Soutenance de thèse - Martina Rovere
Mardi 19 juin 2018 - 14:30 - Sophia Antipolis - Inra PACA - Salle A010
Legume crops are known for their capacities to establish a symbiotic relationship with nitrogen fixing soil bacteria. This mutualism culminates in the formation of a new plant organ, the root nodule, in which the symbiont converts atmospheric nitrogen (N2) into ammonia, which can be directly consumed by plants. In nodules, bacterial nitrogenase enzyme is inhibited by traces of oxygen (O2) so different mechanisms maintain this organ at low O2 level. At the same time, nodules need to maintain a high ATP level for nitrogenase activity, which is highly energy demanding. Thus a balance between a tight defense from O2 and an efficient energy production, referred as the “O2 paradox” of N2-fixing legume nodules, has to be reached. In Arabidopsis thaliana, a direct oxygen sensing mechanism has recently been discovered involving members of the group VII ethylene responsive factors (ERFs). These transcription factors (TFs) possess a characteristic N-terminal amino acid with a cysteine residue at the second position that, under normal O2 conditions, leads to protein degradation following a specific pathway called the N-end rule pathway. Furthermore, it was shown that both O2 and nitric oxide (NO) are required to destabilize the group VII ERFs and that a reduction in the availability of either gas is sufficient to stabilize these proteins. Therefore, the goal of this thesis was to investigated the role of ERF-VII family in O2 sensing and adaptation to hypoxia in M. truncatula and to understand how NO interacts with O2 in hypoxic signalization in the microoxic environment that characterises the nodule.
We identified four genes belonging to the ERF-VII TF family in the M. truncatula genome, which present a strong similarity with ERF-VII of Arabidopsis. The characterization of this family at the transcriptional level revealed that only MtERF-B2.2 is up-regulated by hypoxia stress and during nodule development. The three others, MtERF-B1.1, MtERF-B1.11 and MtERF-B2.3 are found constitutively expressed in leaves, roots and nodules.
To investigated the protein stability of MtERF-B2.1, the closest orthologous to AtRAP2.12 described as O2-sensors in Arabidopsis, in function of O2/NO availability, we realized a fusion protein with the luciferase reporter protein. Our results on Arabidopsis protoplasts indicated that the N-terminal part of MtERF-B2.1 drives its O2-dependent degradation by the N-end rule pathway.
The function of MtERF-B2.1 and MtERF-B2.11 was also investigated both in response to hypoxia stress and during the nodulation process using an RNA interference strategy. Silencing of MtERF-B2.1 and MtERF-2.11 showed a significant lower activation of several core hypoxia-responsive genes such as ADH1, PDC1, nsHb1 and AlaAT. These double knock-down transgenic roots were also affected in symbiotic interaction with a significant reduction of the nodulation capacity and nitrogen fixation activity in mature nodules.
Overall, the results reveal that O2 sensing mechanism is mediated by ERF-VIIs in M. truncatula roots and nodules and that this mechanism, together with downstream targets, is involved in the organ development and ability to efficiently fix nitrogen. Furthermore, results indicated that MtERF-B2.1/B2.11 are positive regulator of the anaerobic metabolism and the Hb-NO cycle–related genes likely in order to activate alternative ATP generation pathways.
Composition du jury
- Présidente du jury : Cécile SABOURAULT, Professeur, Université Nice Sophia Antipolis, Département Science de la vie
- Rapporteur : Luisa LANFRANCO, Professeur Associé Università di Torino, Departement of life science and biological system
- Rapporteur : Andreas NIEBEL, Directeur de Recherche,Unité Mixte de Recherche CNRS-INRA Toulouse
- Examinateur Diana BELLIN , Professeur Associé Università di Verona, Departement of biotecnology
- Directeur de Thèse Renaud BROUQUISSE, Directeur de Recherche, Institut Sophia Agrobiotech - Unité Mixte de Recherche Inra - Université Nice Sophia Antipolis - Cnrs
- Directeur de Thèse Alexandre BOSCARI, Chargé de recherche, Institut Sophia Agrobiotech - Unité Mixte de Recherche Inra - Université Nice Sophia Antipolis - Cnrs