ENOD12 GENE-EXPRESSION AS A MOLECULAR MARKER FOR COMPARING RHIZOBIUM-DEPENDENT AND RHIZOBIUM-INDEPENDENT NODULATION IN ALFALFA

Certain genotypes of alfalfa are able to nodulate in the absence of Rhizobium (Nar), resulting in the formation of non-nitrogen fixing root structures which possess histological features of Rhizobium-elicited nodules. To study the spatio-temporal expression pattern of the early nodulin ENOD12 gene during Nar ontogeny, we have crossed an alfalfa genotype exhibiting a pronounced Nar phenotype with transgenic alfalfa expressing a MtENOD12-GUS chimeric gene fusion. Following nitrogen deprivation, and in the absence of Rhizobium, GUS activity can first be detected in foci of dividing cells in the inner root cortex, corresponding to early stages of Nar development. Such Nar primordia are initiated exclusively in the older part of the root system, in contrast to Rhizobium-elicited nodulation, and the majority of mitotic foci arrest at an early stage of development. At later stages, ENOD12 is transcriptionally active in a sub-meristematic region of the spontaneous nodule, analogous to the localisation of ENOD12 transcripts in N-2-fixing nodules. Based on the spatio-temporal pattern of Nar development throughout the root system, we find no evidence for systemic autoregulation of spontaneous nodulation. These results show that Medicago ENOD12 gene expression can serve as a molecular marker for spontaneous nodule ontogeny in alfalfa and we conclude that, despite certain similarities, significant regulatory and developmental differences exist between Rhizobium-dependent and -independent nodulation.