The symbiosis between Rhizobium leguminosarum and Pisum sativum : regulation of the nitrogenase activity

Inglés. Bacteria of the genus Rhizobium can form a symbiosis with plants of the family Leguminosae. Both bacteria and plant show considerable biochemical and morphological changes in order to develop and carry out the symbiosis. The Rhizobia induce special structures on the legumes, which are called root nodules. In these root nodules, the differentiated bacteria - so-called bacteroids - are localized. Within the root nodule the bacteroids are able to reduce atmospheric N 2 to NH 3 , which - after assimilation - is used by the plant. In turn, the plant supplies the bacteroids with carbon compounds from which the energy required for the N 2 -reduction is derived.The N 2 -reduction within the bacteroids is catalyzed by the enzyme nitrogenase. Nitrogenase requires for activity energy in the form of ATP and a low potential electron donor. An anaerobic environment at the site of nitrogen fixation is a requirement for nitrogenase because O 2 inhibits the activity of this enzyme. However O 2 is necessary for the respiration of the bacteroids. Without bacteroid respiration, no ATP is synthesized and no reducing equivalents are generated, which are both required for nitrogenase activity. This means that the O 2 supply to the bacteroids must be strictly regulated.As a side reaction during N-reduction, H +is reduced. Consequently, by reducing H +ATP and reducing equivalents are consumed. Under optimum condition, about 75 % of the electron flow through the nitrogenase reaction is utilized for the reduction of N2. The remainder is consumed in the reduction of H +. The apparent waste of energy through H +-reduction can be much greater than 25 %. The magnitude of loss is influenced by many factors.The aim of the experiments described in this thesis, is to identify the plant factors which determine the nitrogenase activity and the electron allocation to N 2 and H+ by nitrogenase. The experiments were performed with Rhizobium leguminosarum strain PRE and the host plants Pisum sativum cv. Rondo and Pisum sativum cv. Finale. Different physiological aspects underlying the functioning of the root nodule, were studied, namely:- the role of malate dehydrogenase in the supply of oxidizable substrates to the bacteroids- the role of glutamate oxaloacetate transaminase in the NH 3 assimilation and the exchange of metabolites between the symbionts in the root nodule- the influence of the external pH of bacteroids on bacteroid respiration and nitrogen fixation- the relationships between the bacteroid respiration, the intracellular ATP/ADP ratio and nitrogenase activity.In chapter 2, the presence of root nodule-stimulated forms of malate dehydrogenase is demonstrated. From a comparison of the kinetic properties of the predominant nodule-stimulated form and the main malate dehydrogenase form from uninfected root cells, it is concluded that the nodule-stimulated form is capable of catalyzing a high rate of malate formation from oxaloacetate. The second conclusion drawn from the kinetic data is that under physiological conditions the reduction of oxaloacetate to malate catalyzed by the nodule-stimulated form is inhibited at higher malate concentrations. Only the nodulestimulated form exhibits this kinetic property. This prevents the enzyme from catalyzing the reaction to equilibrium, which would lead to a very low oxaloacetate concentration in the cytoplasm of the root nodule cells. The malate concentration has to be controlled because malate is the main substrate of the bacteroids, it plays a central role in the metabolism of the mitochondria and malate -being a strong acid - affects the pH.In chapter 3, the action of malate/aspartate shuttle between the cytoplasm of the infected plant cell and the bacteroid has been demonstrated. The involvement of a nodule-stimulated glutamate oxaloacetate transaminase, present in the cytoplasm of root nodule cells, in the shuttle is suggested. The shuttle might have the following functions for nitrogen fixation.The shuttle can transport NADH from the cytoplasm of the nodule plant cells to the bacteroid, where NADH can be oxidized by the respiratory chain. The second function of the shuttle is the transamination of oxaloacetate to aspartate in the bacteroid. The aspartate formed in the bacteroid, is transported at high rates to the cytoplasm of the nodule. This is important because labelling studies of other investigators with 14C-labelled aspartate have demonstrated that aspartate is rapidly converted to malate in the cytoplasm of nodule plant cells. The aspartate formed in the bacteroid and transported to the plant cytoplasm by the shuttle, can replenish the loss in aspartate in the plant cytoplasm. The presence of a sufficient concentration of aspartate is necessary for the asparagine synthesis, a reaction of the NH 3 assimilation.In chapter 4, the effect of O 2 on nitrogenase activity and the electron allocation by nitrogenase in the root nodules and in the bacteroids, has been described. Oxygen limitation in bacteroids results in a decreased nitrogenase activity and a decreased electron allocation to N 2 by nitrogenase. In root nodules, the O 2 limitation causes also a decrease in nitrogenase activity, however the electron allocation remains constant. It is shown that the external pH of bacteroids determines the rate of respiration by the bacteroid and consequently the rate of nitrogenase activity, without affecting the electron allocation by nitrogenase. By comparing the electron allocation by nitrogenase in root nodules and that in isolated bacteroids, it is proposed that in the intact root nodule the nitrogenase activity is modulated by the pH.In chapter 5, the mechanism is studied by which the external pH of bacteroids changes the rate of respiration and the rate of nitrogenase activity at low O 2 concentrations. The relationships between the rate of respiration by the bacteroid, the nitrogenase activity and the intracellular ATP/ADP ratio are determined.The results demonstrate that a high rate of respiration of the bacteroids at low free O 2 concentrations is associated with an intracellular ATP/ADP ratio which is lower than ≈1.2 . A high rate of respiration is necessary to achieve maximum nitrogenase activity. When the intracellular ATP/ADP ratio increases above 1.2 , the respiration of the bacteroids decreases and the free O 2 concentration increases, which ultimately results in an inactivation of nitrogenase. From experiments with a H +-conducting ionophore, it is concluded that the lower rate of respiration at higher pH is caused by a higher intracellular ATP/ADP ratio. These observations demonstrate that the intracellular ATP/ADP ratio via the P i potential regulates the rate of respiration. This is similar with the classical mitochondrial respiratory control.In chapter 5 the ATP consumption by nitrogenase is compared with ATP synthesis by oxidative phosphorylation. The calculation shows that under conditions of nitrogen fixation the N 2 -reduction, is a major ATP-consuming process in the bacteroids. About 70 % of the ATP synthesized by oxidative phosphorylation is hydrolyzed by nitrogenase. Thus, nitrogenase by itself keeps the intracellular ATP/ADP ratio low and thereby stimulates the respiration.In chapter 6, the studied biochemical processes of the root nodule, are placed in a broader perspective. Four physiological processes in which malate is involved, are illuminated. A mechanism is postulated, which accounts for the balance between the supply of photosynthates and the supply of O 2 to the bacteroids. A change of the pH of the root nodule cells induced by changes of the malate concentration is the central theme of the proposal. The pH might influence the rate of respiration of the bacteroids and thus nitrogenase activity, but it also might regulate the O 2 influx into the central tissue of the root nodule. The pH changes are determined by the availability of sucrose for the root nodule cells. Finally the comparison between bacteroids and mitochondria is discussed.

Holandés; flamenco. Het doel van de beschreven experimenten is plantenfactoren te identificeren, die de nitrogenase-activiteit en de elektronenverdeling over N2-reduktie en H+-reduktie door nitrogenase bepalen. Er is onderzoek verricht aan verschillende fysiologische aspecten