Cellular expression and regulation of the Medicago truncatula cytosolic glutamine synthetase genes in root nodules

In this paper we have studied the localisation of expression of the two fun ctional cytosolic glutamine synthetase (GS) genes, MtGSa and MtGSb, in root nodules of the model legume Medicago truncatula. We have used a combinatio n of different techniques, including immunocytochemistry, in situ hybridisa tion and promoter beta-glucuronidase (GUS) fusions in transgenic plants, to provide the means of correlating gene expression with protein localisation . These studies revealed that transcriptional regulation (mRNA synthesis) p lays an important part in controlling GS protein levels in nodules of M. tr uncatula. The major locations of cytosolic GS mRNA and protein are the cent ral tissue, the parenchyma and the pericycle of the vascular bundles. These findings indicate that in nodules, GS might be involved in other physiolog ical processes in addition to the primary assimilation of ammonia released by the bacterial nitrogenase. The two genes show different but overlapping patterns of expression with MtGSa being the major gene expressed in the inf ected cells of the nodule. Promoter fragments of 2.6 kb and 3.1 kb of MtGSa and MtGSb, respectively, have been sequenced and primer extension revealed that the MtGSb promoter is expressed in nodules from an additional start s ite that is not used in roots. Generally these fragments in the homologous transgenic system were sufficient to drive GUS expression in almost all the tissues and cell types where GS proteins and transcripts are located excep t that the MtGSa promoter fragment did not express GUS highly in the nodule infected cells. These results indicate that the cis-acting regulatory elem ents responsible for infected-cell expression are missing from the MtGSa pr omoter fragment.