Analysis of nodule meristem persistence and ENOD40 functioning in Medicago truncatula nodule formation

Medicago root nodules are formed as a result of the interaction of the plant with the soil-borne bacterium Sinorhizobium meliloti. Several plant genes are induced during nodule formation and MtENOD40 is one of the earliest genes activated. The precise function as well as the molecule harboring the biological activity of ENOD40, however, remains unknown. In this thesis, we conducted experiments aiming at filling this gap in knowledge concerning ENOD40. As two copies of ENOD40 are present in the genome of Medicago, we used gene-specific knock-down of the two genes to determine whether both genes are involved in nodule formation. This is described in chapter 2. We showed that the number of nodules per root, in case expression of both genes was reduced, was lower than the number of nodules per root in case either of the two genes was reduced. This showed that both genes are involved in initiation of nodule formation and the two genes work in an additive manner in nodule initiation. Furthermore, we showed that reduced expression of either MtENOD40 gene induced premature nodule senescence and that both genes are essential for the development of the bacterium into the nitrogen-fixing bacteroid. Whereas the vast majority of eukaryotic mRNAs code for proteins, a common feature of ENOD40 genes is the absence of a long open reading frame (ORF). Instead, ENOD40s share at the nucleotide level two highly conserved regions, box1 and box2, of which box1, in almost all ENOD40 genes, contains an ORF for a peptide of 10-13 amino acids. In chapter 3, we showed that over-expression of MtENOD40, as well as box1 or box2 only, induced premature nodule senescence. We showed that the box1 activity was mediated by the 13 amino acid peptide encoded within box1, while the box2 activity is not peptide mediated. Using transgenic Medicago lines containing the marker gene coding for RED FLUORESCENT PROTEIN (RFP) with or without box2 sequences in its 3’UTR, we showed that the translation of the mRNA with box2 yielded less RFP than the mRNA lacking box2. This showed that box2 is involved in the regulation of translation of RFP and suggests that box2 functions in a similar way in the regulation of the translation of the peptide encoded by box1 in MtENOD40. Thus our data as described in chapters 2 and 3 propose a role of the peptide in nodule initiation and at a later stage in nodule development, most likely in avoiding senescence. In this latter process the concentration of the peptide is critical as over-expression and reduction in expression of MtENOD40 induces premature nodule senescence. Medicago nodules have a persistent meristem, like roots. In the root meristem the persistence of the meristematic activity is maintained by a group of stem cells that surround the so-called quiescent center cells (QC). These maintain stem cell identity in the surrounding cells and are mitotically inactive themselves. However, it is not known whether a similar mechanism controls the persistence of a nodule meristem. As nodules are root-borne organs, we studied whether promoters of QC and stem cell-specific genes of Arabidopsis were activated in the nodule meristem of Medicago. Our data, as described in chapter 4, showed that three out of the five tested markers for QC and stem cell-specific genes are activated in cells that form a ring at the periphery of the nodule meristem. The activity of the other two markers was restricted to cells that are part of this ring of cells, but only were active in cells abutting on nodule vascular bundles. These data suggest that the nodule meristem contains two different stem cell domains and that the cells in which QC markers are activated may act as organizers and share properties with the QC of the root. As the cells expressing the tested stem cell marker are at the periphery of the nodule meristem, we propose that these cells form the stem cells for nodule peripheral and vascular tissues. Strikingly, none of the tested promoters was activated in cells in the central part of the nodule meristem. This part of the meristem adds cells to the central tissue of the nodule. However, it remains to be determined whether or not the mitotic activity of cells in the nodule meristem is maintained by the QC cells identified at the periphery of the meristem.