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First published online August 7, 2003; 10.1104/pp.102.017830 Plant Physiology 133:243-252 (2003) © 2003 American Society of Plant Biologists Nodule-Specific Modulation of Glutamine Synthetase in Transgenic Medicago truncatula Leads to Inverse Alterations in Asparagine Synthetase Expression1Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal (H.G.C., I.A.L.-C., L.M.L., P.M.M.); and Institut des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique, Boite Postale 27, 31326 Castanet-Tolosan cedex, France (J.V.C.)
Transgenic Medicago truncatula plants were produced harboring chimeric gene constructs of the glutamine synthetase (GS) cDNA clones (MtGS1a or MtGS1b) fused in sense or antisense orientation to the nodule-specific leghemoglobin promoter Mtlb1. A series of transgenic plants were obtained showing a 2- to 4-fold alteration in nodule GS activity when compared with control plants. Western and northern analyses revealed that the increased or decreased levels of GS activity correlate with the amount of cytosolic GS polypeptides and transcripts present in the nodule extracts. An analysis of the isoenzyme composition showed that the increased or decreased levels of GS activity were attributable to major changes in the homo-octameric isoenzyme GS1a. Nodules of plants transformed with antisense GS constructs showed an increase in the levels of both asparagine synthetase (AS) polypeptides and transcripts when compared with untransformed control plants, whereas the sense GS transformants showed decreased AS transcript levels but polypeptide levels similar to control plants. The polypeptide abundance of other nitrogen metabolic enzymes NADH-glutamic acid synthase and aspartic acid amino-transferase as well as those of major carbon metabolic enzymes phosphoenolpyruvate carboxylase, carbonic anhydrase, and sucrose synthase were not affected by the GS-gene manipulations. Increased levels of AS polypeptides and transcripts were also transiently observed in nodules by inhibiting GS activity with phosphinothricin. Taken together, the results presented here suggest that GS activity negatively regulates the level of AS in root nodules of M. truncatula. The potential role of AS in assimilating ammonium when GS becomes limiting is discussed.
Nitrogen is the major nutrient limiting plant growth and crop yield, and thus many studies have been devoted to the mechanisms by which it is taken up and used by plants. Legumes obtain a significant fraction of their nitrogen from atmospheric N2 through symbiotic association with nitrogen fixing bacteria, termed rhizobia. The ammonium produced by nitrogen fixation is mainly released from the rhizobial bacteroids into the infected cells of the root nodules where it is assimilated into the organic pools by plant Gln synthetase (GS; EC 6.3.1.2). GS in conjunction with NADH-Glu synthase (NADH-GOGAT, EC 1.4.1.14) operates the Glu synthase cycle leading to the synthesis of Gln and Glu, which then serve as nitrogen donors for the biosynthesis of essentially all nitrogenous compounds. In temperate legumes, fixed nitrogen is exported from the nodules to the rest of the plant mainly as Asn, which is synthesized by the concerted action of two additional enzymes, Asp aminotransferase (AAT, EC 2.6.1.1) and Asn synthetase (AS; EC 6.3.5.4). The overall pathway of ammonium assimilation to Asn in nodules requires oxaloacetate as carbon skeleton, which is produced by metabolism of photosynthate (involving a nodule-enhanced Suc synthase [SUCS]) in conjunction with nodule CO2 fixation via the enzymes carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC). The metabolic activities of all of these enzymes are tightly linked as supported by their coordinated nodule-enhanced expression during root nodule development (for review, see Gordon et al., 2001  ). Biochemical and molecular analysis indicates that nodule-specific or nodule-enhanced forms of these isoenzymes are responsible for the majority of the increased enzymatic activities (Pathirana et al., 1997; Shi et al., 1997; Hohnjec et al., 1999; Trepp et al., 1999; Yoshioka et al., 1999; Galvez et al., 2000). Being the first enzyme of the pathway, GS is in a key position to regulate nitrogen assimilatory pathways in nodules.
In higher plants, GS is an octameric enzyme of 310 to 350 kD that occurs as a number of isoenzymes, located both in the plastids (GS2) and in the cytosol (GS1). These GS subunits are encoded by small multigene families of generally three to six members (Forde and Cullimore, 1989
A relevant question remaining to be answered is whether GS in nodules plays a regulatory role in controlling the flux through the nitrogen assimilatory pathway that could eventually lead to changes in plant productivity. There is evidence from studies using a GS-inhibitor producing bacterium that the activity of GS in nodules may control the yield of plants growing symbiotically on dinitrogen (Knight and Langston-Unkefer, 1988
Recently, various approaches have been undertaken to modify ammonium assimilation in transgenic plants that have greatly contributed to the understanding of the precise roles and regulation of the enzymes involved (for review, see Hirel and Lea, 2001
Construction of Nodule-Specific Cytosolic GS-Sense/Antisense Chimeric Genes
To alter GS levels specifically in the central infected cells of root nodules, we selected a promoter able to drive a strong and nodule-specific expression. The M. truncatula gene Mtlb1 encoding leghemoglobin has been previously shown to be specifically and strongly expressed in root nodules of M. truncatula by northern analysis (Gallusci et al., 1991
The binary constructs previously described were used to produce primary M. truncatula transformants by Agrobacterium tumefaciens-mediated transformation. We were able to regenerate 36 kanamycin-resistant transgenic M. truncatula plants. These T0 plants were propagated clonally as stem cuttings that when rooted were inoculated with Sinorhizobium meliloti strain 2011. GS activity was determined in root nodules of at least six independent T0 plants from each transformant line. From the 36 regenerated plants, 12 showed alterations in root nodule GS activity, when compared with untransformed control plants (Fig. 2). Four antisense GS1b (LbASB) and four antisense GS1a (LbASA) plants showed 2- to 4-fold reductions in GS activity. Two sense GS1a (LbSA) plants possessed a 2-fold increase in GS activity, whereas two sense GS1b (LbSB) plants showed the opposite effect, a reduction in GS activity. The GS activity levels were unaltered in both leaves and roots of the transgenic plants (data not shown). The transformants showed no visible phenotypic differences when compared with control plants. The number of transgene insertions in the genome of the plants that were showing alterations in activity was analyzed by Southern blot (data not shown), and analysis revealed one to three insertions in different transformants. The number of copies inserted in the genome could not be directly related to the GS activity levels in the nodules. Because all of the analyzed sense or antisense lines behaved in a qualitatively similar way, further studies used the two lines showing the greatest reduction (LbASB-2, antisense) and increase (LbSA-12, sense) in GS activity.
To evaluate whether the alterations in enzyme activity could be correlated with their protein and transcript levels, nodule extracts from the selected transgenic plants were analyzed both by western and northern blots (Fig. 3, A and B). All analyses were performed in two independent plants from the selected lines to assess possible variations between individuals. The variations in enzyme activity could be proportionally correlated with the amounts of cytosolic GS polypeptides (GS1) detected by western-blot analysis of crude nodule extracts, whereas plastid GS (GS2) appeared not to be altered by these manipulations (Fig. 3A). Quantitation of multiple protein blots by image densitometry demonstrated a consistent reduction or increase in cytosolic GS protein content that was proportional to the levels of GS activity in root nodules, i.e. 3-fold reduction in transformant LbASB-2 and a 2-fold increase in transformant LbSA-12.
To assess whether the alterations in GS enzyme activity and polypeptides were resulting from changes in the steady-state levels of the cytosolic transcripts and to determine which GS genes were being affected by the genetic manipulations, we analyzed the levels of each independent GS transcript by northern blots (Fig. 3B). Total nodule RNA from two independent transgenic plants from lines LbASB-2 and LbSA-12 and from the wild-type control plant were separated electrophoretically, blotted, and hybridized with gene-specific probes from the 3'-untranslated regions (UTRs) of MtGS1a, MtGS1b, and MtGS2. The hybridization signal was quantified, and the band intensities were standardized against the intensity of the 25S rRNA bands. MtGS1a was the most affected GS gene showing a remarkable reduction in transcripts in the antisense plants and a 2-fold increase in the GS overexpressing plants (Fig. 3B). Due to the very high homology between MtGS1a and MtGS1b coding sequences, the expression of MtGS1b mRNA in antisense orientation was capable of decreasing both MtGS1a and MtGS1b transcripts. Densitometry analysis suggests that the amount of MtGS1b transcripts remained unaltered in the sense plants and that MtGS2 transcript abundance was not greatly affected by either of the genetic manipulations (Fig. 3B). The GS isoenzyme composition in the transgenic nodules was analyzed by ion-exchange HPLC (IEXHPLC; Fig. 3C). A comparison of the column elution profile of GS activity of nodule extracts from the transgenic plants revealed that homo-octameric GS1a was the major, if not the only, GS holoenzyme affected. Using the plastid GS2 as a reference, which remains unaltered in the transgenic plants, there is a 3-fold decrease in activity of the GS1a homo-octamer (elution at fraction 17) in antisense plants and a 2-fold increase in sense plants, when compared with the non-transformed plants. In all cases, the homo-octamer GS1b represents a minor proportion of GS activity.
Using specific antibodies raised against the nodule-enhanced forms of nitrogen assimilatory enzymes from the closely related species alfalfa (Medicago sativa), we analyzed the levels of NADH-GOGAT, AAT, and AS by western blots (Fig. 4). The antibody raised against the alfalfa root nodule NADH-GOGAT (Anderson et al., 1989
To assess whether the changes observed in the levels of AS polypeptides in the GS-modulated transgenic plants were the result of changes in the steady-state levels of AS transcripts, RNA blots were performed (Fig. 5). A M. truncatula cDNA corresponding to the nodule-enhanced AS of alfalfa (Shi et al., 1997
To assess whether the increased levels of AS detected in the GS-antisense transgenic plants could also be observed by inhibiting total GS activity, we applied PPT to nodulated roots of wild-type plants and analyzed AS and GS content in nodules collected at different times after application of the herbicide. PPT is an analog of Glu and binds to the active site of GS irreversibly inactivating the enzyme (Manderscheid and Wild, 1986 The inhibition of GS activity in root nodules was very effective; GS activity was totally lost after 8 h application of the inhibitor (Fig. 6A), and a similar inhibition of GS activity was observed in roots and in leaves (data not shown). Western-blot analysis revealed that the amount of GS protein was maintained, even though its activity was almost zero, suggesting that GS was inactivated but not yet degraded (Fig. 6B). Interestingly, 48 h after PPT application, a lower Mr GS polypeptide was detected, which probably results from a selective proteolysis of the enzyme. To directly compare the amount of GS and AS, the same membrane was subsequently hybridized with anti-AS antibodies. We could detect an increase in AS polypeptide content in root nodules after 4 and 8 h of PPT treatment, which then decreased after 24 and 48 h.
This increase in AS content in the herbicide-treated nodules appears to occur at the transcript level, because it was also accompanied by an increase in the corresponding mRNA, which appears to be maximal after 4 h of incubation with the herbicide, then decreasing to normal levels (Fig. 6C). MtGS1a transcripts decreased in abundance in the course of the PPT treatment as well as the plastid GS mRNA (Fig. 6C). Interestingly, GDH transcripts showed a clear increase in abundance after 24 and 48 h. This effect was not only observed in nodules but also in roots and leaves (data not shown).
To evaluate whether GS in nodules could play a regulatory role in controlling the flux through the nitrogen assimilatory pathway, we have produced transgenic plants deregulated for this particular step in the metabolic pathway. Making use of the model legume M. truncatula in which we have previously characterized the GS genes (Carvalho et al., 1997 , 2000a, 2000b; Melo et al., 2003), we have succeeded in both increasing and decreasing GS activity specifically in nodules, by expressing GS in sense/antisense orientation under the direction of a strong and nodule-specific promoter. The choice of the Mtlb1 leghemoglobin promoter relied on its nodule-specific expression that follows the same time course of expression as MtGS1a, the nodule-enhanced M. truncatula GS gene (Gallusci et al., 1991; Stanford et al., 1993). By analyzing GUS fusions to this promoter, it is clear that it is also highly specific for the infected cells of the nodule (Fig. 1), thus altering GS levels only in the cells in which ammonium is being produced by rhizobial nitrogen fixation.
Most of the previous attempts to manipulate GS levels by either overexpression or down-regulation of GS genes have used constitutive promoters and heterologous transgenic systems, sometimes leading to alterations in whole-plant metabolism and sometimes leading to lethality (see Temple et al., 1994
Our manipulations resulted in up to 2-fold increases and 4-fold decreases in GS activity specifically in nodules (Fig. 2). Furthermore, the increased or decreased activity could be directly attributable to changes in a single GS holoenzyme, homo-octameric GS1a, which makes up more than 90% of the nodule GS activity in normal plants (Fig. 3C) and is expressed in the same cells as the Mtlb1 promoter (Carvalho et al., 2000a Analysis of these transgenic plants has revealed a remarkable negative regulation of AS by GS in root nodules. Thus when the level of GS is reduced, the expression of AS is increased and vice versa (Figs. 4 and 5). This regulation appears to be specific for AS because no changes were observed in the expression of five other key nitrogen and carbon assimilatory enzymes in nodules (NADH-GOGAT, AAT, CA, PEPC, and SUCS). The mechanism of AS regulation most likely occurs at the transcript level because the abundance of the AS mRNAs were affected, and in most cases, this was accompanied by associated changes in AS protein. However, when GS was increased by the sense strategy, little reduction was seen in AS protein suggesting that the observed lower level of AS mRNA could support the normal level of AS protein synthesis (Fig. 5). The inverse relationship between GS and AS was also seen, albeit transiently, when GS activity was inhibited by PPT (Fig. 6). In this experiment, the abundance of GS protein remained constant during the AS induction, suggesting that the regulation of AS is caused by changes in GS activity rather than in the levels of GS protein.
The finding that the levels of AS transcripts and polypeptides in the transgenic nodules were consistently increased when GS was reduced (Figs. 4 and 5) suggests that AS may compensate for the reduced GS ammonium assimilatory activity. It is known that AS can use ammonium instead of Gln as a nitrogen donor when the level of ammonium is high (Oaks and Ross, 1984
This potential energy savings, through partial assimilation of ammonium by plant AS, could explain the increase in plant productivity that has been observed in certain situations where GS levels were reduced either in genetic variants (Limami et al., 1999
Finally it is noteworthy that in a situation where GS is completely inhibited and the associated increase in AS is transient (addition of PPT; Fig. 6), a remarkable increase in GDH expression was observed. This increase could be viewed as a desperate attempt by the plant to assimilate ammonium using the aminating activity of GDH. However, most evidence suggests that this mitochondrial enzyme usually operates in a deaminating direction and is induced in situations such as carbon starvation where the enzyme plays a role in amino acid degradation and production of organic acids for carbon recycling (Miflin and Habash, 2002 In conclusion, we have obtained transgenic M. truncatula plants with reduced and increased levels of GS in root nodules and have shown using these plants that GS negatively regulates AS expression in nodules. This conclusion was reinforced by studies on plants in which GS was chemically inhibited with PPT. This latter experiment also revealed a role of GS in negatively regulating the expression of GDH and that the effect of GS on AS and GDH expression appears to be due to its enzymatic activity rather than the level of its protein. Many questions remain to be answered concerning the mechanism of this effect and its consequence on the nodule nitrogen and carbon metabolic pathways in the plant. In particular, it needs to be established whether the nitrogen assimilatory pathways in the GS-reduced nodules are more energy efficient and whether this has a positive effect on biomass production. Clearly, these transgenic plants, which are altered in a single GS holoenzyme in a single cell type, will serve as a valuable tool for such investigations.
Plant Material and Growth Conditions
Plants of Medicago truncatula Gaertn. cv Jemalong (genotype H39) used for plant transformation were maintained in in vitro culture in an environmental cabinet at a temperature of 23°C by day and 19°C by night, with 13-h daylength and light intensity of 150 to 200 µmol m-2 s-1. After regeneration, the transgenic plants were grown aeroponically on ammonium nitrate as described by Lullien et al. (1987
Full-length GS cDNAs were isolated from plasmids pTrcGSa and pTrcGSb containing the complete coding sequences of MtGS1a and MtGS1b (accession numbers Y10267 and Y10268, respectively). These plasmids contain an NcoI site at the start codon that has been introduced by PCR (Carvalho et al., 1997
For the construction of the chimeric antisense GS genes, the nos terminator was introduced at the 5' end of pBluescript KS- plasmids containing MtGS1a or MtGS1b full-length cDNAs. The previously described nopaline synthetase blunt fragment was introduced at the SmaI site of the pBluescript KS- GS cDNA clones. The Mtlb1 promoter was isolated from the previously described binary vector as an EcoRV/NcoI fragment, and the NcoI site was made blunt and inserted at the XhoI (Blunt) site of the constructs containing the GS-cDNAs fused to the nos terminator. A NotI (blunt)/SalI fragment containing the 2.1-kb Mtlb1 promoter, full GS1a or GS1b cDNAs in antisense orientation, and the nos terminator was subsequently cloned into SmaI/SalI sites of pBIN19 (Bevan, 1984
The binary constructs LbSA, LbSB, LbASA, and LbASB were introduced into the Agrobacterium tumefaciens strain AGL1 and were used to transform leaf segments of M. truncatula Gaertn. cv Jemalong (genotype H39). Kanamycin-resistant plants were regenerated by somatic embryogenesis as described by Chabaud et al. (1996
Plant material was homogenized at 0°C to 4°C in a mortal and pestle with 2 volumes of an extraction buffer containing 10 mM Tris-HCl, pH 7.5, 5 mM sodium Glu, 10 mM MgSO4, 1 mM dithiothreitol, 10% (v/v) glycerol, and 0.05% (v/v) Triton X-100. The homogenates were centrifuged at 13,000g for 20 min, at 4°C. The extracts were assayed for GS activity by the transferase assay (Cullimore and Sims, 1980
Soluble protein concentration was measured by the Coomassie Blue dye-binding assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as a standard. Proteins were separated by 12.5% (w/v) SDS-PAGE and electroblotted onto nitrocellulose membranes. Immunodetection was performed with the polyclonal antibody raised against GS (Cullimore and Miflin, 1984
Total RNA was isolated from nodules using TRIzol reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer. RNA samples (30 µg per lane) were separated by electrophoresis in 1.5% (v/v) agarose/15% (v/v) formaldehyde gels and were transferred to positively charged nylon (Amersham Biosciences, Uppsala) membranes by capillary blotting. The DNA probes were prepared from plasmid inserts isolated from agarose gels and labeled with 32P by random priming (Sambrook et al., 1989
Soluble protein plant extracts were filtered through a 0.2-µm filter and then applied to a DEAE 5PW column (75-x 7.5-mm diameter, Hewlett Packard, Palo Alto, CA) pre-equilibrated in running buffer. Proteins were eluted at 0.5 mL min-1 with running buffer containing no salt (2 min), 0 to 0.1 M KCl (4 min), 0.1 to 0.4 M KCl (44 min), and 0.6 M KCl (10 min). Fractions of 0.5 mL were collected and assayed for GS activity.
Histochemical staining for GUS activity was performed according to Jefferson et al. (1987
We gratefully acknowledge Dr. David Barker (Institut National de la Recherche Agronomique, Toulouse, France) for providing the Mtlb1 promoter-  -glucuronidase construct. We are also extremely grateful to the following persons for providing antibodies for this work: Dr. Carrol Vance (University of Minnesota, St. Paul) for antibodies against the alfalfa nodule-enhanced forms of NADH-GOGAT, AAT, and AS; Dr. Martin Crespi (Centre National de la Recherche Scientifique-Gif-sur Ivette, France) for antibodies against CA and PEPC; and Dr. Helge Kuster (University of Bielefeld, Germany) for antibodies against SUCS. Received November 15, 2002; returned for revision February 17, 2003; accepted May 7, 2003.
1 This work was part of the Biotechnology Research and Technological Development Shared Cost Project FIXNET supported by the European Union (grant no. CT 97-2319).  * Corresponding author; e-mail mhcarval{at}ibmc.up.pt; fax 351-226099157.
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ABSTRACT
RESULTS
GS gene and the nopaline synthetase terminator, was built in another plasmid, to avoid repeated restriction sites. The 3'-UTR of the gln-
