Plant Science 150 2000 153 – 161
Identification of light- and nitrate-responsive regions of the nitrate reductase promoter from birch
Tim Strater, Wolfgang Hachtel
Botanisches Institut, Uni6ersita¨t Bonn, Kirschallee
1
, D-
53115
Bonn, Germany Received 8 February 1999; received in revised form 19 August 1999; accepted 10 September 1999
Abstract
The regulation of nitrate reductase NR gene expression in higher plants involves both internal and external factors. In birch Betula pendula Roth, the transcription of the NR gene depends on light and nitrate in both leaves and roots. In order to
investigate the transcriptional regulation of the birch NR gene, a 1.6 kb 5 flanking sequence of the NR gene was isolated, sequenced, and the transcription start site was determined. The 1.6 kb sequence and eight 5 deleted versions of the promoter
sequence were each fused with the b-glucuronidase GUS reporter gene uidA and introduced into Nicotiana plumbaginifolia. GUS expression was examined in primary transformants that were transferred from dark to light or from a medium without
nitrate to a nitrate-containing medium. A detailed analysis revealed the presence of light- and nitrate-responsive promoter regions, respectively. Several promoter regions conferred a differential expression of the GUS gene between leaves and roots. © 2000
Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Birch Betula pendula; Light response; Nitrate response; Nitrate reductase; Promoter analysis www.elsevier.comlocateplantsci
1. Introduction
Nitrate reductase NR catalyses the reduction of nitrate to nitrite, the first step of nitrate assimi-
lation. Nitrite is subsequently reduced to ammo- nium by nitrite reductase, and ammonium is then
incorporated into amino acids. The expression of higher plant NR genes is regulated by a number of
factors including nitrate, light, circadian rhythms, carbohydrate levels, nitrogen metabolites, and
phytohormones [1]. Because nitrate assimilation requires both a high input of energy and carbon
skeletons, the complex regulation of NR gene expression can be viewed most simply as a balanc-
ing device between nitrate assimilation and carbon fixation [2].
Despite the extensive study of NR gene expres- sion, little is known about DNA sequences involved
in the regulation of higher plant NR genes [3]. Moreover, molecular approaches to study nitrate
reduction have focused on herbaceous angiosperms while the molecular physiology of nitrate assimila-
tion in woody species virtually has been ignored for a long time. On the other hand, nitrogen has been
recognised to be a critical factor in forest ecosystems and world wide observed forest decline. For these
reasons we decided to investigate NR gene expres- sion in the European white birch Betula pendula
Roth. cDNA clones encoding NR were isolated from birch [4] and used for quantitative Northern
analyses. When young birch plants were grown on ammonium as the nitrogen source, NR mRNA was
found to be very rare in leaves and undetectable in roots. Upon transfer of the plants to a nitrate-con-
taining medium, levels of NR mRNA and activity dramatically increased in leaves and roots in a
strictly light dependent manner. In darkness, no transcripts and activities were found [5].
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the ac-
cession number AJ001725. Corresponding author. Tel.: + 49-228-735-584; fax: + 49-228-
735-502. E-mail address
:
unb142ibm.rhrz.uni-bonn.de W. Hachtel 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 8 2 - X
In order to identify promoter regions involved in the regulation of the birch NR gene expression,
the 1535 bp sequence 5 flanking this gene, and eight sequences obtained by sequential 5 deletion
were fused to the b-glucuronidase GUS reporter gene. The fusion constructs were introduced into
Nicotiana plumbaginifolia via agrobacteria. Trans- formants harbouring a distinct NR promoter GUS
fusion either were grown on a medium containing nitrate, and GUS activity was determined at the
end of both prolonged darkness 3 days and a 3-h light period thereafter, or GUS activity was com-
pared between nitrate-grown and ammonium-fed plants.
2. Materials and methods
