A large proportion of the world’s nitrogen needs is derived from symbiotic nitrogen fixation (SNF), which contributes substantially to agricultural sustainability. The partnership between legumes and rhizobia result in the formation of specialised structures called root nodules. Within these nodules SNF is supported by the sucrose transported from the leaves to the nodules for respiration. The end products of SNF in soybean (Glycine max (L.) Merr.) root nodules, namely ureides, are transported to the upper parts of the plant to supply nitrogen. Symbiotic nitrogen fixation provides a vital advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of the high-protein content in seed and oil. The process of SNF is dramatically affected by drought, salt, cold and heavy metal stresses. Since SNF is such an important yield-determining factor, a lack in understanding these complexes inevitably delays progress towards the genetic improvement of soybean genotypes and also complicates decisions with regard to the suitability of certain genotypes for the various soybean producing areas in South Africa. The largest soybean producing areas in South Africa are situated at high altitudes, with minimum daily temperatures which can be critically low and impeding the production of soybean. Soybean is chilling sensitive, with growth, development and yield being affected negatively at temperatures below 15°C. Dark chilling (low night temperature) stress has proved to be one of the most important restraints to soybean production in South Africa. Among the symptoms documented in dark chilling sensitive soybean genotypes are reduced growth rates, loss of photosynthetic capacity and pigment content, as well as premature leaf senescence and severely inhibited SNF. Existing knowledge about stress-induced nodule senescence is based on fragmented information in the literature obtained in numerous, and often diverse, legume species. The precise nature and sequence of events participating in nodule senescence has not yet been fully explained. The main objectives of this investigation were to characterise the natural senescence process in soybean nodules under optimal growth conditions and to characterise the alteration of the key processes of SNF in a chilling sensitive soybean genotype during dark chilling. Moreover, to establish whether recovery in nodule functionality following a long term dark chilling period occured, or whether nodule senescence was triggered, and if sensitive biochemical markers of premature nodule senescence could be identified. A known chilling sensitive soybean genotype, PAN809, was grown under controlled growth conditions in a glasshouse. To determine the baseline and change over time for key parameters involved in SNF, a study was conducted under optimal growing conditions over a period of 6 weeks commencing 4 weeks after sowing. The cluster of crown nodules were monitored weekly and analysis included nitrogenase activity, ureide content, respiration rate, leghemoglobin content, sucrose synthase (SS) activity and sucrose content. Further investigations focused on induced dark chilling effects on nodule function to determine the alterations in key parameters of SNF. Plants were subjected to dark chilling (6˚C) for 12 consecutive nights and kept at normal day temperatures (26˚C). The induced dark chilling was either only shoot (SC) exposure or whole plant chilling (WPC). These treatments were selected since, in some areas in South Africa cold nights result not only in shoot chilling (SC) but also in low soil temperatures causing direct chilling of both roots and shoots. To determine if premature nodule senescence was triggered, the recovery following 12 consecutive nights of chilling treatment was monitored for another 4 weeks. It was established that the phase of optimum nitrogenase activity under optimal growing conditions occurred during 4 to 6 weeks after sowing where after a gradual decline commenced. This decline was associated with a decline in nitrogenase protein content and an increase in ureide content. The stability of SS activity and nodule respiration showed that carbon-dependent metabolic processes were stable for a longer period than previously mentioned parameters. The negative correlation that was observed between nitrogenase activity and nodule ureide content pointed towards the possible presence of a feedback inhibition trigger on nitrogenase activity. A direct effect of dark chilling on nitrogenase activity and nodule respiration rate led to a decline in nodule ureide content that occurred without any limitations on the carbon flux of the nodules (i.e. stable sucrose synthase activity and nodule sucrose content). The effect on SC plants was much less evident but did indicate that currently unknown shoot-derived factors could be involved in the minor inhibition of SNF. It was concluded that the repressed rates of respiration might have led to increased O2 concentrations in the nodule, thereby inhibiting the nitrogenase protein and so the production of ureides. It was found that long term chilling severely disrupted nitrogenase activity and ureide synthesis in nodules. Full recovery in all treatments occurred after 2 weeks of suspension of dark chilling, however, this only occurred when control nodules already commenced senescence. This points toward reversible activation of the nitrogenase protein with no evidence in support of premature nodule senescence. An increase in intercellular air space area was induced by long term dark chilling in nodules, specifically by the direct chilling of nodules (WPC treatment). The delayed diminishment of intercellular air space area back to control levels following dark chilling may be an important factor involved in the recovery of nitrogenase activity because enlarged air spaces would have favoured gaseous diffusion, and hence deactivation of nitrogenase, in an elevated O2 environment (due to supressed nodule respiration rates). These findings revealed that dark chilling did not close the diffusion barrier, as in the case of drought and other stress factors, but instead opened it due to an increase in air space areas in all regions of the nodule. In conclusion, this study established that dark chilling did not initiate premature nodule senescence and that SNF demonstrated resilience, with full recovery possible following even an extended dark chilling period involving low soil temperatures. / Thesis(PhD (Botany))--North-West University, Potchefstroom Campus, 2013

Dans le contexte de la transition agroécologique en faveur de systèmes de culture plus économes en intrants azotés, la réintroduction des légumineuses a un rôle majeur à jouer pour atteindre la durabilité de ces systèmes. Peu de références sont actuellement disponibles sur les intérêts agronomiques et écologiques des différentes espèces, notamment à l’échelle de la rotation. Dans ce cadre, notre objectif principal consiste à mieux quantifier les flux d’azote impliqués au cours et après culture de légumineuses, et ce pour une gamme élargie d’espèces. Notre travail expérimental porte donc sur la caractérisation des flux d’azote induits dans le sol et dans les cultures de légumineuses aux caractéristiques morphologiques contrastées en parallèle de la mesure des déterminants de ces flux. Les objectifs spécifiques consistent à : i) quantifier la fixation symbiotique en fonction du niveau du stock d’azote minéral du sol, la minéralisation de l’azote des résidus de légumineuses après enfouissement et les pertes d’azote en dehors du système sol-plante (lixiviation, émission de protoxyde d’azote), ii) identifier les « traits de plantes » explicatifs des fonctions liées à ces flux d’azote. Pour atteindre ces objectifs, les différents flux d’azote ont été quantifiés au cours d’une expérimentation au champ avec implantation d’une culture de légumineuses en première année suivie par une culture de blé en année 2 qui a été menée en 2014-2015 et reconduite sur la campagne 2016-2017. En parallèle, les traits des plantes, notamment racinaires, ont été caractérisés plus finement au cours d’expérimentations conduites en conditions contrôlées / In the context of agroecological transition, the reintroduction of legume crops should play a key role in cropping system sustainability by allowing a reduction of nitrogen (N) inputs. But few references are available concerning the agronomical and ecological services provided by a wide range of legume crops, particularly within crops succession scale. Thus, the main objective of our study is to quantify the N fluxes during and after the legume crops taking into account 10 legume crops (peas, lupin, faba bean, soybean...). Our experiment consists in i) quantifying symbiotic N fixation depending on the amount of soil inorganic N, the mineralisation of N present in legume crop residues after soil incorporation and N losses outside of the soil-plant system (leaching, emission of nitrous oxide), ii) identifying plant biological traits associated to N fluxes. Thus, different N fluxes were quantified during a two-year field experiment, i.e. the first year (2014) legume crops were implanted and followed by wheat the second year (14-15) after incorportation of legume residues. This experiment was repeated in 2016-2017.In parallel, plant root traits were characterised during greenhouse hydroponic experiments

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Approximately 10% of sequenced bacterial genomes are multipartite, consisting of two or more large chromosome-sized replicons. This genome organization can be found in many plant, animal, and human pathogens and symbionts. However, the advantage of harbouring multiple replicons remains unclear. One species with a multipartite genome is Sinorhizobium meliloti, a model rhizobium that enters into N2-fixing symbioses with various legume crops. In this work, S. meliloti derivatives lacking one or both of the secondary replicons (termed pSymA and pSymB) were constructed. Phenotypic characterization of these strains, including growth rate, metabolic capacity, and competitive fitness, provided some of the first experimental evidence that secondary replicons evolved to provide a niche specific advantage, improving fitness in a newly colonized environment. These results were further supported by characterizing the symbiotic phenotypes of 36 large-scale pSymA and pSymB deletion mutants. To further this analysis, an in silico S. meliloti genome-scale metabolic network reconstruction was developed and flux balance analysis used to examine the contribution of each replicon to fitness in three niches. These simulations were consistent with the hypothesis that metabolic pathways encoded by pSymB improve fitness specifically during growth in the plant-associated rhizosphere. Phylogenetic analysis of a pSymB region containing two essential genes provided a clean example of how a translocation from the primary chromosome to a secondary replicon can render the secondary replicon essential. Moreover, an experimental analysis of genetic redundancy indicated that 10-15% of chromosomal genes are functionally redundant with a pSymA or pSymB encoded gene, providing an alternative method for how secondary replicons can become essential and influence the evolution of the primary chromosome. Finally, the work presented here provides a novel framework for forward genetic analysis of N2-fixing symbiosis and the identification of the minimal N2-fixing symbiotic genome, which will help facilitate the development of synthetic symbioses. / Thesis / Doctor of Philosophy (PhD) / Many bacteria that enter into symbiotic or pathogenic relationships with plants, animals, and humans contain a genome that is divided into multiple chromosome-like molecules. One example is the N2-fixing legume symbiont Sinorhizobium meliloti, whose genome contains three chromosome-sized molecules. Here, the functions associated with each molecule in the S. meliloti genome were examined through a combination of experimental genetic analyses and computer based simulations. Results from these approaches suggested that adaptation to unique environments selected for the evolution of secondary chromosome-like molecules, with each predominately contributing to growth in a specific environment, including environments associated with an eukaryotic host. The genes on these replicons are therefore prime targets for manipulation of bacterium-host interactions, and represent reservoirs of valuable genes for use in synthetic biology applications. Additionally, the genome reduction approach employed in this study laid out a ground work for identification of the minimal N2-fixing symbiotic genome. This represents a crucial step towards successfully engineering improved nitrogen fixation, and the engineering of synthetic N2-fixing symbioses involving non-legumes and/or non-rhizobia.

Symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. Within nodule cells, N-fixing rhizobia are surrounded by plant-derived symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. First, I investigated the impact of P deprivation on wild-type Medicago truncatula plants. My observations that plants had impaired SNF activity, reduced growth, and accumulated less phosphate in P-deficient tissues (leaves, roots and nodules) is consistent with those of similar previous studies. Galactolipids decreased with increase in phospholipids in all P-starved organs. Matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI-MSI) of phosphatidylcholine (PC) species in nodules showed that under low P environments distributions of some PC species changed, indicating that membrane lipid remodeling during P stress is not uniform across the nodule. Secondly, a metabolomics study was carried out to test the alterations in the metabolic profile of the nodules in P-stress. GC-MS based untargeted metabolomics showed increased levels of amino acids and sugars and decline in amounts of organic acids in P deprived nodules. Subsequently, LC-MS/MS was used to quantify these compounds including phosphorylated metabolites in whole plant. My findings showed strong drop in levels of organic acids and phosphorylated compounds in P deprived leaves with moderate reduction in P deprived roots and nodules. Moreover, sugars and amino acids were elevated in whole plant under P deprivation. Finally, the last project of my thesis involved studying the response of PDIL3 (Phosphate Deficiency-Induced LncRNA-3) a long non-coding RNA (lncRNA) mutant under severe P stress. PDIL3 is known to regulate Pi-deficiency signaling and transport in M. truncatula (Wang et al., 2017). My results confirmed that in P starvation, pdil3 plants showed better shoot growth, accumulated more phosphate in shoots, had impaired SNF and less rhizobial occupancy in nodules than WT. Subsequently, MALDI–MS imaging was used to spatially map and compare the distribution of phosphatidylcholine (PC) species in nodules of pdil3 and WT in P-replete and P-depleted conditions. Several PC species showed changes in distributions in pdil3 nodules compared to WT in both P sufficient and P deprived conditions. These data suggest that PDIL3's role is not just suppression of the Pi transporter, but it may also influence P partitioning between shoots and nodulated roots, meriting further investigation.