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-53115Bonn,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 pendulaRoth) 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.com/locate/plantsci
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 synthethase/glutamate 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.pendulaRoth) 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:[email protected] (W. Hachtel)
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 and/or dark, nitrate and/or ammonium promotes DNA-binding activity to distinct birch NiR promoter sequences.
2. Materials and methods
2.1. DNA and RNA analysis
Techniques for DNA manipulation were as de-scribed in Sambrock et al. [13]. DNA was se-quenced by the dideoxy chain-termination method. DNA for PCR analysis was isolated ac-cording to Cheung et al. [14] from leaves of trans-genic Nicotiana tabacum. RNA was isolated from leaves of young birch plants according to Shirzagedan et al. [15] and used for primer exten-sion analysis [16].
2.2. Electrophoretic mobility shift analysis
Nuclear proteins were prepared from leaves ac-cording to Nelson et al. [17]. The protein prepara-tion was further purified by affinity chromatography using heparin agarose (Sigma-Aldrich, Munic). Proteins were eluted by 250, 550 and 1000 mM KCl, and the fraction between 250 and 550 mM KCl was used for DNA binding assays. DNA fragments of the 5% upstream region flanking the NiR gene were amplified by PCR using specific primers that were complementary to promoter and leader sequences of the birch NiR gene. The primers were designed to obtain an
overlap between adjoining fragments. PCR prod-ucts were purified with PCR purification kit (Quia-gen, Hilden), restricted with HindIII, and 5%
overhangs were filled in by Klenow enzyme (Roche Diagnostics, Mannheim) and dNTPs in-cluding [a-32P]-dATP. Probes were subsequently
purified by the PCR purification kit. To 10 ml of the sample containing the desired amount of protein (4 – 10mg) dissolved in binding buffer (14% glycerol v/v, 15 mM HEPES, 8 mM Tris, 120 mM KCl, 0.14 mM EDTA, 7 mM b-mercaptoethanol, and 0.1 mM PMSF, pH 7.5), 10 ml of a mixture containing 15 000 cpm 32P-labelled DNA probe
and at least 1 mg poly dAdT per 10 mg protein as an non-specific competitor was added. For compe-tition experiments, the appropriate volume of wa-ter was replaced with solutions of the unlabelled DNA probe. The mixture was loaded on a 4% polyacrylamide gel and electrophoresed at 150 V for 2 – 3 h. Following electrophoresis, gels were dried under vacuum on Whatman 3 MM paper, and exposed to Kodak XAR5 film overnight.
2.3. Construction of NiR promoter GUS gene fusions
In order to construct various birch NiR pro-moter GUS gene fusions we used a genomic 885 bp HindIII/EcoRI fragment that contains 735 bp 5%upstream and 150 bp 3%downstream of the NiR
transcription start site. This fragment was am-plified between positions +148 and −735 by PCR using appropriate primers. Five subfrag-ments extending from position +148 each to posi-tion −582, −445, −304, −155 and −56, respectively, were produced in the same way. The primer designed for annealing with the 3% end of the HindIII/EcoRI fragment was equipped with a
BamHI sequence motive to provide a BamHI restriction site to the downstream end of each of the PCR products, and was used in all reactions. The PCR primers designed for annealing with various sequences 5% upstream all were equipped
with a HindIII motive to provide a HindIII re-striction site to the upstream end of each of the PCR products. The produced promoter fragments are listed in Table 1. All promoter fragments were completely sequenced to exclude errors introduced into the sequence during PCR. The BamHI/
HindIII-cut PCR products were inserted into the polylinker of the GUS-encoding plasmid vector
Table 1
PCR fragments containing a NiR 5% untranslated leader
se-quence and a NiR promoter fragment used to construct GUS gene fusions
pBI101 (Clontech, Heidelberg) in fusion to the GUS gene. The plasmid pBI101 is a 12.2 kb derivative of the binary plasmid pBIN19 isolated from Agrobacterium tumefaciens [18]. In pBI101, the GUS gene is flanked at the 3% end by the
polyadenylation signal of the nopaline synthase gene from theA. tumefaciensTi plasmid, and by a multiple cloning site at the 5% end. Further
up-stream is the neomycine phosphotransferase II gene providing resistance against kanamycin fused with a nopaline synthase promoter and termina-tor. Verification of all constructs was carried out by restriction enzyme digests, PCR, and sequencing.
2.4. Transformation of N. tabacum, growth of transformants, and fluorimetric GUS assay
The pBI101-derived recombinant vectors were introduced intoA. tumefaciensstrain LBA4404 by the direct transformation method [19]. Addition-ally, A. tumefaciens cells were transformed using pBI121 (Clontech). In this vector, a cauliflower mosaic virus (CaMV) 35S promoter GUS gene fusion is inserted. For selection of transformants,
A. tumefaciens cells were grown on a medium containing streptomycin and kanamycin. Success-ful transformation was demonstrated by restric-tion analysis of A. tumefaciens DNA, PCR, and sequencing. The infection of leaf discs from N.
tabacum with A. tumefaciens, the selection of transformed calli by the use of kanamycin, and the regeneration of transgenic plants was performed as described [20]. A PCR approach was employed to verify that regenerated kanamycin resistant N. to
-bacum plants contain the desired NiR promoter GUS construct. PCR products were identified by their size and by hybridisation with digoxigenin labelled DNA probes (DIG-DNA-Labeling and Detection Kit, Roche). One of these probes was the promoter fragment −56 spanning from nucle-otide position +148 to position −56, another was an internal GUS gene fragment.
Transgenic plantlets grown on a solidified Mu-rashige and Skoog (MS) medium containing 300 mg ml−1 kanamycin were transferred to pots and
further cultivated in a mixture of swelled clay and quartz sand, and liquid nutrient medium was added as necessary. The medium contained macroelements, microelements, and potassium ni-trate (5 and 10 mM, respectively). In some
experi-ments investigating nitrate induction, plants were cultivated for some weeks without an external nitrogen source. Plants used for electrophoretic mobility shift analysis were grown on either 5 mM KNO3or 5 mM NH4Cl for at least 4 weeks. Plants
were kept under a 16-h light/8-h dark regime at 22 – 24°C in a plant growth chamber. Light was generated by a cool-white fluorescent source with a light intensity at 150 mmol m−2 s−1.
Extraction of GUS from leaves and roots of primary transformants and fluorimetric assays for GUS activity were performed according to Jeffer-son [18] with methylumbelliferone (MU) as a stan-dard. Leaf material was taken from leaf number five counting from the top all leaves of 3 cm and more in length. Leaf number five gave the highest activity among all leaves of a plant. Root material was taken from various parts of the root bale of a plant. In about 75% of all primary transformants, GUS activity was found to be significantly above that of wild type plants. Only plants with GUS activities at least threefold higher than that of the wild type were included in the studies.
2.5. Statistical analysis
Gene expression in populations of first-genera-tion transgenic plants usually does not follow a normal distribution. The measure most suitable to describe the location of an unknown distribution probably is the median [21]. As a distribution-free statistical method we employed the non-paramet-ric Mann – Whitney U-test that does not use the actual measurements, but instead the ranks of the measurements. This method was also used to test proposed hypotheses using a multiplication con-stant [22].
3. Results
3.1. 5% flanking sequences of a birch NiR gene
Fig. 1. DNA sequence of genomic birch DNA comprising part of the N-terminal coding sequence of the NiR gene and the 5%
flanking sequence. The complete NiR-coding sequence as derived from a cDNA clone has been published [22]. The ATG translation start codon and the transcription start (+1) are marked in bold and underlined. AEcoRI restriction site at the 5%end and aHindIII site within the untranslated leader are indicated. The TATA box, GATA/TATC boxes, and AGTCA and TGAGT motifs are underlined. Nucleotides identical between the genomic DNA sequence and the cDNA sequence [22] are given in italics. (EMBL GenBank Accession No. AJ242953 and X60093).
the ATG translation start codon as published [23], and additional 903 bp 5%upstream of the translation start (Fig. 1).
Identity was found between 252 nucleotides out of 254 nucleotides of the amino-terminus of the NiR encoding sequences derived from the genomic clone isolated in this work and the cDNA [23] corresponding to 99.2% identity. Of the 5% untrans-lated sequence, 46 bp adjacent to the translation start are identical between the genomic clone and the cDNA clone [23] whereas there is no similarity further upstream. This is most probably the result of a cloning artefact at the 5%end of the cDNA as
already dicussed [23]. The transcription start site (position +1) of the NiR gene was mapped by primer-extension analysis [16] and found to be an A located 161 bp upstream the translation start (data not shown). This is additional proof that the untranslated leader sequence comprising 441 nucle-otides of the cDNA [23] is not correct.
3.2. Light-regulated expression of NiR promoter GUS gene fusions
The 735 bp sequence 5% upstream of the tran-scirption start site of the birch NiR gene plus 150 bp of the untranslated leader, and five 5% deleted fragments extending to positions −582, −445,
−304, −155 and −56, respectively, were fused to the GUS reporter gene. The fusion constructs were introduced into N. tabacum via agrobacteria.
In order to identify NiR promoter sequences involved into light-dependent GUS gene activa-tion, primary transformants of N. tabacum were transferred to darkness for 72 h to minimise GUS activity. Vitality was reduced when plants were kept in the dark for a longer period. The plants were illuminated again with a light intensity of 150 mmol m−2 s−1 generated by a cool-white
illumi-nation of the roots. GUS activity was measured in extracts obtained from leaves immediately at the end of the dark period and 3, 6, 12 and 15 h after the onset of light. Relative GUS activity after 12 h of illumination was set to 100% for each of the different constructs. At the end of the 72 h dark period, and after 3, 6 and 15 h of exposure to light, 43, 81, 89 and 98% GUS ac-tivity, respectively, was detected in extracts from plants harbouring the −735 NiR-GUS con-structs (mean of ten plants). The induction kinet-ics was the same for all the constructs. Quite a similar time-dependent increase of GUS activity following a dark-to-light transition of the plants was observed in root material. Therefore, we compare the values of GUS activity obtained at the end of a 72 h dark period and after 12 h of light.
The 5%deleted NiR promoter fragments (Table 1)
confer GUS gene expression to both leaves (Table 2) and roots (Table 3) of primary transformants. However, GUS activity in plants harbouring the
−56 construct was very low, particularly in leaves. Significantly enhanced GUS activity after 12 h of light is seen in leaves harbouring at least 304 bp of the birch NiR promoter sequence (P50.004)
(Table 2). The 155 bp fragment, however, does not confer light inducibility of GUS activity in leaves. This is in contrast to the situation found in roots, where a 4.4-fold increased GUS activity was ob-served in plants harbouring the 155 bp NiR pro-moter (P=0.006) (Table 3). Enhanced light induced GUS activity is most pronounced in the presence of the 304 bp NiR promoter in leaves and of the 155 bp NiR promoter in roots, and less pronounced in leaves and roots in the presence of longer promoters in each case.
Table 2
GUS activity in leaves ofN.tabacumplants transformed with various birch NiR promoter GUS fusion constructsa
Statistical significance P Enhancement by light Dark GUS activity
Promoter GUS Light GUS activity median
−304 NiR-GUS 128 1289 0.001
413 0.004 2.8
aGUS activity (pmol MU min−1mg−1protein) was measured in extracts from leaves harvested immediately after a 72-h dark period (dark) and after 12 h of light (light). The number of transformed plants used was between 19 and 40 per promoter GUS construct.
Table 3
GUS activity in roots of N.tabacumplants transformed with various birch NiR promoter GUS fusion constructsa
Enhancement by light n-fold Dark GUS Statistical significance P
Promoter GUS con- Light GUS activity activity
struct median
339 367 1.1
−56 NiR-GUS
560 2469
−155 NiR-GUS 0.006 4.4
1271 2222
−304 NiR-GUS 0.04 1.7
1421 3587
−445 NiR-GUS 0.008 2.5
2742 4680
−582 NiR-GUS 0.05 1.7
1148
−735 NiR-GUS 1944 0.08 1.6
35S-GUS 799 615 0.8
Wildtype 133 28 0.06 0.2
Table 4
Fragments of the NiR promoter used in gel retardation assays
Promoter fragment Nucleotide positiona
I +148 to −155 II −77 to−304
−77 to−155 IIA
IIB −146 to −267
−247 to −304 IIC
−247 to −445 III
−377 to −582 IV
−527 to −735 V
aSee the genomic sequence shown in Fig. 1.
used in gel retardation studies are listed in Table 4. Overlapping fragments were used to avoid that possible binding motives at the fragment borders escape detection. Nuclear proteins were obtained from leaves of wild type tobacco plants that were cultivated in the presence of either 5 mM KNO3or
5 mM NH4Cl as the sole nitrogen source. Leaves
were harvested at the end of a 72-h dark period and after 4 h of illumination. Mobility shifts were observed with all combinations of each of the promoter fragments I – V and each of the four different nuclear protein samples (data not shown). The electrophoretic mobility of each of the fragments I, III, IV and V was reduced in a different manner by each of the four protein sam-ples. In contrast, an identical shift of fragment II was obtained with protein samples from dark-grown plants cultivated on nitrate or ammonium (data not shown). We further analysed subfrag-ments IIA, IIB, and IIC of fragment II (Fig. 2). Again, in the protein samples obtained from leaves of dark-grown plants cultivated on either KNO3
or NH4Cl factors were present that shifted the
mobility of fragment IIB in an identical way 3.3. Gel retardation analysis
The light induced changes in GUS activity in the transgenic tobacco plants may indicate organ-specific enhancer elements on the NiR promoter that are responsible for transcriptional changes in reporter gene activity. In order to test this hypoth-esis we used nuclear protein extracts from leaves and electrophoretic mobility shift assays to see whether proteins recognise and bind to NiR pro-moter sequences. NiR propro-moter DNA fragments
Fig. 3. Competition of NiR promoter fragment IIB binding activity. Lane 1: 0.2 ng 32P-labelled IIB DNA. Lanes 2 – 5: Nuclear proteins from leaves of nitrate grown plants kept in dark for 72 h were tested for binding to 0.2 ng labelled DNA probe IIB in the presence of 0 ng (lane 2), 10 ng (lane 3), 20 ng (lane 4), and 40 ng (lane 5) unlabelled IIB DNA as specific competitor and of 2500 ng poly dAdT. Lane 6: DNA protein binding assay as described for lane 2 except that proteinase K was added. An arrow head indicates DNA – protein com-plexes of fragment IIB that are specific for extracts from leaves of plants kept in the dark.
GUS gene constructs, nitrate grown plants were watered with H2O for 3 weeks, and with full liquid
nutrient medium without a nitrogen source for another 8 days to reduce the nitrate content in the pots and within the plants. At the end of this pretreatment GUS activity had decreased to below 10% of the activity originally observed in the nitrate grown plants. Then 10 mM nitrate was added, and GUS activity was determined immedi-ately before and 3, 6, 24, 48 and 120 h after the addition of nitrate. GUS activity increased in re-sponse to nitrate, and a maximum was reached at 24 h. The nitrate induction kinetics was identical for all NiR-GUS constructs. The response ob-served after 24 h in transformants harbouring different NiR promoter lengths is shown in Table 5 for leaves and Table 6 for roots. A pronounced and significant stimulation by nitrate is conferred to leaves by the NiR promoter sequences with the end point at −304 and further upstream, and to roots by the NiR promoter sequences ending at
−445 and further upstream.
4. Discussion
Light-responsive regions were mapped on the birch NiR promoter by analysing transgenic to-bacco plants transformed with different NiR pro-moter GUS constructs. The data show distinct light responsive promoter elements with different activities in leaves and roots of nitrate grown plants. The promoter region between −56 and
−155 confers a strong upregulation of GUS ex-pression to roots following illumination of the plant shoot (Table 3). In contrast to the situation in roots, a significantly enhanced GUS activity in response to light was observed in leaves harbour-ing at least 304 bp of the birch NiR promoter but not in those with 155 bp or less (Table 2). This indicates the presence of a light responsive element between nucleotide positions −155 and −304, which may bind a leaf specific trans-acting factor. This conclusion is supported by results obtained from gel retardation assays using various DNA fragments of the birch NiR promoter. The elec-trophoretic migration of a 121 bp fragment, lo-cated between positions −146 and −267, was specifically retarded by binding to nuclear proteins present in preparations from plants kept in the dark for 72 h. This dark induced binding activity whereas fragments IIA and IIC formed complexes
of different mobility. The corresponding radioac-tive bands in lanes 9 and 10 of the gel are indi-cated by an arrowhead (Fig. 2). Illuminated leaves obviously did not contain these factors.
In order to verify that these bands are DNA – protein complexes proteinase K was added to the binding assay. The retarded bands disappeared as a result of this treatment, and only the unbound DNA is visible in the gel (Fig. 3, lane 6). In order to test the specificity of the binding between frag-ment IIB and distinct proteins present only in darkened leaves an excess of unlabeled fragment DNA was added as a specific competitor to the protein binding mixture in addition to radioactive labeled IIB fragment DNA. Unlabelled fragment DNA did compete with the radioactive IIB-DNA (Fig. 3, lanes 3 – 5) whereas unlabelled IIA-DNA and IIC-IIA-DNA did not (data not shown).
3.4. Sequences necessary for nitrate-dependent GUS acti6ity
was found with nuclear proteins of both nitrate and ammonium grown plants. It disappeared, when plants were exposed to light for more than 4 h. These data support the following hypothesis: In darkness, gene expression is repressed by binding of atrans-acting protein factor to a promoter sequence between −155 and −267 whereas illumination abolishes repression and initiates transcription of the gene. This hypothesis is based on the yet not proven assumption that the dark induced binding factors just bind to the postulated light responsive sequence between position −155 and −304. A possible candidate for such a repressor might be one of the putative targets of COP1 or other gene
products of the COP and DET loci involved in light-controlled gene expression inA. thaliana[24]. It has been proposed that COP1 modulates the promoter activity of light regulated genes. To achieve this, COP1 must, in darkness, suppress the transcriptional activity of light-inducible promoters (by activation of a repressor or inhibition of an activator), and regulatory photoreceptors must act to reverse this action upon exposure to light. While the light regulation of the Arabidopsis nia2 gene encoding NR appears to be mediated by COP1 [1,25] it is still not known whether COP1 or any other COP or DET protein is also involved in the light-induced expression of NiR genes.
Table 5
GUS activity in leaves ofN.tabacumplants transformed with various birch NiR promoter GUS fusion constructsa
− Nitrogen median of + Nitrate median of Statistical signifi- Enhancement by Promoter GUS
con-nitraten-fold cance P
GUS activity GUS activity struct
11
−56 NiR-GUS 9 0.8
19
−155 NiR-GUS 26 0.7
−304 NiR-GUS 14 345 0.002 24.6
−445 NiR-GUS 18 651 0.001 36.2
25.5 0.003
5720
−582 NiR-GUS 224 99
−735 NiR-GUS 2273 0.002 23.0
672 617 0.9
35S-GUS
aGUS activity (pmol MU min−1 mg −1 protein) was measured in leaves harvested after growth of the plants without an external nitrogen source for about 4 weeks (− nitrogen) (Plants were watered with Aqua dest. for 3 weeks and with dissolved mineral fertiliser without nitrogen for 8 days.); and after further cultivation of the same plants with 10 mM potassium nitrate for 1 day (+nitrate) (The number of transformed plants used was 12 per promoter GUS construct.).
Table 6
GUS activity in roots of N.tabacumplants transformed with various birch NiR promoter GUS fusion constructsa
Promoter GUS − Nitrogen GUS activity + Nitrate GUS activity Statistical signifi- Enhancement by nitrate canceP
median
construct median n-fold
32
−56 NiR-GUS 58 0.06 1.8
54
−155 NiR-GUS 56 1
105 122
−304 NiR-GUS 0.16 1.2
37 272
−445 NiR-GUS 0.001 7.3
243
−582 NiR-GUS 1740 0.02 7.1
−735 NiR-GUS 61 149 0.009 2.4
521 0.9
591 35S−GUS
The difference between transgenic tobacco leaves and roots regarding sequences important for light-dependent GUS expression may be ex-plained by the fact that roots did not receive the light signal directly in our experiments. The nature of the signal(s) transmitted from the shoot to the roots and responsible for enhanced GUS activity in the roots, however, is not known. Whether this is an indirect effect of the increased supply of photosynthate, presumably sucrose, glucose, or fructose needs to be clarified. At least inNicotiana plumbaginifolia, the transcript level of the NiR gene did not increase upon feeding of darkgrown plants with glucose whereas the transcript level of the NR gene did [26].
NiR was found to be regulated at the level of transcript accumulation by light in a number of plants including barley, bean, birch, maize, pine, spinach, and tobacco. A structural and functional analysis of NiR promoter regions has been pub-lished for spinach [6], bean [10], and tobacco [11]. The results obtained in the bean and tobacco NiR-promoter studies are barely comparable to ours because the influence of nitrate was deter-mined only 2 – 3 weeks after replacement of nitrate by ammonium chloride or ammonium succinate [10], and 17 days after growth on either nitrate or glutamine [11], respectively. The NiR promoter from spinach has been characterised most thor-oughly [6]. Deletion analysis of the spinach NiR promoter in cotyledons of transgenic tobacco indi-cate that basic elements required for light-(and nitrate-)dependent expression of the GUS reporter gene are located within the sequence +131 to
−200 relative to the transcription initiation site, and further upstream sequences are required for maximum expression of the reporter gene. A prob-lem with this study was the very low relative level of GUS expression. GUS activity in tobacco plants transformed with spinach NiR promoter GUS fusions did not exceed 5% of GUS activity exhibited by plants transformed with a CaMV-35S promoter GUS fusion. In contrast, in the present investigation the most efficient birch NiR pro-moter sequences conferred a fourfold GUS activ-ity to tobacco plants as compared to the CaMV 35S promoter.
The effect of birch NiR promoter sequences (this study) and birch NR promoter sequences [4] on the expression of the GUS reporter gene in transformedNicotiana plants also deserves a
com-parison. Even with the most efficient NR pro-moter sequences, GUS activity did not exceed 5% of GUS activity exhibited by plants transformed with a CaMV-35S promoter GUS fusion. The enormous difference between NiR promoter medi-ated GUS expression and NR promoter medimedi-ated GUS expression in transgenic Nicotiana may reflect the in situ situation observed in birch since in leaves and roots of young birch plants the relative NiR mRNA pool and NiR activity was about 6- and 10-fold higher than the NR mRNA pool and NR activity, respectively, following a co-activation by light and nitrate [12]. This ap-pears to be a safety measure to prevent nitrite accumulation in the plant.
To investigate the effect of an addition of ni-trate to the growth subsni-trate we added nini-trate to plants grown for several weeks without a nitrogen source. Upon the addition of nitrate GUS activity increased rapidly within a few hours in some of the transformants and reached a maximum after 24 h. A basic element required for the response in leaves is located between −155 and −304, and between −304 and −445 for the response in roots. Our results contrast with those reported by Rastogi et al. [7,8], Neininger et al. [6], and Sivasankar et al. [9] investigating nitrate-mediated spinach NiR-GUS gene expression in whole seedlings of transgenic tobacco. Their data show that the region from +5 to +67, which forms part of the untranslated leader of the spinach NiR gene is important for minimal induction by ni-trate. For a full induction this 62 bp leader se-quence is required together with a −200 to −225 bp region of the 5% flanking sequence. Such a modular regulation is not seen in our transgenic tobacco plants containing NiR promoter and leader sequences from birch.
Some information regarding cis-acting elements necessary for nitrate and light dependent tran-scription of NR and NiR genes is available. It has been demonstrated that 238 and 330 bp of the
promoter region further contains four GATA mo-tifs located between −525 and −735. GATA is a recognition sequence for NIT-2, a regulatory fac-tor of nitrogen metabolism in fungi, most likely involved in the transcriptional regulation of NR genes [28] and NiR genes [8]. Whether the GATA motifs in the birch sequence between −320 and
−535 are functionally important for nitrate regu-lated gene expression remains to be tested. A sequence comparison between the nitrate respon-sive regions of the birch NiR promoter and the NiR promoters of spinach [6 – 9], bean [10] and tobacco [11] did not reveal any obvious common DNA motifs.
Acknowledgements
We wish to thank W. Frank (MPIZ; Cologne, Germany) and D. Bartels (Bonn) for their help with gel retardation analysis. Financial support is acknowledged from Deutsche Forschungsgemein-schaft (grant Ha 817/11-1 to W.H.) and from Konrad-Adenauer-Foundation (to H.O.W.).
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