Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

RESEARCH PAPER

Post-translational regulation of cytosolic glutaminesynthetase of Medicago truncatula

Lıgia Lima1, Ana Seabra1, Paula Melo1, Julie Cullimore2 and Helena Carvalho1,*

1 Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal2 Laboratoire des Interactions Plantes-Microorganismes, INRA-CNRS, BP 27, F-31326 Castanet-Tolosan Cedex,France

Received 30 January 2006; Accepted 26 April 2006

Abstract

It was reported recently that the plastid-located gluta-

mine 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 phosphoryla-

tion 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 phosphory-

lation 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 phos-

phorylation 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 influ-

enced 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.

Introduction

Glutamine synthetase (EC 6.3.1.2) plays a central rolein nitrogen metabolism of higher plants. GS catalysesthe ATP-dependent assimilation of ammonium into glu-tamate to yield glutamine, which is then used for the bio-synthesis of essentially all nitrogenous compounds (Miflinand Lea, 1980). GS in plants occurs as a number of iso-enzymes and, based on its subcellular location, it can bebroadly classified as GS2 (plastid located) and GS1 (cyto-solic located). The isoenzymes are encoded by a smallmultigene 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 celltypes, and assimilate the ammonium produced by differentphysiological processes (Lea et al., 1990).

Due to its key importance for plant growth and de-velopment, the regulatory mechanisms that control plantGS have been the subject of several studies, but the com-plete understanding of the mechanisms controlling GSactivity in plants is complicated by the fact that GS exists asa number of isoenzymes encoded by multiple genes. InMedicago truncatula, the GS gene family consists of onlythree expressed genes: MtGS1a and MtGS1b encoding

* To whom correspondence should be addressed. E-mail: [email protected]: DTT, dithiothreitol; EST, expressed sequence tag; GS, glutamine synthetase; GS1, cytosolic GS; GS2, plastid GS; Ni-NTA, nickel-nitriloaceticacid.

Journal of Experimental Botany, Vol. 57, No. 11, pp. 2751–2761, 2006

doi:10.1093/jxb/erl036 Advance Access publication 9 July, 2006This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

ª 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/) whichpermits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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cytosolic isoenzymes (GS1a and GS1b) and MtGS2 en-coding the precursor to a plastid-located isoenzyme (GS2)(Stanford et al., 1993). In root nodules, MtGS1a showsa nodule-enhanced expression and its encoded isoenzymeis responsible for the rapid assimilation of the ammoniumexcreted by the nitrogen-fixing bacteroids (Stanford et al.,1993; Carvalho et al., 2000a). Primary assimilation ofammonium taken up directly by roots seems to be per-formed by cytosolic GS1b, the predominant GS isoen-zyme in the root cortex (Carvalho et al., 2000b). In matureleaves, the plastid-located GS2 is the major GS isoformresponsible for the reassimilation of photorespiratory am-monia (Wallsgrove et al., 1987; Migge and Becker, 2000;Orea et al., 2002).

Much of the information available suggests that GSactivity in plants is primarily regulated at the transcriptionallevel, and little is known about the regulatory mechanismscontrolling plant GS at the post-translational level. In bac-teria, the regulation of GS by cumulative feedback inhibi-tion, adenylation/deadenylation, and repression/derepressionis well documented (Stadtman, 1990, 2001). In plants,several reports have indicated the involvement of post-translational mechanisms in regulating GS activity (Hoelzleet al., 1992; Temple et al., 1996, 1998; Ortega et al., 1999,2001). Recently, phosphorylation and 14-3-3 interactionhave been implied in the modulation of GS activity inplants (Finnemann and Schjoerring, 2000; Man and Kaiser,2001; Riedel et al., 2001) and in the green alga Chlamy-domonas reihardtii (Pozuelo et al., 2001). The regulationof GS by phosphorylation/14-3-3 interaction in plants hasonly recently been shown, but the involvement of thesemechanisms for the regulation of the activity of otherkey enzymes of carbon and nitrogen metabolism is nowevident (Toroser et al., 1998; Kaiser et al., 2002).

A mechanism for the regulation of M. truncatula GS2by phosphorylation and subsequent interaction with 14-3-3proteins, leading to a selective proteolysis of the enzymeresulting in total inactivation was reported previously(Lima et al., 2006). A major calcium-dependent proteinkinase phosphorylation site at Ser97 in the GS2 proteinwas established. Phosphorylation of this residue createsa 14-3-3-binding motif allowing the formation of the GS2–14-3-3 complex which is recognized by an unknown plantprotease that cleaves the enzyme, resulting in an inactiveGS2 proteolytic fragment of ;40 kDa. These studies onthe regulation of GS2 in M. truncatula by phosphoryla-tion and 14-3-3 interaction have now been extended toinclude the regulation of cytosolic GS. Special attentionwas devoted to root nodules and to the major GS isoformin these organs, cytosolic GS1a. GS phosphorylation hasnever been assessed in root nodules where the enzymeis especially abundant and plays a crucial role in theassimilation of ammonia that is produced in large amountsby nitrogen fixation. It is relevant to know whether GSis phosphorylated in root nodules and what the implications

are of this post-translational modification for the assimila-tion of ammonia resulting from nitrogen fixation by thesymbiotic bacteria.

Materials and methods

Plant material

Plants ofM. truncatula Gaertn. (cv. Jemalong J5) provided by one ofthe authors (JC) were grown in aeroponic conditions at 22 8C, witha relative humidity of 75% and a 14 h light period at 200 lmol m�2

s�1 in the growth medium described by Lullien et al. (1987). Fornodule induction, the growth medium was replaced with freshmedium lacking a nitrogen source 3 d before inoculation with eitherthe wild-type Sinorhizobium meliloti strain RCR2011 or the fixJ S.melilotimutant (GMI347). Nodules, leaves, and roots were harvested14 d after inoculation. All plant material was immediately frozen inliquid nitrogen and stored at –80 8C.

Production of plant GS and 14-3-3 isoenzymes in E. coli

The M. truncatula isoenzymes GS1a and GS2 (without the plastidtargeting signal) were expressed in Escherichia coli using theexpression vector pTrc99A (Amersham Biosciences) as previouslydescribed (Carvalho et al., 1997; Melo et al., 2003). The cloneMtBB06G11 containing a full-length cDNA for a 14-3-3 isoform wasselected from theM. truncatula expressed sequence tag (EST) librarydatabase (Journet et al., 2002; http//medicago.toulouse.inra.fr) andcloned 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 correspondingcDNAs into the expression vector pET 28a (Novagen, Inc.). ThecDNA inserts of pTrc-GS1a, pTrc-GS2, and pTrc-14-3-3 wereremoved from the above-described constructs as NcoI–PstI fragmentsand introduced as blunt fragments into the NheI (blunt) site ofpET28a. The plasmids pET-GS1a, pET-GS2, and pET-14-3-3 wereindependently 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 grownat 37 8C in LB medium supplemented with 100 lg ml�1 ampicillin(for pTrc99A constructs) or kanamycin (for pET28a constructs) untilan OD of 0.5 at 600 nm was reached. The incubation was pro-longed for 3–5 h in the same medium supplemented with 1 mMisopropyl-b-D-galactopyranoside (IPTG) to induce the expressionof the recombinant proteins.

Preparation of soluble protein extracts from E. coli and

plant tissues

Plant material was homogenized at 4 8C using a mortar and pestlewith 2 vols of an extraction buffer containing 10 mM TRIS–HCl pH7.5, 5 mM sodium glutamate, 10 mM MgSO4, 1 mM dithiothreitol(DTT), 10% (v/v) glycerol, 0.05% (v/v) Triton X-100, and a proteaseinhibitor cocktail specific for plant extracts (Sigma Aldrich).Escherichia coli cells were collected by centrifugation (13 000 gfor 15 min) after protein induction. The pellets were frozen in liquidnitrogen and ground with alumina type V (Sigma Aldrich) usinga mortar and pestle with 2 vols of the extraction buffer describedabove. The homogenates were centrifuged at 13 000 g for 20 min at4 8C and the supernatants desalted on P10 Sephadex columns(Amersham Biosciences). Soluble protein concentration was meas-ured with the Bio-Rad dye (Bio-Rad) reagent using bovine serumalbumin (BSA) as a standard. Plant extracts used as the kinase sourcewere obtained by homogenization of the plant tissue in the sameextraction buffer, followed by centrifugation for 20 min at 13 000 g

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and further clarification by ultracentrifugation at 100 000 g for 1 hat 4 8C. Desalted samples containing 20% glycerol were stored at–80 8C until use.

Determination of GS activity and kinetic properties

GS activity was determined using the transferase (Cullimoreand 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 variationsin concentration of the substrates. The Km values were calculatedfrom Lineweaver–Burk plots and the Hill number from Hill plots.

Gel electrophoresis and immunoblotting

Proteins were analysed by SDS–PAGE on 12% gels accordingto Laemmli (1970). For two-dimensional gel electrophoresis, pro-tein samples (200 lg) were applied overnight to 13 cm IPG stripspH 4–7 (Amersham Biosciences) by in-gel rehydration. The rehy-drated gels were subjected to isoelectric focusing in a Multiphor IIunit (Amersham Biosciences) according to the manufacturer’sinstructions. After the first dimension, the strips were incubatedfor 15 min in an equilibration buffer consisting of 50 mM TRIS–HClpH 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 iodo-acetamide (45 mg ml�1) instead of DTT, supplemented with bromo-phenol blue. Second dimensional SDS–PAGE was performed in12% acrylamide gels. Proteins were electroblotted onto a nitrocellulosemembrane (Schleicher & Schuell) using a semi-dry transfer system(Bio-Rad). The membranes were incubated with primary antibodies:polyclonal anti-GS antibody (Cullimore andMiflin, 1984) or anti-14-3-3antibody (Moorhead et al., 1999). The polypeptides were detectedwith secondary peroxidase-conjugated IgGs (Vector Laboratories).

GS immunoprecipitation

GS was immunoprecipitated using a polyclonal anti-GS antibodyraised against the gln-c isoenzyme of Phaseolus GS (Cullimore andMiflin, 1984) in immunoprecipitation (IP) buffer containing 50 mMTRIS–HCl pH 7.5, 150 mMNaCl, 2 mM EDTA, 0.1% IGEPAL (v/v)(Sigma Aldrich), 0.5% (w/v) sodium deoxycholate, 0.5 lM micro-cystin-LR, and protease inhibitor cocktail. Anti-GS antibody wasincubated with protein A–Sepharose, previously washed with 30 mMTRIS–HCl pH 7.5, for 2 h at 4 8C with gentle agitation. Clarifiedextracts containing 2–5 U of GS activity were added to the proteinA–antibody complexes and the immunoprecipitation was allowedto proceed for 2 h. Following several washes with IP buffer, theimmune 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 phosphorusdepleted for 3 d before the radiolabelling experiments were initiated.Nodules (0.5 g) were detached and immediately incubated with1 ml of labelling solution (0.5 mCi of 32Pi, 0.25 mM KCl, 0.25 mMMgSO4, 0.2 mM CaCl2 and 10 mg l�1 monosodium-iron EDTA) for2 h at room temperature with gentle shaking. After washing withdistilled water, proteins were extracted as described except that0.5 lM microcystin-LR and 5 mM NaF were included in the extrac-tion buffer. GS was immunoprecipitated and the polypeptides se-parated by SDS–PAGE. Radiolabelled proteins were analysed usinga Typhoon 8600 phosphor imager (Amersham Biosciences).

In vitro phosphorylation

In vitro phosphorylation assays were performed by incorporation of[c-32P]ATP (11.131013 Bq mmol�1) (Amersham Pharmacia) in aphosphorylation reaction mixture containing 10 mM TRIS–HCl

pH 7.5, 5 mM MgCl2, 0.1 mM CaCl2, 0.5 lM microcystin, 18.5 Bqof [c-32P]ATP, 20–100 lM ATP, and 40 lg of total soluble proteinof extracts of leaves, nodules, or roots used as the kinase source. TheGS proteins produced in E. coli were immunoprecipitated and phos-phorylated for 30 min at 30 8C with gentle shaking. After centrifu-gation, the immune complexes were thoroughly washed with IP buffer,dissociated with SDS loading buffer, and boiled for 10 min. Alter-natively, His-tagged proteins were used for in vitro phosphorylationanalysis. His-tagged proteins were bound to Ni-NTA resin columnsaccording to the manufacturer’s instructions and incubated with thephosphorylation reaction mixture for 30 min at 30 8C. After severalwashing steps to remove non-bound proteins, the bound proteinswere sequentially washed and eluted, as previously described (Limaet 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 witha Typhoon 8600 phosphor imager (Amersham Pharmacia).For stoichiometry determination, purified recombinant His-tagged

GS1a was bound to Ni-NTA resin columns and in vitro phosphory-lated by leaf kinases in the presence of [c-32P]ATP. After intensivewashing to remove plant proteins and non-bound labelled ATP, theproteins were eluted, placed in a scintillation vial containing ;5 mlof scintillation liquid, and the radioactivity (cpm) determined byCerenkov counting.

Results

In vitro phosphorylation of individual GS isoenzymes

It was shown previously that the M. truncatula GS2is regulated by phosphorylation and 14-3-3 interactions(Lima et al., 2006). In order to evaluate whether thecytosolic counterparts are regulated by similar mechanisms,the two M. truncatula cytosolic GS cDNAs, MtGS1a andMtGS1b, were independently expressed in E. coli to pro-duce non-phosphorylated homoctameric isoenzymes, andin vitro phosphorylated by plant kinases in the presence of[c-32P]ATP. The plant GS enzymes expressed in E. coliwere previously shown to be catalytically and physiolog-ically active (Carvalho et al., 1997). In vitro phosphory-lation assays were performed under predetermined optimalphosphorylation conditions (data not shown) in the pre-sence of 0.1 mM CaCl2, 5 mM MgCl2, 100 lM ATP, 0.5lM microcystin, and 40 lg of total protein extracts fromdifferent plant organs (leaves, nodules, and roots) used asa source of kinases (Fig. 1).

To evaluate whether GS phosphorylation was relatedto light/dark transitions and to active nitrogen fixation,extracts from leaves collected during the light and darkperiods, and from effective (Fix+) and ineffective fixJ(Fix�) root nodules were used to phosphorylate in vitrothe GS isoenzymes produced in E. coli. Recombinant GS1aand GS1b isoenzymes were immunoprecipitated and,because plastid-located GS2 is poorly immunoprecipitatedby 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 thepresence of [c-32P]ATP and the absence of plant extracts(–Kin), to ensure that the enzymes produced in E. coli

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become phosphorylated only in the presence of plantkinases (Fig. 1).

A clearly 32P-labelled GS band was detected in all theassays performed with the different plant extracts, beingundetectable in the control samples, demonstrating that thethree M. truncatula GS proteins are susceptible to phos-phorylation by plant kinases present in leaves, nodules, androots (Figs 1, 2). However, the GS isoenzymes appear to bephosphorylated to different extents by kinases present indifferent plant organs and under the different physiologicalconditions tested. Phosphorylation of plastid-located GS2appears to be unaffected by the nitrogen-fixing status ofthe nodule, but interestingly both cytosolic GS isoen-zymes were more strongly phosphorylated by kinasespresent in ineffective nodules relative to wild-type nodules,suggesting that GS1 phosphorylation is related to nitrogen

fixation (Fig. 1). Light also appears to affect cytosolicGS phosphorylation, as an increased phospho-labellingof both GS1a and GS1b isoenzymes was detected inleaves sampled during the light relative to the dark period(Fig. 1). Phosphorylation of the plastid-located GS iso-enzyme does not seem to be highly affected by light(Fig. 1). As equal amounts of GS proteins were loadedon each gel, it can be concluded that cytosolic GS is dif-ferentially phosphorylated by the kinases present in thedifferent plant extracts.

Interestingly, some additional phospho-polypeptideswere co-precipitated with GS1a and GS1b, and co-purifiedwith plastid GS2. These phosphorylated polypeptidesare not detected if GS-immunodepleted plant extractsare used for the in vitro phosphorylation assays (Fig. 2),most probably because in these assays they are co-immunoprecipitated with the endogenous GS and removedfrom solution. These polypeptides are likely to correspondto plant GS-binding proteins, and some of them showedboth isoenzyme and organ specificity. A polypeptide of;52 kDa was detected in almost all situations, but the

Fig. 1. In vitro phosphorylation of GS isoenzymes expressed in E. coli.GS1a (A, B), GS1b (C, D), and His-tagged GS2 (E, F) isoenzymesproduced in E. coli were phosphorylated in vitro by incubation with[c-32P]ATP in the absence (–Kin) or presence of total protein extracts(40 lg) from plants grown under different physiological conditions:leaves collected during the light (L) and dark (D) periods; effective(Fix+) and ineffective (Fix�) nodules; and roots (R). The polypeptideswere separated by SDS–PAGE, visualized by Coomassie staining (A,C, E), and autoradiographed (B, D, F). Molecular weight markers areindicated on the left (kDa).

Fig. 2. In vitro phosphorylation of GS with immunodepleted plantextracts. Plant extracts from roots (R), wild-type nodules (N), and light-grown leaves (L) were GS immunodepleted and used to phosphorylatethe GS1a and GS1b isoenzymes expressed in E. coli. The polypeptideswere resolved by SDS–PAGE and visualized by Coomassie staining(A, C). (B, D) Autoradiography of the corresponding gels.

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polypeptides of ;70 and 72 kDa appear to be specific forthe cytosolic enzymes and were only detected when usingleaf or nodule extracts. When root extracts were used, adifferent pattern was obtained. These putative GS-bindingproteins were found to be differentially labelled under thedifferent physiological conditions, being strongly labelledin ineffective nodules. Note that as these proteins are presentin very low amounts, being hardly visible on Coomassie-stained gels, they must be highly phosphorylated.

In vivo phosphorylation of GS polypeptides in rootnodules of M. truncatula

An unequivocal indication that GS is post-translationallymodified in root nodules was obtained by GS western blotanalysis of two-dimensional gels (Fig. 3). Medicagotruncatula contains only three expressed GS genes,MtGS2 encoding a plastid-located GS polypeptide of42 kDa (GS2), and MtGS1a and MtGS1b encodingtwo cytosolic peptides (GS1a and GS1b) with the samemolecular 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 antibodyrecognized five isoelectric variants of 39 kDa and four42 kDa polypeptides. This result clearly indicates that bothtypes of GS isoenzymes are subjected to post-translationalmodifications, most probably involving regulatory phos-phorylation, in root nodules of M. truncatula.

To examine GS phosphorylation directly in vivo, in situradiolabelling of detached intact M. truncatula noduleswith [32P]orthophosphate was performed. After 2 h of[32P]phosphate incorporation, GS from clarified extractswas immunoprecipitated and analysed by SDS–PAGE and

autoradiography (Fig. 4). In a Coomassie-stained gel ofa GS-immunoprecipitated nodule extract (Fig. 4A) a single39 kDa polypeptide corresponding to cytosolic GS isdetected, reflecting the fact that the antibody has a higheraffinity for cytosolic GS and that it is the predominantisoform in this organ. However, the antibody could recog-nize both isoforms in western analysis (Fig. 4C). Bothcytosolic and plastid GS appear to be phosphorylatedin vivo in root nodules of M. truncatula as two radiolab-elled polypeptides with electrophoretic mobility andimmunoreactivity identical to those of the GS polypep-tides were detected by phosphor imaging analysis of theimmunoprecipitated GS polypeptides (Fig. 4B).

Cytosolic GS1a is phosphorylated at multiple sites

It had been indicated previously that the plastid-locatedGS2 isoenzyme in M. truncatula is phosphorylated atmultiple sites, with Ser97 being identified as a majorregulatory site (Lima et al., 2006). In order to investigatewhether cytosolic GS1a, the most abundant GS isoform inroot nodules, is also phosphorylated at multiple sites,two-dimensional gel electrophoresis of in vitro phosphory-lated GS1a was performed (Fig. 5). Recombinant GS1aproduced with an N-terminal histidine tag was purifiedby Ni-NTA affinity chromatography and was in vitrophosphorylated by leaf kinases under previously deter-mined optimal phosphorylation conditions. After intensivewashing and subsequent purification on Ni-NTA affinitycolumns, the bound proteins were eluted, resolved by two-dimensional gel electrophoresis, and analysed by phosphorimaging (Fig. 5). Two labelled protein spots could beclearly identified, indicating that cytosolic GS1a is phos-phorylated in at least two residues. The higher molecularweight phosphorylated protein spots detected are likely tocorrespond to the 52 kDa GS phospho-binding proteinspreviously described (Fig. 1).

The occurrence of two phosphorylated residues in GS1apolypeptides was confirmed further by stoichiometry de-termination. The Ni-NTA-purified His6-GS1a was in vitrophosphorylated and, after purification by Ni-NTA affinitychromatography, the eluted labelled proteins were analysedby Cerenkov counting. The stoichiometry value obtained

Fig. 3. Two-dimensional SDS–PAGE profile of GS polypeptides in rootnodules. Soluble root nodule proteins (200 lg) were separated by two-dimensional gel electrophoresis in a pH gradient of 4–7. The GSpolypeptides were detected with specific anti-GS antibody by westernanalysis. The positions of the molecular weight markers (kDa) areindicated on the left.

Fig. 4. In vivo phosphorylation of GS in M. truncatula root nodules.Phosphorylation of GS was performed by incubating intact nodules with[32P]orthophosphate for 2 h. Soluble protein extracts were prepared andthe GS polypeptides were immunoprecipitated with anti-GS antibodies,which preferentially precipitate cytosolic forms. The immunoprecipitatedpolypeptides were resolved by SDS–PAGE, and visualized by Coomassiestaining (A) and phosphor imaging (B). (C) Corresponding GS westernblot of crude nodule extract to indicate the positions of GS isoenzymes.

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was 1.6, suggesting that similarly to GS2, the major cyto-solic GS isoform in root nodules is regulated by phos-phorylation at more than one site.

Evaluation of the calcium dependence ofGS1 kinase(s)

To evaluate whether the kinase(s) responsible for cytosolicGS phosphorylation is dependent on calcium, recombinantGS1a and GS1b isoenzymes were independently phos-phorylated in vitro by incubation with desalted plant ex-tracts in the presence of [c-32P]ATP and either 0.2 mMCaCl2 or 0.5 mM EGTA (Fig. 6). In contrast to plastidGS2, which was previously shown to be phosphorylatedby calcium-dependent protein kinase(s) (CDPK) (Limaet al., 2006), phosphorylation of both cytosolic GSisoenzymes was unaffected by the absence of calcium

and thus the two types of GS isoenzymes in M. truncatulaare likely to be phosphorylated by different plant kinases.

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 andSchojoerring, 2000; Man and Kaiser, 2001; Riedel et al.,2001). InM. truncatula, interaction of 14-3-3 proteins withphosphorylated GS2 was shown to result in inactivationof the isoenzyme by selective proteolysis (Lima et al.,2006). This mechanism of GS2 regulation was demon-strated by the occurrence of a specific GS degradationproduct of;40 kDa after purification of leaf GS proteins bya His6-14-3-3 affinity binding strategy.

To evaluate if the M. truncatula cytosolic GS isoen-zymes interact with 14-3-3 proteins, GS extracts fromnodules, the richest source of cytosolic GS, were loaded onaffinity columns containing an M. truncatula His6-14-3-3protein (Fig. 7). This M. truncatula 14-3-3 isoform wasselected from the M. truncatula EST database on the basisof its high levels of expression in mature root nodules. Afterincubation with the plant extracts, non-bound proteins wereremoved, 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, lane2), and the eluted fractions (Fig. 7, lane 3) were analysedby western blot using anti-14-3-3 (Fig. 7A) and anti-GS(Fig. 7B) antibodies. The cytosolic GS was unable tobind this 14-3-3 protein as only the 40 kDa GS2 degrada-tion product could be detected by the GS antibody, indica-ting that the previously reported selective degradation ofGS2 induced by phosphorylation and 14-3-3 interactionis not restricted to leaves but also occurs in root nodules.

To confirm further that GS1a is not a 14-3-3 targetprotein, His6-GS1a recombinant proteins were loaded onNi-NTA columns, incubated with a leaf extract and with14-3-3 (expressed without His6) (Fig. 8). His6-GS2 wasanalysed in parallel as a positive control for 14-3-3 binding.The initial 14-3-3 isoform (Fig. 8, lane 1) and the eluted

Fig. 5. Two-dimensional gel electrophoresis of in vitro phosphorylatedHis-tagged GS1a. His-tagged GS1a was phosphorylated in vitro byincubation with a plant extract used as the kinase source, CaCl2, MgCl2,microcystin, and ATP. After purification by Ni-NTA chromatography,the eluted proteins were resolved by two-dimensional electrophoresisand the labelled proteins detected by phosphor image.

Fig. 6. Evaluation of the calcium dependence of cytosolic GS kinase(s). Recombinant GS1a and GS1b were phosphorylated in vitro by incubation with[c-32P]ATP and 40 lg of desalted extracts from leaves, nodule, or roots in the presence of 0.1 mM CaCl2 or 0.5 mM EGTA. The polypeptides wereseparated by SDS–PAGE and visualized by Coomassie staining and phosphor image.

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fractions from His6-GS2 (Fig. 8, lanes 2 and 4) and His6-GS1a (Fig. 8, lanes 3 and 5) were analysed by western blotwith anti-14-3-3 (Fig. 8A) and anti-GS antibodies (Fig.8B). GS1a failed to interact with theM. truncatula 14-3-3s.The anti-14-3-3 antibody could detect 14-3-3 proteinseluting from the His6-GS2 column (Fig. 8A, lane 2), butnot from the His6-GS1a column (Fig. 8A, lane 3). The GSpolypeptides eluted from the columns correspond toHis6-GS1a with 41.5 kDa (Fig. 8B, lane 5), to non-cleavedHis6-GS2 with 44 kDa (Fig. 8B, lane 4), and to theGS2 degradation product induced by 14-3-3 interactionwith 40 kDa (Fig. 8B, lane 4).

Effect of phosphorylation on GS1a kinetic properties

To identify possible changes in cytosolic GS1a activityresulting from phosphorylation, some kinetic properties ofGS1a in a non-phosphorylated and phosphorylated formwere compared (Table 1). GS1a produced in E. coli andtagged with His6 was phosphorylated by incubation witha desalted plant extract in the presence of 4 mM ATP and0.5 lM microcystin, and purified by Ni-NTA affinitychromatography. Non-phosphorylated GS1a was treatedin the same way except that the plant extract was absent(source of kinases). The two purified GS preparations wereused in parallel for biosynthetic activity determinations in

a series of reactions with varying substrate concentrations.Phosphorylated GS1a was found to have a slightly reducedKm and Vmax for glutamate (Table 1), but a transferase/synthetase ratio slightly higher compared with the non-phosphorylated form. None of the other kinetic propertiesdetermined was found to be significantly altered byphosphorylation.

Discussion

It was shown previously that the plastid-locatedM. truncatula GS2 is regulated by phosphorylation and

Fig. 7. Analysis of GS polypeptides purified by 14-3-3 protein affinitychromatography. The M. truncatula 14-3-3 isoform produced in E. colias a His-tagged protein was bound to an Ni-NTA column and incubatedwith a nodule extract. The initial plant extract (1), the non-bound plantproteins (2), and the eluted fractions (3) were analysed by western blotusing anti-14-3-3 (A) and anti-GS (B) antibodies.

Fig. 8. Evaluation of GS2–14-3-3 and GS1a–14-3-3 interaction by GSaffinity chromatography. GS2 and GS1a produced in E. coli and taggedwith His6 were loaded on Ni-NTA columns and incubated with a leafextract. The bound proteins were subsequently incubated with the M.truncatula 14-3-3 isoform and, after intensive washing, elution wasperformed. The initial 14-3-3 extract (1) and the eluted fractions fromHis-tagged GS2 (2 and 4) and His-tagged GS1a (3 and 5) were analysedby western blot using anti-14-3-3 (A) and anti-GS (B) antibodies.

Table 1. Comparison of some kinetic properties of His-taggedGS1a produced in E. coli in a non-phosphorylated (GS1a) andphosphorylated form (GS1a-P)

Kinetic property GS1a GS1a-P

Km glutamate (mM) 2.8 1.8Km ATP (mM) 3 2.9S0.5 hydroxylamine (mM) 1.2 1.2Hill coefficient for hydroxylamine 1.9 1.9Vmax (glutamate) (lmol min�1) 0.52 0.33Transferase/synthetase ratio 92 120

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interaction with 14-3-3 (Lima et al., 2006). These studieswere extended here to include the regulation of cytosolicGS1. With this study, the entire GS protein family ofM. truncatula has been analysed regarding its susceptibilityto regulatory phosphorylation. All three M. truncatulaGS isoenzymes (GS1a, GS1b, and GS2) are subjected tophosphorylation in vitro by plant kinases, present in roots,leaves, and nodules, but only the plastid-located GS2appears to be able to bind 14-3-3 proteins. GS phosphory-lation and interaction with 14-3-3 were previously shownto occur in leaves of some plant species (Finnemann andSchjoerring, 2000; Man and Kaiser, 2001; Riedel et al.,2001) and in curd extracts from cauliflower (Moorheadet al., 1999). This study established that the regulatoryphosphorylation of this important enzyme also occurs inlegume root nodules. The detection of multiple isoelectricvariants of both cytosolic and plastid GS polypeptideson two-dimensional gels of nodule extracts demonstratesthat GS is post-translationally modified in planta. Insitu [32P]orthophosphate labelling confirmed directly thatboth the cytosolic and plastid GS in detached intactM. truncatula nodules are phosphorylated in vivo.

The three GS isoenzymes were found to be phosphory-lated in vitro by soluble protein kinase(s) present innodules, leaves, and roots, but the kinases present in thedifferent organs showed differential activities against thedifferent GS isoenzymes. Nodule kinases phosphorylatecytosolic GS more strongly than the plastid isoenzyme,whereas root kinases appear to have higher activity againstGS2. As GS1a and GS1b protein sequences, includingpotential phosphorylation sites, are highly conserved (89%amino acid homology), the two isoenzymes are likely tobe phosphorylated by similar kinases. The GS2 polypeptidesequence is more divergent, with ;78% homology at theamino acid level to the cytosolic isoenzymes and, asthe protein is located in a separate organelle, it is likely tobe phosphorylated by different protein kinase(s), locatedinside the plastid. The activity of both GS1a and GS1bphosphorylating kinase(s) was found to be unaffected bythe absence of calcium, whereas phosphorylation of GS2was previously shown to be dependent on calcium (Limaet al., 2006), supporting the notion that the two classes ofenzymes are phosphorylated by different plant kinase(s).

The data presented here indicate that, in contrast toplastidial GS2, the cytosolic GSs of M. truncatula areunable to bind 14-3-3 proteins. The M. truncatula 14-3-3isoform used in this study shows a broad expression inalmost all organs of the plant, but it was selected by its highlevels of expression in root nodules, even though it failed tointeract with the cytosolic GS isoenzymes, highly abundantin this organ. It could be argued that this 14-3-3 isoform isspecific for the plastid enzyme. 14-3-3 isoform specificityis a controversial issue, and the subcellular localizationof these proteins must be a contributing factor (Comparotet al., 2003). It seems unlikely that a differentM. truncatula

14-3-3 isoform interacts with cytosolic GS, as endogenous14-3-3 proteins interacting with cytosolic GS could not bedetected using different approaches which included co-immunoprecipitation, column affinity, and far-westernoverlaid with digoxygenin-labelled 14-3-3s (data notshown). Furthermore, GS2 is the only M. truncatula GSprotein containing a sequence RTIS*KP very similar to thedescribed optimal 14-3-3-binding motif (Yaffe, 2002).Cytosolic GS seems, however, to associate with differentplant proteins. Some phospho-polypeptides of ;70 and72 kDa specifically co-precipitating with cytosolic GScould be detected. Interestingly, these putative GS-bindingproteins were found to be differentially phosphorylatedunder different physiological conditions, being stronglylabelled in ineffective nodules, indicating that their phos-phorylation and binding to GS may be related to thenitrogen-fixing status of the nodule. It is possible that theprotein kinase(s) responsible for GS phosphorylation isamong these proteins since it is known that many proteinkinases are themselves regulated by phosphorylation andsome tend to remain associated with their target enzymes(Ranjeva and Boudet, 1987). The kinases responsible forphosphorylation of the key carbon metabolic enzymessucrose phosphate synthase (Huber and Huber, 1991) andphosphoenolpyruvate carboxylase (Baur et al., 1992)are examples of kinases reported to co-purify with theirtarget enzymes.

As reported for other plant phosphorylated enzymes, itis probable that GS phosphorylation is highly controlledby the respective kinase activity which, in turn, might beregulated by physiological conditions. Light and nitrogennutrition are important factors affecting GS activity inplants; therefore, the GS kinase activities under differentlight and nitrogen regimes were compared. Light/darktransitions were found to affect the phosphorylation statusof cytosolic GS in senescing leaves of Brassica napus,leading the authors to propose a tentative model for GSregulation (Finnemann and Schjoerring, 2000). Accord-ing to this model, in the dark, GS1 would be protectedfrom degradation by phosphorylation and subsequentbinding to 14-3-3 proteins, and in the light GS1 would beunphosphorylated and more susceptible to degradation(Finnemann and Schjoerring, 2000). The phosphorylationcapacity of kinases present in M. truncatula leaf extractssampled during the dark and light periods was thereforecompared. The results contrast with those obtained forBrassica; both GS1a and GS1b appear to be more highlyphosphorylated by kinases present in leaves sampledduring the light period and the two isoenzymes failedto interact with 14-3-3 proteins. It is intriguing thatthe present results are different from those reportedby Finnemann and Schjoerring (2000), suggesting thateither there are species-specific differences in the wayGS is modulated or that senescence influences GS1phosphorylation, since the studies performed in Brassica

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used senescing leaves whereas the present work with M.truncatula used mature leaves.

Due to the interest in the physiological significance ofGS phosphorylation for controlling nitrogen fixation andassimilation, the phosphorylation capacity of the kinasespresent in active, nitrogen-fixing nodules versus ineffective(Fix�) nodules was compared. Interestingly, cytosolic GSphosphorylation appears to be linked to the nitrogen-fixingstatus of the nodule; both GS1a and GS1b appeared morehighly labelled in ineffective nodules whereas the plastid-located GS2 was poorly phosphorylated by nodule kinasesand appears to be unaffected by the nitrogen-fixing statusof the nodules.

Although phosphorylation of GS in root nodules hasnot been assessed before, there is evidence suggestingthat nodule GS is likely to be regulated by phosphorylationand protein–protein interaction. Temple et al. (1996)detected a slow migrating GS complex in native gels ofsoybean nodule extracts that was not present in roots,strongly suggesting that the nodule isoenzymes are associ-ated with other proteins. Furthermore, the authors haveshown that wild-type nodules contained higher levels ofGS activity when compared with ineffective (Nif�) nod-ules, but the level of GS subunits was equivalent inboth types of nodules, indicating a connection betweennitrogen fixation and GS activity. The GS isoenzymesin the ineffective nodules were found to be more unstablein vitro when compared with other GS isoenzymes, andthe authors suggested that nitrogen-fixed ammonium ora product of ammonium assimilation is responsible forthe stabilization of the GS holoprotein in nodules(Temple et al., 1996). In the present study, the degree ofcytosolic GS phosphorylation appears to be greater inineffective nodules than in nitrogen-fixing nodules, andthree phosphorylated polypeptides were co-purified withphosphorylated GS1 isoenzymes. Interestingly, these puta-tive GS-binding proteins were found to be more highlylabelled in ineffective nodules, meaning that in theseconditions either more protein molecules bind to GS orthat the binding proteins are more highly phosphorylated.It is tempting to speculate that these proteins couldbe involved in the regulation of GS activity in root nodulesof M. truncatula as an adaptation to changes in thenitrogen-fixing status of the nodule. Whether these proteinscan only bind phospho-GS and whether they need to be ina phosphorylated state to be able to bind GS are importantquestions that will be the subject of future studies.

Phosphorylation of the nodule-enhanced M. truncatulaGS1 isoenzyme (GS1a) resulted in an increase in theaffinity for the substrate glutamate which may confer anadaptability of the isoenzyme to certain physiologicalconditions where the levels of this substrate could belimiting. However the Vmax of the enzyme was decreased.Interestingly, in ineffective alfalfa nodules, the levels ofNADH-GOGAT transcripts, protein, and activity were

found to be several-fold less than those for GS (Vanceet al., 1995). As a result, glutamate, the product of GOGATactivity, is presumably present in very low amounts in thesenodules. It is therefore conceivable that under non-fixingconditions, phosphorylation functions to reduce the overallGS activity but still allows assimilation of the ammoniumproduced by other metabolic pathways, by increasing theaffinity of the enzyme for the limiting substrate glutamate.Although at the present stage any consideration regardingthe physiological implication of GS phosphorylation fornodule metabolism can only remain highly speculative, thefinding that the M. truncatula GS is phosphorylated innodules and that its phosphorylation status is related tonitrogen fixation opens up new possibilities to explore theinvolvement of this post-translational mechanism for thefunctioning of the nodule.

To our knowledge, this is the first evidence that GS isphosphorylated in root nodules, where its activity isessential for the assimilation of the high amounts ofammonium that are released by nitrogen fixation. How-ever, it is known that the key carbon metabolic enzymesphosphoenolpyruvate carboxylase and sucrose synthaseare regulated by phosphorylation in root nodules (Zhanget al., 1995, 1999). With this finding, GS represents onlythe third metabolic enzyme known to undergo phosphory-lation in legume root nodules. This finding is significantbecause GS is the first enzyme in the nitrogen assimilatorypathway, and regulatory phosphorylation may contributeto the co-ordination of the carbon and nitrogen assimilationpathways and have important implications for the controlof the nitrogen and carbon fluxes in root nodules.

Acknowledgements

We gratefully acknowledge Michel Rossignol and Giselle Borderie(IFR40, Toulouse, France) for expert assistance with two-dimensionalelectrophoresis, and Dr Carol Mackintosh (MRC Unit, Universityof Dundee, UK) for providing the anti-14-3-3 antibody. We arealso grateful to Jorge Azevedo and Pedro Pereira (IBMC, Porto,Portugal) for helpful discussions. This work was supported bythe Fundacxao para a Ciencia e Tecnologia (Projects no. POC/PI/41433/2001 and POCTI/AGG/39079/2001).

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