Structure and expression of glutamine synthetase in root nodules of Phaseolus vulgaris L

• Main Author: Bennett, Malcolm John
• Published: University of Warwick 1989
• Subjects: 572; QK Botany
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235512

Glutamine synthetase (GS) catalyses the assimilation of ammonia with glutamate, to form glutamine. In higher plants GS is an octameric enzyme of Mr 360,000 located in plastids and the cytosol. In the legume Phaseolus vulgaris L., GS is encoded by four nucleur genes (g1n-α, g1n-β, g1n-γ and g1n-δ) which encode the cytosolic α, β, and γ GS subunits of Mr 39,000, and the plastid δ GS polypeptides of Mr 44,000, accordingly. The aim of the work is to study the regulation of expression and structure of P. vulgaris root nodule GS. This thesis has reported the construction of a P. vulgaris nodule cDNA library, and the isolation and characterisation of a full length nodule GS cDNA clone. The polypeptide encoded by this cDNA and two previously identified GS cDNA clones, pR-1 and pR-2 (see Gebhardt et al, 1986, EMBO J. 5, 1429-1435) have been produced in vitro by transcription/translation and shown to co-migrate on two-dimensional gels with the cytosolic α, β, and γ GS polypeptides respectively. An RNase protection technique has been used to specifically and quantitatively determine the abundance of the g1n-β, g1n-γ and leghaemoglobin (Lhb) mRNAs. Differences in the relative abundances of g1n-β and g1n-γ mRNAs at different stages of nodulation suggest that these two genes are divergently regulated, whereas the g1n-γ and Lhb mRNAs are coordinately expressed during nodule development, detectable at least one day prior to the onset of dinitrogen fixation. Furthermore, the detection of the γ polypeptide, although at a reduced level, in nodules grown in the absence of dinitrogen (under an atmosphere of 80% Argon: 20% oxygen) suggests that a product of dinitrogen fixation does not provide the primary signal for, but may have a role in the level of, g1n-γ nodule expression. A study of the abundance of the g1n-γ and Lhb mRNAs from a variety of P. vulgaris organs has identified that in contrast with the nodule specific expression of Lhb mRNA, g1n-γ mRNA is also detectable, albeit at a lower abundance, in stems, petioles, and green cotyledons. The structure of P. vulgaris nodule and plumule GS has been studied through the resolution of the component GS isoforms, and the determination of their respective GS polypeptide compositions by ion exchange fast protein liquid chromatography and two-dimensional western blotting respectively. These studies have identified that both α + β, and β + γ GS subunits may assemble, perhaps randomly, to produce a heterogenous mixture of GS isoforms. However, a study of the changes in GS isoforms during nodulation has shown that the β8 isoform is present at higher activity than would be expected, which could reflect the differences in the temporal and/or spatial expression of the γ and β GS subunits within the nodule. The three cytosolic GS cDNAs have been expressed individually In Escherichia coli, synthesising GS subunits of the correct Mr, which appear for α, γ and β to be mainly soluble, intermediate in solubility and insoluble respectively. The soluble α, β and γ GS subunits are able to assemble into kinetically active oligameric isoenzymes, in the absence of any specific plant assembly factors. In addition, the α and γ cDNAs have been shown to complement an E.coli g1nA mutation, suggesting that the recombinant α and γ higher plant GS enzymes can effectively function in the E.coli nitrogen assimilatory pathway.