CHARACTERIZATION OF A NODULE-ENHANCED GLUTAMINE-SYNTHETASE FROM ALFALFA - NUCLEOTIDE-SEQUENCE, IN-SITU LOCALIZATION, AND TRANSCRIPT ANALYSIS

We have characterized two glutamine synthetase (GS) cDNA clones (pGS13 and pGS100) representing mRNA from root nodules of alfalfa, pGS13 is a full-length version of a previously isolated partial cDNA from an alfalfa nodule cDNA library, while pGS100 was previously isolated from an alfalfa suspension culture cDNA library. Using the 3' untranslated region of the two cDNAs as gene-specific probes, we have shown that the GS genes represented by pGS100 and pGS13 are expressed in all organs tested, although at varying levels. pGS13, however, represents the nodule-enhanced GS gene class. Genomic Southern blot analysis using gene-specific probes shows multiple hybridizing bands, in each case suggesting multiple genes and/or alleles for each class of cytoplasmic GS genes. In situ hybridization of alfalfa nodule sections with gene-specific antisense RNA probes has shown that the nodule-enhanced GS genes are induced in the invasion zone and that their expression is limited to the symbiotic zone, while the GS genes represented by pGS100 are induced in the early symbiotic zone and are expressed throughout the symbiotic and senescent zones. Transcripts for both sets of GS genes are localized in the infected cells and based on the spatial expression pattern it would appear that the two gene classes are induced independently of the onset of nitrogen fixation.