Glutamine synthetase activity of the endosperm, embryo and pedicel-placento-chalazal regions of developing maize (Zea mays) kernels

Glutamine synthetase (GS, EC 6.3.1.2) activity in homogenates of the maize ( Zea mays L. hybrid A619 X W64A) kernel pedicel-placento-chalazal (PPCh), endosperm regions was characterized in order to optimize assay (hydroxylamine-dependent γ-glutamyl hydroxymate formation) conditions for quantitating maize kernel GS in crude extracts. The GS activities of all three tissue extracts exhibited optima at pH 7.0 with ATP:Mg²⁺ of 1:1.6. Assays of kernel tissue GS activity required relatively high concentrations of substrates to achieve saturation compared to GS from other plant tissue sources, a point which has not been considered in previous reports of maize kernel GS activity. When measured under optimal assay conditions. PPCh-GS increased to a peak of 51 nmol γ-glutamyl hydroxymate kernel⁻¹ min⁻¹ at 25 days after pollination and then declined throughout the remainder of kernel development. Embryo GS activity increased steadily throughout development to a maximum of 24 nmol γ-glutamyl hydroxymate embryo⁻¹ min⁻¹ by 50 days after pollination. In contrast, endosperm GS activity, which was 25 nmol γ-glutamyl hydroxymate endosperm⁻¹ min⁻¹ at 25 days after pollination, exhibited no discernable pattern of change during kernel development. These findings are discussed with respect to the possible roles PPCh, endosperm and embryo GS play in kernel development.     

Response of Zea mays to the Inoculation with Azospirillum on Nitrogen Metabolism under Greenhouse Conditions

The maize (Zea mays L.) plants inoculated by N₂-fixing bacterium Azospirillum showed increased activity of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in root cells free extracts over uninoculated control plants. Maximum differences in NADH-GDH activity were observed during the second and third weeks after sowing. The specific activity of GS showed a greater increase at the end of the assay. The percentage of nitrogen in leaves, root and foliage length, total fresh mass and nitrogenase activity were higher in inoculated plants than in the control ones.     

Membrane stabilization by abscisic acid under cold aids proline in alleviating chilling injury in maize (Zea mays L.) cultured cells

Previous studies of maize suspension‐cultured cells showed that abscisic acid (ABA) treatment at warm temperatures improved the tolerance of cells to subsequent chilling. In the present study, it is shown that both ABA‐treated and untreated maize cells accumulated proline in response to chilling. However, ABA‐treated cells displayed less lipid peroxidation during chilling, and thus, unlike untreated cells, were able to retain the accumulated proline intracellularly. Proline application experiments indicate that an intracellular proline level higher than 2 µmole (g FW)^?1 prior to chilling was needed to meaningfully reduce chilling‐enhanced lipid peroxidation and significantly improve chilling tolerance. The results suggest that total proline accumulation in ABA‐treated as well as untreated cells during chilling was enough to potentially improve chilling tolerance, but proline leakage rendered the control cells unable to benefit from the endogenous synthesis of proline in relation to the alleviation of chilling injury. Proline participated in chilling tolerance improvement in ABA‐treated maize cells, as evidenced by: (1) the inhibition of proline accumulation by l ‐methionine‐d , l ‐sulphoximine (MSO), an inhibitor of glutamine synthetase, reduced ABA‐improved chilling tolerance, and (2) the addition of glutamine into the medium prevented the MSO‐induced reduction in chilling tolerance. The revised relationship between proline accumulation and membrane stability at cold is discussed in the light of these current findings.     

Isoforms of glutamine synthetase in asparagus spears: the cytosolic enzyme increases after harvest

Two distinct forms of glutamine synthetase (GS) have been identified in the spear tip tissues of harvested asparagus (Asparagus officinalis L. cv. Limbras 10). The GS activities were separated by anion exchange chromatography. They have distinct kinetic properties and contain polypeptides of different sizes, and the abundances of the GS isoforms change differently after harvest. Plastid GS has a 44 kD polypeptide, and during the post-harvest period the abundance of this polypeptide declined dramatically. After 5 d, the activity of plastid GS had declined to just 20% of that at harvest. Cytosolic GS has a 40 kD polypeptide and is the major constituent of the GS activity present at harvest (73% of total). After harvest, cytosolic GS activity declined by half and then, at 3 or 4 d after harvest, rose to 80% of the cytosolic GS activity present at harvest. The nitrogen metabolism of asparagus spears is significantly altered as the tissues deteriorate rapidly after harvest. We demonstrate that cytosolic GS activity increases during the post-harvest period and is likely to be a critical feature of the physiology of the tip of a harvested asparagus spear.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

Modelling postsilking nitrogen fluxes in maize (Zea mays) using 15N-labelling field experiments

In maize (Zea mays), nitrogen (N) remobilization and postflowering N uptake are two processes that provide amino acids for grain protein synthesis. To study the way in which N is allocated to the grain and to the stover, two different 15N-labelling techniques were developed. 15NO(3-) was provided to the soil either at the beginning of stem elongation or after silking. The distribution of 15N in the stover and in the grain was monitored by calculating relative 15N-specific allocation (RSA). A nearly linear relationship between the RSA of the kernels and the RSA of the stover was found as a result of two simultaneous N fluxes: N remobilization from the stover to the grain, and N allocation to the stover and to the grain originating from N uptake. By modelling the 15N fluxes, it was possible to demonstrate that, as a consequence of protein turnover, a large proportion of the amino acids synthesized from the N taken up after silking were integrated into the proteins of the stover, and these proteins were further hydrolysed to provide N to the grain.     

Targeting of glutamine synthetase to the mitochondria of transgenic tabacco

Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) 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 -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 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 -F₁ ATPase has successfully targeted the GS 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 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 polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.     

高等植物谷氨酰胺合成酶研究进展

谷氨酰胺合成酶（GS）是参与高等植物氮同化过程的关键酶。介绍了高等植物谷氨酰联合成酶及其同工酶的分布、性质、生理作用及分子生物学等方面的研究进展。     

The role of glutamine synthetase in regulation of nitrogen metabolism within the soil microbial community

Recent advances in our understanding of the enzymology and regulatory systems involved in microbial metabolism of N hold promise to elucidate some of the underlying factors controlling metabolism of N in soil ecosystems. A review of recent work is used to construct a paradigm for N metabolism regulation in soil based on the central role of glutamine synthetase (GS) in such regulation within the soil microbial community. The studies involved use of GS inhibitors to elucidate the role of GS activity in regulation of soil N metabolism. Such studies have shown that the glutamine formed by microbial assimilation of NH₄ ⁺ via GS activity influences the regulatory mechanisms controlling both the production and activity of enzymes involved in N metabolism. For example, these studies showed that the inhibition of GS activity within the soil microbial community relieved the repression of urease production caused by microbial assimilation of inorganic N and blocked the short-term regulation of assimilatory nitrate reductase (ANR) by NH₄ ⁺ assimilation. Other studies have indicated that common environmental factors in soil may influence GS activity in microorganisms and thereby may influence metabolism of N within the soil microbial community. The paradigm for N metabolism regulation in soil that has emerged from such studies should lead to a better understanding of the mechanisms controlling fate of N in soil ecosystems.     

Synthesis of glutamine and glutamate by intact bundle-sheath cells of maize (Zea mays L.)

Intact bundle-sheath cells with functional plasmodesmata were isolated from leaves of Zea mays L. cv. Mutin, and the capacity of these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence of added 8 mM ATP. At lower concentrations of ATP a higher rate of glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities of glutamine synthetase (located mainly in the cytosol) and of glutamate synthase (located in the stroma) are high enough to meet the demands of whole maize leaves.Abbreviations Gln glutamine - Glu glutamate - GOGAT glutamate synthase - GS glutamine synthetase - 2-OG 2-oxoglutarate This work was supported by the Bundesminister für Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1.     

Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves

Localization of two isoforms of glutamine synthetase (GS; EC 6.3.1.2) was investigated in different cell types, mesophyll cells and bundle sheath cells, of corn ( Zea mays L. var. W64A × W182E) leaves by using ion exchange chrotnatography. In whole leaf extracts, relative activities of GS1 (cytosolic GS) and GS2 (chloroplastic GS) were almost equal. Purified mesophyll protoplasts and bundle sheath strands also showed similar proportions of GS1 and GS2. Methionine sulfoximine (1 mM ) enhanced the accumulation of ammonia when mesophyll protoplasts were incubated with nitrite or when bundle sheath strands were incubated with glycine. This clearly indicates a spatial separation of metabolism of NH⁺₄ derived from photorespiration and from reduction of NOJ.     

Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1

Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function.     

The role of cytosolic glutamine synthetase in wheat

The role of glutamine synthetase (GS; EC 6.3.1.2) was studied in wheat. GS isoforms were separated by HPLC and the two major leaf isoforms (cytosolic GS1 and chloroplastic GS2) were found to change in content and activity throughout plant development. GS2 dominated activity in green, rapidly photosynthesising leaves compared to GS1 which was a minor component. GS2 remained the main isoform in flag leaves at the early stages of grain filling but GS1 activity increased as the leaves aged. During senescence, there was a decrease in total GS activity which resulted largely from the loss of GS2 and thus GS 1 became a greater contributor to total GS activity. The changes in the activities of the GS isoforms were mirrored by the changes in GS proteins measured by western blotting. The changes in GS during plant development reflect major transitions in metabolism from a photosynthetic leaf (high GS2 activity) towards a senescencing leaf (relatively high GS1 activity). It is likely that, during leaf maturation and subsequently senescence, GS1 is central for the efficient reassimilation of ammonium released from catabolic reactions when photosynthesis has declined and remobilisation of nitrogen is occurring. Preliminary analysis of transgenic wheat lines with increased GS1 activity in leaves showed that they develop an enhanced capacity to accumulate nitrogen in the plant, mainly in the grain, and this is accompanied by increases in root and grain dry matter. The possibility that the manipulation of GS may provide a means of enhancing nitrogen use in wheat is discussed.     

Zinc deficiency and pollen fertility in maize (Zea mays)

Zinc deficiency decreased pollen viability in maize (Zea mays L. cv. G2) grown in sand culture. On restoring normal zinc supply to zinc-deficient plants before the pollen mother cell stage of anther development, the vegetative yield of plants and pollen fertility could be recovered to a large extent, but the recovery treatment was not effective when given after the release of microspores from the tetrads. If zinc deficiency was induced prior to microsporogenesis it did not significantly affect vegetative yield and ovule fertility, but decreased the fertility of pollen grains, even of those which visibly appeared normal. If the deficiency was induced after the release of microspores from the tetrads, not only vegetative yield and ovule fertility but pollen fertility also remained unaffected.     

Purification and properties of glutamine synthetase from Douglas fir roots

Glutamine synthetase (GS. EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl chromatography, affinity chromatography on Matrex Gel Red A. and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40-fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non-denaturing gradient-PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 m M . respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K_m values which were 11 and 75 μ M in the presence of ammonium concentrations lower and higher than 1.3 m M , respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and bio-synthetic activities, respectively.