Coordinated transcriptional regulation of the divinyl ether biosynthetic genes in tobacco by signal molecules related to defense

In tobacco, 9-divinyl ethers (DVEs) produced by the lipoxygenase NtLOX1 and DVE synthase NtDES1 are important for full resistance to pathogens. In this work, the regulation of NtLOX1 and NtDES1 expression by signal molecules was investigated in LOX1 promoter-reporter transgenic plants and by RT-qPCR. Methyl jasmonate, ACC and elicitor were shown to coordinately trigger the DVE pathway. Induction was strongly attenuated in the presence of salicylic acid, which seems to act as a negative regulator of the 9-DVE biosynthetic enzymes. Our data suggest that, in tobacco, DVE biosynthesis is cross-regulated by jasmonates, and by other hormonal and signal molecules such as ethylene and SA.     

The allotetraploid invasive weedBromus hordeaceus L. (Poaceae): Genetic diversity, origin and molecular evolution

Bromus hordeaceus (sectionBromus, Poaceae), a predominantly self-fertilizing tetraploid (2n=28), is an annual weed native to the Mediterranean Basin, which now has a world-wide distribution. High morphological variation led to the recognition of four subspecies, three of which correlated with habitat-type. We examined genetic diversity at enzyme loci in 15 populations from the Mediterranean and the Atlantic region. Although sampled over a larger range of ecological and geographical conditions, the North-African populations appeared less genetically differentiated than populations from Brittany, suggesting higher levels of gene flow among the first ones (Nm=3.756 and 1.066 respectively). No genetic differentiation was encountered among the four subspecies. The populations were homozygous at homologous loci, suggesting high rates of selfing, but they frequently exhibited fixed intergenomic heterozygosity. The meiotic chromosome behaviour and disomic inheritance encountered are in accordance with the previously proposed allopolyploid origin of the species. The diploidsB. arvensis andB. scoparius have been previously implicated in the parentage ofB. hordeaceus on the basis of morphology and serology. We comparedB. hordeaceus with related diploid species belonging to the same section (sectionBromus) using different sources of data (flow cytometry, karyotypes, RAPD and DNA sequences). Molecular phylogeny based on internal transcribed spacer sequences of nuclear ribosomal genes provided the first clear scheme of relationships among monogenomic species of the section. A new hypothesis is proposed concerning the origin ofB. hordeaceus: We found that it diverged earlier than all other species of sectionBromus excluding the diploidB. caroli-henrici which is basal in this group. The 13 autapomorphies accumulated byB. hordeaceus, and the absence of intra-individual sequence heterogeneity are also consistent with the relatively ancient origin of the species within the section.     

Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters

The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluorophenylacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous esters (o-nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m-(acetamido) N,N,N-trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (alpha > beta > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric 'cross-talk' between the peripheral anionic site and the catalytic centre.     

A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the Cu(A) center of cytochrome c oxidase aa(3). This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O(2) reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD(+)(P) from ferrous iron (uphill pathway) required for CO(2) fixation and other anabolic processes. Besides the proteins involved in the O(2) reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc(42)), suggesting a possible physical link between these two pathways.     

The POK/AtVPS52 protein localizes to several distinct post-Golgi compartments in sporophytic and gametophytic cells

The organization and dynamics of the plant endomembrane system require both universal and plant-specific molecules and compartments. The latter, despite the growing wealth of information, remains poorly understood. From the study of an Arabidopsis thaliana male gametophytic mutant, it was possible to isolate a gene named POKY POLLEN TUBE (POK) essential for pollen tube tip growth. The similarity between the predicted POK protein sequence and yeast Vps52p, a subunit from the GARP/VFT complex which is involved in the docking of vesicles from the prevacuolar compartment to the Golgi apparatus, suggested that the POK protein plays a role in plant membrane trafficking. Genetic analysis of Arabidopsis mutants affecting AtVPS53 or AtVPS54 genes which encode putative POK partners shows a transmission defect through the male gametophyte for all lines, which is similar to the pok mutant. Using a combination of biochemical approaches and specific antiserum it has been demonstrated that the POK protein is present in phylogenetically divergent plant species, associated with membranes and belongs to a high molecular weight complex. Combination of immunolocalization studies and pharmacological approaches in different plant cells revealed that the POK protein associates with Golgi and post-Golgi compartments. The role of POK in post-Golgi endomembrane trafficking and as a member of a putative plant GARP/VFT complex is discussed.     

Caracterisation de la structure d'un processus de croissance

Resume L'analyse d'une cinétique de croissance y(t) est conduite à partir du modèle logistique généralisé de Richards-Nelder. On distingue 2 types de processus dits mono- et multi-logistique.Dans le cas mono-logistique, le phénomène est correctement décrit par une seule fonction logistique. La cinétique de croissance est alors caractérisée par lea propriétés de chacune des phases G ₁ à G ₄ , délimitées par les points singuliers _max, V _max et _min (Buis, 1991, 1993). On appelle structure de croissance (structure temporelle on diachronique) la contribution relative de ces différentes phases à l'expression de la croissance totale (durée, quantité de croissance, en valeurs relatives par phase, indépendamment de y _max). Cette distribution temporelle de l'activité de croissance est une représentation discrétisée de la trajectoire y ₀ y _max.On appelle processus multi-logistique toute cinétique de croissance correspondant à une somme de 2 ou plusieurs logistiques généralisées. II existe alors un plus grand nombre de points singuliers (extremums locaux de V et ) et donc un plus grand nombre de phases G _i que dans le cas monologistique. La structure de croissance définit avec précision la cinetique du phénomène, ainsi que l'importance relative des différentes composantes logistiques au cours du temps.Une application en est donnée avec le grandissement des cellules pleuridiophores de l'Algue Antithamnion plumula (Rhodophyceae, Ceramiales). L'allongement de ces cellules est toujours de type bi-logistique. Les caractéristiques de la structure de croissance permettent une discrimination nette du sporophyte et des gametophytes mâle et femelle. Tandis que la composante 1 (la plus importante en début de grandissement) est pen différente, la composante 2 varie fortement selon la ploïdie du thalle. En revanche, il y a pen de différences entre les deux gamétophytes.Cette notion de structure de croissance est ainsi susceptible d'apporter des résultats originaux. Audelà de cet example, cette approche est proposée comme une méthode de caractérisation simple mais précise de toute cinétique de croissance basée sur lea variations temporelles de l'activité de croissance [couple (V, )].

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Characterization of the structure of a growth processApplication à la croissance des cellules pleuridiophores de l'Algue Antithamnion plumula Application to the growth of the pleuridiophoral cells of the alga Antithamnion plumula

The analysis of a growth kinetics y(t) is carried out using the generalized logistic model of Richards — Nelder. Two types of processes, termed mono- and multi-logistic, can be distinguished.In a mono-logistic process, the phenomenon is adequately described by only one logistic function. The growth kinetics is then characterized by the properties of each of phases G ₁ to G ₄, with boundaries defined by the singular points _max, V _max and _min (Buis, 1991, 1993). The growth structure (temporal or diachronic structure) is defined by the relative contribution of the various phases to the expression of the total growth (duration, growth amount, in relative values per phase, independently of y _max). This temporal distribution of the growth activity is a discretized representation of the trajectory y ₀ y _max.A multi-logistic process corresponds to any growth kinetics which is the sum of 2 or several generalized logistics. The number of singular points (local extrema of V and ), and therefore the number of phases G, are then higher than in the mono-logistic case. The growth structure defines precisely the kinetics of the phenomenon as well as the relative importance of the various logistic components in the course of time.The application presented concerns the growth of the pleuridiophoral cells of the alga Antithanurion plumula (Rhodophyceae, Ceramiales). Cells elongation is of the bi-logistic type. The elongation growth structure characteristics afford a clear discrimination between the sporophyte and the male and female gametophytes. Whereas component 1 (which is the more important at the beginning of growth) hardly changes, component 2 varies dramatically with the thallus ploïdy. In contrast, there are few differences between the two gametophytes.The growth structure concept is thus liable to lead to original results. Beyond the example selected, the approach considered is put forward as a simple but precise characterization method for any growth kinetics based on temporal variations of the growth activity [(V, ) couple].

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Ce travail a été réalisé dans le cadre du programme de recherche du Groupe Physiologie, Phylogénie et Morphogénèse des Algues.     

Polymer-oligonucleotide conjugate synthesis from an amphiphilic block copolymer. Applications to DNA detection on microarray

An amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-N-acryloxysuccinimide)) (poly(TBAm-b-(NAM/NAS)) and a random copolymer poly(NAM/NAS), synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization process, have been used as support for oligonucleotide (ODN) synthesis, to elaborate polymer-oligonucleotide conjugates. In a first step, starters of ODN solid-phase synthesis were coupled to activated ester functions of polymers, and second, resulting functionalized polymers were covalently grafted onto hydroxylated controlled pore glass (CPG) support to further accomplish ODN synthesis. An efficient capping of residual hydroxyl functions of CPG was performed before synthesis, with both acetic anhydride and diethoxy-N,N-diisopropyl-phosphoramidite reagents, to suppress parasite-free ODN population present in conjugate crude material and resulting from syntheses directly initiated on silica beads. After purification, conjugates were evaluated in a DNA hybridization assay on a microarray, as macromolecules being able to favor capture of the target. Conjugate coating conditions were studied on the dT25/dA25 model. The role of the hydrophobic part (poly(TBAm)) of the conjugate synthesized with the block copolymer in the orientation of the conjugate after coating was revealed by spotting experiments achieved in a mixed solvent (DMF/H(2)O). The use of block copolymer-dT25 conjugate afforded a significant sensitivity improvement of the hybridization assay.     

A quick solution structure determination of the fully oxidized double mutant K9-10A cytochrome c7 from Desulfuromonas acetoxidans and mechanistic implications

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c₇, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c₇, which is paramagnetic with three paramagnetic centers, has been determined via ¹H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 ± 0.17 Å and 1.05 ± 0.14 Å for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short -sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 ± 0.07 Å and 0.92 ± 0.06 Å for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c₇ with [Fe] hydrogenase, inducing a change in K _m. This finding is in agreement with the identification of the protein area around heme IV as the interacting site.