Etude des relations structure-fonction de la protéine ORF138 responsable de la stérilité mâle cytoplasmique Ogura

*INRA. Unité de Génétique et d'Amélioration des Plantes (FRA) Diffusion du document : INRA. Unité de Génétique et d'Amélioration des Plantes (FRA) Diplôme : Dr. d'Universit

Etude des relations structure-fonction de la protéine ORF 138 responsable de la stérilité mâle cytosplasmique Ogura

La protéine ORF138 (codée par le génome mitochondrial) est une protéine membranaire mitochondriale responsable de la stérilité mâle cytoplasmique (SMC) Ogura. Le phénotype des plantes stériles se manifeste par une dégénérescence prématurée du tapis de l'anthère selon un mécanisme encore inconnu alors que la protéine ORF138 est présente dans tous les tissus. Les objectifs du travail de thèse étaient de développer des outils moléculaires et biochimiques pour obtenir des informations pertinentes sur cette protéine, et permettre l'élaboration d'hypothèses solides quant au mécanisme de stérilité mâle.Nous avons montré que l'association des domaines hydrophobe et hydrophile de la protéine est indispensable à son effet. Un domaine de 15 acides aminés dont la structure secondaire est conservée entre 3 protéines de SMC non apparentées a été mis en évidence et joue également un rôle dans cet effet.Nous avons montré que l'ORF138 est enchâssée dans la membrane interne mitochondriale de colza sous des formes homooligomériques. Un complexe protéique de taille comprise entre 750 et 900 kDa impliquant l'ORF138 a également été identifié. Nous avons aussi prouvé que l'ORF138 peut être co-purifiée avec des composants nucléoprotéiques mitochondriaux de plantes mâle stériles.Une plus forte accumulation de l'alternative oxydase mitochondriale probablement accompagnée d'une activité accrue ont été observées chez les plantes stériles. La liaison entre stress oxydant et activité alternative oxydase est bien connue, et un lien possible entre un stress oxydant et la mort prématurée du tapis constitue une des pistes les plus prometteuses pour l'élucidation du mécanisme de stérilité.The ORF138 protein (encoded by the mitochondrial genome) is a mitochondrial membrane protein responsible of Ogura cytoplasmic male sterility (CMS) in Brassicaceae. In sterile plants microspore abortion is apparently due to premature degeneration of the anther tapetum, by a still unknown mechanism. This is the only induced phenotype whereas ORF138 is present in all tissues. The objectives of this work were to develop molecular and biochemical tools for the study of this protein, and to obtain relevent information for setting up hypotheses about the mechanism of male sterility.We showed that the association of the hydrophobic and hydrophilic domains of the protein is essential for its effect. A domain of 15 amino acids, sharing predicted secondary structure with two other proteins of unrelated CMS's, was highlighted and showed to play a role in this effect. We showed that ORF138 was integrated in the mitochondrial inner membrane of rapeseed and formed homooligomers. A proteic complex of size ranging between 750 and 900 kDa implying ORF138 was also identified. We also proved that ORF138 can be copurified with mitochondrial nucleoproteic components of male sterile plants. An increased accumulation of mitochondrial alternative oxydase probably accompanied by an increased activity, was observed in the sterile plants compared to fertiles. The connection between oxidative stress and alternative oxydase activity is well-known, and a possible bond between an oxidizing stress and the premature death of the tapetum constitutes one of the most promising tracks for the elucidation of the sterility mechanism.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The kinesin AtPSS1 promotes synapsis and is required for proper crossover distribution in meiosis.

Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes.

International audienceLand plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells

Genetic recombination in wild type and <i>Atpss1-1</i>.

<p>Genetic distances in six intervals using tetrad analysis with fluorescent-tagged lines (FTL), were calculated with the Perkins equation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004674#pgen.1004674-Perkins1" target="_blank">[67]</a> and are given in centiMorgans. I1b and I1c are adjacent intervals on chromosome 1 and so on for the other pairs of intervals as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004674#pgen.1004674-Berchowitz2" target="_blank">[46]</a> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004674#pgen.1004674.s006" target="_blank">Table S1</a>).</p

MLH1 immunolocalization.

<p>Immunolocalization of MLH1 at diakinesis is shown (A) in wild type and (B) in <i>Atpss1-1</i>. (C, D). Scatter plot of MLH1 foci number per cell at diplotene and diakinesis. (E) Distribution of chromosomes according to their MLH1 foci number at diakinesis. Cells were prepared according to Chelysheva <i>et al.</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004674#pgen.1004674-Chelysheva2" target="_blank">[47]</a>. Scale bar = 10 µm.</p

Average number of bivalents (blue) and pairs of univalents (red) per male meiocyte.

<p>Number of metaphase I cells analyzed is indicated in brackets.</p