Molecular aspects of the nitrogen fixing system in pea root nodules

This thesis deals with research on symbiotic nitrogen fixation of Pisumsativum and Rhizobium Leguminosarum. Nitrogen fixation takes place in the Rhizobium bacteroids which are located within root nodule cells. Two important proteins in nitrogen fixation are nitrogenase and leghemoglobin. Nitrogenase, the enzyme that reduces N 2 , is synthesized by Rhizobium. Leghemoglobin, which has a function in the O 2 supply of the bacteroids is synthesized by the plant. The molecular biology of symbiotic nitrogen fixation has hardly been the subject of investigation. The regulation of the synthesis in root nodules of e.g. nitrogenase and leghemoglobin is not at all completely clear.In this thesis some general aspects of nodule formation and the regulation of nitrogenase and leghemoglobin (Lb) synthesis have been studied.General aspectsThe transformation of Rhizobium bacteria into nitrogen fixing bacteroids was studied by investigating the DNA content of bacteroid cells by means of cytofluorometry (chapter I). Furthermore the RNA content and the quality of the RNA of R. leguminosarum (PRE) bacteroids were followed during pea development (chapter IV) and the protein synthesis in bacteroids and in the plant fraction of pea root nodules during nodule development was studied (chapter IV). The regulation of nitrogenase and leghemoglobin synthesis were studied in more detail.Regulation of nitrogenase and Lb synthesisNitrogenase and Lb synthesis were studied by 35SO42-labeling of intact pea plants. Intact pea plants are a rather complicated system to study Lb or nitrogenase synthesis, but in more simple systems like e.g. detached root nodules, nitrogenase and Lb synthesis are repressed. We did not succeed in developing a more simple system suitable for studying nitrogenase or Lb synthesis (chapter VII). The regulation of nitrogenase and Lb synthesis were studied by culturing pea plants under conditions that diminished in vivo nitrogenase activity and by determining nitrogenase and Lb synthesis during nodule formation and development. Growth conditions used to diminish in vivo nitrogenase activity were: 1. the addition of NH4+to the growth medium (chapter II) and 2. waterlogging (chapter III). The synthesis of the two nitrogenase components and Lb during nodule formation and development was studied in two different ways: 1. the synthesis of these three proteins was followed by 35SO42-labeling of pea plants of different ages, and analysis of bacteroid and plant proteins by polyacrylamide gel electrophoresis (chapter IV), 2. the sequence of appearance of the two nitrogenase components and Lb during pea nodule formation was determined with specific radioimmunoassays for these three proteins (chapter V).In principle, turnover of proteins can be an important factor in the regulation of protein composition in the bacteroid or plant fraction of root nodules, e.g. during nodule development or after changes in the environmental conditions. Therefore turnover rates of bacteroid and plant proteins of pea root nodules, especially nitrogenase and Lb, were determined (chapter VI).