Plant Science 155 2000 141 – 151
Functional analysis of a nitrite reductase promoter from birch in transgenic tobacco
,
Hans Olaf Warning, Wolfgang Hachtel
Botanisches Institut, Uni6ersita¨t Bonn, Karlrobert-Kreiten-Str.
13
, D-
53115
Bonn, Germany Received 8 December 1999; received in revised form 27 January 2000; accepted 31 January 2000
Abstract
Nitrate assimilation is a highly regulated process in higher plants, and the regulatory cues governing gene expression in this pathway include both external and internal factors. In birch Betula pendula Roth the expression of nitrate reductase NR and
nitrite reductase NiR genes is co-regulated by light and nitrate at the transcriptional level. In order to identify cis-acting DNA-elements involved in light and nitrate induction of the birch NiR gene, a 0.9 kb 5 flanking region of the NiR gene was
isolated, analysed on the DNA level, and the transcription start site was determined. Deletion analysis of the birch NiR promoter region fused to the GUS reporter gene uidA in transgenic tobacco Nicotiana tabacum revealed the presence of light- and
nitrate-responsive promoter fragments. The responsive fragments showed different activities in leaves and roots. Further, gel mobility shift assays using nuclear proteins from leaves detected a specific DNA-binding activity to the sequence between − 146
and − 267 bp that was induced in darkness and disappeared in the light. The deletion analysis has shown that this region is critical for light inducibility of the birch NiR gene in leaves. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Birch Betula pendula; DNA-binding activity; Light response; Nitrate response; Nitrite reductase; Promoter analysis www.elsevier.comlocateplantsci
1. Introduction
Nitrate is the major source of nitrogen available to plants. The assimilation of nitrate involves the
following reactions: its uptake from the external medium by specific translocators, its reduction to
nitrite in the cytosol by nitrate reductase NR, the reduction of nitrite to ammonium in the plastids
by nitrite reductase NiR, and the incorporation of ammonium into amino acids by the glutamine
synthethaseglutamate synthase cycle. Nitrate as- similation is a highly regulated process, and the
regulatory cues governing gene expression and enzyme activity in this pathway include nitrate,
end-products of nitrogen assimilation, light, su- crose, and cytokinins reviewed in [1].
Whereas the molecular mechanisms underlying the induction of nitrate assimilation in higher
plants remain to be elucidated, promoter analyses of the NR genes of Arabidopsis thaliana [2,3]and
Betula pendula [4]and of the NiR gene of spinach [5 – 9], Phaseolus 6ulgaris [10] and tobacco [11]
have revealed cis-acting DNA elements involved in nitrate induction.
While molecular approaches to study nitrate assimilation in plants have focused on herbaceous
species, much less is known for woody species. We decided to investigate the nitrate assimilation sys-
tem of the European white birch B. pendula Roth for two reasons: nitrogen has been recognised to
be a critical factor in forest ecosystems and world wide forest decline, and B. pendula is involved in
forest disease. In birch, the rate of transcription of
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the ac-
cession number AJ242953. In memoriam Dr Klaus Brinkmann, Professor of Botany at the
University of Bonn, who died on February 11, 2000. He encouraged me Wolfgang Hachtel almost 10 years ago to work on molecular
aspects of nitrate assimilation in birch. Corresponding author. Tel.: + 49-228-735584; fax + 49-228-
731697. 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 0 0 0 0 2 1 1 - 9
the NR and the NiR gene was shown to be co-regulated by nitrate and light [12]. In the
present investigation we analyse various promoter regions of the birch NiR gene in an attempt to
identify their role in light and nitrate induction. Further we examine whether light andor dark,
nitrate andor ammonium promotes DNA-binding activity to distinct birch NiR promoter sequences.
2. Materials and methods
