A histochemical study of root nodule development

In cooperation with soil bacteria of the genera Rhizobium , Bradyrhizobium or Azorhizobium , many members of the legume family are able to form specialized organs on their roots, called root nodules. The bacteria, wrapped up inside a plant membrane, are accomodated in large parenchymatic cells located centrally in these root nodules. For this, they reward their host by converting atmospheric nitrogen into a form usable for the plant. The central infected tissue of the nodule is surrounded by a peripheral tissue provided with vascular bundles through which metabolites are exchanged with the other parts of the plant.

In the interaction with the bacteria, the host plant expresses specific genes that are not transcribed at a detectable level in other parts of the plant. The products of several of these genes are made during the formation of the nodule and are named early nodulins.

The present study aims at elucidating the role of these early nodulins in the formation and infection of the root nodules. For that purpose, we set out to combine the molecular approach of studying gene expression with the microscopical approach of studying the structural development of the nodule.

To provide a background to these studies, chapter 11 summarizes existing knowledge about nodule development from an anatomical/cytological point of view, supplemented with data on already described nodulins and with brief excursions into physiological phenomena relevant to the rest of our study.

In chapter III and IV, nodulin gene expression is analysed in common vetch ( Vicia sativa ) nodules elicited by a panel of bacterial strains with various defined genetic changes. Such nodules were blocked at different stages in the development of the central tissue depending on the bacterium involved; the precise stage at which the blockade occurred was determined by light- and electron-microscopical observations. In that way, insight could be gained in the diverse genetic information supplied by the bacterium for nodule development to proceed through the successive developmental stages and the induction of the appropriate nodulin genes going with it. Furthermore, the start of the expression of individual nodulin genes, for instance the early nodulin Nps-40', could be related to certain stages of central tissue development. In the case of the leghemoglobin genes, such a correlation between nodulin gene expression and specific developmental stages could be confirmed by the direct localization of the leghemoglobin proteins in pea ( Pisum sativum ) nodule sections comprising different consecutive developmental stages, by immunolabeling.

Such direct approach of studying nodulin gene expression in nodule sections was further pursued in the chapters V, VI, VII and VIII. In chapters V, VI and VII early nodulin gene transcripts for which sequenced cDNA clones had become available were localized by in situ hybridization: in chapter V, ENOD2 in soybean ( Glycine max ) and pea nodules, respectively; in chapter VI, ENOD2 in alfalfa ( Medicago sativa ) nodules; and in chapter VII, PsENOD12 in pea. In chapter VIII an attempt to localize the Nps-40' protein by immunolabeling in pea nodules is described. By these in situ localization methods, different temporal and spatial patterns of gene expression for each early nodulin were determined. Speculations about the functions of the individual nodulins are made based upon the gene expression patterns and the amino acid sequences of the nodulins as deduced from the nucleotide sequence of the corresponding cDNA clones.

In addition, in chapter VI, in situ localization of MsENOD2 transcripts was performed on alfalfa nodules induced by certain engineered bacterial strains or by auxin transport inhibitors. Such nodules do not have bacteria in their central tissue and also differ in other structural details from effective nodules, but nevertheless were shown to exhibit a tissue- specific expression pattern of the MsENOD2 gene similar to effective nodules. In chapter VII the results of further experiments are reported pertaining to the influence of the bacterium on nodulin gene expression, particularly the involvement of bacterial factors and the bacterial nod genes in the induction of the expression of the PsENOD12 genes.

Finally, chapter IX summarizes the results of the in situ localization of early nodulin gene products. In the light of these results, the significance of our histochemical approach to elucidating the role of nodulins in root nodule development is discussed.