Plant Molecular Biology42: 741–756, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Cellular expression and regulation of theMedicago truncatulacytosolicglutamine synthetase genes in root nodules

Helena Carvalho1,∗, Nicole Lescure2, Françoise de Billy2, Mireille Chabaud2, Ligia Lima1,Roberto Salema1 and Julie Cullimore21Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-Porto, Portugal (∗author for corre-spondence);2Laboratoire de Biologie Mol´eculaire des Relations Plantes-Microorganismes INRA-CNRS, BP 27,31326 Castanet-Tolosan Cedex, France

Received 2 September 1999; accepted in revised form 3 January 2000

Key words:gene expression, glutamine synthetase, legume-Rhizobiumsymbiosis, nitrogen assimilation, rootnodules

Abstract

In this paper we have studied the localisation of expression of the two functional cytosolic glutamine synthetase(GS) genes,MtGSaandMtGSb, in root nodules of the model legumeMedicago truncatula. We have used a combi-nation of different techniques, including immunocytochemistry,in situhybridisation and promoterβ-glucuronidase(GUS) fusions in transgenic plants, to provide the means of correlating gene expression with protein localisation.These studies revealed that transcriptional regulation (mRNA synthesis) plays an important part in controlling GSprotein levels in nodules ofM. truncatula. The major locations of cytosolic GS mRNA and protein are the centraltissue, the parenchyma and the pericycle of the vascular bundles. These findings indicate that in nodules, GSmight be involved in other physiological processes in addition to the primary assimilation of ammonia releasedby the bacterial nitrogenase. The two genes show different but overlapping patterns of expression withMtGSabeing the major gene expressed in the infected cells of the nodule. Promoter fragments of 2.6 kb and 3.1 kb ofMtGSaandMtGSb, respectively, have been sequenced and primer extension revealed that theMtGSbpromoteris expressed in nodules from an additional start site that is not used in roots. Generally these fragments in thehomologous transgenic system were sufficient to drive GUS expression in almost all the tissues and cell typeswhere GS proteins and transcripts are located except that theMtGSapromoter fragment did not express GUShighly in the nodule infected cells. These results indicate that thecis-acting regulatory elements responsible forinfected-cell expression are missing from theMtGSapromoter fragment.

Introduction

Glutamine synthetase (GS, EC.6.3.1.2) plays a cen-tral role in the nitrogen metabolism of higher plants.GS catalyses the assimilation of ammonium into glu-tamine, which then serves both as a nitrogen donorin the biosynthesis of essentially all nitrogenous com-pounds and as a major nitrogen transport compound(Miflin and Lea, 1980). The ammonium for GS activ-ity is derived from the plant’s primary nitrogen sources(soil nitrate, and atmospheric nitrogen in the case oflegumes) as well as from a number of other metabolicpathways such as photorespiration, phenylpropanoid

metabolism and deamination of amino acids (Leaet al., 1990).

The various roles of GS are undertaken by differ-ent GS isoenzymes located in both the cytosol andthe plastids, and these are derived from the differen-tial expression of a small family of genes. Generallythere is a single gene, per haploid genome, that en-codes the plastid-located GS and several genes (2 to 6)that encode cytosolic GS (Forde and Cullimore, 1989;McGrath and Coruzzi, 1991; Peterman and Good-man, 1991; Liet al., 1993). The individual membersof plant GS gene families are differentially expressedduring plant growth and development and at least in

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some cases have different metabolic roles (Edwardset al., 1990).

The regulation of plant GS in nodules is of majorinterest due to the key location of this enzyme in thepathways between the nitrogen-fixing bacteria and theplant and the importance of legumes in the global ni-trogen cycle and agricultural systems. Moreover, thelegume root nodule is one of the richest sources of GSin higher plants. The activity of this enzyme increasesduring nodule development and this is attributed to theincreased expression of certain GS genes that leads tothe appearance of specific cytosolic GS polypeptidesand isoenzymes. Immunolocalisation,in situhybridis-ation and promoter-fusion studies have all been used tostudy the location and regulation of expression of thesegenes and proteins in various legumes (Bennettet al.,1989; Fordeet al., 1989; Walker and Coruzzi, 1989;Edwardset al., 1990; Brearset al., 1991; Miaoet al.,1991; Marsolieret al., 1993; Stanfordet al., 1993).The general picture emerging from these studies is thatGS genes exhibit a degree of tissue-specific expressionin nodules and that this expression appears to be underboth developmental and/or metabolic control, depend-ing on the species. However a complete picture of theexpression pattern in a single legume is lacking.

Medicago truncatulais a legume which is increas-ingly being used for genetic and molecular studiesparticularly for symbiotic interactions for whichAra-bidopsisis a poor model (Barkeret al., 1990; Blondonet al., 1993). We have shown in addition that thisspecies contains the smallest GS gene family so fardescribed in higher plants with a single gene en-coding plastid-located GS and only two functionalgenes (MtGSa and MtGSb) encoding cytosolic GS(Stanfordet al., 1993). These two genes were foundto be induced during nodule development, but non-coordinately. In addition, their protein products mayassemble either homologously or heterologously toproduce the active octameric isoenzymes (Carvalhoet al., 1997). In this report we have studied the lo-calisation of expression of each of these cytosolic-GSgenes and of the GS gene products in root nodules ofM. truncatula. This work forms the basis of a largerstudy, using this model legume, to understand theregulation of plant GS in the nodule and whether itplays a role in the regulation of the nitrogen flux fromsymbiotic nitrogen fixation.

Materials and methods

Plant material and growth conditions

Plants ofMedicago truncatulaGaertn. cv. Jemalongwere grown from surface-sterilised seeds under 13 hlight (23 ◦C) / 11 h dark (19◦C) in vermiculite wa-tered with the growth medium described by Lullienet al. (l987). For nodule induction the plants were wa-tered with fresh medium lacking ammonium nitrate,7 days before inoculation withRhizobium melilotistrain RCR 2011 (GMI51). Plants used forin vitroculture (genotype H39) were maintained in an envi-ronmental cabinet at a temperature of 23◦C by dayand 19◦C by night, 13 h day length and light intensityof 150 to 200µmol m−2 s−1.

Preparation of plant tissues for microscopy

Plant material was fixed and embedded as described inde Billy et al. (1991) except that paraffin was used forimmunolocalisation of GS and paraplast forin situhy-bridisation and the fixative contained 0.5% glutaralde-hyde. The embedded tissues were sectioned (5 to 7µmthick) and affixed to poly-L-lysine-coated slides. Forimmuno-transmission electron microscopy the mate-rial was embedded in LR white resin (Polysciences,Warrington, USA) and sectioned as described in Car-valhoet al. (1992).

Immunolocalisation

For light microscopy, the paraffin was removed andthe sections were rehydrated and then washed in wa-ter and twice in Tris-buffered saline (TBS). To inhibitthe endogenous peroxidases the sections were incu-bated for 30 min in 0.5% H2O2 in methanol, followedby a 15 min incubation in 3% H2O2 in water. Afterwashing twice in TBS, the sections were blocked for30 min with swine serum (5%) in 12.5% bovine serumalbumin (BSA) in TBS. The binding of the specificanti-GS antibody (Cullimore and Miflin, 1984) wasdone at a concentration of 1µg/ml freeze-dried serumin TBS containing 2.5% BSA and 0.02% Tween 20,overnight at 4◦C. After washing in TBS, the sectionswere incubated with secondary antibody, biotinylatedswine anti-rabbit immunoglobulin-G (Dako, Den-mark) diluted 1:200 in the same buffer. After twowashes in TBS, the tissue sections were incubatedwith the Vectastain ABC reagent kit (Vector Lab,USA), which contains peroxidase-linked streptavadin,followed by incubation in colour substrate solution

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(0.005% diamino-benzidine; 0.03% H2O2 in TBS).The reaction was stopped by washing with water andphotographs were taken using a Nikon Microscope.Control sections were treated as experimental sec-tions, except that the primary antibody was replacedwith rabbit non-immune serum and photographs of thesections were taken under dark field.

For immunofluorescence in confocal microscopythe experimental procedure was essentially as de-scribed above omitting the treatment with H2O2 andwith swine serum. The antigens were visualised usinga goat anti-rabbit antibody linked to the fluorochromeCy5 (Vector Laboratories, UK) under a Nikon confo-cal microscope. Visualisation of the plant tissues wasobtained by staining the nucleic acids and cell wallswith propidium iodine, which has a red-emitting fluo-rescence. Immunogold labelling of GS was performedas described in Carvalhoet al. (1992).

In situhybridisation

Total GS sense and antisense RNA probes were pre-pared byin vitro transcription of the whole cDNAinserts ofMtGSaand MtGSbcloned in pBluescriptSK+ (Stratagene, California). Equal quantities of thea and b probes were mixed together. For the gene-specific sense and antisense probes the 3′-untranslatedregions corresponding to 1145–1405 and 1216–1414of MtGSaandMtGSbcDNA sequences, respectively(Carvalhoet al., 1997), were amplified by PCR andsubcloned into pBluescript. Probes were prepared asdescribed by de Billyet al. (1991) to a specific ac-tivity of 108 cpm perµg RNA and their specificitywas ascertained by Southern blot hybridisation. Hy-bridisation was performed essentially as described byde Billy et al. (1991) except that 6× 104 cpm perµl of probe was used and the hybridisation stringencywas determined by 50% formamide in the buffer at40◦C for the GS total probes and 40% formamide and37 ◦C for the specific probes. After autoradiographyand staining with Toluidine Blue (de Billyet al., 1991)the sections were observed by bright-field or dark-fieldmicroscopy using a Zeiss Axiophot II microscope.

Construction of transcriptionalM. truncatulaGSgene promotergusAfusions

Transcriptional fusions betweenMtGSa and MtGSbgene promoters and theEscherichia coli gusAgenewere constructed by using the intermediate cloningvector pOGUS (Axeloset al., 1989) which has anNcoI

restriction site (CCATGG) at the position of transla-tion initiation. NcoI sites were introduced at the startcodon positions of the genesMtGSaand MtGSbbymeans of polymerase chain reaction (PCR) using theproof-reading polymerase Vent (Biolabs, New Eng-land). Primers specific for start codon positions (5′-GCAAAGCCATGGTGATG-3′ for MtGSa and 5′-GTACTGCAGGAGAGCCATGGTTTCG-3′ for Mt-GSb; the bases underlined in the primer sequences cor-respond to substitutions with respect to the genomicsequences) were used in combination with primerscorresponding to upstream promoter regions contain-ing the restriction sitesEcoRI for MtGSaand BglIIfor MtGSb to amplify MtGSa (2.6 kb) andMtGSb(200 bp) promoter fragments. The amplified promoterfragment ofMtGSawas subsequently cloned betweentheEcoRI andNcoI sites of pOGUS. Partial sequenc-ing of this construct confirmed the promoter-gusAjunction and showed that no errors were present withinthe 400 bp lying immediately upstream of the ATGcodon. The PCR-amplified 200 bpMtGSbpromoterfragment was cloned into pBluescript KS (Stratagene)and fully sequenced to assure that no mistakes wereintroduced. The full-lengthMtGSb3.1 kb promoterfragment was subsequently reconstructed by cloningtheBglII-PstI PCR-amplified fragment into a plasmidcontaining theSacI-BglII upstream promoter region.The complete promoter fragment was then cloned be-tween theSacI andNcoI sites of pOGUS and partiallysequenced to confirm the promoter-gusAjunction. Fi-nally, the two gene fusions were cloned into the binaryvector pBinl9 (Bevan, 1984) using the flankingEcoRIand HindIII sites to obtain pBin-prMtGSa-GUS, orusing theSacI and HindIII sites to obtain plasmidpBin-prMtGSb-GUS.

Transformation ofM. truncatulaand recovery oftransgenic plants

The binary vectors pBin-prMtGSa-GUS and pBin-prMtGSb-GUS were introduced into theAgrobac-terium tumefaciensstrain LBA4404 and used to trans-form leaf segments ofM. truncatulaH39. Kanamycin-resistant plants were regenerated by somatic embryo-genesis as described by Chabaudet al. (1996) andpropagatedin vitro.

Histochemical localisation of GUS activity

Histochemical staining for GUS activity was per-formed according to Jeffersonet al. (1987). Whole-plant fragments were fixed by vacuum infiltration for

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Figure 1. Immunocytochemical localisation of GS polypeptides in 21-day oldM. truncatula nodules. A. Longitudinal section incubatedwith anti-GS antibodies and detected with peroxidase-conjugated anti-rabbit IgG. There is no other staining besides the immunostaining.B. Dark-field image of the section shown in A. C. Dark-field image of a non-immune control section. D-G. Detection by immunofluorescencein confocal microscopy using Cy5-conjugated secondary antibody (green colour) and anti-GS (D and F) or non-immune (E and G) serum.Visualisation of the plant tissues was obtained by staining the nucleic acids with propidium iodine (red-emitting fluorescence). D and E.Longitudinal sections through zone III. F and G. Transversal sections through a vascular bundle and parenchyma cells. The histologicallydefined zones of the nodule (I to IV) are indicated on A. Abbreviations: a, amyloplast; IC, infected cell; M, meristem; Pa, parenchyma;PaC, parenchyma cell; PC, pericycle cell; UC, uninfected cell; VB, vascular bundle; VBE, vascular bundle endodermis; XV, xylem vessel.Bar= 100µm in A–C and 10µm in D-G.

5 min in an ice-cold solution of 0.5%p-formaldehydein 0.1 M phosphate buffer at pH 7.0, followed byincubation on ice for 1 h and two washes in phos-phate buffer. The material was cut into 80µm thicksections using a vibratome (Leica VT 1000) and thesections immersed in the GUS substrate solution con-taining 1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium; Biosynth,Switzerland), 5 mM EDTA, 0.5 mM potassium fer-rocyanide, 0.5 mM potassium ferricyanide and 0.1 Mpotassium phosphate buffer, pH 7.0. The immersedtissues were incubated in the dark at 37◦C for 1–16 h, depending on the intensity of the coloration. Thesections were briefly cleared with NaOCl to improvethe contrast between stained and non-reactive tissues.Samples were mounted in water between a slide anda coverslip and observed by bright-field or dark-fieldmicroscopy using an Olympus BX50 light microscopeand photographs were taken using a digital camera.

Determination of the transcriptional start sites byprimer extension

The transcription start sites of GS genes were mappedby primer extension. For this purpose two 26-meroligonucleotides were synthesised, complementary to−33 to−7 of MtGSaand−32 to−6 of MtGSb. Theoligonucleotides were labelled with [γ -32P]-ATP. Af-ter annealing with 10µg of M. truncatula poly(A)mRNA extracted from roots and nodules, the primerextension reaction was performed at 42◦C for 1 h.The [γ -32P]-labelled cDNA product was analysed ona 6% polyacrylamide sequencing gel, in parallel witha sequencing reaction generated with the same primersand the cloned promoter region of each gene as DNAtemplates.

Results

Immunocytochemical localisation of GS polypeptidesin M. truncatularoot nodules

The distribution of GS polypeptides in root nod-ules was studied by light, confocal and electron mi-croscopy coupled to immunocytochemistry. The an-tibody used recognises both the plastid and cytosolicpolypeptides ofMedicago truncatulaas ascertainedby western blots (Carvalhoet al., 1997) but not rhi-zobial GS proteins (Cullimore and Miflin, 1984) andcan thus provide an overall view of plant GS distribu-tion in the nodule. In a first approach to investigate thespatial localisation of GS we have performed an im-munolocalisation in root nodules ofM. truncatulabylight microscopy, using a peroxidase detection asso-ciated with the secondary antibody. Nodule structureis classified based on the nomenclature of Vasseet al.(1990).

In a longitudinal section through a 21-day oldM. truncatulanodule (Figure 1A) the immunostain-ing, detected as a brown colour, was found in mostparts of the nodule including both central and periph-eral tissues. Staining was strong in the meristematiczone I, poor in the infection zone II and then strongestin the infected and uninfected cells of the nitrogen-fixing zone III, particularly in the distal region. Theparenchyma layer surrounding the nodule and thevasculature also showed high levels of GS. Controlsections could be viewed under dark-field microscopy(Figure 1C) but under bright-field microscopy theywere hardly visible, indicating the absence of non-specific immunostaining (data not shown).

GS localisation was investigated at higher mag-nification by both immunofluorescence in confocalmicroscopy (Figure 1D–G) and electron microscopy(Figure 2) in order to determine the cellular and sub-cellular distribution of GS polypeptides and to identifyspecific cell types. For confocal microscopy the sec-ondary antibody was linked to the fluorochrome Cy5,which is visualised in Figure 1 as a green colour. The

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Figure 2. Immunogold localisation of GS in root nodules ofM. truncatula. A. Section through the central tissues of the nitrogen fixing zoneshowing two infected cells (IC) and one uninfected cell (UC). B. Similar non-immune control section. C. Light microscopy of a transversalsection through a vascular bundle (thin section) from which the subsequent ultrathin sections were made. D. Section through the parenchymacells (PC). E. Pericycle transfer cells using non-immune serum. F. Section through the pericycle showing transfer cells with their characteristiccell wall ingrowths (cwi) next to the xylem vessels (XV) (see also inset). G. Pericycle cells with plasmodesmatal connections (arrows).Abbreviations: b, bacteroids; cw, cell wall; cwi, cell wall ingrowths; m, mitochondria; n, nucleus; pl, plastids; E, endodermis; IC, infected cell;P, pericycle; Pa, parenchyma; PaC, parenchyma cell; PC, pericycle cell; TC, transfer cell; UC, uninfected cell; XV, xylem vessel. Bar= 1µmexcept C= 100µm.

choice of this fluorophore relies on its far-red/infraredemission wavelength (670 nm), which avoids interfer-ence by the auto-fluorescence of plant tissues, whichwas negligible at this wavelength. Visualisation ofthe plant tissues was obtained by staining the nucleicacids and cell walls with propidium iodine, which hasa red-emitting fluorescence. For electron microscopyimmuno-gold labelling was used.

Both techniques showed that GS, in the centraltissues of the nitrogen fixing zone, is clearly locatedin the cytosol and amyloplasts of both the infectedand uninfected cells (Figures 1D and 2A and otherobservations). The homogeneous distribution of goldparticles in the ground cytosol of both cell types(Figure 2A) suggests that the enzyme is at similarconcentrations. No specific labelling was observed inbacteroids, bacteroid envelopes, peribacteroid spacesor the peribacteroid membranes nor in similar nodulesections treated with non-immune serum (Figures 1Eand 2B).

In a transverse section through a nodule vascularbundle (Figure 1F), particularly heavy staining wasobserved in the cytosol of the pericycle cells. Theultrastructure of the nodule vascular bundles was stud-ied by EM (Figure 2E–G). These studies revealed thatthe pericycle cells are enlarged and interconnected bynumerous plasmodesmata (Figure 2G). Some of thepericycle cells have wall ingrowths, particularly welldeveloped on the walls contiguous with the xylem el-ements (Figure 2E, F), and thus correspond to transfercells. Immuno-EM shows that a striking abundanceof gold particles was detected in the cytosol of thesetransfer cells and also in the plastids (Figure 2F). Peri-cycle transfer cells incubated with non-immune serumshowed no labelling (Figure 2E). Immunolabellingwith the GS antibody was also evident in the amylo-plasts and cytosol of parenchyma cells (Figures 1F and2D), but not with non-immune serum (Figure 1G).

Distribution of cytosolic GS transcripts inM. truncatularoot modules byin situhybridisation

In situ hybridisation was used to determine the cellu-lar locations ofMtGSaandMtGSbtranscripts. Serialsections ofM. truncatula14-day old nodules were hy-bridised with35S-labelled sense and antisense probesand the sites of hybridisation were visualised by au-toradiography, appearing as white silver grains in darkfield and black spots in bright field (Figure 3). Initiallya total GS probe was used which hybridises with thetranscripts of bothMtGSaandMtGSb(Figures 3A-C).This probe clearly indicates that the central tissue ofzone III is the major location of GS transcripts, withthe intensity of signal being highest in the distal partof this zone (Figure 3A). Transcripts were also de-tected in the meristem (not shown), the parenchymalayer and associated with the vascular bundles (Fig-ure 3A). Higher magnification and observation underbright field, in order to distinguish the two cell types,revealed that expression of GS in zone III occurs inboth the infected and uninfected cells (Figure 3C).

Gene-specific probes forMtGSaandMtGSbwereobtained from the 3′-untranslated regions of the cDNAclones and their specificity were verified by Southernblotting (not shown). TheMtGSamessenger is abun-dantly present in the central infected cells of zone III(Figure 3D) and higher magnification revealed thatthis expression started in the amyloplast-containingcells of interzone II/III (Figure 3G, H). Uninfectedcells also seem to show the presence ofMtGSatran-scripts, although at considerably lower levels. In theperipheral tissues,MtGSatranscripts were clearly de-tected in the vascular bundles (Figure 3F) and to amuch lower extent in the parenchyma.

MtGSb transcripts were much less abundant thanMtGSa. However, a specificMtGSbsignal was ob-served in the meristem and in the uninfected cellsof zone III (Figure 3I, J). Some of the infected cellsin this zone also seem to express these transcripts.In the peripheral tissues a clear expression was seenin the parenchyma tissue especially surrounding thevascular traces (Figure 3K). In all these experiments,

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Figure 3. Localisation of GS transcripts in longitudinal sections of 14-day old root nodules byin situ hybridization. A–C. Hybridised with atotal GS35S-labelled antisence RNA probe which hybridises to all cytosolic GS transcripts. Dark-field (A) and bright-field (B) micrograph ofsame section. C. Detail of B showing infected and uninfected cells of zone III. D–H. Hybridised with aMtGSa-specific35S-labelled antisenseRNA probe. Dark-field (D) and bright-field (E) micrograph of the same section. F. Detail of D showing a vascular bundle, the parenchymaand neighbouring infected tissues. Dark-field (G) and phase contrast (H) micrograph of a higher magnification of the section in D/E showinginterzone II/III. I–K. Hybridised with aMtGSb-specific35S-labelled antisense RNA probe. Dark-field (I) and bright-field (J) micrograph of thesame nodule section. K. Detail of a peripheral region from I containing a vascular bundle. I–III, histologically defined zones; IC, infected cells;UC, uninfected cells; Pa, nodule parenchyma; VB, vascular bundles; M, meristem. Bars; 100µm in A, B, D, E, I, J; 50µm in F, G, H, K;15µm in C.

control sections, hybridised with the correspondingsense probes, showed no labelling above background(not shown).

Analysis of the expression driven by the 5′-upstreamregions ofMtGSaandMtGSb in transgenicM. truncatulaplants

To complement thein situ hybridisation studies andto analyse the expression directed by theMtGSaandMtGSb promoters, transgenic plants expressing theE. coli reporter genegusAencodingβ-glucuronidaseunder the direction of each GS promoter were pro-duced. The 2.6 kb and 3.1 kb of DNA lying imme-diately upstream of the translation initiation codonof MtGSa and MtGSb, respectively, were fused togusAin a way to generate precise transcriptional fu-sions. These chimeric constructions were then intro-duced intoM. truncatula by means of anAgrobac-terium tumefaciensleaf disc transformation protocol(Chabaudet al., 1996). Regeneration of whole plantsvia somatic embryogenesis led to the isolation of 14MtGSa-gusAand 17MtGSb-gusAtransformed plants.Of these plants 3 and 6 respectively were GUS-negative whereas the GUS activity patterns in theothers were qualitatively identical within a construct.

The following general features relating to the his-tological expression patterns of the gene fusions havebeen deduced from the microscopic observation ofa large number of longitudinal and/or transverse vi-bratome slices of nodules collected at several devel-opmental stages. Reporter gene expression driven bythe MtGSapromoter was found both in central andsome peripheral nodule tissues at all stages of devel-opment analysed. In the central tissues GUS activitywas restricted to uninfected cells (Figure 4A, C, D)with the most intense expression located in the dis-tal part of zone III. Some expression was also seenin the proximal region of zone III and in zone II.GUS activity driven by this promoter was not ob-served in the bacteria-free cells of the meristematiczone I. In peripheral tissues, GUS activity was located

in nodule parenchyma and particularly strikingly in thevascular bundles (Figure 4C). Blue staining was nei-ther observed in the nodule cortex nor the endodermis(Figure 4A, C).

The expression driven by theMtGSbpromoter wasquite similar to that observed forMtGSa in periph-eral tissues, but in central tissues the two promotersshowed a different pattern of expression (Figure 4B).In zone III of the central tissues the GUS staining waslocalised to some of the infected cells (Figure 4E, F),particularly in the distal region and in the uninfectedcells, especially in the proximal region (Figure 4B).GUS activity driven by this promoter could also bedetected in the bacteria-free cells of the meristematiczone I and in zone II (Figure 4B).

Analysis of theMtGSaandMtGSbpromotersequences

The promoter fragments directly upstream of the initi-ating methionine codons of genesMtGSaandMtGSbwere completely sequenced. The promoter fragmentsequences (2641 bp forMtGSaand 3148 bp forMt-GSb) have an elevated content of A+T residues, 73%for MtGSaand 75%MtGSb. A computer analysis re-vealed a TATA box motif (TTATAAATA at−130 rel-ative to the translational start site forMtGSa, and po-sition−144 TATAAATAAA for MtGSb) (Figure 5A).The functionality of these TATA boxes was confirmedby primer extension analysis (Figure 5B, C). The re-sults indicated a major transcriptional start site forMtGSa, both in nodules and roots, located at−98,about 20 bp downstream of the canonical TATA boxsequence (Figure 5B). In the case ofMtGSb, primerextension revealed the presence of two major tran-scripts about 40 nucleotides apart in nodules, whereasin roots only the shorter transcript was detected (Fig-ure 5C). The shorter transcript start site is located at−107 about 30 bp downstream of the canonical TATAbox. However, for the longer transcript whose start siteis located on the TATA box of the shorter one (−143)no related TATA box consensus sequence could be

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Figure 4. Histochemical localisation of GUS activity in longitudinal (A, B, C) or transversal (D, E, F) sections of 16-day old root nodules oftransgenicM. truncatulaplants expressing theMtGSa-GUS (A, C, D) orMtGSb-GUS (B, E, F) construct. Nodules were sectioned (80µmthick), stained as described in Materials and methods and photographed by dark-field microscopy. Blue precipitate indicates the location ofGUS activity. I–III, histologically defined zones; IC, infected cells; UC, uninfected cells; P, parenchyma; C, cortex; VB, vascular bundle. Bars:100µm in A and B, 50µm in C–F.

found. The most related sequence to a TATA box isTAAAGAATA found about 40 bp upstream. The ex-istence of this longer transcript cannot be an artefactof the primer extension technique as we have isolatedtwo cDNA clones from a nodule cDNA library startingat position−143, thus proving that the correspondingmRNA exists in nodules.

Comparison of theMtGSasequence to a−1321promoter fragment of its orthologous pea gene whichhas been used in GUS fusion analyses (Brearset al.,1991) revealed a region of 88 bp showing 82% iden-

tity located around the TATA-box motif of both genes.This region does not comprise the putativecis-actingelement binding a protein factor, identified in theGS3A promoter fragment. Another region of high ho-mology (77%) is present in similar positions in thetwo promoters but located about 60 bp upstream of theminimal−132 GS3A promoter fragment.

A computer search was performed on both se-quences in order to identify DNA motifs suggested tobe involved in the transcriptional regulation of GS ornodulin genes from other plants. The results of this

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Figure 5. Structure of the upstream regions of theMtGSaandMtGSbgenes. A. Distribution of nodulin putative regulatory elements inMtGSaandMtGSbupstream regions: NB1-Nodulin box 1 and NB2-Nodulin box 2 (Sandalet al., 1987), PNF1/NAT2 binding site (Fordeet al., 1990).The orientation of the motifs in the promoters are indicated by arrows. The cDNA sequences of the regions immediately upstream of thetranslational start sites (ATG) are shown. The transcriptional start sites are indicated with dots and the putative TATA boxes are boxed. B, C.Mapping of the 5′ ends ofMtGSa(B) of MtGSb(C) transcripts by primer extension in nodules (N) and roots (R). Size markers were obtainedby carrying out a set of 4 dideoxy sequencing reactions (lanes A, T, G and C) using the same primers as used for the primer extension and thecloned promoter regions as DNA templates. The transcriptional start sites are indicated with arrows.

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analysis are schematically represented in Figure 5A.The nodulin consensus motifs CTCTT and AAAGAT(Sandalet al., 1987) were found at a higher frequencyin MtGSathan inMtGSb(Figure 5A).

The A/T-rich consensus sequence TATTTWATknown to bind the nodule nuclear factor PNF1 initiallyidentified in the promoter region of thePhaseolus vul-garis gln-γ gene (Fordeet al., 1990) is present inboth MtGSa and MtGSb. However this motif is lo-cated in distal regions of the promoters (−1691 forMtGSaand−2148 and−2351 forMtGSb), whereasin other nodulin promoters it was located in proximalregions, between 500 and 100 bp upstream of the startof transcription.

Discussion

This study set out to investigate the cell-specific ex-pression of the complete cytosolic GS gene familyin root nodules ofM. truncatula using a combi-nation of different techniques which permitted thecorrelation of individual gene expression with theircorresponding transcription and translation products.GS protein localisation was analysed by immunocy-tochemistry and gene expression was evaluated bothby GS promoter-GUS fusions in transgenic plantsand in situ hybridisation using gene-specific probes.The possibility to transformM. truncatulaenabled thestudy of promoter gene fusions in transgenic plants inthe homologous system, where alltrans-acting fac-tors involved in gene expression are available. Allother studies concerning GS gene fusions in transgenicplants have used heterologous hosts, which might notreflect what is happening in the native background.For example, the soybean GS21 was found to be-have differently in its native background than as GUSfusions in three heterologous hosts,Lotus cornicula-tus, Nicotiana tabacumand alfalfa (Miaoet al., 1991;Marsolieret al., 1995; Carrayolet al., 1997).

Initially we used non-specific GS probes to mea-sure the overall distribution and abundance of cy-tosolic GS polypeptides and mRNAs in nodules byimmunolocalisation andin situ hybridisation respec-tively. Generally the two techniques gave similar re-sults indicating that transcriptional regulation (mRNAsynthesis) plays an important part in controlling GSprotein levels in nodules. Although GS is an essentialenzyme and is probably expressed in all cells of theplant, three tissues of the nodule showed particularlyhigh expression: the central tissues, the surrounding

parenchyma and the vascular bundles (Figures 1, 2 and3).

Several studies have shown previously that GS isexpressed highly in the cytosol of the central-tissueinfected cells (Brangeonet al., 1989; Fordeet al.,1989; Brearset al., 1991; Dattaet al., 1991). This lo-cation is entirely consistent with the primary functionof plant cytosolic GS in nodules being the assimila-tion of ammonium that is produced at high rates bythe infecting rhizobia. In agreement within situ hy-bridisation studies on alfalfa, we also see a higherexpression of this enzyme in the distal part of the in-fected zone, thus consistent with this being the mostactive nitrogen-fixing zone (Treppet al., 1999a).

In M. truncatula, our immunolocalisation andin situ hybridisation studies have suggested that theabundance of GS polypeptides and mRNA in the cy-tosol of the central-tissue uninfected cells may be ashigh as in infected cells. However as these cells arehighly vacuolated and make up only a small proportionof the central tissue volume, they are quantitativelymuch less important in ammonium assimilation thaninfected cells. In determinate nodules these uninfectedcells play an important part in the synthesis of urei-des (Verma, 1986) and have been shown to expresscytosolic GS (Kouchiet al., 1988; Miaoet al., 1991).In amide exporters such asMedicago, their role is notso clear but the expression in these cells of NADH-GOGAT (Vanceet al., 1995; Treppet al., 1999b),aspartate aminotransferase (Robinsonet al., 1994),asparagine synthetase (Shiet al., 1997), and PEPcarboxylase (Robinsonet al., 1996; Pathiranaet al.,1997) suggests that they contain the complete com-plement of enzymes for glutamine and asparaginesynthesis. Whether the ammonium is directly derivedfrom the rhizobia by diffusion from the neighbouringinfected cells or from some other pathway is not clear.

In the peripheral tissues ofM. truncatula nod-ules, GS is notably abundant in two locations: theparenchyma and the vasculature. The parenchyma ex-pression of GS has not been reported previously butpromoter-GUS fusions have shown that GS is ex-pressed in the vasculature of pea (Brearset al., 1991)andPhaseolus vulgaris(Fordeet al., 1989) althoughthe exact cell types have not been described. Using acombination of immunofluorescence in confocal mi-croscopy and immunogold labelling we have shownhere that GS protein is abundantly present in the vas-culature pericycle cells, especially in transfer cells(Figures 2 and 3). These cells possess cell wall in-growths consisting of branched filiform protuberances

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which increase enormously the surface-to-volume ra-tio of the cells and can be clearly identified in electronmicroscopy (Figure 2E, F). They have been detectedin the nodule vasculature of many amide-exportingspecies and are considered to play a role in transport-ing amino acids away from the infected nodule tissueto the xylem and in supplying photosynthate from thephloem to the infected cells (Pateet al., 1969; Pate andAtkins, 1983).

One possibility therefore for the expression of GSin the vasculature pericycle cells and perhaps alsothe parenchyma could be the assimilation of ammo-nium into organic combination and subsequent exportfrom the nodule. Supporting this hypothesis, sev-eral enzymes involved in nodule nitrogen and carbonmetabolism have recently been localised in the noduleparenchyma and vasculature including PEP carboxy-lase (Pathiranaet al.1997), carbonic anhydrase (de laPenaet al., 1997) and malate dehydrogenase (Milleret al., 1998) although perhaps significant asparaginesynthetase has not been detected in the vasculature(Shi et al., 1997). It is noteworthy that GS in thephloem ofNicotiana tabacumandSolanum tuberosumand pea has previously been interpreted in terms ofa functional role of this enzyme in providing gluta-mine for long-distance transport (Edwardset al., 1990;Carvalhoet al., 1992; Pereiraet al., 1992).

A second possibility could be a role in the regu-lation of the parenchyma-located oxygen barrier. Al-terations in the permeability of the oxygen barrier areconsidered to be the major means of regulating ni-trogen fixation and is believed to be effectuated byosmotic changes in the parenchyma layer cells (Huntand Layzell, 1993). Recently it has been shown thatthe nitrogen composition of the phloem, particularlythe glutamine content, affects the permeability of theoxygen barrier, thus providing a means of regulatingnitrogen fixation according to the nitrogen status ofthe plant (Neo and Layzell, 1997; see also Parsonset al., 1993). GS in the vasculature and parenchymacould thus be involved in detecting the nitrogen statusof the plant, transmitting a signal to the parenchyma,and perhaps in the synthesis of organic compounds in-volved in osmotic changes important for the regulationof the oxygen diffusion barrier. At present the majorosmoticants in the parenchyma are unknown althoughinorganic ions, nitrogen metabolites and sucrose haveall been suggested (Hunt and Layzell, 1993). Recentlyit has been shown using antisense technology that GS,in the phloem, regulates proline levels (Brugièreet al.,1999). As proline is a major osmoticant in plants (De-

launey and Verma, 1993; Taylor, 1996) and is clearlypresent in alfalfa nodules (Fougèreet al., 1991), it ispossible that a function of GS in the peripheral nod-ule tissues could be in the regulation of the oxygendiffusion barrier, via the synthesis of proline. The hy-pothesis of GS being involved in osmoticant synthesisis further supported by the detection of GS in twoother plant tissues known to be involved in osmoticprocesses: stomatal guard cells and pulvini (Marsolieret al., 1993, H. Carvalho, unpublished work).

The gene-specificin situ hybridisation and GUSfusion studies have revealed thatMtGSaandMtGSbshow different but partially over lapping patterns ofexpression.MtGSa is at least 10-fold more highlyexpressed in nodules thanMtGSb(at both the RNAand protein levels; Stanfordet al., 1993; Carvalhoet al., 1997).In situ hybridisation has shown thatMt-GSais predominantly expressed in the infected cellsof the central tissue similar to its alfalfa orthologue(Temple et al., 1995), with expression starting inthe interzone II/III similar to late nodulins such asleghaemoglobin (de Billyet al., 1991) and NADH-GOGAT (Trepp et al., 1999b). It is also highlyexpressed in the vasculature including around theirapices and, to a lesser extent, in the uninfected cells ofthe central tissue and in the parenchyma. Bothin situhybridisation and GUS fusion work suggest thatMt-GSb is uniformly expressed in the uninfected cellsof the central tissue and is the major GS gene ex-pressed in the parenchyma and the meristem. It alsoshows expression in some but not all infected cells,suggesting that there is some variable component inthese cells which affects its expression. In alfalfa theMtGSbhomologue appears to be expressed only in in-fected cells (Templeet al., 1995) suggesting that theremay be some differences in closely related species.The presence of principally homooctameric isoen-zymes of GSa and GSb in the nodule, when the twosubunits are clearly able to assemble heterologously(Carvalhoet al., 1997), is consistent with essentiallydifferent nodule expression patterns. The presence ofsome heterooctameric isoenzymes confirms howeverthe partially overlapping expression of the two genes.

The location of expression of theMtGSb-GUSfusion is generally consistent with thein situ hybridi-sation results and suggests that the 3.1 kb promoterfragment contains all the information necessary forcorrect expression of this gene in nodules. It is in-teresting to note that theMtGSbgene appears to beexpressed from two different start sites one of whichis not used in roots and in which a TATA box is not

754

identifiable. Two transcription initiation sites have alsobeen reported for cytosolic GS genes of alfalfa,P. vul-garisandLotus japonicus(Tischeret al., 1986; Fordeet al., 1989; Thykjaeret al., 1997) and for the plastidicGS genes ofP. vulgarisand pea (Cocket al., 1991;Tjaden et al., 1995) but their significance remainsunknown.

The MtGSapromoter fragment directs expressionessentially in the same peripheral tissues as revealedby in situ hybridisation, with strong expression in thevasculature and some expression in the parenchyma.However a striking anomaly was the high abundanceof MtGSatranscripts in the central infected cells (Fig-ure 3A) and the virtual absence of GUS activity drivenby theMtGSapromoter fragment in these cells (Fig-ure 4A). This result indicates that theMtGSapromoterfragment does not contain essential elements for theexpression in the infected cells, but contains elementsfor the correct expression in other nodule cell types.This is surprising as (1) this gene contains a higherfrequency of occurrence of nodulin consensus motifsthanMtGSb(Figure 5A) and (2) the pea orthologue ofthis gene, GS3A, has been reported to require only ashort DNA element of 132 bp to confer cell-specificand developmentally regulated expression patterns inalfalfa nodules (Brearset al., 1991). However the re-gion in this pea promoter fragment that binds a proteinfactor is poorly conserved in theM. truncatula or-thologue although there are considerable regions ofhomology around the TATA box and in a region furtherupstream. Our results clearly suggest that the regula-tory regions for the expression ofMtGSa in noduleinfected cells may lie elsewhere in the gene, per-haps further upstream, as shown for a soybean GSgene (Tercé-Laforgueet al., 1999) or downstream ofthe translational start as occurs in some other genes(see Taylor, 1997) including the nodulin gene ENOD2(Chenet al., 1998).

In conclusion, this work has shown that the twocytosolic GS genes ofM. truncatulaexhibit complexexpression patterns in nodules suggesting that theirencoded proteins are involved in other physiologicalprocesses in addition to the assimilation of ammoniumfrom nitrogen fixation. TheMtGSa gene is clearlymostly involved in this latter role but the regions re-quired for expression of this gene are not located inthe 2.6 kb upstream of the promoter. This work also il-lustrates the necessity of using a variety of techniquesto study gene expression and that misleading resultsmay be obtained by using GUS fusions alone.

Acknowledgements

We would like to thank Sylvie Camut for mainte-nance of the transgenic plants and Claudio Sunkelfor helpful discussions. We acknowledge the Univer-sity of Newcastle Research Associates (TUNRA) inAustralia for providingM. truncatulaJemalong 2HA(H39) seeds. We gratefully acknowledge funding fromthe European Union Biotechnology programme (con-tract BIO4-97-2319, project Fixnet), the Fundaçãopara a Ciência e Tecnologia (PRAXIS XXI) and theLuso-Français Programme of Scientific and TechnicalCooperation.

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