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Bacteroids of B. japonicum from nodules of soybean roots were isolated using differential centrifugation (the standard bench method) and density gradient centrifugation methods (either sucrose- or Percoll-) under anaerobic conditions in which N2 fixation was preserved. The relationships between N2 fixation and respiration, O2 supply, O2 demand, substrate (mainly malate) transport and metabolism in bacteroids were investigated using the flow chamber system. In related experiments, the primary products of N2 fixation which leave the bacteroids were investigated using a 15N-labelling technique in a closed shaken system and other biochemical methods.¶ In the flow chamber experiments, the rates at which O2 was supplied to bacteroids in the chamber were varied by (a) changing the flow rate of reaction medium through the chamber; (b) by changing the [O2 free] in the inflowing reaction medium by using either 3-5% (v/v) or 100% air in the gas mixture above the stirred reaction medium in two reservoir flasks; (c) by successively withdrawing bacteroids from the chamber, thus increasing the supply of O2 per bacteroid to those remaining in the chamber. The results showed that the rate of O2 supply regulates respiratory demand for O2 by bacteroids rather than the O2 concentration present in the reaction system. Respiration is always coupled to N2 fixation. ¶ Uptake of malate by bacteroids withdrawn from the flow chamber was measured under microaerobic conditions. Malate uptake by these N2-fixing bacteroids was lower than that by bacteroids isolated under aerobic conditions, which eliminate N2 fixation of bacteroids, but is closely correlated with bacteroid respiration rates. When respiration was increased by an increase in O2 supply, malate uptake by bacteroids was also increased. This suggested that transport of malate through the bacteroid membrane is also regulated by O2 supply, but indirectly. Higher uptake by bacteroids under aerobic conditions was observed because respiration was enhanced by the high availability of O2, but the fast uptake of malate by bacteroids driven by the abnormal respiration rates may not reflect the reality of malate demand in vivo by bacteroids when N2 fixation by bacteroids is fully coupled. ¶ The results of 15N labelling experiments and other biochemical assays once again demonstrated that ammonia is the principal significant 15N labelled product of N2 fixation accumulated during 30 min in shaken assays with 0.008-0.01 atm O2. Alanine although sometimes found in low concentrations in the flow chamber reactions, was not labelled with 15N in shaken closed system experiments. No evidence could be obtained from the other biochemical assays, either. Therefore, it is concluded that these and earlier results were not due to contamination with host cytosolic enzymes as suggested by Waters et al. (Proc. Natl. Aca. Sci. 95, 1998, pp 12038-12042). ¶ Malate transported into bacteroids is oxidized in a modified TCA cycle present in bacteroids. The results of flow chamber experiments with a sucA mutant (lacking a-ketoglutarate dehydrogenase) showed that respiratory demand for O2 by the mutant bacteroids is regulated by O2 supply in the same way as the wild-type. Despite differences in other symbiotic properties, rates of nitrogen fixation by the mutant bacteroids, based on the bacteroid dry weight, appeared to be the same as in the wild-type. Also N2 fixation was closely coupled with respiration in the same manner in both mutant bacteroids and wild type bacteroids. These results and other supporting data, strongly support the conclusion that there is an alternative pathway of the TCA cycle in bacteroids, which enables the missing step in the mutant to be by-passed with sufficient activity to support metabolism of transported malate.

Medicago truncatula forme une association symbiotique avec Sinorhizobium meliloti qui conduit à la formation de nodosités fixatrices d’azote. Les cellules symbiotiques végétales accueillent des centaines de bactéries qui restent viables dans la nodosité et se différencient en bactéroïdes fixateurs d’azote. Dans le but de mieux comprendre les mécanismes moléculaires nécessaires à la mise en place de cette interaction, nous avons recherché de nouveaux gènes de plante requis pour une symbiose effective en utilisant des approches de génétique directe et inverse. Des méthodes de biologie cellulaire et moléculaire ont été utilisées pour caractériser le phénotype des mutants et mieux comprendre la fonction biologique de ces gènes.Le gène symbiotique DNF2 code une phosphatidylinositol phospholipase C putative. Les nodosités formées par le mutant dnf2 contiennent une zone de fixation qui est réduite et dans laquelle les rhizobia ne se différencient pas complètement en bactéroïdes. De plus ces nodosités sénescent rapidement et présentent des réactions similaires à des réponses de défense. Sous certaines conditions d’expérimentation, le phénotype sauvage peut être restauré chez ce mutant ce qui montre le caractère conditionnel du phénotype.Le gène symbiotique SYMCRK code un récepteur kinase riche en cystéine. Le phénotype du mutant symCRK est similaire à celui de dnf2, ce qui suggère que ces deux gènes sont impliqués dans des processus aboutissant à des réponses similaires, probablement la persistance des bactéries dans les cellules végétales ou l’inhibition des réactions de défense de la plante. Les phénotypes Fix- atypiques des mutants dnf2 et symCRK suggèrent que les gènes correspondants sont impliqués dans les processus de répression des défenses de la plante et de persistance des bactéroïdes. / Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. In the nodules, symbiotic plant cells home and maintain hundreds of viable bacteria which are differentiated into bacteroids, the nitrogen-fixing form of rhizobia. In order to better understand the molecular mechanism sustaining this phenomenon, we used a combination of forward and reverse genetics approaches to identify genes required for nitrogen fixation. In addition we have used cell and molecular biology to characterize the phenotype of the corresponding mutants and to gain an insight into the genes functions.The symbiotic gene DNF2 encodes a putative phosphatidylinositol phospholipase C-like protein. Nodules formed by the mutant contain a zone of infected cells reduced to a few cell layers. In this zone, bacteria do not differentiate properly into bacteroids. Mutant nodules senesce rapidly and they exhibit defense-like reactions. The dnf2 symbiotic phenotype has been shown to be dependent on the experimental conditions.The symbiotic gene SYMCRK encodes a cystein-rich receptor kinase. The symCRK phenotype is similar to dnf2 suggesting that the two genes SYMCRK and DNF2 are participating in similar processes. This atypical phenotype amongst Fix- mutants unravels DNF2 and SYMCRK as new actors of bacteroid persistence inside symbiotic plant cells and repression of plant defense.

Philosophiae Doctor - PhD / Growth of symbiotic legume hosts is P limited, because of the high energetic requirements associated with N2 fixation. Attempts to overcome P deficiency in soils where legumes are grown involve addition of P-based fertilisers. However, these are produced from fmite, non-renewable resources that could be exhausted in the next 50-80 years. For this and other prudent reasons, viable alternatives are sought that include producing genetically enhanced plants with better P use efficiency (PUE). There exist some inter- and intraspecific genetic variation for associated traits of PUE in various legumes and these will have to be exploited to realize the development of P efficient cultivars. With the advent of sophisticated molecular tools, good progress has been made to understand the molecular response of some common physiological and morphological functions observed under LP. The research aims here were to investigate the energy costs and the alternative metabolic routes associated with C and N metabolism under LP in legumes, which is very scant in literature. We also investigated the recovery responses of nodulated roots upon P alleviation. Consequently, improvement strategies to produce legume varieties for better adaptation in poor P soils are envisaged. We have demonstrated varying degrees of sensitivity between the amide and ureide legume systems being investigated under short-term LP. The species-specific responses were ascribed to differences related to the agro-climatic origins, nodule morphologies and the type of N containing export product of the different legume types. These different responses also underscore possible different regulatory mechanisms under LP. Lupins were probed further, because of its apparent tolerance to P deficiency. Lupin nodules had between 3 to 5-fold higher Pj concentrations compared with soybeans under LP and HP, respectively. The maintenance of Pj levels, as oppose to a decline in the total P pool, is discussed in relation to its role in maintaining N2 fixation in lupins. Under LP, an effective Pj recycling mechanism in nodules is proposed to occur via the induction of the PEPc- MDH-ME route. This route also enhanced the capacity of root nodules to procure high malate concentrations that are used to fuel bacteroid respiration and N2 fixation. Two distinctly different cMDH proteins, one corresponding to HP and another corresponding to LP, were identified. The high malate concentrations reported here are speculated to have arisen through LP-induced cMDH. Metabolically available Pj decline developed gradually as P deficiency progressed. This coincided with a 15% decline in the %Ndfa. Moreover, under prolonged P deficiency the disproportionate synthesis of organic acids, most notably malate, that occurred at the expense of amino acids was proposed to account for this decline. The recovery in response to alleviation from LP involved alterations in the allocation of respiratory costs to growth and nutrient acquisition. Under LP, smaller nodules were formed and nodule metabolism revolved around accentuating PUE. Thus, there is considerable potential for improvement of P efficiency in legumes through manipulation of root: shoot partitioning.

En cas de carence azotée, les légumineuses sont capables de mettre en place une symbiose avec des bactéries du sol fixatrices d’azote appelées rhizobia. Cette symbiose a lieu dans un organe appelé nodosité où les bactéries sont endocytées et appelées bactéroïdes. Certains clades de légumineuses imposent un processus de différenciation à leurs bactéroïdes qui agrandissent considérablement et deviennent polyploïdes, menant à des morphotypes bactériens allongés ou sphériques. Au cours de cette thèse, j’ai étudié la différenciation des bactéroïdes de Bradyrhizobium spp. en association avec Aeschynomene spp.. Les bactéroïdes de ces plantes présentent des degrés de différenciation distincts qui dépendent de l’espèce hôte. Mes données suggèrent que les bactéroïdes les plus différenciés sont aussi les plus efficaces. J’ai cherché à savoir quels facteurs procaryotes pourraient être impliqués dans les adaptations des bactéroïdes au processus de différenciation et à leurs divers hôtes, le tout en lien avec cette différence d’efficacité symbiotique au travers d’approches globales sans a priori de type -omiques. Les conditions considérées sont des bactéroïdes de différents morphotypes et des cultures libres de référence. Les fonctions activées en conditions symbiotiques ont été identifiées, ainsi que les gènes spécifiques d’un hôte donné. Des analyses fonctionnelles des gènes d’intérêt ont également été menées. Les mutants bactériens n’ont toutefois pas présenté de phénotype symbiotique drastique, montrant ainsi l’existence de réseaux de gènes complexes menant à la résilience des génomes de rhizobia. / In case of nitrogen starvation, legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. This interaction takes place in nodules where the symbionts are internalized and become bacteroids. Some legume clades also impose a differentiation process onto the bacteroids which become enlarged and polyploid, leading to elongated or spherical morphotypes. During my PhD work, I have studied bacteroid differentiation of Bradyrhizobium species in association with Aeschynomene spp.. These bacteroids display distinct differentiation levels depending on the plant host, and my analyses suggest that the most differentiated ones are also the most efficient. I investigated the bacterial factors potentially involved in the adaptations to differentiation and host-specificity, and related to the higher efficiency of the most differentiated bacteroids using global-omics approaches without a priori. The analyzed conditions were bacteroids of distinct morphotypes and free-living reference cultures. Activated functions under symbiotic conditions were identified, as well as host-specific ones. Functional analyses were performed on genes of interest. However, the bacterial mutants did not display drastic symbiotic phenotypes, showing the existence of complex gene networks leading to high resilience of rhizobial genomes.