The differential expression of the genes encoding glutamine synthetase in developing root modules

Abstract

Glutamine synthetase (GS) is one of the key enzymes involved in the assimilation of ammonia into organic nitrogen in plants. It is important in legume root nodules where ammonia, produced by the Rhizobium-legume symbiosis, is converted to organic nitrogen before it can be transported to other parts of the plant. In Phaseolus vulgaris three cytosolic and one plastidic GS polypeptide have been identified. One or more of these polypeptides assemble to form distinct octameric GS isoenzymes. GS activity increases significantly in P. vulgaris during nodulation and this is associated with the Increased (or repressed) expression of the three cytosclic polypeptide genes jln-a, gln-β and gln-γ. The temporal and spatial pattern of mRNA and protein distribution of these genes has been investigated using in situ hybridation and immunocytochemistry. An in situ hybridization protocol has been established using photobiotinylated cRNA probes, visualised with alkaline phosphatase, or streptavidin gold with silver enhancement. The fixation, embedding, section pretreatments and hybridization conditions have all been optimized for legume root nodule sections, the mRNA distributions corresponding to the gln-α, gln-β and gln-γ genes within P. vulgaris root-nodule sections indicate that the the assembly of the GS isoenzymes is at least partially controlled by the differential temporal and spatial expression of these genes throughout the nodule tissues during nodulation. These results have been compared with the expression of the β- glucuronidase (GUS) gene fused with the 5' flanking regions of the P. vulgaris GS genes in chimaeric Lotus corniculatus plants. The GUS expression was demonstrated by the optimized in situ hybridization tecniques in conjunction with immunocytoeheinica1 and GUS histochemical localization tecniques. Results indicate the control of GS gene expression is at the transcriptional level and at least partially determined by the 5' flanking regions of these genes.