringenin, (R)‐naringenin was 2‐fold more potent for CYP2C9 and CYP3A. Chiral flavanones like naringenin are difficult to separate into their enantiomeric forms, but enantioselective effects may be observed that ultimately impact clinical effects. Inhibition of specific drug metabolizing enzymes by naringenin observed in vitro may be exploited to understand pharmacokinetic changes seen in vivo. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

An Eco‐Friendly Enantioselective Access to (R)‐Naringenin as Inhibitor of Proinflammatory Cytokine Release

(RS)‐Naringenin is a flavanone well‐known for its beneficial health‐related properties, such as its anti‐inflammatory activity. The preparative enantioselective chromatographic resolution of commercial (RS)‐naringenin was performed on a Chiralpak AD‐H column (500×50 mm i.d., d_p 20 μm) using MeOH as eluent. The developed method is in accordance with the principles of green chemistry, since the environmental impact was lowered by recycling of the eluent, and allowed the production of gram amounts of each enantiomer with high purity (chemical purity >99%, enantiomeric excess (ee) >94%). Racemic and enantiomeric naringenin were subjected to an exhaustive in vitro investigation of anti‐inflammatory activity, aimed at evaluating the relevance of chirality. The assay with cultured human peripheral blood mononuclear cells (hPBMC) activated by phytohemagglutinin A revealed that (R)‐naringenin was more effective in inhibiting T‐cell proliferation than the (S)‐enantiomer and the racemate. Moreover, (R)‐naringenin significantly reduced proinflammatory cytokine levels such as those of TNF‐α and, with less potency, IL‐6. These results evidenced the anti‐inflammatory potential of naringenin and the higher capacity of (R)‐naringenin to inhibit both in vitro hPBMC proliferation and cytokine secretion at non toxic doses. Thus, (R)‐naringenin is a promising candidate for in vivo investigation.     

An efficient way from naringenin to carthamidine and isocarthamidine by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Biotransformation of naringenin with Aspergillus niger CGMCC 3.4628 yielded two hydroxylation products which were identified unambiguously as 6-hydroxylnaringenin (carthamidin) and 8-hydroxylnaringenin (isocarthamidin) by ESI–MS and ¹H-NMR. Both products simultaneously arrived at high level after 48 h in the biotransformation process. The highest conversion efficiency of carthamidin was 0.38 mg/mg of naringenin and that of isocarthamidin was 0.43 mg/mg of naringenin. Antioxidant property assay using a thin layer chromatography-bioautographic-based DPPH scavenging method demonstrated that both hydroxylation metabolites exhibited much stronger activity than naringenin. The high efficiency and convenient procedure makes the biotransformation with A. niger described in current work a potential way to produce carthamidin and isocarthamidin.     

Determination of naringin and naringenin in human plasma by high-performance liquid chromatography

An HPLC method for determining a flavonoid, naringin, and its metabolite, naringenin, in human plasma is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using genistin (for naringin) or daidzein (for naringenin) as an internal standard and solid-phase extraction using a Sep-Pak t C₁₈ cartridge. For the determination, HPLC was carried out using an Inertsil ODS-2 column (250x4.6 m I.D., 5 μm particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v; pH 7.1) for naringin and acetonitrile-0.1 M ammonium acetate solution-acetic acid (30:69:1, v/v; pH 4.9) for naringenin. The flow-rate was 1 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 280 nm for naringin and at 292 nm for naringenin. The detection limits on-column were about 0.2 ng for the two flavonoids.     

Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction

An HPLC method for determining a flavonoid naringin and its metabolite, naringenin, in human urine is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using hesperidin for naringin or hesperetin for naringenin as internal standard and solid-phase extraction using a strong anion exchanger, Sep-Pak Accell QMA cartridge. The HPLC assay was carried out using an Inertsil ODS-2 column (250×4.6 mm I.D., 5 μm particle size). The mobile phases were acetonitrile–0.1 M ammonium acetate–acetic acid (18:81:1, v/v; pH 4.7) for naringin and acetonitrile–0.1 M ammonium acetate–triethylamine (25:75:0.05; v/v; pH 8.0) for naringenin. The flow-rate was 1.0 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 282 nm for naringin and at 324 nm for naringenin. The lower limits of quantification were ca. 25 ng/ml for naringin and naringenin with R.S.D. less than 10%. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 5 ng for naringin and 1 ng for naringenin. A preliminary experiment to investigate the urinary excretion of naringin, naringenin and naringenin glucuronides after oral administration of 500 mg of naringin to a healthy volunteer demonstrated that the present method was suitable for determining naringin and naringenin in human urine.     

Controlling selectivity and enhancing yield of flavonoid glycosides in recombinant yeast

Flavonoid glycosides are known for their medicinal properties and potential use as natural sweeteners. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase from Dianthus caryophyllus was used as a whole-cell biocatalyst. The yeast system’s performance was characterized using the flavanone naringenin as a model substrate for the production of naringenin glycosides. It was found that final naringenin glycoside yields increased in a dose-dependent manner with increasing initial naringenin substrate concentrations. However, naringenin concentrations >0.5 mM did not give further enhancements in glycoside yield. In addition, a method for controlling overall selectivity was discovered where the glucose content in the culture medium could be altered to control the selectivity, making either naringenin-7-O-glucoside (N7O) or naringenin-4′-O-glucoside (N4O) the major products. The highest yields achieved were 87 mg/L of N7O and 82 mg/L of N4O using 40MSGI and 2xMSGI media, respectively. The effects of two intermediates involved in UDP-glucose biosynthesis, uridine 5′-monophosphate (UMP) and orotic acid, on glycoside yields were also determined. Addition of UMP to the culture medium significantly decreased glycoside yield. In contrast, addition of orotic acid to the culture medium significantly enhanced the glycoside yield and shifted the selectivity toward N7O. The highest naringenin glycoside yield achieved using 10 mM orotic acid in the 40MSGI media was 155 mg/L, a 71% conversion of substrate to product.     

Molecular characterization of flavonol synthase from poplar and its application to the synthesis of 3-<Emphasis Type="Italic">O</Emphasis>-methylkaempferol

Biosynthesis of flavonoid derivatives requires enzyme(s) having high reactivity as well as regioselectivity. We have synthesized 3-O-kaempferol from naringenin using two enzymes. The first reaction, in which naringenin is converted to kaempferol, is mediated by flavonol synthase (FLS). An FLS (PFLS) with strong catalytic activity was cloned and characterized from the genome sequence of the poplar (Populus deltoides). PFLS consists of a 1,008 bp ORF encoding a 38 kDa protein. PFLS was expressed in Escherichia coli with a glutathione-S-transferase (GST) tagging. The purified recombinant PFLS was characterized. Catalytically, it was more efficient than the previously characterized FLSs. A mixture of two E. coli transformants harboring either PFLS or ROMT9 (a kaempferol 3-O-methyltransferase) converted naringenin into 3-O-methylkaempferol.     

Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster

For the fermentative production of plant-specific flavanones (naringenin, pinocembrin) by Escherichia coli, a plasmid was constructed which carried an artificial biosynthetic gene cluster, including PAL encoding a phenylalanine ammonia-lyase from a yeast, ScCCL encoding a cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor A3(2), CHS encoding a chalcone synthase from a licorice plant and CHI encoding a chalcone isomerase from the Pueraria plant. The recombinant E. coli cells produced (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. When the two subunit genes of acetyl-CoA carboxylase from Corynebacterium glutamicum were expressed under the control of the T7 promoter and the ribosome-binding sequence in the recombinant E. coli cells, the flavanone yields were greatly increased, probably because enhanced expression of acetyl-CoA carboxylase increased a pool of malonyl-CoA that was available for flavanone synthesis. Under cultural conditions where E. coli at a cell density of 50 g/l was incubated in the presence of 3 mM tyrosine or phenylalanine, the yields of naringenin and pinocembrin reached about 60 mg/l. The fermentative production of flavanones in E. coli is the first step in the construction of a library of flavonoid compounds and un-natural flavonoids in bacteria.     

Exploration of glycosylated flavonoids from metabolically engineered <Emphasis Type="Italic">E. coli</Emphasis>

Flavonoids glycosylated with UDP-glucuronic acid and UDP-xylose are spatially distributed in nature. To produce these glycosides, E. coli was engineered to overexpress biosynthetic gene clusters of UDP-sugars (galU from E. coli K12, UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) from Micromonospora echinospora spp. calichensis). Flavonoids were glycosylated by overexpression of the glycosyltransferase gene (atGt-5) from Arabidopsis thaliana. Finally, metabolically engineered host E. coli (US89Gt-5) was generated. Production of flavonoid glycosides was observed in a biotransformation system consisting of flavonoids (naringenin and quercetin) exogenously fed to host cells. The glycosylated derivatives 7-O-glucuronyl naringenin (m/z⁺ 449), 7-O-xylosyl naringenin (m/z⁺ 405), and 7-O-glucuronyl quercetin (m/z⁺ 479) were detected and confirmed by ESI-MS/MS, ESI-MS/MS and LC/MS-MS analysis, respectively.     

关键酶泛素化位点对柚皮素生物合成的影响

黄酮类化合物具有多种生物活性,在食品、药品、化妆品等领域都有重要应用.柚皮素是多种重要黄酮类化合物生物合成的平台化合物.泛素化是蛋白质翻译后修饰的重要一环,参与调控细胞的生命活动.泛素化的蛋白质通过泛素-蛋白酶体系统降解,对维持细胞正常生理活动具有重要意义,对外源蛋白的表达和积累也可能具有显著影响.文中利用荧光双分子互...     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C₂-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7–4.2% of naringenin) and naringenin production by STS (1.4–2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Production of naringenin from D‐xylose with co‐culture of E. coli and S. cerevisiae

Heterologous production of naringenin, a valuable flavonoid with various biotechnological applications, was well studied in the model organisms such as Escherichia coli or Saccharomyces cerevisiae. In this study, a synergistic co‐culture system was developed for the production of naringenin from xylose by engineering microorganism. A long metabolic pathway was reconstructed in the co‐culture system by metabolic engineering. In addition, the critical gene of 4‐coumaroyl‐CoA ligase (4CL) was simultaneously integrated into the yeast genome as well as a multi‐copy free plasmid for increasing enzyme activity. On this basis, some factors related with fermentation process were considered in this study, including fermented medium, inoculation size and the inoculation ratio of two microbes. A yield of 21.16 ± 0.41 mg/L naringenin was produced in this optimized co‐culture system, which was nearly eight fold to that of the mono‐culture of yeast. This is the first time for the biosynthesis of naringenin in the co‐culture system of S. cerevisiae and E. coli from xylose, which lays a foundation for future study on production of flavonoid.     

Genetic mapping identifies a rice naringenin O-glucosyltransferase that influences insect resistance

Naringenin, the biochemical precursor for predominant flavonoids in grasses, provides protection against UV damage, pathogen infection and insect feeding. To identify previously unknown loci influencing naringenin accumulation in rice (Oryza sativa), recombinant inbred lines derived from the Nipponbare and IR64 cultivars were used to map a quantitative trait locus (QTL) for naringenin abundance to a region of 50 genes on rice chromosome 7. Examination of candidate genes in the QTL confidence interval identified four predicted uridine diphosphate-dependent glucosyltransferases (Os07g31960, Os07g32010, Os07g32020 and Os07g32060). In vitro assays demonstrated that one of these genes, Os07g32020 (UGT707A3), encodes a glucosyltransferase that converts naringenin and uridine diphosphate-glucose to naringenin-7-O-β-d -glucoside. The function of Os07g32020 was verified with CRISPR/Cas9 mutant lines, which accumulated more naringenin and less naringenin-7-O-β-d -glucoside and apigenin-7-O-β-d -glucoside than wild-type Nipponbare. Expression of Os12g13800, which encodes a naringenin 7-O-methyltransferase that produces sakuranetin, was elevated in the mutant lines after treatment with methyl jasmonate and insect pests, Spodoptera litura (cotton leafworm), Oxya hyla intricata (rice grasshopper) and Nilaparvata lugens (brown planthopper), leading to a higher accumulation of sakuranetin. Feeding damage from O. hyla intricata and N. lugens was reduced on the Os07g32020 mutant lines relative to Nipponbare. Modification of the Os07g32020 gene could be used to increase the production of naringenin and sakuranetin rice flavonoids in a more targeted manner. These findings may open up new opportunities for selective breeding of this important rice metabolic trait.     

Whole-cell bioconversion of naringenin to high added value hydroxylated compounds using Yarrowia lipolytica 2.2ab in surface and liquid cultures

The bioconversion process of bioactive naringenin by whole-cells of Yarrowia lipolytica 2.2ab for the production of increased value-added compounds is successfully achieved in surface and liquid cultures. This approach is an alternative to the commercial production of these bioactive compounds from vegetable sources, which are limited due to their low concentrations and the complexity of the purification processes. The experimentation rendered seven value-added compounds in both surface and liquid bioconversion cultures. Some of the compounds produced have not been previously reported as products from the bioconversion processes, such as the case of ampelopsin. Biosynthetic pathways were suggested for the naringenin bioconversion using whole-cells of Y. lipolytica 2.2ab. Finally, the extracts obtained from the naringenin bioconversion in liquid cultures showed higher percentage of inhibition of DPPH· and ABTS· radicals up to 32.88 and 2.08 times, respectively, compared to commercial naringenin.

     

The Differential Tissue Distribution of the Citrus Flavanone Naringenin Following Gastric Instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [³H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2?h post-gavage. After 18?h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18?h post-gavage. Total identified metabolites detected after 18?h in most tissues were only 1–5% of the levels detected after 2?h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2?h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18?h versus 2?h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18?h are retained as smaller decomposition molecules which cannot yet be identified.     

Influence of lime on nitrogen fixation by tropical and temperate legumes

Summary A nitrogen deficient Oxisol which had been fertilized with P, K, Zn and Mo received CaCO₃ at rates which increased continuously from zero to 22 tons/ha. Liming produced a range of pH in the saturation paste from 4.7 to 7.1; a range of calcium in the saturation extract from 0.3 to 3 meq/l; and a significant decline in available manganese. Responses of 9 tropical and 7 temperate legumes were compared across the pH variable.For Stylosanthes spp. nodule numbers and weight and plant yield declined above pH 5.5. Arachis hypogaea and Vigna sinensis, whose yield increased by only 30%, showed no clear improvement in nodulation or nodule effectiveness (acetylene reduction rate per unit nodule weight).Increased nodule effectiveness could account for most of the growth increase in Dolichos axillaris and Glycine max var. Kahala. Both the number and effectiveness of nodules increased for Desmodium intortum, Glycine wightii, Medicago sativa, and Trifolium subterraneum. Nodule size and effectiveness increased for G. max var. Kanrich. Only in one species, Coronilla varia, could increase in nodule numbers alone account for the increased growth associated with liming, although increased numbers of nodules probably accounted for much of the response by Lotus corniculatus, and by Desmodium canum and D. intortum up to pH 5.3.Increased nodulation and plant N contents were consistent with the conclusion that for most species improved N₂-fixation was the cause of growth improvement associated with liming. However, percent N was high in leaves of Vigna and Phaseolus vulgaris at all lime levels. In Phaseolus, variation in nodulation and growth were unrelated. The growth improvement was associated with decline in leaf N, suggesting that something other than N nutrition was limiting.Journal Series No. 1957 of the Hawaii Agricultural Experiment Station.Department of Soils and Plant Nutrition, University of California, Davis.Department of Agronomy and Soil Science, University of Hawaii, Honolulu, Hawaii.