Root nodule organogenesis : molecular characterization of the zonation central tissue

Legume plants form root nodules by interacting with the soil bacterium, Rhizobium. In these nodules bacteria are able to convert atmospheric nitrogen into ammonia which is used by the host plants as nitrogen source. Therefore symbiotic nitrogen fixation in root nodules is of great importance for agriculture.Root nodule formation involves several developmental stages, namely are: induction of cell divisions in the root cortex, formation of nodule primordium and meristern, and finally differentiation of the meristern into nodule tissues. A mature nodule is composed of a central tissue where bacteria are hosted and several peripheral tissues. The induction of nodule specific genes of the host plants as well as the bacteria in a temporally and spatially controlled manner regulates the development of root nodules. The aim of the research described in this thesis was to investigate mechanisms that control nodule development. For this purpose genes of interest have been isolated and their expression was studied by means of the in situ hybridization technique.In chapter 2 a general introduction summarizing what we know about nodule development at present is given with an emphasis on gene expression and exchange of signals between the host plant and the rhizobia.Early studies of Allen et al. (1953) and more recently Hirsch et al. (1989) on polar auxin transport inhibitors (ATIs) provided evidences that exogenously applied ATIs cause the formation of nodule-like structures on several legume plants. These studies showed that auxin plays a major role in nodule development. Since certain flavonoids, e.g. quercetin, are endogenous ATIs, we studied the expression of chalcone synthase (CHS) genes, which encode a key enzyme in flavonoid biosynthesis, in situ during nodule development. The results are presented in chapter 3.To study gene expression during nodule development, two nodulin genes, ENOD40 and NOD6, were isolated and their expression during nodule development was studied by in situ hybridization. In chapter 4, a cDNA clone of the early nodulin gene ENOD40 was characterized. The pattern of expression of ENOD40 during soybean and pea nodule development suggested that it may play an important role in nodule formation. In chapter 5, the isolation of the late nodulin gene NOD6 was described and its expression pattern was compared with that of other nodulin genes.In chapters 6 and 7, the expression patterns in pea nodules of several bacterial genes were studied. These genes are nif A and nif H , and rop A The expression pattern of nif A and nif H in nodules is described in chapter 6. The expression of the rop A gene, which encodes a bacterial outer membrane protein, is described in chapter 7. The expression pattern of rop A in nodules is compared with that of nif H . The rop A protein was localized at a ultrastructural level by immunocytochemistry.In chapter 8, the results reported in this thesis are discussed with respect to the mechanisms that controls the induction of cortical cell divisions, meristern formation and formation of zones in the nodule central tissue.