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.     

Does lowering glutamine synthetase activity in nodules modify nitrogen metabolism and growth of Lotus japonicus?

A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L. japonicus via transformation with Agrobacterium tumefaciens. Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity. Three of these lines were selected and their T(1) progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions. Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots. The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content. Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g. asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L. japonicus. Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated.     

Biochemical and molecular characterization of transgenic<Emphasis Type="Italic"> Lotus japonicus</Emphasis> plants constitutively over-expressing a cytosolic glutamine synthetase gene

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS₁) or in the chloroplast (GS₂). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS₁ gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS₁ polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.Abbreviations GS Glutamine synthetase - UTR Untranslated region     

Does root glutamine synthetase control plant biomass production in Lotus japonicus L.?

To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two different approaches were conducted using the model legume Lotus japonicus. In the first series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to a decrease in plant biomass production. Using ¹⁵N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using a series of recombinant inbred lines issued from the crossing of two different Lotus ecotypes, Gifu and Funakura. It was confirmed that a negative relationship exists between root GS expression and plant biomass production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least 13% of plant growth variation can be accounted for by variation in GS activity. Received: 24 September 1998 / Accepted: 14 April 1999     

Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

The Effect of Nitrogen Nutrition on the Cellular Localization of Glutamine Synthetase Isoforms in Barley Roots

Glutamine synthetase (GS) was detected by immunogold localization in the cytosol and plastids of roots of 7-d-old barley (Hordeum vulgare L. cv Klaxon) seedlings grown in the presence or absence of NO3- (15 mM) or NH4+ (30 mM). The number of GS polypeptides changed during root development, and this was affected by N nutrition. There was no evidence of a NO3--inducible root plastid GS.In apical 5- to 10-mm regions of the root the concentration of immunogold labeling of cytosolic GS was higher in the cortical parenchyma than in the vascular cells of the stele, irrespective of N nutrition. This labeling was at least 50% higher in both cell types in N-free compared with N-grown (either NO3- or NH4+) seedlings. In contrast, GS specific activity was highest in roots of NO3--grown seedlings. It is suggested that this indicates the presence of inactive GS in roots grown without N. This study has identified both cell- and development-specific responses of GS to N nutrition.     

Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn.

Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants.     

Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Enhanced tolerance to and accumulation of mercury, but not arsenic, in plants overexpressing two enzymes required for thiol peptide synthesis

Arsenic and mercury are among the most toxic elemental pollutants in the environment, endangering human health and ecological integrity. Both elements are found in highly thiol-reactive forms, arsenite and Hg(II), respectively, in plant tissues. Overexpression of Escherichia coli γ-glutamylcysteine synthetase (ECS) or glutathione synthetase (GS) in Arabidopsis thaliana plants provided significant increases in the thiol peptides glutathione (GSH) and γ-glutamylcysteine (γ-EC), and/or phytochelatins (PCs), and some resistance to arsenic and mercury, but no substantial increases in the levels of these elements in above-ground tissues. In contrast, the co-expression of ECS and GS in ECS × GS lines produced significant increases in tolerance to toxic levels of mercury. The ECS × GS co-expression line accumulated 35-fold more biomass and three-fold more mercury aboveground than the wild type (WT) when grown on Hg(II). No increases in arsenic accumulation were detected in the ECS × GS line. Increased resistance to and accumulation of mercury apparently resulted from enhanced root concentrations of PCs in ECS × GS co-expression lines not seen in the wild type or lines expressing ECS or GS alone. Correlations between the levels of arsenic and mercury resistance and accumulation and increases in the accumulation of the various thiol peptides in the ECS, GS and ECS × GS transgenic plant lines are discussed.