Structural and functional characterization of the 5' upstream region of a glutamine synthetase gene from Scots pine

Ann. For. Sci. 59 (2002) 669–673
669
© INRA, EDP Sciences, 2002
C. Avila
Scots
et
pine al.
GS1a promoter
DOI: 10.1051/forest:2002054
Original article
Structural and functional characterization of the 5’ upstream region
of a glutamine synthetase gene from Scots pine
Concepción Avilaa, Francisco R. Cantóna, Pilar Barnesteina,
María-Fernanda Suáreza, Pierre Marraccinia**, Manuel Reyb, Jaime M. Humarac,
Ricardo Ordásc and Francisco M. Cánovasa*
a Departamento de Biología Molecular y Bioquímica, Instituto Andaluz de Biotecnología, Unidad Asociada UMA-CSIC Facultad de Ciencias,
Universidad de Málaga, 29071 Málaga, Spain
b Laboratorio Fisiología y Biotecnología Vegetal, Facultad de Ciencias, Universidad de Vigo, 36200 Vigo, Spain
c Laboratorio Fisiología Vegetal, Departamento BOS, Universidad de Oviedo, 33071 Oviedo, Spain
(Received 5 July 2001; accepted 25 January 2002)
Abstract – We report here the isolation and characterization of a genomic clone encoding Scots pine (P. sylvestris) cytosolic glutamine synthe-
tase GS1a. The clone contains the 5’ half of the gene including part of the coding region organized in seven exons, interrupted by 6 introns and
980 bp upstream of the translation initiation codon. Earlier experiments carried out in our lab have shown that the GS1a gene is expressed in a
light dependent fashion during the initial stages of Scots pine development. These data suggest a specific role for GS1a in ammonia assimilation
in photosynthetic tissues of pine seedlings similar to the physiological role of GS2 in angiosperms. We have used a transcriptional fusion to uidA
to transform pine cotyledons and Arabidopsis and demonstrated the ability of this 5’-upstream sequence to drive gene expression in both species
and light regulation in Arabidopsis.
cytosolic glutamine synthetase / conifer / gene expression / N metabolism
Résumé – Caractérisation structurale et fonctionnelle de la région 5’ du gène de la glutamine synthetase du pin sylvestre. Un clone géno-
mique codant la glutamine synthetase cytosolique GS1a de pin sylvestre (P. sylvestris) a été isolé et caractérisé. Ce clone contient la moitié 5’ du
gène comprenant une partie de la séquence codante organisée en 7 exons séparés par 6 introns et également une séquence de 980 pb en amont du
codon d’initiation de la traduction. Des expériences préliminaires menées dans notre laboratoire ont montré que la lumière régule l’expression du
gène GS1a pendant les étapes initiales du développement du pin sylvestre. Ces données suggèrent un rôle spécifique de GS1a dans l’assimilation
des ions ammonium par les tissus photosynthétiques des plantules de pin analogue au rôle physiologique de GS2 chez les angiospermes. Nous
avons préparé une fusion transcriptionnelle avec le gène uidA pour transformer des cotylédons de pins ainsi qu’Arabidopsis. Nous avons ainsi
démontré la capacité de cette séquence 5’ de 980 pb à diriger (1) l’expression du gène chez ces deux espèces et (2) sa régulation par la lumière
chez Arabidopsis.
glutamine synthetase cytosolique / conifère / expression génique / métabolisme de l’azote
1. INTRODUCTION
ammonia released by photorespiration and other metabolic
processes. The various roles of GS in plant metabolism are
Glutamine synthetase (GS) plays a central role in nitrogen
undertaken by different isoforms encoded by a small
metabolism of higher plants. GS is responsible for the pri-
multigene family [10]. As occurs in angiosperms it seems
mary assimilation of ammonia produced by nitrate reduction
that a small multigene family could be operative in gymno-
or fixation of dinitrogen as well as the reassimilation of
sperms [12]. In the last years our studies have focused on
* Correspondence and reprints
Tel.: 3452131942; fax: 3452132000; e-mail: canovas@uma.es
** Current address: Nestlé Research Center Tours, Plant Science and Technology, 101 Gustave Eiffel, BP 9716, 37097 Tours Cedex 2, France

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670
C. Avila et al.
ammonia assimilation in pine and studying regulation of the
vacuum infiltrated as is described elsewhere [5]. T1 seeds were har-
genes involved in the process [7]. Two distinct but homolo-
vested in bulk and transformed seeds were selected in MS plates
gous nuclear genes for GS have been detected and
containing 50 µg mL–1 kanamycin. T2 seeds were harvested individ-
ually and kept for further analysis.
colocalized in the pine genome [2] both of them encoding
cytosolic isoforms in conifers, GS1a and GS1b [1], but differ-
Histochemical GUS assays in bombarded cotyledons were per-
formed as described by Rey et al. [19] whereas the fluorometric as-
entially expressed in pine seedlings [3].
say of gus in extracts of transgenic Arabidopsis were performed as
Molecular data derived from the characterization of a
described by Jefferson [14]. A 35S-promoter derivative pBI121 and
GS1a cDNA clone showed that the gene is actively expressed
promoter-less pBI101 plasmids were used as controls.
in chloroplast containing tissues of developing seedlings
and the level of the transcript was affected by developmental and
2.4. Gel retardation analysis
light conditions [9]. Here, we present the DNA sequence of a
partial genomic clone containing 7 exons of the coding region
A DNA fragment used for gel retardation analysis containing a
of GS1a gene. The clone includes 980 bp upstream of the
sequence from the 5’-untranslated region of GS1a was obtained by
functional ATG. So far, very few studies involving genomic
cleavage with restriction enzymes of the genomic clone pGS217.
The fragment containing the A/T– rich region of 173 bp long was
clones from gymnosperms have been reported in the litera-
electrophoresed in 5% acrylamide gels excised and eluted by diffu-
ture [4,16,18], and none of them correspond to nitrogen me-
sion into 0.5 M NH OAc. Binding was carried out in 15 µl of 10 mM
4
tabolism. We have studied the promoter activity of the
Tris (pH 8), 1 mM EDTA, 100 mM NaCl, 2 mM DTT, 10% glycerol
5’-untranslated region using fusions with the reporter gene
and 2 µg of denatured salmon sperm DNA (binding buffer). The
uidA and the presence of DNA-protein interactions in the
DNA (1–2 ng) labeled by filling in reaction with Klenow was incu-
5’flanking region of GS1a gene from Scots pine.
bated with 4 µg of crude nuclear extract as a source of protein as de-
scribed previously [11]. Mixes were incubated for 30 min on ice. In
non specific competition experiments 0 to 0.5 µg of poly dI-dC was
also included in the mixes. At the end of the incubation period 1/10th
2. MATERIALS AND METHODS
of the mix volume of loading buffer was added and samples were
loaded on a 5% polyacrylamide 2% glycerol pre-electrophoresed gel.
Running buffer was 0.5 × TBE. Gels were run in the cold room at
2.1. Isolation of a genomic clone containing GS1
10 V cm–1 for 2–5 h.
sequences in pine
Scots pine genomic DNA was digested with EcoRI and size frac-
tionated by electrophoresis. Fragments were ligated to ?gt10 and re-
3. RESULTS AND DISCUSSION
combinant clones containing the GS1a gene were identified by
screening using the 5’end of the cDNA clone previously isolated
[8].
3.1. Sequencing and structural characteristics
The fragment released from one of these clones by enzyme di-
of the pine GS1 genomic clone
gestion was subcloned into the plasmid pGEM-3Z to generate the
clone pGS217 and used for analysis and sequencing.
A ?gt10 subgenomic library of Scots pine was screened
for GS clones using the previously isolated pGSP114 pine GS
2.2. Fusions of the 5’region of GS1a to the GUS
cDNA [8]. About 1 × 106 recombinant clones were screened
reporter gene
and four positives were isolated. One of these, pGS217 was
subcloned and further characterized. As an initial step the
The 981 bp sequence upstream of the translation codon was iso-
genomic clone was entirely sequenced and determined to be
lated from the clone by Hae III digestion. The resulting fragment
2543 bp in length. The comparison of nucleotide sequences
was subcloned into the vector pBI101 [15] creating a GS1: uidA
between the gene and the cDNA [8] showed that the fragment
gene fusion. The GS1 T-DNA construct, and also two controls,
contained the 5’ half of the gene including part of the coding
which were the original plasmid pBI121 containing the CaMV 35S
promoter and pBI101, a plasmid containing a promoter-less 1.87 Kb
region organized in seven exons, interrupted by 6 introns and
GUS cassette in the binary vector pBin19, were transformed indi-
980 bp upstream the translation initiation codon (figure 1).
vidually in Agrobacterium tumefaciens LBA4104.
The sizes of introns in the GS1 genomic clone were be-
tween 91 bp and 282 bp as shown in table I, all of them having
2.3. Transient and stable transformation
the usual range size for angiosperm introns, which are typi-
with the gene constructs
cally shorter than most mammalian introns [23]. The AT per-
centage in higher plants introns is usually between 70%
A Biolistic PDS-1000/He apparatus from Bio-Rad was used for
described for dicot plants and around 60% for monocot plants
particle bombardment of P. pinea cotyledons excised from embryos
germinated for one day. After bombardment, cotyledons were main-
[22]. Unfortunately not many data are available for gymno-
tained in the same medium where they were bombarded until GUS
sperm genes, however the introns in the GS1 clone showed an
assays were performed 24 h after as described before [19].
average AT percentage around 64%, which is within the
For stable transformation, Arabidopsis thaliana WS ecotype
range reported for angiosperms. We have also analyzed the
plants were grown at 24 oC under a 16 h light/8 h dark regime and
sequences of 5’and 3’splice sites in all 6 pine cytosolic GS

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Scots pine GS1a promoter
671
Table II. Effect of ammonium, light and dark treatments on GUS ex-
pression in transgenic Arabidopsis grown at 24 ºC under a 16 h
light/8 h dark regime. Plants at the rosette stage were used. GUS ac-
tivity was undetectable in roots and only data from the shoot apex are
showed. Activities of C1 plants from 7 independent transformed lines
were determined individually. The average +/– SD data of at least
3 different experiments are shown. Plants grown in a 16–h light/8–h
dark regime were transferred to a medium containing 10 mM NH4Cl
(C1/N), continous light (C1/light) or continuous dark (C1/dark) for
3 days. A promoter-less derivative pBI101 was used as control.
Sample
MU (pmol mg–1 protein min–1)
Control (–)
1.47
Figure 1. Comparative diagram of the pGS217 genomic clone and the
C1
31.6 +/–1.05
full-length cDNA corresponding to the GS1a clone. Closed boxes
C1/N
24.6 +/–3.16
represent coding regions. Exons are denoted by roman numbers from
C1/light
60.9 +/–2.57
I to VII. Untranslated regions including introns are represented by a
C1/dark
3.05 +/–0.75
bar. The nucleotide sequence data reported will appear in the EMBL
data bank under the accession number AJ 225121.
gene was absent or very low at the seedling and rosette stages,
but apparent in adult plants with floral stems. These data
Table I. Characteristics of introns in the pGS217 pine genomic clone.
therefore show that the 5’upstream region of pine GS1a gene
Intron nº
Size (bp)
(%) A/T
(%) Pyrimidines
is able to drive gene expression in an heterologous system.
1
107
62.6
40
Moreover, our results are consistent with the report of
2
91
63.7
65
Kojima et al. [17] indicating that a pine gene promoter can be
3
282
69.8
50
operative in angiosperms and therefore suggesting that
4
96
63.5
40
transcriptional machinery is well conserved between angio-
5
236
60.4
70
sperms and gymnosperms.
6
151
63.6
60
GS1a abundance determined in pine seedlings was un-
changed when they were supplied with either inorganic nitro-
gen, nitrate or ammonium [6], however illumination
increased the amount of the GS1 transcript [9]. In order to de-
introns and compared them with monocot and dicot plants,
termine whether or not the expression driven by the 980 bp
yeast consensus and vertebrate splice consensus sequences.
sequence from the GS1a gene is affected by these external
The strict requirement in both sides of the intron for: G/GT in
stimuli in an heterologous system, GUS activity was mea-
the 5’site and AG/G in the 3’end indicates a general use in all
sured in seven C1 independent transgenic lines following ei-
compared organisms.
ther supply with ammonium or light/dark treatments. As
We have also analyzed the presence of putative elements
shown in table II, no meaningful changes were observed in
in the 5’ region of the gene. There is a canonical TATA box at
NH +-treated plants with regard to controls. By contrast, GUS
4
–35 bp from the transcription start site and a putative CAAT
activity levels were highly influenced by light in close agree-
box at –138 bp. The 5’ region also contains two A/T-rich se-
ment with light-enhanced GS transcript abundance in pine
quences starting at –720 and –540 and 173 and 190 bp long
cotyledons.
respectively.
3.3. Analysis of DNA-protein interactions
3.2. GUS expression in pine cotyledons and transgenic
in the 5’ region of GS1a gene from Scots pine
Arabidopsis
We have carried out an in vitro study of interactions
A transcriptional construct (C1) containing the complete
between nuclear factors from Scots pine cotyledons and an
980 bp upstream the translation initiation codon fused to the
A/T-rich sequence in the upstream region of GS1a gene
GUS gene was created. The chimeric gene was used to test
using the technique of gel retardation analysis. The frag-
transient expression in P. pinea cotyledons. According to
ment was end labeled with 32P and incubated with crude nu-
GUS histochemical assays, the 5’upstream region of the pine
clear
extracts
from
Scots
pine
cotyledons.
The
gene was able to drive gene expression in pine cotyledons. To
concentration of salmon sperm DNA and poly dI-dC
further characterize the function of the 5’upstream region of
needed to eliminate non specific binding was first estab-
the pine GS1a gene, stable GUS expression was studied in
lished (2 µg and 0.5 µg per assay, respectively). The binding
transformed Arabidopsis plants. Expression of the reporter
reactions were electrophoresed on acrylamide gels to resolve

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672
C. Avila et al.
21]. We have identified the presence of cis elements in a light
responsible promoter of a conifer GS1 gene, but still experi-
mental work is necessary to characterize further if the puta-
tive cis elements present in AT–1 region are functionally
involved in regulation of the GS1a gene expression by light.
Acknowledgements: We would like to thank Remedios
Crespillo (Universidad de Málaga) for her excellent technical assis-
tance and the research facilities of the Molecular Biology Labora-
tory, Research Services, Universidad de Málaga.
The nucleotide sequence data reported are available in the EMBL,
GenBank and DDBJ Nucleotide Sequence Database under the ac-
cession number AJ225121.
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