Plant Molecular Biology 39: 551–564, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
551
A strong constitutive positive element is essential for the
ammonium-regulated expression of a soybean gene encoding cytosolic
glutamine synthetase
Th
´
er
`
ese Terc
´
e-Laforgue
1
, Elisa Carrayol
1
,Mich
`
ele Cren
1,2
, Guilhem Desbrosses
1
,Val
´
erie
Hecht
1
and Bertrand Hirel
1,∗
1
Laboratoire du M´etabolisme et de la Nutrition des Plantes, INRA, Centre de Versailles, Route de St Cyr,
78026 Versailles Cedex, France (
∗
author for correspondence);
2
Universit´e de Versailles-St Quentin-en-Yvelines,
D´epartement de Biologie, 45 avenue des Etats Unis, 78035 Versailles Cedex, France
Received 7 May 1998; accepted in revised form 26 September 1998
Key words: ammonium, gene expression, glutamine synthetase, nodules, positive element, promoter
Abstract
In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean
gene GS15 encoding cytosolic glutamine synthetase, a series of 5
promoter deletions were fused to the GUS
reportergene. To allow thedetection of positiveand negativeregulatoryelements,a seriesof 3
deletions were fused
to a −90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of constructwere introduced
into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both
spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and
shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the
promoter distal region is indispensable for the ammonium-regulated expression of GS15. Interestingly, this SCPE
was able to direct constitutive expression in both a legume and non-legume background to a level similar to that
driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located
in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression.
This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or
nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence
was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-
specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that
these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA
for trans-acting factor accessibility.
Introduction
In contrast to procaryotes [19, 59, 61], and fungi [39],
metabolic regulation of gene expression has not been,
up to now, thoroughly investigated in higher plants
because of the relatively few examples of enzymatic
activity controlled by carbon and nitrogen metabolites
[30]. However, in a number of plant species, such reg-
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession number AJ011009.
ulation has been extensively described for the reaction
catalysed by the enzyme nitrate reductase for which
both gene transcription and neosynthesis of the pro-
tein are under control of nitrate availability [7, 14, 27].
More recently, an increasing number of studies have
shown that, in addition to external factors or devel-
opmental regulation, many genes can be induced by
the metabolic status of the plant and that either soluble
carbohydrates and/or nitrate can exert specific [2, 10,
27, 60] or pleiotropic control on the major carbon or