Pleiotropic effect of a pelargonidin-hydroxylation gene in Silene dioica?

In petals of Silene dioica a gene P has been identified, which controls the 3′-hydroxylation of the B-ring of pelargonidin to cyanidin. Another gene Ac controls the acylation of the terminal sugar at the 3-position of anthocyanin 3-rhamnosylglucoside-5-glucosides. In p/p plants the bound acyl group is p-coumaric acid; in P/P plants, however, it is caffeic acid. Gene P seems to exert a pleiotropic effect: it not only controls the hydroxylation of the B-ring of pelargonidin but also that of the acyl group.     

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.     

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.     

Differences in the incorporation pattern of labelled cinnamic acid and l-phenylalanine into the anthocyanins of Petunia hybrida

The incorporation of phenylalanine-[¹⁴C] into anthocyanins of petals of Petunia hybrida is greater than that of cinnamic acid-[¹⁴C]. Moreover, there is a preferential incorporation of phenylalanine-[¹⁴C] into delphinidin 3-monoglucoside, as compared with the incorporation into cyanidin and peonidin 3-monoglucosides.     

Genetic control of chalcone synthase activity in flowers of Antirrhinum majus

Chalcone synthase activity was demonstrated in flower extracts of defined genotypes of Antirrhinum majus. Independent of the genetic state of the g     

Biosynthesis and genetic control of isovitexin 7-O-xyloside in the petals of melandrium album

An enzyme was detected in petal extracts of Melandrium album which catalysed the transfer of the xylose moiety of UDP-xylose to the 7-hydroxyl group of isovitexin. Genetical analysis revealed that the presence of the dominant allele g^x was necessary for enzymic activity. This activity was independent of the residual genetic background. Xylosyltransferase activity is also present in extracts of g^Gg^x plants, in which the product of the enzyme is not detectable. Maximal activity was found between pH 7·0 and 7·5; MnCl₂ inhibited this transfer. The enzyme had an ‘apparent K_m' value of 1·0 mM for UDP-xylose and of O·04 mM for isovitexin.     

Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures, was not observed with the H. gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation.     

Biosynthesis of pterocarpan and isoflavan phytoalexins in Medicago sativa: the biochemical interconversion of pterocarpans and 2′-hydroxyisoflavans

Feeding experiments have shown that 2′-7-dihydroxy-4′-methoxy-isoflavone-[Me-¹⁴C] and -isoflavanone-[Me-¹⁴C] are efficient precursors of the phytoalexins demethylhomopterocarpin, sativan and vesitol in CuCl₂-treated lucerne (Medicago sativa) seedlings. Demethylhomopterocarpin-[Me-¹⁴C] was also incorporated into sativan and vestitol, and vestitol-[Me-¹⁴C] was incorporated into demethylhomopterocarpin and sativan. Thus, the pterocarpan demethylhomopterocarpin and the 2′-hydroxy-isoflavan vestitol are interconvertible in M. sativa, but incorporation data, and the results of kinetic feeding experiments with l-phenylalanine-[U-¹⁴C] suggest that these compounds are synthesized simultaneously from a common intermediate, which could be involved in the interconversion. A carbonium ion, derived from an isoflavanol, a likely intermediate in the biosynthetic reductive sequence from 2′,7-dihydroxy-4′-methoxy-isoflavone and -isoflavanone, is proposed as this common intermediate. 7-Hydroxy-2′,4′-dimethoxyisoflavone-[4′-Me-¹⁴C] was a very poor precursor of all three phytoalexins. Sativan, then, is most probably derived by methylation of vestitol. The incorporation of vestitol-[Me-¹⁴C] into demethylhomopterocarpin, but not into maackiain, pterocarpan phytoalexins of red clover (Trifolium pratense), is also demonstrated.     

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid–general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid–general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.     

Salt sensitivity of the vegetative and reproductive stages in chickpea (Cicer arietinum L.): Podding is a particularly sensitive stage

Soil salinity is an increasing problem, including in regions of the world where chickpea is cultivated. Salt sensitivity of chickpea was evaluated at both the vegetative and reproductive phase. Root-zone salinity treatments of 0, 20, 40 and 60 mM NaCl in aerated nutrient solution were applied to seedlings or to older plants at the time of flower bud initiation. Even the reputedly tolerant cultivar JG11 was sensitive to salinity. Plants exposed to 60 mM NaCl since seedlings, died by 52 d without producing any pods; at 40 mM NaCl plants died by 75 d with few pods formed; and at 20 mM NaCl plants had 78-82% dry mass of controls, with slightly higher flower numbers but 33% less pods. Shoot Cl exceeded shoot Na by 2-5 times in both the vegetative and reproductive phase, and these ions also entered the flowers. Conversion of flowers into pods was sensitive to NaCl. Pollen from salinized plants was viable, but addition of 40 mM NaCl to an in vitro medium severely reduced pollen germination and tube growth. Plants recovered when NaCl was removed at flower bud initiation, adding new vegetative growth and forming flowers, pods and seeds. Our results demonstrate that chickpea is sensitive to salinity at both the vegetative and reproductive phase, with pod formation being particularly sensitive. Thus, future evaluations of salt tolerance in chickpea need to be conducted at both the vegetative and reproductive stages.     

Pseudocitrobacter gen. nov., a novel genus of the Enterobacteriaceae with two new species Pseudocitrobacter faecalis sp. nov., and Pseudocitrobacter anthropi sp. nov,isolated from fecal samples from hospitalized patients in Pakistan

Four isolates of Gram-negative facultatively anaerobic bacteria, three of them producing NDM-1 carbapenemase, were isolated from hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and studied for their taxonomic position. Initially the strains were phenotypically identified as Citrobacter species. Comparative analysis of 16S rRNA gene sequences then showed that the four strains shared >97%, but in no case >98.3%, 16S rRNA gene sequence similarities to members of the genera Citrobacter, Kluyvera, Pantoea, Enterobacter and Raoultella, but always formed a separate cluster in respective phylogenetic trees. Based on multilocus sequence analysis (MLSA) including partial recN, rpoA, thdF and rpoB gene sequence and respective amino acid sequence analysis it turned out that the strains also here always formed separate clusters. Based on further comparative analyses including DNA–DNA hybridizations, genomic fingerprint analysis using rep- and RAPD-PCRs and physiological tests, it is proposed to classify these four strains into the novel genus Pseudocitrobacter gen. nov. with a new species Pseudocitrobacter faecalis sp. nov. with strain 25 CIT^T (= CCM 8479^T = LMG 27751^T) and Pseudocitrobacter anthropi sp. nov. with strain C138^T (= CCM 8478^T = LMG 27750^T), as the type strains, respectively.     

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr_Z were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr_Z/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr_Z by P₆₈₀^+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn₄CaO₅ cluster in the S₂ and S₃ states could weaken the strength of the H-bond interaction between Tyr_Z^● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr_Z.