JXB Advance Access originally published online on July 10, 2006
Journal of Experimental Botany 2006 57(11):2751-2761; doi:10.1093/jxb/erl036
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula
1Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
2Laboratoire des Interactions Plantes-Microorganismes, INRA-CNRS, BP 27, F-31326 Castanet-Tolosan Cedex, France
*To whom correspondence should be addressed. E-mail: mhcarval{at}ibmc.up.pt
Received 30 January 2006; Accepted 26 April 2006
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Abstract |
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It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.
Key words: Glutamine synthetase, Medicago, phosphorylation, 14-3-3 proteins
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Introduction |
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Glutamine synthetase (EC 6.3.1.2 [EC] ) plays a central role in nitrogen metabolism of higher plants. GS catalyses the ATP-dependent assimilation of ammonium into glutamate to yield glutamine, which is then used for the biosynthesis of essentially all nitrogenous compounds (Miflin and Lea, 1980). GS in plants occurs as a number of isoenzymes and, based on its subcellular location, it can be broadly classified as GS2 (plastid located) and GS1 (cytosolic located). The isoenzymes are encoded by a small multigene family showing distinct patterns of expression (Forde et al., 1989; Sakakibara et al., 1992; Li et al., 1993; Dubois et al., 1996) in different organs and cell types, and assimilate the ammonium produced by different physiological processes (Lea et al., 1990).
Due to its key importance for plant growth and development, the regulatory mechanisms that control plant GS have been the subject of several studies, but the complete understanding of the mechanisms controlling GS activity in plants is complicated by the fact that GS exists as a number of isoenzymes encoded by multiple genes. In Medicago truncatula, the GS gene family consists of only three expressed genes: MtGS1a and MtGS1b encoding cytosolic isoenzymes (GS1a and GS1b) and MtGS2 encoding the precursor to a plastid-located isoenzyme (GS2) (Stanford et al., 1993). In root nodules, MtGS1a shows a nodule-enhanced expression and its encoded isoenzyme is responsible for the rapid assimilation of the ammonium excreted by the nitrogen-fixing bacteroids (Stanford et al., 1993; Carvalho et al., 2000a). Primary assimilation of ammonium taken up directly by roots seems to be performed by cytosolic GS1b, the predominant GS isoenzyme in the root cortex (Carvalho et al., 2000b). In mature leaves, the plastid-located GS2 is the major GS isoform responsible for the reassimilation of photorespiratory ammonia (Wallsgrove et al., 1987; Migge and Becker, 2000; Orea et al., 2002).
Much of the information available suggests that GS activity in plants is primarily regulated at the transcriptional level, and little is known about the regulatory mechanisms controlling plant GS at the post-translational level. In bacteria, the regulation of GS by cumulative feedback inhibition, adenylation/deadenylation, and repression/derepression is well documented (Stadtman, 1990, 2001). In plants, several reports have indicated the involvement of post-translational mechanisms in regulating GS activity (Hoelzle et al., 1992; Temple et al., 1996, 1998; Ortega et al., 1999, 2001). Recently, phosphorylation and 14-3-3 interaction have been implied in the modulation of GS activity in plants (Finnemann and Schjoerring, 2000; Man and Kaiser, 2001; Riedel et al., 2001) and in the green alga Chlamydomonas reihardtii (Pozuelo et al., 2001). The regulation of GS by phosphorylation/14-3-3 interaction in plants has only recently been shown, but the involvement of these mechanisms for the regulation of the activity of other key enzymes of carbon and nitrogen metabolism is now evident (Toroser et al., 1998; Kaiser et al., 2002).
A mechanism for the regulation of M. truncatula GS2 by phosphorylation and subsequent interaction with 14-3-3 proteins, leading to a selective proteolysis of the enzyme resulting in total inactivation was reported previously (Lima et al., 2006). A major calcium-dependent protein kinase phosphorylation site at Ser97 in the GS2 protein was established. Phosphorylation of this residue creates a 14-3-3-binding motif allowing the formation of the GS2–14-3-3 complex which is recognized by an unknown plant protease that cleaves the enzyme, resulting in an inactive GS2 proteolytic fragment of 
40 kDa. These studies on the regulation of GS2 in M. truncatula by phosphorylation and 14-3-3 interaction have now been extended to include the regulation of cytosolic GS. Special attention was devoted to root nodules and to the major GS isoform in these organs, cytosolic GS1a. GS phosphorylation has never been assessed in root nodules where the enzyme is especially abundant and plays a crucial role in the assimilation of ammonia that is produced in large amounts by nitrogen fixation. It is relevant to know whether GS is phosphorylated in root nodules and what the implications are of this post-translational modification for the assimilation of ammonia resulting from nitrogen fixation by the symbiotic bacteria.
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Materials and methods |
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Plant material
Plants of M. truncatula Gaertn. (cv. Jemalong J5) provided by one of the authors (JC) were grown in aeroponic conditions at 22 °C, with a relative humidity of 75% and a 14 h light period at 200 µmol m–2 s–1 in the growth medium described by Lullien et al. (1987). For nodule induction, the growth medium was replaced with fresh medium lacking a nitrogen source 3 d before inoculation with either the wild-type Sinorhizobium meliloti strain RCR2011 or the fixJ S. meliloti mutant (GMI347). Nodules, leaves, and roots were harvested 14 d after inoculation. All plant material was immediately frozen in liquid nitrogen and stored at –80 °C.
Production of plant GS and 14-3-3 isoenzymes in E. coli
The M. truncatula isoenzymes GS1a and GS2 (without the plastid targeting signal) were expressed in Escherichia coli using the expression vector pTrc99A (Amersham Biosciences) as previously described (Carvalho et al., 1997; Melo et al., 2003). The clone MtBB06G11 containing a full-length cDNA for a 14-3-3 isoform was selected from the M. truncatula expressed sequence tag (EST) library database (Journet et al., 2002; http//medicago.toulouse.inra.fr) and cloned as described in Lima et al. (2006).
GS and 14-3-3 proteins containing an N-terminal extension of six histidines (His6) were produced by subcloning the corresponding cDNAs into the expression vector pET 28a (Novagen, Inc.). The cDNA inserts of pTrc-GS1a, pTrc-GS2, and pTrc-14-3-3 were removed from the above-described constructs as NcoI–PstI fragments and introduced as blunt fragments into the NheI (blunt) site of pET28a. The plasmids pET-GS1a, pET-GS2, and pET-14-3-3 were independently transformed into the bacterial strain BL21 (DE3) (Novagen, Inc.).
XL1-Blue and BL21 (DE3) competent cells transformed with pTr99A- and pET28a-derived constructs, respectively, were grown at 37 °C in LB medium supplemented with 100 µg ml–1 ampicillin (for pTrc99A constructs) or kanamycin (for pET28a constructs) until an OD of 0.5 at 600 nm was reached. The incubation was prolonged for 3–5 h in the same medium supplemented with 1 mM isopropyl-ß-D-galactopyranoside (IPTG) to induce the expression of the recombinant proteins.
Preparation of soluble protein extracts from E. coli and plant tissues
Plant material was homogenized at 4 °C using a mortar and pestle with 2 vols of an extraction buffer containing 10 mM TRIS–HCl pH 7.5, 5 mM sodium glutamate, 10 mM MgSO4, 1 mM dithiothreitol (DTT), 10% (v/v) glycerol, 0.05% (v/v) Triton X-100, and a protease inhibitor cocktail specific for plant extracts (Sigma Aldrich). Escherichia coli cells were collected by centrifugation (13 000 g for 15 min) after protein induction. The pellets were frozen in liquid nitrogen and ground with alumina type V (Sigma Aldrich) using a mortar and pestle with 2 vols of the extraction buffer described above. The homogenates were centrifuged at 13 000 g for 20 min at 4 °C and the supernatants desalted on P10 Sephadex columns (Amersham Biosciences). Soluble protein concentration was measured with the Bio-Rad dye (Bio-Rad) reagent using bovine serum albumin (BSA) as a standard. Plant extracts used as the kinase source were obtained by homogenization of the plant tissue in the same extraction buffer, followed by centrifugation for 20 min at 13 000 g and further clarification by ultracentrifugation at 100 000 g for 1 h at 4 °C. Desalted samples containing 20% glycerol were stored at –80 °C until use.
Determination of GS activity and kinetic properties
GS activity was determined using the transferase (Cullimore and Sims, 1980) and the semi-biosynthetic assay (Cullimore et al., 1982). The kinetic determinations were all made using the semi-biosynthetic assay; a series of reactions were made with variations in concentration of the substrates. The Km values were calculated from Lineweaver–Burk plots and the Hill number from Hill plots.
Gel electrophoresis and immunoblotting
Proteins were analysed by SDS–PAGE on 12% gels according to Laemmli (1970). For two-dimensional gel electrophoresis, protein samples (200 µg) were applied overnight to 13 cm IPG strips pH 4–7 (Amersham Biosciences) by in-gel rehydration. The rehydrated gels were subjected to isoelectric focusing in a Multiphor II unit (Amersham Biosciences) according to the manufacturer's instructions. After the first dimension, the strips were incubated for 15 min in an equilibration buffer consisting of 50 mM TRIS–HCl pH 7.5, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and DTT (3.5 mg ml–1) and for 15 min in the same buffer containing iodoacetamide (45 mg ml–1) instead of DTT, supplemented with bromophenol blue. Second dimensional SDS–PAGE was performed in 12% acrylamide gels. Proteins were electroblotted onto a nitrocellulose membrane (Schleicher & Schuell) using a semi-dry transfer system (Bio-Rad). The membranes were incubated with primary antibodies: polyclonal anti-GS antibody (Cullimore and Miflin, 1984) or anti-14-3-3 antibody (Moorhead et al., 1999). The polypeptides were detected with secondary peroxidase-conjugated IgGs (Vector Laboratories).
GS immunoprecipitation
GS was immunoprecipitated using a polyclonal anti-GS antibody raised against the gln-
isoenzyme of Phaseolus GS (Cullimore and Miflin, 1984) in immunoprecipitation (IP) buffer containing 50 mM TRIS–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% IGEPAL (v/v) (Sigma Aldrich), 0.5% (w/v) sodium deoxycholate, 0.5 µM microcystin-LR, and protease inhibitor cocktail. Anti-GS antibody was incubated with protein A–Sepharose, previously washed with 30 mM TRIS–HCl pH 7.5, for 2 h at 4 °C with gentle agitation. Clarified extracts containing 2–5 U of GS activity were added to the protein A–antibody complexes and the immunoprecipitation was allowed to proceed for 2 h. Following several washes with IP buffer, the immune complexes were dissociated in SDS sample buffer, boiled, and separated by SDS–PAGE.
In vivo 32P labelling
The plants used for in vivo phosphorylation assays were phosphorus depleted for 3 d before the radiolabelling experiments were initiated. Nodules (0.5 g) were detached and immediately incubated with 1 ml of labelling solution (0.5 mCi of 32Pi, 0.25 mM KCl, 0.25 mM MgSO4, 0.2 mM CaCl2 and 10 mg l–1 monosodium-iron EDTA) for 2 h at room temperature with gentle shaking. After washing with distilled water, proteins were extracted as described except that 0.5 µM microcystin-LR and 5 mM NaF were included in the extraction buffer. GS was immunoprecipitated and the polypeptides separated by SDS–PAGE. Radiolabelled proteins were analysed using a Typhoon 8600 phosphor imager (Amersham Biosciences).
In vitro phosphorylation
In vitro phosphorylation assays were performed by incorporation of [-32P]ATP (11.1x1013 Bq mmol–1) (Amersham Pharmacia) in a phosphorylation reaction mixture containing 10 mM TRIS–HCl pH 7.5, 5 mM MgCl2, 0.1 mM CaCl2, 0.5 µM microcystin, 18.5 Bq of [-32P]ATP, 20–100 µM ATP, and 40 µg of total soluble protein of extracts of leaves, nodules, or roots used as the kinase source. The GS proteins produced in E. coli were immunoprecipitated and phosphorylated for 30 min at 30 °C with gentle shaking. After centrifugation, the immune complexes were thoroughly washed with IP buffer, dissociated with SDS loading buffer, and boiled for 10 min. Alternatively, His-tagged proteins were used for in vitro phosphorylation analysis. His-tagged proteins were bound to Ni-NTA resin columns according to the manufacturer's instructions and incubated with the phosphorylation reaction mixture for 30 min at 30 °C. After several washing steps to remove non-bound proteins, the bound proteins were sequentially washed and eluted, as previously described (Lima et al., 2006). The phosphorylation products were resolved by SDS–PAGE and the gels stained with Coomassie brilliant blue R-250, thoroughly destained, and dried. The dried gels were analysed with a Typhoon 8600 phosphor imager (Amersham Pharmacia).
For stoichiometry determination, purified recombinant His-tagged GS1a was bound to Ni-NTA resin columns and in vitro phosphorylated by leaf kinases in the presence of [-32P]ATP. After intensive washing to remove plant proteins and non-bound labelled ATP, the proteins were eluted, placed in a scintillation vial containing 5 ml of scintillation liquid, and the radioactivity (cpm) determined by Cerenkov counting.
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In vitro phosphorylation of individual GS isoenzymes
It was shown previously that the M. truncatula GS2 is regulated by phosphorylation and 14-3-3 interactions (Lima et al., 2006). In order to evaluate whether the cytosolic counterparts are regulated by similar mechanisms, the two M. truncatula cytosolic GS cDNAs, MtGS1a and MtGS1b, were independently expressed in E. coli to produce non-phosphorylated homoctameric isoenzymes, and in vitro phosphorylated by plant kinases in the presence of [
-32P]ATP. The plant GS enzymes expressed in E. coli were previously shown to be catalytically and physiologically active (Carvalho et al., 1997). In vitro phosphorylation assays were performed under predetermined optimal phosphorylation conditions (data not shown) in the presence of 0.1 mM CaCl2, 5 mM MgCl2, 100 µM ATP, 0.5 µM microcystin, and 40 µg of total protein extracts from different plant organs (leaves, nodules, and roots) used as a source of kinases (Fig. 1).
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To evaluate whether GS phosphorylation was related to light/dark transitions and to active nitrogen fixation, extracts from leaves collected during the light and dark periods, and from effective (Fix+) and ineffective fixJ (Fix–) root nodules were used to phosphorylate in vitro the GS isoenzymes produced in E. coli. Recombinant GS1a and GS1b isoenzymes were immunoprecipitated and, because plastid-located GS2 is poorly immunoprecipitated by anti-GS antibody, it was expressed containing a His6 tag, allowing purification by Ni-NTA affinity chromatography (Lima et al., 2006). Control assays were performed in the presence of [
-32P]ATP and the absence of plant extracts (–Kin), to ensure that the enzymes produced in E. coli become phosphorylated only in the presence of plant kinases (Fig. 1). A clearly 32P-labelled GS band was detected in all the assays performed with the different plant extracts, being undetectable in the control samples, demonstrating that the three M. truncatula GS proteins are susceptible to phosphorylation by plant kinases present in leaves, nodules, and roots (Figs 1, 2). However, the GS isoenzymes appear to be phosphorylated to different extents by kinases present in different plant organs and under the different physiological conditions tested. Phosphorylation of plastid-located GS2 appears to be unaffected by the nitrogen-fixing status of the nodule, but interestingly both cytosolic GS isoenzymes were more strongly phosphorylated by kinases present in ineffective nodules relative to wild-type nodules, suggesting that GS1 phosphorylation is related to nitrogen fixation (Fig. 1). Light also appears to affect cytosolic GS phosphorylation, as an increased phospho-labelling of both GS1a and GS1b isoenzymes was detected in leaves sampled during the light relative to the dark period (Fig. 1). Phosphorylation of the plastid-located GS isoenzyme does not seem to be highly affected by light (Fig. 1). As equal amounts of GS proteins were loaded on each gel, it can be concluded that cytosolic GS is differentially phosphorylated by the kinases present in the different plant extracts.
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Interestingly, some additional phospho-polypeptides were co-precipitated with GS1a and GS1b, and co-purified with plastid GS2. These phosphorylated polypeptides are not detected if GS-immunodepleted plant extracts are used for the in vitro phosphorylation assays (Fig. 2), most probably because in these assays they are co-immunoprecipitated with the endogenous GS and removed from solution. These polypeptides are likely to correspond to plant GS-binding proteins, and some of them showed both isoenzyme and organ specificity. A polypeptide of
52 kDa was detected in almost all situations, but the polypeptides of 70 and 72 kDa appear to be specific for the cytosolic enzymes and were only detected when using leaf or nodule extracts. When root extracts were used, a different pattern was obtained. These putative GS-binding proteins were found to be differentially labelled under the different physiological conditions, being strongly labelled in ineffective nodules. Note that as these proteins are present in very low amounts, being hardly visible on Coomassie-stained gels, they must be highly phosphorylated.
In vivo phosphorylation of GS polypeptides in root nodules of M. truncatula
An unequivocal indication that GS is post-translationally modified in root nodules was obtained by GS western blot analysis of two-dimensional gels (Fig. 3). Medicago truncatula contains only three expressed GS genes, MtGS2 encoding a plastid-located GS polypeptide of 42 kDa (GS2), and MtGS1a and MtGS1b encoding two cytosolic peptides (GS1a and GS1b) with the same molecular mass of 39 kDa but different pIs: 5.54 and 5.36, respectively (Carvalho and Cullimore, 2003). However, after separation of root nodule GS polypeptides by two-dimensional electrophoresis, the GS-specific antibody recognized five isoelectric variants of 39 kDa and four 42 kDa polypeptides. This result clearly indicates that both types of GS isoenzymes are subjected to post-translational modifications, most probably involving regulatory phosphorylation, in root nodules of M. truncatula.
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To examine GS phosphorylation directly in vivo, in situ radiolabelling of detached intact M. truncatula nodules with [32P]orthophosphate was performed. After 2 h of [32P]phosphate incorporation, GS from clarified extracts was immunoprecipitated and analysed by SDS–PAGE and autoradiography (Fig. 4). In a Coomassie-stained gel of a GS-immunoprecipitated nodule extract (Fig. 4A) a single 39 kDa polypeptide corresponding to cytosolic GS is detected, reflecting the fact that the antibody has a higher affinity for cytosolic GS and that it is the predominant isoform in this organ. However, the antibody could recognize both isoforms in western analysis (Fig. 4C). Both cytosolic and plastid GS appear to be phosphorylated in vivo in root nodules of M. truncatula as two radiolabelled polypeptides with electrophoretic mobility and immunoreactivity identical to those of the GS polypeptides were detected by phosphor imaging analysis of the immunoprecipitated GS polypeptides (Fig. 4B).
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Cytosolic GS1a is phosphorylated at multiple sites
It had been indicated previously that the plastid-located GS2 isoenzyme in M. truncatula is phosphorylated at multiple sites, with Ser97 being identified as a major regulatory site (Lima et al., 2006). In order to investigate whether cytosolic GS1a, the most abundant GS isoform in root nodules, is also phosphorylated at multiple sites, two-dimensional gel electrophoresis of in vitro phosphorylated GS1a was performed (Fig. 5). Recombinant GS1a produced with an N-terminal histidine tag was purified by Ni-NTA affinity chromatography and was in vitro phosphorylated by leaf kinases under previously determined optimal phosphorylation conditions. After intensive washing and subsequent purification on Ni-NTA affinity columns, the bound proteins were eluted, resolved by two-dimensional gel electrophoresis, and analysed by phosphor imaging (Fig. 5). Two labelled protein spots could be clearly identified, indicating that cytosolic GS1a is phosphorylated in at least two residues. The higher molecular weight phosphorylated protein spots detected are likely to correspond to the 52 kDa GS phospho-binding proteins previously described (Fig. 1).
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The occurrence of two phosphorylated residues in GS1a polypeptides was confirmed further by stoichiometry determination. The Ni-NTA-purified His6-GS1a was in vitro phosphorylated and, after purification by Ni-NTA affinity chromatography, the eluted labelled proteins were analysed by Cerenkov counting. The stoichiometry value obtained was 1.6, suggesting that similarly to GS2, the major cytosolic GS isoform in root nodules is regulated by phosphorylation at more than one site.
Evaluation of the calcium dependence of GS1 kinase(s)
To evaluate whether the kinase(s) responsible for cytosolic GS phosphorylation is dependent on calcium, recombinant GS1a and GS1b isoenzymes were independently phosphorylated in vitro by incubation with desalted plant extracts in the presence of [-32P]ATP and either 0.2 mM CaCl2 or 0.5 mM EGTA (Fig. 6). In contrast to plastid GS2, which was previously shown to be phosphorylated by calcium-dependent protein kinase(s) (CDPK) (Lima et al., 2006), phosphorylation of both cytosolic GS isoenzymes was unaffected by the absence of calcium and thus the two types of GS isoenzymes in M. truncatula are likely to be phosphorylated by different plant kinases.
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Evaluation of GS1a interaction with 14-3-3 proteins
Cytosolic and plastid GS were found to be 14-3-3-interacting proteins in some plant species (Finneman and Schojoerring, 2000; Man and Kaiser, 2001; Riedel et al., 2001). In M. truncatula, interaction of 14-3-3 proteins with phosphorylated GS2 was shown to result in inactivation of the isoenzyme by selective proteolysis (Lima et al., 2006). This mechanism of GS2 regulation was demonstrated by the occurrence of a specific GS degradation product of
40 kDa after purification of leaf GS proteins by a His6-14-3-3 affinity binding strategy. To evaluate if the M. truncatula cytosolic GS isoenzymes interact with 14-3-3 proteins, GS extracts from nodules, the richest source of cytosolic GS, were loaded on affinity columns containing an M. truncatula His6-14-3-3 protein (Fig. 7). This M. truncatula 14-3-3 isoform was selected from the M. truncatula EST database on the basis of its high levels of expression in mature root nodules. After incubation with the plant extracts, non-bound proteins were removed, the columns were washed, and His6-14-3-3-interacting proteins were eluted. The initial plant extracts (Fig. 7, lane 1), the non-bound plant proteins (Fig. 7, lane 2), and the eluted fractions (Fig. 7, lane 3) were analysed by western blot using anti-14-3-3 (Fig. 7A) and anti-GS (Fig. 7B) antibodies. The cytosolic GS was unable to bind this 14-3-3 protein as only the 40 kDa GS2 degradation product could be detected by the GS antibody, indicating that the previously reported selective degradation of GS2 induced by phosphorylation and 14-3-3 interaction is not restricted to leaves but also occurs in root nodules.
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To confirm further that GS1a is not a 14-3-3 target protein, His6-GS1a recombinant proteins were loaded on Ni-NTA columns, incubated with a leaf extract and with 14-3-3 (expressed without His6) (Fig. 8). His6-GS2 was analysed in parallel as a positive control for 14-3-3 binding. The initial 14-3-3 isoform (Fig. 8, lane 1) and the eluted fractions from His6-GS2 (Fig. 8, lanes 2 and 4) and His6-GS1a (Fig. 8, lanes 3 and 5) were analysed by western blot with anti-14-3-3 (Fig. 8A) and anti-GS antibodies (Fig. 8B). GS1a failed to interact with the M. truncatula 14-3-3s. The anti-14-3-3 antibody could detect 14-3-3 proteins eluting from the His6-GS2 column (Fig. 8A, lane 2), but not from the His6-GS1a column (Fig. 8A, lane 3). The GS polypeptides eluted from the columns correspond to His6-GS1a with 41.5 kDa (Fig. 8B, lane 5), to non-cleaved His6-GS2 with 44 kDa (Fig. 8B, lane 4), and to the GS2 degradation product induced by 14-3-3 interaction with 40 kDa (Fig. 8B, lane 4).
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Effect of phosphorylation on GS1a kinetic properties
To identify possible changes in cytosolic GS1a activity resulting from phosphorylation, some kinetic properties of GS1a in a non-phosphorylated and phosphorylated form were compared (Table 1). GS1a produced in E. coli and tagged with His6 was phosphorylated by incubation with a desalted plant extract in the presence of 4 mM ATP and 0.5 µM microcystin, and purified by Ni-NTA affinity chromatography. Non-phosphorylated GS1a was treated in the same way except that the plant extract was absent (source of kinases). The two purified GS preparations were used in parallel for biosynthetic activity determinations in a series of reactions with varying substrate concentrations. Phosphorylated GS1a was found to have a slightly reduced Km and Vmax for glutamate (Table 1), but a transferase/synthetase ratio slightly higher compared with the non-phosphorylated form. None of the other kinetic properties determined was found to be significantly altered by phosphorylation.
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Discussion |
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It was shown previously that the plastid-located M. truncatula GS2 is regulated by phosphorylation and interaction with 14-3-3 (Lima et al., 2006). These studies were extended here to include the regulation of cytosolic GS1. With this study, the entire GS protein family of M. truncatula has been analysed regarding its susceptibility to regulatory phosphorylation. All three M. truncatula GS isoenzymes (GS1a, GS1b, and GS2) are subjected to phosphorylation in vitro by plant kinases, present in roots, leaves, and nodules, but only the plastid-located GS2 appears to be able to bind 14-3-3 proteins. GS phosphorylation and interaction with 14-3-3 were previously shown to occur in leaves of some plant species (Finnemann and Schjoerring, 2000; Man and Kaiser, 2001; Riedel et al., 2001) and in curd extracts from cauliflower (Moorhead et al., 1999). This study established that the regulatory phosphorylation of this important enzyme also occurs in legume root nodules. The detection of multiple isoelectric variants of both cytosolic and plastid GS polypeptides on two-dimensional gels of nodule extracts demonstrates that GS is post-translationally modified in planta. In situ [32P]orthophosphate labelling confirmed directly that both the cytosolic and plastid GS in detached intact M. truncatula nodules are phosphorylated in vivo.
The three GS isoenzymes were found to be phosphorylated in vitro by soluble protein kinase(s) present in nodules, leaves, and roots, but the kinases present in the different organs showed differential activities against the different GS isoenzymes. Nodule kinases phosphorylate cytosolic GS more strongly than the plastid isoenzyme, whereas root kinases appear to have higher activity against GS2. As GS1a and GS1b protein sequences, including potential phosphorylation sites, are highly conserved (89% amino acid homology), the two isoenzymes are likely to be phosphorylated by similar kinases. The GS2 polypeptide sequence is more divergent, with 78% homology at the amino acid level to the cytosolic isoenzymes and, as the protein is located in a separate organelle, it is likely to be phosphorylated by different protein kinase(s), located inside the plastid. The activity of both GS1a and GS1b phosphorylating kinase(s) was found to be unaffected by the absence of calcium, whereas phosphorylation of GS2 was previously shown to be dependent on calcium (Lima et al., 2006), supporting the notion that the two classes of enzymes are phosphorylated by different plant kinase(s).
The data presented here indicate that, in contrast to plastidial GS2, the cytosolic GSs of M. truncatula are unable to bind 14-3-3 proteins. The M. truncatula 14-3-3 isoform used in this study shows a broad expression in almost all organs of the plant, but it was selected by its high levels of expression in root nodules, even though it failed to interact with the cytosolic GS isoenzymes, highly abundant in this organ. It could be argued that this 14-3-3 isoform is specific for the plastid enzyme. 14-3-3 isoform specificity is a controversial issue, and the subcellular localization of these proteins must be a contributing factor (Comparot et al., 2003). It seems unlikely that a different M. truncatula 14-3-3 isoform interacts with cytosolic GS, as endogenous 14-3-3 proteins interacting with cytosolic GS could not be detected using different approaches which included co-immunoprecipitation, column affinity, and far-western overlaid with digoxygenin-labelled 14-3-3s (data not shown). Furthermore, GS2 is the only M. truncatula GS protein containing a sequence RTIS*KP very similar to the described optimal 14-3-3-binding motif (Yaffe, 2002). Cytosolic GS seems, however, to associate with different plant proteins. Some phospho-polypeptides of 70 and 72 kDa specifically co-precipitating with cytosolic GS could be detected. Interestingly, these putative GS-binding proteins were found to be differentially phosphorylated under different physiological conditions, being strongly labelled in ineffective nodules, indicating that their phosphorylation and binding to GS may be related to the nitrogen-fixing status of the nodule. It is possible that the protein kinase(s) responsible for GS phosphorylation is among these proteins since it is known that many protein kinases are themselves regulated by phosphorylation and some tend to remain associated with their target enzymes (Ranjeva and Boudet, 1987). The kinases responsible for phosphorylation of the key carbon metabolic enzymes sucrose phosphate synthase (Huber and Huber, 1991) and phosphoenolpyruvate carboxylase (Baur et al., 1992) are examples of kinases reported to co-purify with their target enzymes.
As reported for other plant phosphorylated enzymes, it is probable that GS phosphorylation is highly controlled by the respective kinase activity which, in turn, might be regulated by physiological conditions. Light and nitrogen nutrition are important factors affecting GS activity in plants; therefore, the GS kinase activities under different light and nitrogen regimes were compared. Light/dark transitions were found to affect the phosphorylation status of cytosolic GS in senescing leaves of Brassica napus, leading the authors to propose a tentative model for GS regulation (Finnemann and Schjoerring, 2000). According to this model, in the dark, GS1 would be protected from degradation by phosphorylation and subsequent binding to 14-3-3 proteins, and in the light GS1 would be unphosphorylated and more susceptible to degradation (Finnemann and Schjoerring, 2000). The phosphorylation capacity of kinases present in M. truncatula leaf extracts sampled during the dark and light periods was therefore compared. The results contrast with those obtained for Brassica; both GS1a and GS1b appear to be more highly phosphorylated by kinases present in leaves sampled during the light period and the two isoenzymes failed to interact with 14-3-3 proteins. It is intriguing that the present results are different from those reported by Finnemann and Schjoerring (2000), suggesting that either there are species-specific differences in the way GS is modulated or that senescence influences GS1 phosphorylation, since the studies performed in Brassica used senescing leaves whereas the present work with M. truncatula used mature leaves.
Due to the interest in the physiological significance of GS phosphorylation for controlling nitrogen fixation and assimilation, the phosphorylation capacity of the kinases present in active, nitrogen-fixing nodules versus ineffective (Fix–) nodules was compared. Interestingly, cytosolic GS phosphorylation appears to be linked to the nitrogen-fixing status of the nodule; both GS1a and GS1b appeared more highly labelled in ineffective nodules whereas the plastid-located GS2 was poorly phosphorylated by nodule kinases and appears to be unaffected by the nitrogen-fixing status of the nodules.
Although phosphorylation of GS in root nodules has not been assessed before, there is evidence suggesting that nodule GS is likely to be regulated by phosphorylation and protein–protein interaction. Temple et al. (1996) detected a slow migrating GS complex in native gels of soybean nodule extracts that was not present in roots, strongly suggesting that the nodule isoenzymes are associated with other proteins. Furthermore, the authors have shown that wild-type nodules contained higher levels of GS activity when compared with ineffective (Nif–) nodules, but the level of GS subunits was equivalent in both types of nodules, indicating a connection between nitrogen fixation and GS activity. The GS isoenzymes in the ineffective nodules were found to be more unstable in vitro when compared with other GS isoenzymes, and the authors suggested that nitrogen-fixed ammonium or a product of ammonium assimilation is responsible for the stabilization of the GS holoprotein in nodules (Temple et al., 1996). In the present study, the degree of cytosolic GS phosphorylation appears to be greater in ineffective nodules than in nitrogen-fixing nodules, and three phosphorylated polypeptides were co-purified with phosphorylated GS1 isoenzymes. Interestingly, these putative GS-binding proteins were found to be more highly labelled in ineffective nodules, meaning that in these conditions either more protein molecules bind to GS or that the binding proteins are more highly phosphorylated. It is tempting to speculate that these proteins could be involved in the regulation of GS activity in root nodules of M. truncatula as an adaptation to changes in the nitrogen-fixing status of the nodule. Whether these proteins can only bind phospho-GS and whether they need to be in a phosphorylated state to be able to bind GS are important questions that will be the subject of future studies.
Phosphorylation of the nodule-enhanced M. truncatula GS1 isoenzyme (GS1a) resulted in an increase in the affinity for the substrate glutamate which may confer an adaptability of the isoenzyme to certain physiological conditions where the levels of this substrate could be limiting. However the Vmax of the enzyme was decreased. Interestingly, in ineffective alfalfa nodules, the levels of NADH-GOGAT transcripts, protein, and activity were found to be several-fold less than those for GS (Vance et al., 1995). As a result, glutamate, the product of GOGAT activity, is presumably present in very low amounts in these nodules. It is therefore conceivable that under non-fixing conditions, phosphorylation functions to reduce the overall GS activity but still allows assimilation of the ammonium produced by other metabolic pathways, by increasing the affinity of the enzyme for the limiting substrate glutamate. Although at the present stage any consideration regarding the physiological implication of GS phosphorylation for nodule metabolism can only remain highly speculative, the finding that the M. truncatula GS is phosphorylated in nodules and that its phosphorylation status is related to nitrogen fixation opens up new possibilities to explore the involvement of this post-translational mechanism for the functioning of the nodule.
To our knowledge, this is the first evidence that GS is phosphorylated in root nodules, where its activity is essential for the assimilation of the high amounts of ammonium that are released by nitrogen fixation. However, it is known that the key carbon metabolic enzymes phosphoenolpyruvate carboxylase and sucrose synthase are regulated by phosphorylation in root nodules (Zhang et al., 1995, 1999). With this finding, GS represents only the third metabolic enzyme known to undergo phosphorylation in legume root nodules. This finding is significant because GS is the first enzyme in the nitrogen assimilatory pathway, and regulatory phosphorylation may contribute to the co-ordination of the carbon and nitrogen assimilation pathways and have important implications for the control of the nitrogen and carbon fluxes in root nodules.
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Acknowledgements |
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We gratefully acknowledge Michel Rossignol and Giselle Borderie (IFR40, Toulouse, France) for expert assistance with two-dimensional electrophoresis, and Dr Carol Mackintosh (MRC Unit, University of Dundee, UK) for providing the anti-14-3-3 antibody. We are also grateful to Jorge Azevedo and Pedro Pereira (IBMC, Porto, Portugal) for helpful discussions. This work was supported by the Fundação para a Ciência e Tecnologia (Projects no. POC/PI/41433/2001 and POCTI/AGG/39079/2001).
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Abbreviations |
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DTT, dithiothreitol; EST, expressed sequence tag; GS, glutamine synthetase; GS1, cytosolic GS; GS2, plastid GS; Ni-NTA, nickel-nitriloacetic acid.
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