Identification of Novel Putative Regulatory Genes Induced During Alfalfa Nodule Development with a Cold-Plaque Screening Procedure

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Abstract
Until now very few plant genes with possible regulatory functions during nodule development have been isolated. We have used a modified cold-plaque screening method to identify new transcripts expressed at low levels that are induced during nodulation. Several clones were isolated and characterized by their mRNA expression patterns during nodule development and in spontaneous nodules. Sequence homology with known genes of other organisms indicated that transcripts corresponded to (i) “basic” genes probably required during the growth of the nodule organ (e.g., structural proteins), (ii) genes related to the metabolic adaptations taking place during nodule morphogenesis and function (e.g., carbonic anhydrase), and (iii) genes containing regulatory motifs and/or homologies (three clones out of the 20 identified). The latter genes encode a zinc-finger-containing protein, a putative protein kinase, and a Wilm's tumor (WT) suppressor homologue, respectively. Expression of the kinase and WT suppressor homologues was induced early in nodulation, although the latter was activated transiently. Accumulation of the Zn-finger gene transcripts was detected at a later stage of development and seems to be regulated in a complex manner. Hence, using a cold-plaque screening procedure, we could identify genes that may play regulatory roles in the signal transduction pathways activated during nodule development.