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 ¶ Uptake of malate by bacteroids withdrawn from the flow chamber was measured under microaerobic conditions.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.

Actinorhizal plants are in symbiosis with the nitrogen-fixating soil bacterium Frankia, which forms nodules in the plant root. However, several studies also report other endophytic bacteria appearing in the nodules, but their function and interaction with the host plant or Frankia is not yet understood. This thesis used a bioinformatic approach to investigate the metagenomes of eighteen actinorhizal nodule samples to find out which bacteria are present, how the microbiomes differed from each other, and if the genomes of non-Frankia inhabitants could give indications of any functions. The results showed that the bacterial composition, richness, and diversity differed among the samples, especially between the samples sequenced from the field versus those primarily cultivated in a greenhouse. All samples had a substantial number of sequencing reads belonging to potential endophytes, such as strains of Enterobacteria, Pseudomonas, Streptomyces, Micromonospora, Mycobacteria and Pseudonocardia. There seemed to be a common microbial community shared among the plants on a family level, since no significant difference was found in the core microbiomes between the field and greenhouse groups. Some sequences found in the metagenomes were annotated as potential functions of the fellow travellers, such as antibiotic synthesis, proteins involved in regulating abiotic stresses, but also probable plant damaging compounds rather associated with pathogens than symbionts.

Thesis (PhD(Agric) (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: Salinity is one of the major limiting abiotic stresses on legume plant yield, leading to early senescence of root nodules. This occurs because of accumulation of reactive oxygen species (ROS) in plant cells under salinity stress. Concurrent with the increase in cellular reactive oxygen species levels is the increase in cellular antioxidants and corresponding antioxidant enzymes. This feature is observed mostly in the shoots and roots of more tolerant genotypes compared to the susceptible genotypes. It is accepted that the mechanism of plant tolerance to stress is dependent upon the response of the antioxidant systems. Most studies carried out on shoot tissues suggest that scavenging of ROS by the plant antioxidant system is modulated by nitric oxide (NO). However, the pathways by which NO mediates such antioxidant responses are not fully understood. For legumes, salinity stress has adverse effects on yield and this is in part due to inhibition of nitrogen fixation in the root nodules of the legumes, which causes severe nitrogen starvation in nitrogen-deficient soils. Nodules are specialized organs comprising of both the rhizobia and the plant tissue, hence the physiological aspects may vary from the findings from the leaves. It was therefore deemed necessary to establish the role of NO on the nodule antioxidant system in the absence and presence of salinity stress. For the purposes of this study, the effect of both exogenously applied NO and endogenous NO on superoxide dismutase, glutathione peroxidase and glutathione content was determined. The studies involved the use of nitric oxide donors like sodium nitroprusside (SNP) and diethylenetriamine/nitric oxide adduct (DETA/NO), their respective fixed controls potassium ferricyanide and diethylenetriamine (DETA), plus a nitric oxide synthase inhibitor (to inhibit nitric oxide production by the enzyme nitric oxide synthase) on nodulated roots. The data obtained in this work points out specifically at roles played by nitric oxide in regulating superoxide dismutases, glutathione peroxidase and glutathione during salinity stress and proposes a link between nitric oxide-mediated changes in these antioxidant systems and salinity stress tolerance. Both the exogenously applied and endogenous nitric oxide increases the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GR). However, there is both time dependency and nitric oxide concentration dependency on the enzyme activities. The total SOD enzyme activity increases upon nitric oxide exposure and with time of exposure. The individual SOD isoforms identified and studied in the root nodules all contribute to this increase in SOD activity upon nitric oxide treatment except for MnSOD I. This increase in activity is regulated at transcriptional level as the RT-PCR results targeting the individual isoforms reveals an increase in transcript levels after 6 hours of nitric oxide treatment. However, the CuZn SOD I isoform transcripts are reduced upon nitric oxide treatment. A similar response was also observed in GPX enzyme activity in which nitric oxide increased the GPX activity above all the controls. The GR enzyme activity exhibits an opposite response because the activity decreases with time of exposure to NO and concentration of NO. In order to determine the effect of NO under saline conditions, an experiment was set up that involved incubation of nodulated roots in solutions containing 150 mM NaCl. The stressed nodules exhibited generally higher levels of enzyme activities than the non-stressed nodules. Furthermore, exposure to nitric oxide donor in combination with NaCl induced even higher activities of SOD and GPX than NaCl or nitric oxide donor alone. There were also higher levels of reduced glutathione and total glutathione recorded under stress compared to optimal conditions. Nitric oxide increased the concentration of these forms of glutathione, suggesting an improved redox status based on the GSH/GSSG ratios under salinity stress in the presence of nitric oxide. Attenuation of nitric oxide synthesis with L-Nω-Nitroarginine methyl ester (L-NAME) reverses all the recorded effects of nitric oxide on antioxidant enzymes and glutathione pool. This was observed in salinity stressed nodules and non-stressed nodules. This work further establishes that NO plays a pivotal role in modulating the enzymatic activities through a pathway that is mediated by guanosine 3,5-cyclic monophosphate (cGMP). The experiment involving the inhibition of soluble guanylyl cyclase (sCG) (an enzyme that catalyzes the biosynthesis of cGMP), cell-permeable cGMP anaologue and L-NAME revealed that GPx activity is modulated through a cGMP-dependent pathway and NO is positioned up-stream of cGMP in the pathway leading to improved GPX activity. Cyclic GMP also modulates the GPX activity in a concentration dependent manner. NO improves the redox status of the cell under both saline conditions and non-saline conditions and this effect is modulated through a cGMP-dependent pathway. It is thus rational to conclude that; in the root nodules of legumes, like in other plant tissues, the increased accumulation of antioxidants and the increased activity of their corresponding enzymes, as modulated through the cGMP-dependent pathway by nitric oxide, confer root nodule tolerance to salinity. This concept directly points out at an attractive strategy for developing legumes that are genetically improved for enhanced root nodule tolerance to salinity; via differential regulation of antioxidants and antioxidant enzyme genes in the root nodules under abiotic stress. Towards attaining the goal for such genetic improvement, experiments involving construction of an abiotic stress-responsive and nodule-specific chimeric promoter were carried out. By fusing the 5-untranslated (5-UTR) region of the LEA gene that contains an abiotic stress-responsive cis-acting element (from theGmPM9 promoter) to the nodulin N23 promoter bearing the highly functional cluster of motifs for nodule specificity, the candidate nodule specific promoter that is abiotic stress responsive (ASREF/NSP) was constructed. The construct harbouring this ASREF/NSP chimeric promoter was fused to the -glucuronidase (GUS) reporter gene so as to study the functionality of the promoter in Medigaco truncatula plants. The construct was delivered into the Medicago plants through Agrobacterium rhyzogenes mediated transformation to produce composite Medicago plants. The transgenic roots have been cultured for futher manipulation and to confirm the functionality of the promoter. Furthermore several strategies can be deployed via the use of this chimeric promoter so as to enhance the nodular antioxidant system. This would involve either gene regulator-chimeric promoter fusion or the use of a single gene approach. As part of this work, the MtNOA gene homologous to AtNOAs, has been cloned from Medicago trancatula and put as ASREF/NSP fusion in a binary vector pBINPLUS and delivered into Medicago trancatula for nodule-specific and abiotic stress-induced nitric oxide synthesis. Since there is no plant NOS identified to date, the possibility of the use of a regulatory gene in this aspect is still limited. There are other options involving the use of the chimeric promoter with the individual genes encoding the antioxidant enzyme genes such as genes encoding SOD, GPX and the glutathione synthatase to enhance the plant antioxidant system during abiotic stress. / AFRIKAANSE OPSOMMMING: Geen opsomming was ingedien met die tesis

No description available.

Red alder (Alnus rubra Bong.) is the only tree in British Columbia and the Northwest US to engage in actinorhizal symbiosis to fix atmospheric nitrogen. This study was conducted to explore the plasticity in growth and physiology among 58 17-year-old red alder families in response to variation in climate in two common garden plots, one at Bowser, BC and one at Terrace, BC. Physiological assessments included height and diameter growth, bud flush, water use efficiency as measured by δ13C, cold hardiness as measured by controlled freezing and electrolyte leakage, autumn leaf senescence, and instantaneous and seasonally integrated rates of nitrogen fixation as measured by acetylene reduction and natural abundance δ15N isotope analysis, respectively. Significant differences were identified among families for growth (height and diameter), bud burst stage, leaf senescence, cold hardiness, and bud nitrogen content. No significant differences among families were identified for water use efficiency as measured by δ13C, or for rates of nitrogen fixation as measured by either acetylene reduction or natural abundance δ15N. This study identified possible adaptive differences among red alder genotypes, especially in traits such as bud flush timing, cold hardiness, or nitrogen fixation and their respective contributions to growth. These differences often reflected a tradeoff between growth and the ability to tolerate an extreme environment. Cold hardiness results indicate that red alder families are well adapted to their climate of origin, and may not be able to acclimate sufficiently to a northward assisted migration of genotypes. Nitrogen fixation results demonstrated gaps in our current knowledge of Frankia distribution and impact on the actinorhizal symbiosis in British Columbia. / Graduate

Les légumineuses sont connues pour leurs capacités à établir une relation symbiotique avec des bactéries du sol fixatrices de l'azote atmosphérique. Cette interaction aboutit à la formation d'un nouvel organe au niveau des racines, la nodosité, au sein duquel le symbiote convertit l'azote atmosphérique (N2) en ammoniac, qui peut être directement consommé par les plantes. A l’intérieur de cette nodosité, la concentration en oxygène (O2) est maintenue à un très faible niveau car la réaction de réduction du N2 par l’enzyme bactérienne nitrogénase est inhibée par des traces d’oxygène. Un mécanisme de perception directe de l'O2 impliquant des membres de la famille des facteurs de transcription « Ethylene Responsive Factors » (ERFs) du groupe VII a récemment été découvert chez Arabidopsis thaliana. Ces facteurs de transcription (FT) possèdent une extrémité N-terminale caractéristique avec un résidu de cystéine à la seconde position. Dans des conditions normales d'O2, les FT sont conduit à la dégradation suivant une voie spécifique du protéasome. En condition de stress hypoxique, les TFs sont stabilisés et peuvent activer l’expression des gènes de réponse à l'hypoxie. Il a été démontré que la présence d’O2 et de NO était nécessaire pour déstabiliser ces protéines, et qu'une réduction de la disponibilité de l'un ou l'autre des gaz est suffisante pour protéger le résidu cystéine N-terminale de l'oxydation. L’objectif de cette thèse a été d'étudier le rôle de la famille ERF-VII dans la perception et l'adaptation au manque d'O2 chez M. truncatula. Des travaux ont aussi été menés pour déterminer l’importance du NO dans le fonctionnement en microoxie de la nodosité. Quatre gènes codant pour des facteurs de transcription de la famille ERF-VII ont été identifiés dans le génome de M. truncatula. La caractérisation de cette famille au niveau transcriptionnel a révélé que seul MtERF-B2.2 était induit par le stress hypoxique et au cours du développement des nodosités. Les trois autres, MtERF-B1.1, MtERF-B1.11 et MtERF-B2.3, sont constitutivement exprimés dans les feuilles, les racines et les nodosités. Pour étudier la stabilité de la protéine MtERF-B2.1, l’orthologue de RAP2.12 principal ERF-VII décrit dans la perception de l’O2 chez Arabidopsis, en fonction de la disponibilité de O2/NO, nous avons réalisé une protéine de fusion entre l’extrémité N-terminale de notre protéine et la protéine rapporteur luciférase. Les résultats obtenus sur des protoplastes d'Arabidopsis montrent l’implication la partie N-terminale de MtERF-B2.1 dans la régulation de la stabilité de la protéine, mais en contradiction avec les résultats obtenus en plantes composites de M. truncatula. La fonction de MtERF-B2.1 et MtERF-B2.11 a également été étudiée dans le cadre de la réponse au stress hypoxique et au cours du processus de nodulation en utilisant une stratégie d'interférence ARN. Des racines transgéniques dérégulées sur l’expression de MtERF-B2.1 et MtERF-B2.11 ont montré un défaut d’activation de plusieurs gènes de réponses à l'hypoxie tels que l’alcool déshydrogénase (ADH1) ou la pyruvate décarboxylase (PDC1). Ces racines transgéniques ARNi-MtERF-B2.1/B2.11 sont également affectées dans l'interaction symbiotique avec une réduction significative de la capacité de nodulation et de l'activité de fixation de l'azote dans les nodules matures. En conclusion, ces travaux révèlent que le mécanisme de détection d'O2 est médié par les ERF-VII dans les nodosités de M. truncatula et que ce mécanisme, associé aux cibles moléculaires régulées en aval, participe au développement de cet organe et au maintien de la capacité de fixatrice de celui-ci. De plus, les résultats indiquent que MtERF-B2.1/B2.11 sont des régulateurs positifs du métabolisme anaérobie et que les gènes associés au cycle hémoglobine-NO sont susceptibles d'activer d'autres voies de génération d'ATP. / 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 to support the nitrogenase activity, which is highly energy demanding. Thus, a balance between a tight protection 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 ethylene responsive factors (ERFs) group VII. 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 ERFs VII 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, model plant for legumes, and to understand how NO interacts with O2 in hypoxic signalization in the microoxic environment that characterizes 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 MtERFB2.1 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.