Biosynthesis and genetic control of isovitexin 2″-O-arabinoside in petals of Silene dioica

In petals of Silene dioica plants, an enzyme has been demonstrated which catalyses the transfer of the arabinose moiety of UDP-arabinose to the hydroxyl group on the 2″-position of the carbon-carbon bound glucose of isovitexin. The presence of this arabinosyltransferase activity is controlled by the dominant allele gl^A. Maximal activity takes place at pH 7.2–7.5; the reaction is stimulated by the divalent metal ions Mg and Mn. For optimal solubilization of the enzyme, Triton X-100 is necessary. Substrate specificity and kinetic behaviour have been investigated.     

Identification, properties and genetic control of UDP-glucose: isovitexin 7-O-glucosyltransferase isolated from petals of Melandrium album

Summary In extracts of petals of M. album, an enzyme has been demonstrated which catalyzes the transfer of the glucosyl moiety of UDP-glucose to the 7-hydroxylgroup of isovitexin.This enzyme is controlled by a dominant gene G; in plants with the recessive genotype no glucosyltransferase activity could be detected.The enzyme was purified 16-fold by (NH₄)₂SO₄ fractionation and Sephadex-chromatography.The glucosyltransferase had a pH optimum of pH 7.5, was not stimulated by divalent metal ions, and had a true Km value of 1.2x10^-4 M for UDP-glucose and a true Km value of 4.6x10^-4 M for isovitexin.Several flavones with an apigenin hydroxylation pattern could serve as glucosyl acceptors. The highest activity was found, however, with isovitexin.The enzyme was unable to catalyze the transfer of xylose from UDP-xylose to the 7-hydroxylgroup of isovitexin, although isovitexin 7-O-xyloside has been found in petals of M. album plants.ADP-glucose could not serve as a glucosyl donor.The transferase activity was also present in leaves and calyces of GG plants. In these organs the transferase uses another flavone as a substrate. Neither isovitexin 7-O-glucoside nor isovitexin could be detected in these organs.     

Variation in the substrate specificity of allozymes catalyzing flavone-O-glucoside biosynthesis in Silene plants

The 7-O- and 2-O-glycosylation of the flavone isovitexin (6-C-glucosylapigenin) in the petals of Silene plants is accomplished by allozymes which differ in their specificity toward the sugar to be transferred. The g locus controls the 7-O-glycosylation; allele gG controls the binding of glucose, and allele gX that of xylose. In the present paper it is shown that at least two different forms of gG exist. The enzyme activities encoded by these two different alleles differ with respect to the flavone acceptor to which glucose is transferred. Allele gGm encodes a 7-O-glucosyltransferase that transfers glucose to isovitexin but that is not able to glycosylate isovitexin 2-O-rhamnoside. The 7-O-glucosyltransferase encoded by allele gGd preferentially transfers glucose to isovitexin 2-O-rhamnoside and not to isovitexin. The allozymes encoded by gGm and gGd were partly purified. Linearity of incorporation, pH optimum, effect of divalent cations and EDTA, apparent molecular weight, substrate specificity, and Michaelis enzyme kinetic parameters were determined for both enzyme activities. The simultaneous presence within a plant of gene glR, which controls the biosynthesis of isovitexin 2-O-rhamnoside, with either gGm or gGd leads to different glycosylation types. In gGm/glR plants two monoglycosides accumulate in the petals, isovitexin 7-O-glucoside and isovitexin 2-O-rhamnoside, respectively, whereas in gGd/glR plants the corresponding diglycoside, isovitexin 7-O-glucose 2-O-rhamnoside, is synthesized. The distribution of the two alleles over chemical races of Silene pratensis in Europe is described; possible evolutionary relations between the various glycosyltransferases in Silene are discussed.These investigations were supported by the Foundation for Fundamental Biological Research (BION; Grant 14-15-01), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

The pH dependent substrate specificity of udp-glucose: isovitexin 2″-O-glucosyltransferase in Silene alba

In Silene alba plants the dominant allele of gene Fg controls an enzyme which catalyses the formation of isovitexin 2″-O-glucoside both in petals and green parts. Both isovitexin and isoorientin can act as substrate. K_mvalues for the isovitexin glucosylation are 0.09 mM for isovitexin and 0.3 mM for UDP-glucose, V_max 0.17 nmol min^?1 mg protein^?1. For the isoorientin glucosylation K_m values of 0.45 mM for isoorientin, of 0.75 mM for UDP-glucose and V_max of 0.27 nmol min^?1 mg protein^?1 are found. The pH optima for both substrates differ markedly. For the substrate with one hydroxyl in the B-ring, isovitexin, the pH optimum is pH 8.5. For isoorientin, which has two hydroxyls in the B-ring, a pH optimum of 7.5 is found. These results suggest that the B-ring hydroxylation pattern influences the pH at which the substrate has optimal affinity for the enzyme. The location of the carbon-carbon bound glucose on a the flavonoid skeleton is of importance for enzyme activity as well. Vitexin, which has glucose at the 8-position, was not a substrate. The glucosylation of vitexin could, however, be demonstrated in enzyme extracts of petals of plants, grown from seed collected in Armenia; in these petals apart from isovitexin glycosides, vitexin glycosides are found as well.     

The geographic distribution of flavone-glycosylating genes in Silene pratensis (Rafn.) Godron and Gren. (Caryophyllaceae)

In Silene pratensis three loci (g, gl and fg) control the glycosylation of isovitexin. Three alleles are known for both the g-locus (g, g ^G and g ^X ) and the gl-locus (gl, gl ^A and gl ^R ); for the fg-locus there are only two alleles (fg and Fg). The distribution of these alleles over 285 European populations of S. pratensis has been investigated. It was concluded that there are three different chemical races within S. pratensis in Europe. The first race contains the populations in western and southern Europe and displays high frequencies of g ^G , gl and fg. The frequencies of g ^G and gl ^R are very high in the second chemical race, which can be found in the USSR, Scandinavia and eastern Poland. The third chemical race occurs in central Europe and in this race the frequencies of both g and gl ^R are high, Fg has low to moderate frequencies in the second and third groups. The alleles gl ^A and g ^X are seldom found in S. pratensis, but are present in the closely related S. dioica. They do occur with low frequencies in some populations of S. pratensis, possibly as a result of hybridization with S. dioica.     

The 2″-O-glucosylation of vitexin and isovitexin in petals of Silene alba is catalysed by two different enzymes

Two separate genes, Fg and Vg, which govern the presence of isovitexin 2″-O-glucoside and vitexin 2″-O-glucoside respectively in the petals of Silene alba control different glucosyltransferases. In Vg/Vg,fg/fg plants no isovitexin 2″-O-glucosyltransferase was present and in vg/vg,Fg/Fg plants no vitexin 2″-O-glucosyltransferase activity could be detected. The Fg-controlled UDP-glucose: isovitexin 2″-O-glucosyltransferase has a pH optimum of8.5, while the Vg-controlled vitexin 2″- O-glucosyltransferase has a pH optimum of7.5. Both glucosyltransferases are stimulated by the divalent cations Ca²⁺, Co²⁺, Mn²⁺ and Mg²⁺. For isovitexin 2″-O-glucosylation, however, much higher concentrations are needed than for vitexin 2″-O-glucosylation.For UDP-glucose a ‘true K_m’ value of0.3 mM with the Fg-controlled and of 0.2 mM with the Vg-controlled enzyme was found. For isovitexin and vitexin these values are respectively 0.09 and 0.01 mM.     

Genetic control and biosynthesis of two new flavone-glycosides in the petals of Melandrium album

In a chemicogenetic analysis of the geographical distribution of flavone-glycosides in the petals of Melandrium album, we found two unknown flavone-glycosides in ten Hungarian and four German populations. By means of classical techniques for the identification and structure determination of flavonoids, the structure of these flavones turned out to be 6-C-glucosylglucosylapigenin and 7-O-glucosyl-6-C-glucosylglucosylapigenin, respectively. Genetic analysis showed that the coupling of glucose to the carbon-carbon bound glucose of isovitexin (6-C-glucosylapigenin) was controlled by a single dominant gene, Fg. Fg controls a UDP-glucose: isovitexin 6-C-glucosylglucosyltransferase. By means of ammonium sulfate fractionation and Sephadex chromatography, the enzyme was purified sixfold. The partly purified enzyme had a pH optimum between 8.0 and 8.5. The apparent K _m value for UDP-glucose in the presence of 1.0 mm isovitexin was 2.2 mm. The apparent K _m value for isovitexin in the presence of 1.8 mm UDP-glucose was 0.08 mm. The glucosyltransferase activity was stimulated by the divalent cations Mn, Mg, Co, and Ca. Neither 7-O-glucosyl nor 7-O-xylosyl isovitexin could serve as an enzyme substrate. Therefore, the biosynthesis of 7-O-glucosyl-6-C-glucosylglucosylapigenin found in the petals of M. album proceeds in a sequential manner: first the formation of 6-C-glucosylglucosylapigenin, followed by 7-O-glucosylation. Isovitexin 6-C-glucosylglucosyltransferase activity controlled by gene Fg could also be demonstrated in leaves of Fg plants. The enzyme probably uses another substrate in these green parts of the plant, because both isovitexin and isovitexin-glycosides are absent.     

Acinetobacter strains carry two functional oligosaccharyltransferases,one devoted exclusively to type IV pilin,and the other one dedicated to O‐glycosylation of multiple proteins

Multiple species within the Acinetobacter genus are nosocomial opportunistic pathogens of increasing relevance worldwide. Among the virulence factors utilized by these bacteria are the type IV pili and a protein O‐glycosylation system. Glycosylation is mediated by O‐oligosaccharyltransferases (O‐OTases), enzymes that transfer the glycan from a lipid carrier to target proteins. O‐oligosaccharyltransferases are difficult to identify due to similarities with the WaaL ligases that catalyze the last step in lipopolysaccharide synthesis. A bioinformatics analysis revealed the presence of two genes encoding putative O‐OTases or WaaL ligases in most of the strains within the genus Acinetobacter. Employing A. nosocomialis M2 and A. baylyi ADP1 as model systems, we show that these genes encode two O‐OTases, one devoted uniquely to type IV pilin, and the other one responsible for glycosylation of multiple proteins. With the exception of ADP1, the pilin‐specific OTases in Acinetobacter resemble the TfpO/PilO O‐OTase from Pseudomonas aeruginosa. In ADP1 instead, the two O‐OTases are closely related to PglL, the general O‐OTase first discovered in Neisseria. However, one of them is exclusively dedicated to the glycosylation of the pilin‐like protein ComP. Our data reveal an intricate and remarkable evolutionary pathway for bacterial O‐OTases and provide novel tools for glycoengineering.     

Large and unexpected variation in expression and substrate specificity of allelic glycosyltransferase gens on three loci in Silene dioica

A pilot study with regard to the distribution and expression of isovitexin glycosylation genes in Silene dioica showed that in this species, like in S. pratensis, a large variation in expression and substrate specificty exists for these genes. From S. dioica populations from Finland, Poland, Czechoslovakia, W. Germany, France and England a single plant was tested for allelic variation on the isovitexin glycosylation loci g, f and gl. In the English population a new allele of the g-locus, gA, with a different substrate specificity was identified. In the plant taken from the population in Poland a new allele glR1 was identified which was hypostatic to the allele glR known from S. pratensis. In Germany, a new allele gX1 was identified which is hypostatic over gX from S. pratensis. With regard to the isovitexin-glycoside phenotype the populations from Finland, Poland, Czechoslovakia, and England were homogeneous, whereas the populations of W. Germany and France showed variation. The mechanisms leading to this variation and their significance were discussed.     

Deletion-mapping of resistance against chlorate in Klebsiella aerogenes

Summary Chlorate-resistant mutants with deletions have been isolated from Klebsiella aerogenes. Mutants with deletions in the gal-bio region of the genetic map can be divided into 9 groups. From the properties of the various groups of mutants the gene order nic A-aro G-gal-chl D-hut-bio-uvr B-chl A is deduced. Furthermore deletions have been observed in a segment of the chromosome containing nic B, thi B, inl B, and chl G. Thi B and chl G are adjacent but no more information about the gene order in this segment can be given. In both segments a great homology is observed with the corresponding regions of the genetic maps of E. coli and S. typhimurium. Deletions of chl A, chl D or chl G have a pleiotropic effect. Mutants with a deletion of one of these genes do not produce nitrite from nitrate or gas from glucose.     

UV-microscopic studies on the vacuolar changes caused by the flavone “aglycone” isovitexin inSilene pratensis plants

Summary UV-microscopic and chromatographic studies have been performed on the variation in contents and configuration of the flavones present in epidermal cells of the petals, stem leaves, rosette leaves and cotyledons ofSilene pratensis plants. Most of the flavone contents is located in the vacuole of the upper epidermis cells, the concentration depending on the light intensity at which the plants were grown. In plants able to glycosylate isovitexin in the petals (genotypegG/. gl/gl fg/fg, accumulating isovitexin 7-O-glucoside) the vacuole is completely filled with the UV absorbing flavone. In plants which are unable to glycosylate isovitexin in their petals (genotypeg/g gl/gl fg/fg, accumulating only isovitexin) the upper epidermal cells of stem leaves and petals contain droplet like structures in their vacuoles. At high light intensities these structures increase in mass and become detectable in the visible light. These denser structures often condense to structures with radiating threads.As compared with the accumulation of isovitexin in upper epidermal cells of stem leaves and petals in genotypeg/g gl/gl fg/fg, the cotyledons and the rosette leaves contain two isovitexin glycosides. In the latter organs the upper epidermal cells are very similar to the upper epidermal cells fromgG/. gl/gl fg/fg plants, having a vacuole filled with UV absorbing material. It appears therefore that isovitexin itself causes the formation of the structurés in the cells. It was shown by varying the light intensity that a relative high concentration of isovitexin is necessary for the droplet like structures to appear. Still higher concentrations are needed for the formation of the structures with radiating threads. It is hypothesized that isovitexin interferes with the energy supply of the cells, which therefore are not able to maintain their turgor.     

Identification and mapping of pm2026: a recessive powdery mildew resistance gene in an einkorn (Triticum monococcum L.) accession

Triticum monococcum accession TA2026 showed resistance to wheat powdery mildew. To identify the resistance gene and transfer it to common wheat, genetic analysis and molecular mapping were conducted using an F₂ population and derived F₃ families from the cross of TA2026 × M389. The results indicated that TA2026 possessed a recessive powdery mildew resistance gene. This gene was mapped to the terminal portion of chromosome 5A^mL and flanked by SSR marker loci Xcfd39 and Xgwm126. Eight RFLP markers previously mapped to the terminal chromosome 5A^mL were converted into STS markers. Three loci, detected by MAG1491, MAG1493 and MAG1494, the STS markers derived from RFLP probes CDO1312, PSR164 and PSR1201, respectively, were linked to this resistance gene with Xmag1493 only 0.9 cM apart from it. In addition, the STS marker MAG2170 developed from the tentative consensus wheat cDNA encoding the Mlo-like protein identified a locus co-segregating with Xmag1493. This is the first recessive powdery mildew resistance gene identified on chromosome 5A^m, and is temporarily designated pm2026. We have successfully transferred it to a tetraploid background, and this resistance stock will now be used as the bridge parent for its transfer to common wheat.     

At the nexus of three kingdoms: the genome of the mycorrhizal fungus Gigaspora margarita provides insights into plant,endobacterial and fungal interactions

As members of the plant microbiota, arbuscular mycorrhizal fungi (AMF, Glomeromycotina) symbiotically colonize plant roots. AMF also possess their own microbiota, hosting some uncultivable endobacteria. Ongoing research has revealed the genetics underlying plant responses to colonization by AMF, but the fungal side of the relationship remains in the dark. Here, we sequenced the genome of Gigaspora margarita, a member of the Gigasporaceae in an early diverging group of the Glomeromycotina. In contrast to other AMF, G. margarita may host distinct endobacterial populations and possesses the largest fungal genome so far annotated (773.104 Mbp), with more than 64% transposable elements. Other unique traits of the G. margarita genome include the expansion of genes for inorganic phosphate metabolism, the presence of genes for production of secondary metabolites and a considerable number of potential horizontal gene transfer events. The sequencing of G. margarita genome reveals the importance of its immune system, shedding light on the evolutionary pathways that allowed early diverging fungi to interact with both plants and bacteria.     

Quantitative disease resistance to the bacterial pathogen Xanthomonas campestris involves an Arabidopsis immune receptor pair and a gene of unknown function

Although quantitative disease resistance (QDR) is a durable and broad‐spectrum form of resistance in plants, the identification of the genes underlying QDR is still in its infancy. RKS1 (Resistance related KinaSe1) has been reported recently to confer QDR in Arabidopsis thaliana to most but not all races of the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc). We therefore explored the genetic bases of QDR in A. thaliana to diverse races of X. campestris (Xc). A nested genome‐wide association mapping approach was used to finely map the genomic regions associated with QDR to Xcc12824 (race 2) and XccCFBP6943 (race 6). To identify the gene(s) implicated in QDR, insertional mutants (T‐DNA) were selected for the candidate genes and phenotyped in response to Xc. We identified two major QTLs that confer resistance specifically to Xcc12824 and XccCFBP6943. Although QDR to Xcc12824 is conferred by At5g22540 encoding for a protein of unknown function, QDR to XccCFBP6943 involves the well‐known immune receptor pair RRS1/RPS4. In addition to RKS1, this study reveals that three genes are involved in resistance to Xc with strikingly different ranges of specificity, suggesting that QDR to Xc involves a complex network integrating multiple response pathways triggered by distinct pathogen molecular determinants.     

5‐HT2A Receptor Gene Promoter Polymorphism in Relation to Abdominal Obesity and Cortisol

Objective: There is considerable evidence that cortisol secretion is associated with obesity. The regulation of the 5‐hydroxytryptamine receptor 2A (5‐HT_2A) gene might play an essential role because it is involved in the control of cortisol secretion. Therefore, we examined the potential impact of the 5‐HT_2A ?1438G/A promoter polymorphism on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol, in 284 unrelated Swedish men born in 1944. Research Methods and Procedures: The subjects were genotyped by using polymerase chain reaction amplification of the promoter region of the gene for 5‐HT_2A followed by digestion of the reaction product with the restriction enzyme MspI. Results: The frequencies were 0.39 for allele ?1438A and 0.61 for allele ?1438G. Homozygotes for the ?1438G allele had, in comparison with ?1438A/A subjects, higher body mass index, waist‐to‐hip ratio, and abdominal sagittal diameter. Moreover, cortisol escape from 0.25‐mg dexamethasone suppression was found in subjects with the ?1438A/G genotype. Serum leptin, fasting insulin, and glucose, as well as serum lipids, were not different across the ?1438G/A genotype groups. Discussion: From these results, we suggest the possibility that an abnormal production rate of the 5‐HT_2A gene product might lead to the development of abdominal obesity. The pathophysiology could involve stress factors that destabilize the serotonin‐hypothalamic‐pituitary‐adrenal system in those with genetic vulnerability in the serotonin receptor gene.     

The two genes encoding protein synthesis initiation factor eIF-5A in Saccharomyces cerevisiae are members of a duplicated gene cluster

Summary Translation initiation factor eIF-5A is an abundant protein in which a lysine residue is modified by spermidine to form the amino acid derivative, hypusine. The factor is encoded by two genes in Saccharomyces cerevisiae, called TIF51A and TIF51B, which are regulated reciprocally by oxygen and by heme. TIF51B, also called ANBI, is located on chromosome X in a region called COR. We physically mapped TIF51A and its associated serine tRNA₂ gene by the method of chromosome fragmentation and pulsed-field gel electrophoresis. TIF5IA maps 90 kb from the left end of chromosome V in a region called ARC. The COR and ARC regions contain CYCI and CYC7, respectively, and appear to be duplications carrying numerous related genes. The arrangements of related genes in the two regions are incompatible with a duplication mechanism involving a circular intermediate.