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We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.

The gln-delta gene, which encodes the plastid-located glutamine synthetase of Phaseolus vulgaris, was cloned and its promoter region was sequenced. Primer extension analysis was used to map the two major transcription initiation sites which are about 90 nucleotides apart. A fusion of 2.3 kb of the upstream region of the gln-delta gene to the reporter gene uidA encoding beta-glucuronidase was shown to be expressed in the chlorophyllous cell types of leaves and stems and in the root meristem region of transgenic tobacco. Analysis of a series of three 5' promoter deletion fusions revealed the presence of a region essential for promoter activity between -786 and -327 and regions involved in tissue-specific regulation and light regulation between -786 and +43.

Glutamine synthetase (GS) activity increased over three-fold in developing primary leaves of Phaseolus vulgaris L. This increase was shown to be the result of differential expression of three members of the GS gene family: gln-alpha and gln-beta, which encode cytosolic GS polypeptides, and gln-delta, which encodes the chloroplast-located GS. The gln-delta gene was the most highly expressed GS gene and was regulated in a complex manner with two different transcripts accumulating differentially during leaf development. This gene was expressed weakly in the dark and was induced strongly by light; this induction was shown not to be an indirect effect of photorespiration. In the long term, gln-delta showed increased expression in photorespiring compared with non-photorespiring leaves. However, in the short term, there was no induction of gln-delta following transfer of plants to photorespiratory conditions. These results suggest that regulation of gln-delta by photorespiration was the result of indirect, long-term effects on cellular metabolism. In general, in all these experiments, analysis of cytosolic versus chloroplastic GS polypeptides and of the GS isoenzyme profiles showed the same pattern of changes in abundance as that observed for the mRNAs suggesting that regulation of GS gene expression occurred primarily at the mRNA level. However, it was noteworthy that the delta isoenzyme remained at a high abundance in older leaves, grown in both light and dark, despite a decrease in abundance of gln-delta mRNA.

Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) gamma polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia beta-F1 ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS gamma polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the beta-F1 ATPase has successfully targeted the GS gamma polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the gamma polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the gamma polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.

In this paper we have examined whether the four glutamine synthetase (gln) genes, expressed in roots and nodules of Phaseolus vulgaris are substrate-inducible by ammonium. Manipulation of the ammonium pool in roots, through addition and removal of exogenous ammonium, did not elicit any changes in the abundances of the four mRNAs thus suggesting that the gln genes in roots of this legume are neither substrate-inducible by ammonium nor derepressed during nitrogen starvation. In nodules the effect of the ammonium supply on expression of the gln genes has been examined by growing nodules under argon/oxygen atmospheres, or with a number of Fix- Rhizobium mutants, and following addition of exogenous ammonium. The results of these experiments suggest that the expression of the gln-gamma gene, which is strongly induced during nodule development, is primarily under a developmental control. However nitrogen fixation appears to have a quantitative effect on expression of gln-gamma as the abundance of this mRNA is about 2 to 4-fold higher under nitrogen-fixing conditions. This effect could not be mimicked by addition of exogenous ammonium and moreover is not specific to the gln-gamma gene as mRNA from a leghaemoglobin gene was similarly affected. Taken together these results have failed to find an effect of ammonium on specifically inducing the expression of glutamine synthetase genes in roots and nodules of P. vulgaris.

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit β also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.

We report the sequence of an essentially full-length glutamine synthetase (GS) cDNA clone (pcGS-γ1) isolated from a root nodule library ofPhaseolus vulgaris L. The polypeptide encoded by this cDNA has been producedin vitro by transcription/translation and shown to co-migrate on two-dimensional gels with the previously identified major cytosolic GS polypeptide (γ) of nodules. Two previously identified GS cDNA clones, pR-2 and pR-1 (see Gebhardtet al., EMBO J 5: 1429-1435, 1986) have similarly been shown to encode the α and β cytosolic GS polypeptides respectively. An RNase protection technique has been used to analyse specifically and quantitatively the abundance of mRNA related to these three GS cDNAs and to the cDNA (pcGS-δ1) encoding the chloroplast-located GS, during nodulation. Differences in the abundances of these mRNAs at different times suggest that they are not coordinately regulated. Moreover, using this technique mRNA specifically related to pcGS-γ1 was found at high levels in nodules but not in roots or leaves. Surprisingly the expression of this gene is not nodule-specific as previously suggested, as its mRNA was also detected, but at lower levels, in stems, petioles and in green cotyledons. By comparison, mRNA related to a leghaemoglobin gene was detected only in nodules. Comparisons of the relative abundances of the pcGS-γ1 mRNA and the γ polypeptide in different organs and at different stages during nodulation, suggest that the appearance of the γ polypeptide is largely under transcriptional control.