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 Rh izobium (Nar), resulting in the formation of non-nitrogen fixing root structures which possess histological features of Rhizobium-elicited n odules. To study the spatio-temporal expression pattern of the early n odulin ENOD12 gene during Nar ontogeny, we have crossed an alfalfa gen otype exhibiting a pronounced Nar phenotype with transgenic alfalfa ex pressing a MtENOD12-GUS chimeric gene fusion. Following nitrogen depri vation, and in the absence of Rhizobium, GUS activity can first be det ected in foci of dividing cells in the inner root cortex, correspondin g to early stages of Nar development. Such Nar primordia are initiated exclusively in the older part of the root system, in contrast to Rhiz obium-elicited nodulation, and the majority of mitotic foci arrest at an early stage of development. At later stages, ENOD12 is transcriptio nally active in a sub-meristematic region of the spontaneous nodule, a nalogous to the localisation of ENOD12 transcripts in N-2-fixing nodul es. Based on the spatio-temporal pattern of Nar development throughout the root system, we find no evidence for systemic autoregulation of s pontaneous nodulation. These results show that Medicago ENOD12 gene ex pression can serve as a molecular marker for spontaneous nodule ontoge ny in alfalfa and we conclude that, despite certain similarities, sign ificant regulatory and developmental differences exist between Rhizobi um-dependent and -independent nodulation.