Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.     

Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum

In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or “twin” GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the “twin” GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD⁻ strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development.     

Differential expression within the glutamine synthetase gene family of the model legume Medicago truncatula.

The glutamine synthetase (GS) gene family of Medicago truncatula Gaertn. contains three genes related to cytosolic GS (MtGSa, MtGSb, and MtGSc), although one of these (MtGSc) appears not to be expressed. Sequence analysis suggests that the genes are more highly conserved interspecifically rather than intraspecifically: MtGSa and MtGSb are more similar to their homologs in Medicago sativa and Pisum sativum than to each other. Studies in which gene-specific probes are used show that both MtGSa and MtGSb are induced during symbiotic root nodule development, although not coordinately. MtGSa is the most highly expressed GS gene in nodules but is also expressed to lower extents in a variety of other organs. MtGSb shows higher levels of expression in roots and the photosynthetic cotyledons of seedlings than in nodules or other organs. In roots, both genes are expressed in the absence of an exogenous nitrogen source. However the addition of nitrate leads to a short-term, 2- to 3-fold increase in the abundance of both mRNAs, and the addition of ammonium leads to a 2-fold increase in MtGSb mRNA. The nitrogen supply, therefore, influences the expression of the two genes in roots, but it is clearly not the major effector of their expression. In the discussion section, the expression of the GS gene family of the model legume M. truncatula is compared to those of other leguminous plants.     

Effects of the overexpression of a soybean cytosolic glutamine synthetase gene (GS15) linked to organ-specific promoters on growth and nitrogen accumulation of pea plants supplied with ammonium.

A soybean cytosolic glutamine synthetase gene (GS15) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC(3)), or a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies (Lines 1, 7, 8 and 9) and four lines with two copies each of GS15 (Lines 2, 4, 6 and 11) were compared to the wild-type (WT) parental line for levels of cytosolic glutamine synthetase (GS1), glutamine synthetase (GS) activity, N accumulation, N derived form the atmosphere (NDFA), and biomass of plants grown on 0.0, 0.1, 1.0 or 10.0 mM NH(4)(+). Enhanced levels of GS1 were detected in leaves of one of the two lines transformed with the 35S-GS15 construct, and all three lines containing the rolD-GS15 construct. All three lines containing the LBC(3)-GS15 construct had increased levels of GS1 in nodules. Despite the increased levels of GS1 in many transformants, only the roots of lines containing the rolD-GS15 construct consistently demonstrated enhanced levels of GS activity (up to 12-fold). Positive responses in plant N content, NDFA, and biomass were rare, but increases in plant biomass and N content of up to 17% and 54%, respectively, occurred in some of the rolD-GS15 lines at certain levels of ammonium. In general, GS15 copy number did not seem to differentially affect phenotype of the transformants, and transformants respond to ammonium concentrations in similar patterns to that previously observed with nitrate. Despite the fact that the rolD-GS15 transformants consistently resulted in increased GS activity in roots and resulted in some occurrences of increases in biomass and plant N content, the lack of consistent positive growth effect across all transformants indicates that the generalized overexpression of GS1 in tissues holds little potential for positive growth responses in pea.     

Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate

A glutamine synthetase gene ( GS15) coding for soybean cytosolic glutamine synthetase (GS1) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC(3)) and a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies of GS15 (one 35S-GS15 line, one LBC (3) -GS15 line, and two rolD-GS15 lines) were tested for the expression of GS15, levels of GS1, GS activity, N accumulation, N(2) fixation, and plant growth at different levels of nitrate. Enhanced levels of GS1 were detected in leaves of three transformed lines (the 35S-GS15 and rolD-GS15 transformants), in nodules of three lines (the LBC (3) -GS15 and rolD-GS15 transformants), and in roots of all four transformants. Despite increased levels of GS1 in leaves and nodules, there were no differences in GS activity in these tissues or in whole-plant N content, N(2) fixation, or biomass accumulation among all the transgenic lines and the wild-type control. However, the rolD-GS15 transformants, which displayed the highest levels of GS1 in the roots of all the transformants, had significantly higher GS activity in roots than the wild type. In one of the rolD-GS15 transformed lines (Line 8), increased root GS activity resulted in a lower N content and biomass accumulation, supporting the findings of earlier studies with Lotus japonicus (Limami et al. 1999 ). However, N content and biomass accumulation was not negatively affected in the other rolD-GS15 transformant (Line 9) and, in fact, these parameters were positively affected in the 0.1 mM treatment. These findings indicate that overexpression of GS15 in various tissues of pea does not consistently result in increases in GS activity. The current study also indicates that the increase in root GS activity is not always consistent with decreases in plant N and biomass accumulation and that further investigation of the relationship between root GS activity and growth responses is warranted.     

Cytosolic glutamine synthetase in soybean is encoded by a multigene family, and the members are regulated in an organ-specific and developmental manner.

Gln synthetase (GS) is the key enzyme in N metabolism and it catalyzes the synthesis of Gln from glutamic acid, ATP, and NH4+. There are two major isoforms of GS in plants, a cytosolic form (GS1) and a chloroplastic form (GS2). In leaves, GS2 functions to assimilate ammonia produced by nitrate reduction and photorespiration, and GS1 is the major isoform assimilating NH3 produced by all other metabolic processes, including symbiotic N2 fixation in the nodules. GS1 is encoded by a small multigene family in soybean (Glycine max), and cDNA clones for the different members have been isolated. Based on sequence divergence in the 3'-untranslated region, three distinct classes of GS1 genes have been identified (alpha, beta, and gamma). Genomic Southern analysis and analysis of hybrid-select translation products suggest that each class has two distinct members. The alpha forms are the major isoforms in the cotyledons and young roots. The beta forms, although constitutive in their expression pattern, are ammonia inducible and show high expression in N2-fixing nodules. The gamma1 gene appears to be more nodule specific, whereas the gamma2 gene member, although nodule enhanced, is also expressed in the cotyledons and flowers. The two members of the alpha and beta class of GS1 genes show subtle differences in the expression pattern. Analysis of the promoter regions of the gamma1 and gamma2 genes show sequence conservation around the TATA box but complete divergence in the rest of the promoter region. We postulate that each member of the three GS1 gene classes may be derived from the two ancestral genomes from which the allotetraploid soybean was derived.     

农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究

 谷氨酰胺合成酶（Glutamine synthetase,GS,E.C. 6.3.1.2）是植物氨同化过程中的关键酶,对植物的氮素吸收和代谢起着至关重要的作用。谷氨酰胺合成酶还是除草剂草胺膦(Phosphinothricin　(PPT)或Basta)的靶标酶。前期工作已从我国特有的豌豆(Pisum satium)品种中克隆了细胞质型谷氨酰胺合成酶（GS1）cDNA和叶绿体型谷氨酰胺合成酶（GS2）cDNA。为了验证谷氨酰胺合成酶的功能,构建了同时含有GS1 cDNA和GS2 cDNA的植物表达载体p2GS。以该表达载体通过农杆菌介导法,转化小麦(Triticum aestivum)的未成熟胚愈伤组织,经PPT筛选及分化再生培养,获得了抗PPT的转基因小麦植株41株。PCR和基因组Southern 杂交分析证实了GS1 和GS2基因已经整合到转基因小麦的基因组。用除草剂草胺膦Basta溶液涂抹转p2GS小麦叶片,结果证明GS转基因植株可以抗高达0.3%的 Basta溶液,而对照植株叶片逐渐变黄直至枯死。转基因小麦植株能正常结实。上述实验结果表明：1) GS基因在小麦植株中获得了有效表达,从而赋予小麦植株抗PPT特性;2) GS基因能够作为研究小麦遗传转化的筛选标记基因。     

The effects of nitrate on the enzymes involved in nitrogen assimilation in nodulated pea roots

Pea Plants ( Pisum sativaum L. ev. Little Marvel) were grown in N-free medium and when well nodulated (28 days) were supplied for 8 days with nitrate or ammonium. Over the 8 days of nitrate treatment, total amino and amide N in sap declined, and the proportion of aspartate relative to the other amino acids increased. After 8 days of treatment, nitrogenase (EC 1.18.2.1) activity in nitrate-treated plants declined to about 30% of the activity in controls even though nodules were not directly in contact with nutrient solution. Nitrogenase activity was also decreased by the addition of ammonium chloride (10 m M ). With addition of nitrate or ammonium. clear signs of senescence began to show in the nodules after 4 days. Nitrate reductase (EC 1.6.6.1) activity was induced in roots by nitrate, but decreased sharply in nodules. In response to nitrate addition, newly formed root tissues showed 3- to 5-times higher glutamine synthetase (GS. EC 6.3.1.4) activity than newly formed tissues of control plants, expressed on a protein or weight basis. In complementary experiments, when ammonium salts were used instead of nitrates, the increase in GS activity was significantly lower. GS activity decreased in nodules of treated plants and total extractable protein was 3 times lower in nodules of nitrate-treated plants than in controls at day 8 of treatment.     

Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin

Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP(+)-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities (El-Khatib et al., 2004).     

Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.)

A major source of inorganic nitrogen for rice plants grown in paddy soil is ammonium ions. The ammonium ions are actively taken up by the roots via ammonium transporters and subsequently assimilated into the amide residue of glutamine (Gln) by the reaction of glutamine synthetase (GS) in the roots. The Gln is converted into glutamate (Glu), which is a central amino acid for the synthesis of a number of amino acids, by the reaction of glutamate synthase (GOGAT). Although a small gene family for both GS and GOGAT is present in rice, ammonium-dependent and cell type-specific expression suggest that cytosolic GS1;2 and plastidic NADH-GOGAT1 are responsible for the primary assimilation of ammonium ions in the roots. In the plant top, approximately 80% of the total nitrogen in the panicle is remobilized through the phloem from senescing organs. Since the major form of nitrogen in the phloem sap is Gln, GS in the senescing organs and GOGAT in developing organs are important for nitrogen remobilization and reutilization, respectively. Recent work with a knock-out mutant of rice clearly showed that GS1;1 is responsible for this process. Overexpression studies together with age- and cell type-specific expression strongly suggest that NADH-GOGAT1 is important for the reutilization of transported Gln in developing organs. The overall process of nitrogen utilization within the plant is discussed.