Copyright © Physiologia Plantarum 2001PHYSIOLOGIA PLANTARUM 112: 388–396. 2001Printed in Ireland—all rights reser�ed ISSN 0031-9317

The promoter of a cytosolic glutamine synthetase gene from the coniferPinus syl�estris is active in cotyledons of germinating seeds andlight-regulated in transgenic Arabidopsis thaliana

Concepcion Avilaa, Francisco R. Cantona, Pilar Barnesteina, Marıa-Fernanda Suareza, Pierre Marraccini1a, Manuel Reyb,Jaime M. Humarac, Ricardo Ordasc and Francisco M. Canovasa,*

aDepartamento de Biologıa Molecular y Bioquımica, Instituto Andaluz de Biotecnologıa, Unidad Asociada UMA-CSIC, Uni�ersidad deMalaga, E-29071, Malaga, SpainbLaboratorio Fisiologıa y Biotecnologıa Vegetal, Facultad de Ciencias, Uni�ersidad de Vigo, E-36200, Vigo, SpaincLaboratorio Fisiologıa Vegetal, Departamento BOS, Uni�ersidad de O�iedo, E-33071 O�iedo, Spain1 Present address: Nestle Research Center Tours, Plant Science and Technology, 101 Gusta�e Eiffel, B.P. 9716, F-37097, Tours Cedex 2,France*Corresponding author, e-mail: cano�as@uma.es

Received 11 September 2000; revised 30 January 2001

expressed in the cotyledons of germinating pine seeds trans-We have isolated and characterized a genomic clone encodingformed by microprojectile bombardment. Stable transforma-Scots pine (Pinus syl�estris) cytosolic glutamine synthetase

(GS). The clone contains the 5� end half of the gene including tion of Arabidopsis thaliana revealed the shoot apicalmeristem as the major region of heterologous permanentpart of the coding region and 980 bp upstream of the transla-

tion initiation codon. The major transcription start site (+1) expression in Arabidopsis, in agreement with the expression ofthe GS gene in Pinus. Moreover, quantitative data derivedwas mapped around 180 nucleotides upstream of the transla-

tion initiation codon. Sequence analysis of the 5�-upstream from fluorometric �-glucuronidase assays in control and con-region of the gene reveals the presence of putative regulatory tinuous light-grown transgenic Arabidopsis plants indicate

that the isolated upstream region of the gene contains regula-elements including a poly-CT consensus sequence, a purine-tory sequences involved in the response to light.rich tandem repeat and two AT-rich regions. Fusions of the

upstream gene region to uidA were shown to be transiently

mation is available. cDNA clones encoding cytosolic GShave been isolated from Scots (Canton et al. 1993, Elmlingeret al. 1994) and loblolly pine (Kinlaw et al. 1996) but nocharacterization of a genomic clone has been reported.

Previous work indicated that cytosolic GS (GS1) is thepredominant GS isoform in conifer seedlings (Canovas et al.1991, Canton et al. 1996, Garcıa-Gutierrez et al. 1998). GSpolypeptides are present in all organs of the seedlings in-cluding photosynthetic and nonphotosynthetic: cotyledons,needles and roots. Molecular data derived from the charac-terization of a GS cDNA clone showed that GS1 gene isactively expressed in chloroplast containing tissues of devel-oping seedlings (Canton et al. 1996) and the level of thetranscript was affected by developmental and light condi-

Introduction

Glutamine synthetase (GS EC 6.3.1.2) is the enzyme respon-sible for the primary assimilation of ammonia in higherplants (Miflin and Lea 1976). GS catalyzes the assimilationof ammonia into glutamine, which then serves as a majornitrogen transport vehicle and donor in the biosynthesis ofessentially all nitrogenous compounds. The enzyme is activein a number of organs (McNally et al. 1983) where it isinvolved in assimilation or reassimilation of ammonium.Depending on site location (Hirel et al. 1993), the variousroles of GS are undertaken by different GS isoforms en-coded by a small multigene family (Cullimore et al. 1984,Tingey et al. 1988, Sakamoto et al. 1989).

Most of these molecular studies have been carried out inannual crop plants; however, in woody plants limited infor-

Abbre�iations – GS, glutamine synthetase; GUS, �-glucuronidase; NOS, nopaline synthase; NPT, neomycin phosphotransferase; LB, leftborder; RB, right border.

Physiol. Plant. 112, 2001388

tions (Canton et al. 1999). Thus, to go further in thisstudy, we present the DNA sequence of a partial genomicclone containing 7 exons of the coding region of GS1 gene.The clone includes 980 bp upstream of the functionalATG. So far, very few studies involving genomic clonesfrom gymnosperms have been reported in the literature(Kojima et al. 1992, Barrett et al. 1994, Loopstra andSederoff 1995) and none of them correspond to nitrogenmetabolism. Consequently, studying a GS genomic clonegives us the opportunity to increase our knowledge notonly on GS gene families in higher plants, but also ingymnosperm gene organization and function. We havestudied the promoter activity of this 5�-untranslated regionusing fusions with the reporter gene uidA encoding �-glu-curonidase (GUS). The transient expression of the reportergene has been analyzed in Pinus pinea cotyledons aftermicroprojectile bombardment. Gene fusions were alsotransferred to Arabidopsis thaliana via Agrobacterium andstable gene expression examined in transgenic plants.

Materials and methods

Construction of a Scots pine subgenomic library andcharacterization of GS1 clones

Partially EcoRI digested fragments of Scots pine (Pinussyl�estris) genomic DNA were size fractionated by elec-trophoresis and cloned into �gt10 vector from Promega(Madison, WI, USA). Recombinant phages carrying theGS1 gene were identified by screening 1×106 primaryphage plaques with a 5�-fragment corresponding to 500 bpof the previously isolated cDNA clone, pGSP114 (Cantonet al. 1993). Positive clones were amplified, phage DNApurified and genomic inserts released by EcoRI enzymedigestion. Restriction fragments were subcloned into theplasmid vector pGEM-3Z (Promega) for detailed restric-tion mapping and DNA sequencing using the method ofSanger et al. (1977).

Primer extension analysis

Primer extension analysis was carried out essentially asdescribed in Sambrook et al. (1989) by using an oligonucle-otide whose sequence, 5�-GGTTGAGAAGGTCTGT-TAAT-3�, matched from nucleotides 12 to 32 downstreamof the ATG initiation codon. The primer was allowed tohybridize for 1 h at 65°C. Total RNA (10 �g) extractedfrom green cotyledons of 2-week-old seedlings was used asa template for the experiment.

Constructions containing the GS1 putative promoter andGUS reporter gene

The 981-bp sequence upstream of the translation startingpoint was removed from the clone by HaeIII digestion.The resulting fragment and a truncated HpaII fragmentwith 64 bp less as well as a third DdeI-generated fragmentcontaining 37 bp of the coding region, respectively, wereeach subcloned into the vector pBI101 (Jefferson et al.

1987), creating GS1::uidA gene fusions. pBI101 is a binaryvector based on pBin19 and carries, within the T-DNAborders, a kanamycin-resistant gene and the polyadenyla-tion signal from the nopaline synthase gene. The GS1T-DNA constructs, and also two controls, which were theoriginal plasmid pBI121 containing the CaMV 35S pro-moter and pBI101, a plasmid containing a promoter-less1.87 kb GUS cassette in the binary vector pBin19, wereindividually transferred to Agrobacterium tumefaciensLBA4404. The constructs were also checked for rearrange-ments in A. tumefaciens by Southern blotting.

Bombardment of pine cotyledons with the GS1 putativepromoter-GUS reporter gene constructs

Optimized bombardment parameters for transient uidAgene expression in P. pinea cotyledons excised from 1-day-old embryos are described elsewhere (Rey et al. 1996, Hu-mara et al. 1998). A Biolistic PDS-1000/He apparatus fromBio-Rad (Madrid, Spain) was used. Each bombardmentdelivered approximately 0.8 �g of plasmid DNA associatedwith 500 �g of gold particles and each Petri dish wasbombarded twice under a pressure in the shooting chamberof 85 kPa. After bombardment, cotyledons were main-tained in the same medium where they were bombardeduntil GUS assays were performed. All cultures were incu-bated in a growth chamber at 26�2°C with a 16-h pho-toperiod. GUS histochemical assays (Humara et al. 1998)were carried out 24 h after bombardment immersingcotyledons in 50 mM sodium phosphate buffer pH 7,0.01% Triton X-100, 0.5 mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 1 mM X-Gluc, and incubat-ing for 48 h at 37°C.

Fluorogenic assay of GUS in extracts from bombardedcotyledons as well as transgenic Arabidopsis was performedas described by Jefferson (1987). Protein concentration wasdetermined by the method of Bradford (1976). Assays wereperformed with the constructs shown in Fig. 3 subclonedinto plasmid pBI101. A 35S-promoter derivative pBI121and promoter-less pBI101 plasmids were used as controls.

Arabidopsis thaliana vacuum infiltration and stabletransformation with the gene constructs

Arabidopsis thaliana WS ecotype plants were grown at24°C under a 16 h light/8 h dark regime and vacuuminfiltrated as described elsewhere (Bechtold et al. 1993). T1seeds were harvested in bulk and transformant seeds wereselected in MS (Murashige and Skoog 1962) plates contain-ing 50 �g ml−1 kanamycin. T2 seeds were harvested indi-vidually and kept for further analysis. GUS histochemicalassay of the transformed plants was carried out as de-scribed above with additional processing steps. After incu-bation with the GUS substrate, plants were cleared ofchlorophyll by incubation with a solution of 5% formalde-hyde/5% acetic acid/20% ethanol for 10 min followed by2-min incubations with 50 and 100% ethanol. Quantitativeanalyses were performed as described before.

Physiol. Plant. 112, 2001 389

In situ hybridization

Hybridized transcripts of GS from pine samples were de-tected essentially as described (Canton et al. 1999) usinganti-digoxigenin antibodies conjugated to alkaline phos-phatase according to the supplier’s directions (Amersham,Barcelona, Spain). In situ hybridization studies withparaffin-embedded pine root tip sections were carried outessentially as described by Canton et al. (1999). Tissues werefixed in 4% formaldehyde, 0.1% glutaraldehyde. Digoxi-genin-labelled riboprobes were generated by in vitro tran-scription of a full-length cDNA of P. syl�estris GS (Cantonet al. 1993). Both sense and antisense probes were used inparallel hybridizations of transverse and longitudinal tissuesections.

Results and discussion

Isolation, sequencing and structural characteristics of thepine GS1 genomic clone

A �gt10 subgenomic library of Scots pine was screened forGS clones using the previously isolated pGSP114 pine GScDNA (Canton et al. 1993). A PstI-EcoRI fragment derivedfrom the 5� region of the cDNA was chosen as hybridizationprobe in order to increase the chance to specifically identifygenomic clones containing regulatory sequences of the gene.A number of 1×106 recombinant clones were screened and4 positive lambda clones were isolated. These 4 clones wereshown to have a similar insert size and, by restrictionmapping, all resulted to be derived from the same region ofthe P. syl�estris genome (data not shown). One of these,GS217, was subcloned and further characterized. As aninitial step, the genomic clone was entirely sequenced anddetermined to be 2543 bp in length. The comparison ofnucleotide sequences between the gene and the cDNA (Can-ton et al. 1993) showed that the fragment contained the 5�end of the gene including part of the coding region orga-nized in 7 exons, interrupted by 6 introns, and 980 bpupstream of the translation initiation codon. The sequenceof GS217 was 100% homologous to the correspondingsequences in the cDNA clone (pGSP114) (Canton et al.1993).

In spite of the evolutionary distance, all exons in pine andother cytosolic genes previously described in literature (Tis-cher et al. 1986, Walker et al. 1995) are the same in lengthexcept for the first pine GS exon, which is one codon longer,all of them keeping the same processing sites interruptingthe coding region and always in homologous positions. Thiscoincidence suggests the existence of domains in the proteinwhich have been conserved along the evolutionary scale dueto their importance in the structure and/or function of theGS subunit/holoenzyme. The sizes of introns in the GS1genomic clone varies between 91 bp the shorter and 282 bpthe longer, all of them in the usual range size for angiospermintrons, which are typically shorter than most mammalianintrons (Simpson and Filipowicz 1996). The AT percentagein higher plants introns is usually between 70% described fordicot plants and around 60% for monocot plants (Simpsonet al. 1993). However, the introns in the GS1 clone showed

an average AT percentage around 64% in the range ofreported in angiosperms. We have also analyzed the consen-sus sequences at the 5� and 3� splice sites (data not shown).We have designed consensus sequences for 5� and 3� splicesites in all 6 pine cytosolic GS introns and compared themwith monocot and dicot plants, yeast consensus and verte-brate splice consensus sequences. The strict requirement inboth sides of the intron for G/GT in the 5� site and AG/Gin the 3� end indicates a general use in all compared organ-isms. Otherwise, modifications of these sequences can affectboth the efficiency and accuracy of splicing, as well as theabundance of detectable transcripts (Simpson and Filipow-icz 1996).

Transcription start site analysis of the pine GS1 gene

Primer extension analysis was performed to identify thetranscription start site in the pine GS gene. An antisense20-bp oligonucleotide (GSP2) starting at 32 bp downstreamof the ATG was annealed to total RNA prepared fromScots pine cotyledons and extended with reverse transcrip-tase. Results shown in Fig. 1 indicate that transcription ofthe GS1 gene initiates at a major site located around 180nucleotides upstream of the translation initiation codon.The tentative transcription start site corresponds to a purine(adenine) as occurs in most eukaryotic genes. Althoughtranscription start sites determined for most other GS genesare located between 46 and 90 bp upstream of the ATG(Forde et al. 1990, Brears et al. 1991, Marsolier et al. 1995),transcription initiation has been mapped at 130 bp in analfalfa GS1 gene (Tischer et al. 1986). However, we cannotalso rule out the existence of alternative transcription startsites in other tissues and/or under other growing and devel-opmental conditions since we used total RNA from cotyle-dons of light-grown seedlings. Northern blotting analysesshowed a high level of GS1 transcript in these experimentalconditions (Canton et al. 1999).

Analysis of putative elements in the 5�-flanking region

Figure 2 shows the sequence of the 5�-flanking region of thepine GS1 gene extending from −800 relative to the start oftranscription to +180. Studies based on sequence analysisof the 5�-untranscribed region allowed us to define a canon-ical TATA box at −35 bp from the transcription startingsite similar to other plant genes in which it has beendescribed to be placed at position −32 (�7) (Joshi 1987,Forde et al. 1990, Marsolier et al. 1995). A putative CAATbox (GGTCAAGACTT) at −138 bp is also present. Theportion of the 5�-flanking region between positions −331and −224 contains a purine-rich sequence that is a candi-date for a regulatory element. The sequence contains atandem repeat centred around the position −278. The5�-flanking region also contains two A/T-rich regions be-tween −721 and −670 and −504 and −459. A/T-richregions have been described in other GS promoters (Fordeet al. 1990, Brears et al. 1991) acting as regulatory ciselements involved in gene expression. A poly-CT sequencemotif was identified at position +144 in the transcribed but

Physiol. Plant. 112, 2001390

untranslated region of the gene. A similar element has beendescribed in the gene encoding HMG from Arabidopsis to beinvolved in the regulation at transcriptional level of thegenes where it is present (Enjuto et al. 1995, Lumbreras etal. 1995). No significant similarities were observed with the5�-flanking regions of GS genes in angiosperms, except forthe presence of AT-rich sequences described above.

Transient GUS expression in pine cotyledons

To examine the capability of the 5�-untranslated region ofthe isolated DNA fragment as a promoter, we decided todevelop fusions to the uidA gene and determine GUS activ-

ity in transformed plants. Agrobacterium mediated stabletransformation of conifer species is not a trivial task (Huanget al. 1991). Bombardment with high velocity microprojec-tiles is an alternative gene transfer system to conifer cells(Ellis et al. 1993, Charest et al. 1996) which has been provedto be particularly efficient for studying transient gene ex-pression in pine species (Charest et al. 1993, Rey et al.1996). In this work, pine GS promoter-GUS chimeric con-structions were transferred to pine cotyledons (P. pinea) bymicroprojectile bombardment and transient GUS gene ex-pression studied in the target tissues. In addition, A. thalianaplants were stably transformed with the same chimericconstructs via Agrobacterium and promoter-directed GUSactivity analyzed in the transgenics. Three constructs (Fig.3) containing fragments derived from the 5�-upstream regionof the GS1 clone fused to the GUS gene were created. TheC1-HaeIII construct contained the complete 980-bp regionupstream of the translation initiation codon, C2-HpaII con-tained 918 bp from the 5� region with a deletion of 64 bpthat included the poly-CT sequence, and C3-DdeI containedan addition of 37 bp from the coding region correspondingto the first 12 amino acid residues. We included this lastconstruct because the presence of cis elements located withinthe 5� region of the message has been described in somegenes (Dickey et al. 1992). All 3 chimeric genes were used totest transient expression in P. pinea cotyledons. To establishtransformation efficiency we used, as a positive control, thepBI121 plasmid (Jefferson et al. 1987) which contains theconstitutive 35S promoter and, as a negative control, thepromoter-less pBI101 plasmid. As shown in Table 1, thehighest expression level was obtained for the constitutivepromoter in pBI121, with almost all of the bombardedtissues presenting GUS activity, even considering thatCaMV35S is not particularly efficient in conifers (Charest etal. 1993). By contrast, no GUS activity was visualized withpBI101 and when it was quantified it represented about 10%of control activity with pBI121. According to quantitativefluorimetric data, the higher level of expression obtainedwas for C1, confirming the function of the 5�-upstreamregion and indicating the presence of regulatory elementsdriving gene expression on it. The C2 showed a 40% de-crease, suggesting a possible role of the deleted region on thepositive control of transcriptional activity. With regard tothe C3 construct, only basal GUS activity was detected,indicating that additional nucleotides corresponding to thecoding region of the gene could reduce GUS enzyme activityas a result of the N-terminal extension.

The pine GS promoter can direct expression in transgenicArabidopsis

To further characterize the function of the 5�-upstreamregion of the pine GS1 gene, stable GUS expression wasstudied in transformed plants with the C1 construct. Indi-vidual transgenic plants were harvested at several develop-mental stages and analyzed for GUS expression. Figure 4illustrates the developmental- and tissue-specific location ofGUS activity in Arabidopsis plants. Expression of the re-porter gene was absent or very low at the seedling and

Fig. 1. Identification of the GS1 transcription start site. Primerextension was carried out using 10 �g of total RNA from cotyle-dons and the GSP2 oligonucleotide (32P-labelled with �-32P-ATPand polynucleotide kinase) which hybridizes to the N-terminalcoding region of the GS cDNA gene (exon I). The reverse transcrip-tion reaction was as described in Materials and methods and theproducts were separated in a 6% polyacrylamide 8 M urea sequenc-ing gel. The sequence of the noncoding strand shown in the figure(lanes AGCT) is indicated next to the gel. The A labelled with anasterisk indicates the transcription starting site at this position.

Physiol. Plant. 112, 2001 391

Fig. 2. Sequence of the 5�-upstream region in thepGS217 clone. Nucleotides are numbered relative tothe transcription starting site (position +1). Theputative TATA box placed at −35 and the possibleCAAT box placed at −138 are noted in bold.Poly-CT element placed between +144 and +161is double underlined. A repeated purine-richsequence placed between −331 and −224 isunderlined as well as A/T-rich regions between−721 and −670 and −504 and −459. Codingsequence is indicated in capital letters starting withthe ATG codon in bold.

rosette stages (Fig. 4A,B) but apparent in adult plantswith floral stems (Fig. 4C). These data therefore confirmthe functionality of the 5�-upstream region of pine GSgene in a heterologous system. Moreover, our results areconsistent with the report of Kojima et al. (1994) indicat-ing that a pine gene promoter can be operative inangiosperms and therefore suggesting that transcriptional

machinery is well conserved between angiospermsand gymnosperms. With regard to the specific location inadult plants, GUS activity was mainly detected in theshoot apex and leaf primordia. Figure 4D–F clearlyshows that C1 construction exhibited the higher levels ofGUS expression in the transgenic and a minimum for C3,in close agreement with the results of transient expression

Fig. 3. Diagram of transcriptional fusionsof GUS gene and 5�-flanking region ofpGS217 genomic clone. The upperdiagram shows pBI101 vector used thatcontains a kanamycin-resistant gene and amulticloning site where the putative GSpromoter region has been inserted.C1-HaeIII contains the complete5�-upstream region, C2-HpaII constructhas a deletion of the poly-CT element andC3-DdeI construction includes 37nucleotides of the coding region asindicated. NPT, neomycinphosphotransferase; GUS,�-glucuronidase; NOS, nopaline synthase;RB, right border; LB, left border.

Physiol. Plant. 112, 2001392

Fig. 4. Gene expression analysisin transgenic Arabidopsis andpine seedlings. (A–C)Developmental analysis of GUSexpression in A. thaliana plantstransformed with the C1-HaeIIIconstruction. (A) seedling, (B)rosette, (C) adult plant withfloral stem. (D–F) Sections ofthe shoot apex showing GUSgene expression. (D) C1-HaeIII,(E) C2-HpaII, (F) C3-DdeI. (G,H) Localization of GS1 mRNAin the shoot apical meristem ofdeveloping pine seedlingsanalyzed by in situ hybridization.(G) Tissue section hybridizedwith the sense GS1 riboprobe(pGSP114). (H) Tissue sectionhybridized with the antisenseGS1 riboprobe. (I) GUSexpression in the root tips ofC1-HaeIII plants. The arrowsindicate the area of maximalexpression. (J,K) Detection ofGS1 mRNA in the root tips ofdeveloping pine seedlings. (J)Control sections followinghybridization with the sensecontrol probe. (K) A view of theroot tip showing the specificlocation of the GS transcripts(arrow) in a few cells. Barsrepresent 200 �m.

obtained in pine cotyledons by microprojectile bombard-ment (Table 1). Moreover, this observation is consistentwith GS1 transcript location in pine seedlings (Canton et al.1999). Thus, in situ hybridization experiments performedwith specific GS sense and antisense probes (Fig. 4G,H)determined that most abundant expression of the GS1 genewas localized in the shoot apical meristem adjacent to the

position where the cotyledons arose, in good agreement withthe performed GUS assay in Arabidopsis (Fig. 4D). GUSexpression was also detected but at a minor extent in theroot meristems (Fig. 4I). Interestingly, in situ hybridizationexperiments (Fig. 4J,K) done in pine root tips showed thepresence of GS1 transcripts although expression was re-stricted to a small numberof cells. These findings suggest

Physiol. Plant. 112, 2001 393

Table 1. Transient expression in P. pinea cotyledons after bombardment with pBI121 and pBI101 as positive and negative controls,respectively, and the 3 GS-GUS fusions used in this study (Fig. 3). Results are average�SD of at least 3 separate experiments. ND, notdetectable.

Construction Number of assayed MU (pmol mg−1 proteinCotyledons expressing GUS activity Spot number permin−1)cotyledons cotyledon(%)

pBI121 128 97.7�6.1 93.0�6.1 9.6�2.1C1-HaeIII 98 4.1�1.163.3�4.9 12.1�1.7C2-HpaII 135 34.8�4.1 3.5�0.6 2.3�0.4C3-DdeI 174 19.4�3.0 1.1�0.1 1.6�0.2pBI101 68 1.1�0.7ND ND

conservation of gene expression in Arabidopsis relative toP. syl�estris. GUS expression associated with the rootmeristem has also been reported in the promoter character-ization of several GS genes in angiosperms (Brears et al.1991, Miao et al. 1991, Cock et al. 1992, Marsolier et al.1995).

Pine GS promoter activity is stimulated by light intransgenic Arabidopsis

GS abundance determined in pine seedlings was unchangedwhen they were supplied with inorganic nitrogen, eithernitrate or ammonium (Canovas et al. 1991); however, illu-mination increased the amount of the GS1 transcript (Can-ton et al. 1999). In order to determine whether or not theGS promoter contains regulatory sequences involved in theresponse to these external stimuli, GUS activity was mea-sured in 7 C1 independent transgenic lines of Arabidopsis,following either supply with ammonium or light/dark treat-ments. As shown in Fig. 5, no significant changes were

observed in NH4+-treated plants with regard to C1 con-

trols. By contrast, GUS activity levels were highly increasedby light in close agreement with light-enhanced GS tran-script abundance in pine cotyledons. Photoresponsive re-gions involving light-regulated plant promoters presentsome motifs conserved in phylogenetically distant plants(Arguello-Astorga and Herrera-Estrella 1996), and the lightresponsive elements seem to be located in the proximalregion of the promoter (Arguello-Astorga and Herrera-Es-trella 1998) as may occur in the pine GS1 gene. All thesedata strongly suggest that the 5�-upstream region of thegene characterized in this work contains a light responsiveelement on it.

Summarizing, in this paper we report for the first timethe isolation of a genomic clone corresponding to a cytoso-lic GS gene from a gymnosperm, P. syl�estris. Sequenceanalysis of the clone revealed the high homology existing insplicing sites corresponding to other GS genes previouslyreported from other sources (Tischer et al. 1986, Walker etal. 1995) indicating possible conserved functional domainsalong the evolution of GS genes in plants. Furthermore, wehave studied promoter functionality of the 5�-untranslatedregion of the gene in homologous and heterologous sys-tems. In both approaches the promoter region regulatedexpression of GUS reporter gene, although the level ofexpression of GUS was not very high. This can be due tothe fact that some cis distal regulatory elements might bemissed (Terce-Laforgue et al. 1999). However, the pine GS1promoter characterized in this work appears to contain theregulatory sequences involved in the transcriptional activa-tion by light.

Location of expression in shoot apical meristem in trans-genic plants together with main transcript location by insitu experiments in pine seedlings (Canton et al. 1999)could be related with a high rate of cellular division. In thiscontext, the nitrogen requirement for cellular metabolismor transport to other organs is quantitatively very impor-tant. Current work is in progress to further analyze thepossible cis elements present in the 5�-flanking region andits potential involvement in regulation of gene expression.

Acknowledgements – We would like to thank Remedios Crespillo(Universidad de Malaga) for her excellent technical assistance andthe research facilities of the Molecular Biology Laboratory, Re-search Services, Universidad de Malaga. This work was supportedby grants from the Spanish Ministry of Education (PB95-0482,PB98-1396 and AG95-0960-C02-01), Junta de Andalucıa (ResearchGroup CVI-0114) and Universidad de Oviedo (DF95.217-1). Thenucleotide sequence data reported are available in the EMBL,GenBank and DDBJ Nucleotide Sequence Database under theaccession number AJ225121.

Fig. 5. Effect of ammonium, light and dark treatments on GUSexpression in transgenic Arabidopsis. GUS activity in roots tipextracts was undetectable and only data derived from the shootapex are showed. Relative activities, normalized to control plants(C1) from 7 independent transformed lines, are the average�SD ofat least 3 different experiments. A relative GUS activity value of100 correspond to 30 pmol of 4-methylumbelliferone per mg ofprotein per min. Plants grown in a 16-h light/8-h dark regime weretransferred to a medium containing 10 mM NH4Cl (C1/N), contin-uous light (C1/LIGHT) or continuous dark (C1/DARK) for 3 days.A promoter-less derivative pBI101 was used as the control.

Physiol. Plant. 112, 2001394

ReferencesArguello-Astorga GR, Herrera-Estrella LR (1996) Ancestral multi-

partite units in light-responsive plant promoters have structuralfeatures correlating with specific phototransduction pathways.Plant Physiol 112: 1151–1166

Arguello-Astorga GR, Herrera-Estrella LR (1998) Evolution oflight-regulated plant promoters. Annu Rev Plant Physiol PlantMol Biol 49: 525–555

Barrett JW, Beech RN, Dancik BP, Strobeck C (1994) A genomicclone of a type I cab gene encoding a light harvesting chloro-phyll a/b binding protein of photosystem II identified fromlodgepole pine. Genome 31: 166–172

Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacteriummediated gene transfer by infiltration of adult Arabidopsisthaliana plants. CR Acad Sci Paris Life Sci 816: 1194–1199

Bradford MM (1976) A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal Biochem 72: 248–254

Brears T, Walker EL, Coruzzi GM (1991) A promoter sequenceinvolved in cell-specific expression of the pea glutamine syn-thetase GS3A gene in organs of transgenic tobacco and alfalfa.Plant J 1: 235–244

Canovas FM, Canton FR, Gallardo F, GarcIa-Gutierrez A, deVicente A (1991) Accumulation of glutamine synthetase duringearly development of maritime pine (Pinus pinaster) seedlings.Planta 185: 372–378

Canton FR, Garcıa-Gutierrez A, Gallardo F, de Vicente A,Canovas FM (1993) Molecular characterization of a cDNAclone encoding glutamine synthetase from a gymnosperm Pinussyl�estris. Plant Mol Biol 22: 819–828

Canton FR, Garcıa-Gutierrez A, Crespillo R, Canovas FM (1996)High-level expression of Pinus syl�estris glutamine synthetase inEscherichia coli. Production of polyclonal antibodies against therecombinant protein and expression studies in pine seedlings.FEBS Lett 393: 205–210

Canton FR, Suarez M-F, Jose-Estanyol M, Canovas FM (1999)Expression analysis of a cytosolic glutamine synthetase gene incotyledons of Scots pine seedlings: Developmental, light regula-tion and spatial distribution of specific transcripts. Plant MolBiol 40: 623–634

Charest PJ, Calero N, Lachance D, Datla RSS, Duchesne LC,Tsang EWT (1993) Microprojectile-DNA delivery in coniferspecies: Factors affecting assessments of transient gene expres-sion using �-glucuronidase reporter gene. Plant Cell Rep 12:189–193

Charest PJ, Devautier Y, Lachance D (1996) Stable genetic trans-formation of Picea mariana (black spruce) via particle bombard-ment. Cell Dev Biol 32: 91–99

Cock JM, Hemon P, Cullimore JV (1992) Characterization of thegene encoding the plastid located glutamine synthetase ofPhaseolus �ulgaris : Regulation of �-glucuronidase gene fusionsin transgenic tobacco. Plant Mol Biol 18: 1141–1149

Cullimore JV, Gebhardt C, Saarelainen R, Miflin BJ, Idler KB,Baker RF (1984) Glutamine synthetase of Phaseolus �ulgaris L.:Organ-specific expression of a multigene family. J Mol ApplGenet 2: 589–599

Dickey LF, Gallo-Meagher M, Thompson WF (1992) Light regula-tory sequences are located within the 5�-portion of the Fed-1message sequence. EMBO J 1: 2311–2317

Ellis DD, McCabe DE, McInnis S, Ramachandran R, Russell DR,Wallace BJ, Martinell BJ, Roberts DR, Raffa KF, McCown BH(1993) Stable transformation of Picea glauca by particle acceler-ation. Biotechnology 11: 84–89

Elmlinger MW, Bolle C, Batschauer A, Oelmuller R, Mohr H(1994) Coaction of blue light and light absorbed by phy-tochrome in control of glutamine synthetase gene expression inScots pine (Pinus syl�estris L.) seedlings. Planta 192: 189–194

Enjuto M, Lumbreras V, Marin C, Boronat A (1995) Expression ofthe Arabidopsis HMG 2 gene, encoding 3-hydroxy-3-methylglu-taryl coenzyme A reductase, is restricted to meristematic andfloral tissue. Plant Cell 7: 517–527

Forde BG, Freeman J, Oliver JE, Pineda M (1990) Nuclear factorsinteract with conserved AT-rich elements upstream of a nodule-enhanced glutamine synthetase gene from French bean. PlantCell 2: 925–939

Garcıa-Gutierrez A, Dubois F, Canton FR, Gallardo F, SangwanRS, Canovas FM (1998) Two different modes of early develop-ment and nitrogen assimilation in gymnosperm seedlings. PlantJ 13: 187–199

Hirel B, Miao GH, Verma DPS (1993) Metabolic and developmen-tal control of glutamine synthetase genes in legumes and non-legume plants. In: Verma DPS (ed) Control of Plant GeneExpression. CRC Press Inc, Boca Raton, FL, pp 443–458.ISBN 0-8493-8866-X

Huang Y, Diner AM, Karnosky DF (1991) Agrobacterium rhizoge-nes-mediated genetic transformation and regeneration of aconifer: Larix decidua. In Vitro Cell Dev Biol 27: 201–207

Humara JM, Lopez M, Ordas RJ (1998) Modifying transient�-glucuronidase expression in pine species using introns. PlantCell Tissue Organ Cult 52: 183–187

Jefferson RA (1987) Assaying chimeric genes in plants: The GUSgene fusion system. Plant Mol Biol Rep 5: 387–405

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions:�-glucuronidase as a sensitive and versatile gene fusion markerin higher plants. EMBO J 6: 3901–3907

Joshi CP (1987) An inspection of the domain between putativeTATA box and translation start site in 79 plant genes. NucleicAcids Res 12: 6643–6653

Kinlaw CS, Ho T, Gerttula SM, Gladstone E, Harry DE, QuintanaL, Baysdorfer C (1996) Gene discovery in loblolly pine throughcDNA sequencing. In: Ahuja MR, Boerjan W, Neale D (eds)Somatic Cell Genetics and Molecular Genetics of Trees. KluwerAcademic Publishers, Dordrecht, pp 175–182. ISBN 0-7923-4179-1

Kojima K, Yamamoto N, Sasaki S (1992) Structure of the pine(Pinus thunbergii ) chlorophyll a/b-binding protein gene ex-pressed in the absence of light. Plant Mol Biol 19: 405–410

Kojima K, Sasaki S, Yamamoto N (1994) Light-independent andtissue-specific expression of a reporter gene mediated by the pinecab-6 promoter in transgenic tobacco. Plant J 6: 591–596

Loopstra CA, Sederoff RR (1995) Xylem-specific gene expression inloblolly pine. Plant Mol Biol 27: 277–291

Lumbreras V, Campos N, Boronat A (1995) The use of an alterna-tive promoter in the Arabidopsis thaliana HMG1 gene generatesan mRNA that encodes a novel 3-hydroxy-3-methylglutarylcoenzyme A reductase isoform with an extended N-terminalregion. Plant J 8: 541–549

Marsolier MC, Debrosses G, Hirel B (1995) Identification of severalsoybean cytosolic glutamine synthetase transcripts highly orspecifically expressed in nodules: Expression studies using one ofthe corresponding genes in transgenic Lotus corniculatus. PlantMol Biol 27: 1–15

McNally SF, Hirel B, Gadal P, Mann F, Stewart GR (1983)Glutamine synthetase isoforms of higher plants. Plant Physiol72: 22–25

Miao G-H, Hirel B, Marsolier MC, Ridge RW, Verma DPS (1991)Ammonia-regulated expression of a soybean gene encoding cy-tosolic glutamine synthetase in transgenic Lotus corniculatus.Plant Cell 3: 11–22

Miflin BJ, Lea PJ (1976) The pathway of nitrogen assimilation inplants. Phytochemistry 15: 873–885

Murashige T, Skoog F (1962) A revised medium for rapid growthand bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

Rey M, Humara JM, Lopez M, Gonzalez MV, Rodrıguez R,Tavazza R, Ancora G, Ordas RJ (1996) Foreign gene expressionin Pinus nigra, P. radiata and P. pinea following particle bom-bardment. In: Ahuja MR, Boerjan W, Neale D (eds) SomaticCell Genetics and Molecular Genetics of Trees. Kluwer Aca-demic Publishers, Dordrecht, pp 113–117. ISBN 0-7923-4179-1

Sakamoto A, Ogawa M, Masumura T, Shibata D, Takeba G,Tanaka K, Fujii S (1989) Three cDNA sequences coding forglutamine synthetase polypeptides in Oryza sati�a L. Plant MolBiol 13: 611–614

Sambrook J, Fritsch E, Maniatis T (1989) Molecular Cloning aLaboratory Manual. Cold Spring Harbor Press, Cold SpringHarbor, NY. ISBN 0-97969-577-3

Sanger F, Miklen S, Coulson AR (1977) DNA sequencing withchain termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467

Physiol. Plant. 112, 2001 395

Simpson CG, Leader DJ, Brown JWS (1993) Characteristics of plantpre-mRNA introns and transposable elements. In: Croy RRD(ed) Plant Molecular Biology Labfax. BIOS Scientific Publishers,Oxford, pp 183–251. ISBN 0-12-198370-6

Simpson GG, Filipowicz W (1996) Splicing of precursors to mRNAin higher plants: Mechanism, regulation and sub-nuclear organi-zation of the spliceosomal machinery. Plant Mol Biol 32: 1–41

Terce-Laforgue T, Carrayol E, Cren M, Desbrosses G, Hecht V, HirelB (1999) A strong constitutive positive element is essential for theammonium-regulated expression of a soybean gene encodingcytosolic glutamine synthetase. Plant Mol Biol 39: 551–564

Tingey SV, Tsai F-Y, Edwards JW, Walker EL, Coruzzi GM (1988)Chloroplast and cytosolic glutamine synthetase are encoded byhomologous nuclear genes which are differentially expressed invivo. J Biol Chem 263: 9651–9657

Tischer E, Dasbarma S, Goodman HM (1986) Nucleotide sequenceof an alfalfa glutamine synthetase gene. Mol Gen Genet 203:221–229

Walker E, Weeden N, Taylor C, Green P, Coruzzi G (1995)Molecular evolution of duplicate genes encoding cytosolicglutamine synthetase in Pisum sati�um. Plant Mol Biol 29:1111–1125

Edited by J. K. Schjo�rring

Physiol. Plant. 112, 2001396