Biotransformation of soybean isoflavones by a marine <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 060524 and cytotoxicity of the products

A marine Streptomyces sp. 060524 capable of hydrolyzing the glycosidic bond of isoflavone glycosides, was isolated by detecting its β-glucosidase activity. 5 isoflavone aglycones were isolated from culture filtrates in soybean meal glucose medium. They were identified as genistein (1), glycitein (2), daidzein (3), 3′,4′,5,7-tetrahydroxyisoflavone (4), and 3′,4′,7-trihydroxyisoflavone (5), based on UV, NMR and mass spectral analysis. The Streptomyces can selectively hydroxylate at the 3′-position in the daidzein and genistein to generate 3′-hydroxydaidzein and 3′-hydroxygenistein, respectively. The Strain biotransformed more than 90% of soybean isoflavone glycosides into their aglycones within 108 h. 3′-hydroxydaidzein and 3′-hydroxygenistein exhibited stronger cytotoxicity against K562 human chronic leukemia than daidzein and genistein.     

A new perfusion system for the measurement and characterization of potential rates of soil nitrification

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.     

Acetophenone derivatives from Chilean isolate of Trichoderma pseudokoningii Rifai

The novel acetophenone derivative 2′,4′-dihydroxy-3′-methoxymethyl-5′-methylacetophenone and the known 2′,4′-dihydroxy-3′,5′-dimethylacetophenone (clavatol) were isolated from the culture filtrate of a Chilean strain of Trichoderma pseudokoningii. This revised version was published online in July 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

Algal Carotenoids 51. Secondary carotenoids 2.Haematococcus pluvialis aplanospores as a source of (3S, 3′S)-astaxanthin esters

Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,¹H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.     

Detection and characterization of anS-adenosyl-l-methionine: Flavonoid 7-O-methyltransferase from leaves ofPrunus x yedoensis matsum. with special reference to prunetrin formation

An enzyme,S-adenosyl-l-methionine: flavonoid 7-O-methyltransferase (F7OMT), catalyzing the transfer of the methyl group fromS-adenosyl-l-methionine (SAM) to the 7 position of sophoricoside (5, 7, 4′-trihydroxyisoflavone 4′-O-glucoside) and some of the other flavonoids, was detected in extracts from leaves ofPrunus x yedoensis, and it was partially purified (about 203-fold) by a combination of gel filtration and ion-exchange column chromatographies. F7OMT was isolated as a soluble enzyme with a pH optimun of 7.5 in K-phosphate buffer. The molecular mass of F7OMT, which had an isoelectric point at pH 4.1, was estimated by elution from a column of Sephadex G-100 to be about 36 kDa. The activity of F7OMT was stimulated by 14 mM 2-Co²⁺ and reagents that react with sulfhydryl groups. The apparentKm values for sophoricoside, its aglycone genistein (5, 7, 4′-trihydroxyisoflavone) and quercetin were 1.49, 2.19 and 1.89 μM, respectively. The apparentKm value for SAM as methyl donor was 2.08 mM. The specificity of F7OMT for methyl acceptors was not strict; flavonols, flavanones and flavanonols in addition to isoflavones served as methyl acceptor. An examination ofP. x yedoensis leaves during spring and autumn showed variations in the activities of F7OMT and UDP-glucose: isoflavone 4′-O-glucosyltransferase (I4′ GT). The activities of F7OMT and I4′GT increased in enlarging leaf tissues and then markedly declined when the leaves approached maturation. In autumn leaves F7OMT activity was scarcely detected, but a small peak of I4′GT activity was observed during autumnal reddening.     

Rhizospheric exudation of Eriophorum vaginatum L. — Potential link to methanogenesis

Root exudates are a direct link between primary production in higher plants and methanogenesis. The relationship has been widely studied on rice paddies, but less is known about its role in wetlands populated by naturally occurring species. This study provides information about the amount and composition of root exudates produced by a widespread mire plant, Eriophorum vaginatum L. For this purpose, E. vaginatum plants were grown in quartz sand in pots from April to October, and root exudates were collected once a month by percolation of the cultivation substrate. In June and October, a set of plants was labelled with ¹⁴CO₂ for two days and subsequently harvested for the determination of dry weight and for root exudates collected by the dipping method. The study supports earlier findings that natural wetland plants can enhance methanogenesis in their rhizosphere via active and seasonally varying exudation, but that the amount of exuded carbon (C) is many times lower than that delivered via litter formation. At both harvests in June and October, the proportion of incorporated radioactivity in shoots, roots and exudates was 92–96%, 4–8%, and 0.2%, respectively. New C was primarily fixed in the metabolically important carbohydrates, as well as acid anions that composed the main compounds of the new exudates. However, microbes seemed to rapidly metabolise the exudates into other substances like acetate. This was the dominant compound in the rhizoplane and rhizosphere, and it was the only detected substance that occurred in higher amounts outside the roots than inside them. Further studies in the field, including the quantification of gaseous end products, are necessary to complete our understanding of the carbon cycling in E. vaginatum-soil-microbe-system.     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.     

Mycorrhizal associations in Hong Kong Fagaceae

Polyphenols histochemically detected in fresh uninfected roots of Quercus, Castanopsis and Lithocarpus growing in Hong Kong and shown to be condensed tannins were found mainly as intracellular material in the cells of the root cap, the epidermal layer and the endodermis. The cell walls of the outer cortex and the endodermis also contained suberin. Following invasion by compatible ectomycorrhizal symbionts, condensed tannins disappeared from cells of the root cap and the epidermal layer but hyphae were prevented from colonizing the cortex presumably due to suberin barriers. In vitro experiments indicated that a number of broad-host ectomycorrhizal fungi could utilise various polyphenolic compounds, including tannins found in the root exudates of the host trees, with different degrees of efficiency.     

Lithospermum officinale callus produces shikalkin

To study biosynthetic abilities of Lithospermum officinale, callus formation from young leaves and stems of the plant was induced on Linsmaier-Skoog medium supplemented with 2,4-D (10⁻⁶ M) and kinetin (10⁻⁵ M). Maintaining the calli on this medium resulted in polyphenolic compounds production. Their transfer onto White medium containing IAA (10⁻⁷ M) and kinetin (10⁻⁵ M) resulted in the production of a red naphthoquinonic pigment named shikalkin. Shikalkin production from callus cultures was suppressed on the White medium containing NAA instead of IAA. This observation indicates that both shikalkin and polyphenolic acids biosynthetic pathways exist in the L. officinale callus cells and a regulatory system counterbalances the ratio of shikalkin to polyphenolic acids.     

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc⁺⁺, Nod ^−/+ phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.     

Dissolution of ferric phosphate by alfalfa (Medicago sativa L.) root exudates

Alfalfa (Medicago sativa L.) was grown in hydroponic culture to investigate adaptation to Fe-deficiency. Root exudates released into the nutrient solution from Fe-deficient plants were trapped and condensed on an amberlite XAD-4 resin column. The diethyl ether fraction of these exudates dissolved ferric phosphate remarkably. The dissolving capability was about 62 times higher than that of root exudates obtained from Fe-sufficient plants in complete nutrient solution. The Fe-dissolving compound was separated and identified. It was a new natural compound with molecular formula C₁₄H₁₀O₅ and was identified as 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran by means of mass spectrometry and ¹H-nuclear magnetic resonance. This new compound worked as a phytoalexin and inhibited completely the fungal growth of Fusarium oxysporum f. sp. phaseoli.     

Development of a simple molecular marker specific for detecting the self-compatible<Emphasis Type="Italic">S4′</Emphasis> haplotype in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

In sweet cherry (Prunus avium L.), theS4′ haplotype, characterized by a self-incompatibility (SI) defect in pollen, is self-compatible and is derived from the self-incompatibleS4 haplotype by x-ray mutagenesis.SFBs (S haplotype-specific F-box protein genes) have been found to associate with pollen determinant of SI. This report identified theSFB4′ of the self-compatibleS4′ haplotype. The alignment of the sequences ofSFB4′ andSFB4 by the BLAST program revealed a 4-bp deletion inSFB4′, which is TTTA. The sequence polymorphism generated by the TTTA deletion inSFB4′ was exploited to develop a simple molecular marker specific for detecting theS4′ but not theS4 haplotype. The simple marker specific to theS4′ haplotype can be visualized directly on an agarose gel, so it can be immediately applied to a marker-assistant cherry-breeding program. Thus, this work provides a practical molecular marker for cherry breeding. Principal author. An erratum to this article is available at .     

Temporal and spatial patterns of soil water following wildfire-induced changes in plant communities in the Great Basin in Nevada,USA

Infection of tall fescue (Festuca arundinacea Schreb.) with its endemicNeotyphodium coenophialum-endophyte (Morgan-Jones and Gams) Glenn, Bacon and Hanlin appears to reduce copper (Cu) concentrations in forage and serum of grazing animals, contributing to a range of immune-related disorders. A greenhouse experiment was conducted to identify effects of novel endophyte strains on Cu acquisition by tall fescue (Festuca arundinacea Schreb.) varieties Grasslands Flecha and Jesup infected with a novel, non ergot producing endophyte strain AR542, and two perennial ryegrass (Lolium perenne L.) varieties Aries and Quartet infected with a novel, non lolitrem B producing strain AR1, and their noninfected (E−) forms. Individual endophyte/grass associations were cultivated in nutrient solutions at 1.0 (P+) and 0.0 mM (P−) phosphorus concentrations. The Cu²⁺-binding activity of extracellular root exudates, and concentrations of Cu and other heavy metals in roots and shoots were measured. Extracellular root exudates of AR542-infected vs. E− tall fescue had higher Cu²⁺-binding activity only in P− nutrient solution as shown by lower concentration of free Cu²⁺ (0.096 vs. 0.188 mmol Cu²⁺ g⁻¹ root DM, respectively). The Cu²⁺-binding activity by root exudates of perennial ryegrass was not affected by endophyte infection, but was higher (i.e., lower concentration of free Cu²⁺) in P− vs. P+ nutrient solution (0.068 vs. 0.114 mmol Cu²⁺ g⁻¹ root DM). In this hydroponic experiment, Cu concentrations in shoots of both grasses were not a function of Cu²⁺-binding activity and endophyte effects on heavy metal concentrations in shoots and roots were specific for each variety. The Cu²⁺-binding activity of extracellular root exudates may affect Cu accumulation by field-grown, endophyte-infected tall fescue under P-limiting growth conditions and warrants verification by more specific methods.     

Facile synthesis of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N 6-ethenoadenosine nucleosides

Facile synthetic methods of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N ⁶-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthetic products were isolated and purified by preparative HPLC and their structures were confirmed by¹H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.     

Isolation of a new flavonol glycoside and its effects on the blue color of seed coats ofOphiopogon jaburan

From the blue seed coats ofOphiopogon jaburan, a new flavonol glycoside was isolated as needles and determined to be kaempferol 3-O-β-d-galactoside-4′-O-β-d-glucoside (OK-2) by UV and NMR spectral analyses. OK-2 and kaempfrol 3, 4′-di-O-β-d-glucoside (OK-1), which was detected previously, in the blue seed coat were present in a molar ratio of about 13:7. OK-2 was newly found as a factor causing the blueing effects on ophionin which is a main anthocyanin in the blue seed coats. The mixture of 4.8×10⁻³ M OK-2 and 2.5×10⁻³ M ophionin in Mcllvaine's buffer solution (pH 5.6) showed stable blue color, and the absorption spectrum of the mixture showed two absorption peaks and a shoulder in visible reasion, coinciding with that of the fresh blue seed coat. The effect of ophionin and OK-2 co-pigmentation on the blue color of seed coat ofO. jaburan was discussed.     

A transformation booster sequence (<Emphasis Type="Italic">TBS</Emphasis>) from <Emphasis Type="Italic">Petunia hybrida</Emphasis> functions as an enhancer-blocking insulator in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Several matrix-attachment regions (MARs) from animals have been shown to block interactions between an enhancer and promoter when situated between the two. Since a similar function for plant MARs has not been discerned, we tested the Zea mays ADH1 5′ MAR, Nicotiana tabacum Rb7 3′ MAR and a transformation booster sequence (TBS) MAR from Petunia hybrida for their ability to impede enhancer–promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP)::β-glucuronidase (GUS) fusion were assayed for GUS expression in vegetative tissues. We demonstrate that the TBS MAR element, but not the ADH1 or Rb7 MARs, is able to block interactions between the 35S enhancer and AGIP without compromising the function of either with elements from which they are not insulated. Accession numbers: TBS from Petunia hybrida cultivar V26, GenBank accession number EU864306.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.