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 al falfa nodule cDNA library, while pGS100 was previously isolated from a n alfalfa suspension culture cDNA library. Using the 3' untranslated r egion 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 nod ule-enhanced GS gene class. Genomic Southern blot analysis using gene- specific probes shows multiple hybridizing bands, in each case suggest ing multiple genes and/or alleles for each class of cytoplasmic GS gen es. In situ hybridization of alfalfa nodule sections with gene-specifi c 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 induc ed in the early symbiotic zone and are expressed throughout the symbio tic and senescent zones. Transcripts for both sets of GS genes are loc alized in the infected cells and based on the spatial expression patte rn it would appear that the two gene classes are induced independently of the onset of nitrogen fixation.