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 the A. tumefaciens Ti 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 into A. tumefaciens strain 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 KNO
3
or 5 mM NH
4
Cl for at least 4 weeks. Plants were kept under a 16-h light8-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 In a birch genomic library [4] we identified a
recombinant phage clone that hybridises to a birch NiR cDNA clone [23]. DNA from this recombi-
nant clone was digested with EcoRI, and a 2 kb EcoRI fragment hybridising to the NiR cDNA
was isolated, cloned into pUC19, and partially sequenced. It was found to contain the amino-ter-
minal coding sequence of the NiR gene including
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. A EcoRI restriction site at the 5 end and a HindIII site within the untranslated leader are indicated. The TATA box, GATATATC 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 5upstream 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 fluores- cence source. Plant pots were wrapped in light-im-
permeable foil to prevent an uncontrolled 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 5deleted 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 P 5 0.004 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 of N. tabacum plants transformed with various birch NiR promoter GUS fusion constructs
a
Statistical significance P Enhancement by light
Dark GUS activity Promoter GUS
Light GUS activity median
median n-fold
construct 15
13 −
56 NiR-GUS 1.1
291 330
− 155 NiR-GUS
1.1 10
− 304 NiR-GUS
128 1289
0.001 413
0.004 2.8
− 445 NiR-GUS
1186 2461
0.001 1.9
− 582 NiR-GUS
4643 2.3
0.003 1091
− 735 NiR-GUS
471 1.5
35S-GUS 738
1094 0.088
4 4
1 Wildtype
a
GUS activity pmol MU min
− 1
mg
− 1
protein 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. tabacum plants transformed with various birch NiR promoter GUS fusion constructs
a
Enhancement by light n-fold Dark GUS
Statistical significance P Promoter GUS con-
Light GUS activity activity
struct median
339 1.1
367 −
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 1.7
0.05 1148
− 735 NiR-GUS
1944 0.08
1.6 35S-GUS
799 615
0.8 Wildtype
133 0.06
28 0.2
a
GUS activity pmol MU min
− 1
mg
− 1
protein was measured in extracts from roots harvested immediately after a 72-h dark period dark and after 12 h of illumination of the shoots light. The number of transformed plants used was between 14 and 40
per promoter GUS construct.
Table 4 Fragments of the NiR promoter used in gel retardation assays
Promoter fragment Nucleotide position
a
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
a
See 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 KNO
3
or 5 mM NH
4
Cl 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 KNO
3
or NH
4
Cl 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 promoter DNA fragments
Fig. 2. Autoradiograph of a gel retardation assay using radioactive labelled NiR promoter fragments IIC lanes 1 – 5, IIB lanes 6 – 10, and IIA lanes 11 – 15, and nuclear proteins obtained from leaves of plants cultivated with two different nitrogen sources
and kept under the following dark light regime: 72-h dark – 4-h light L, 5 mM NH
4 +
lanes 2, 7 and 12; 72-h dark – 4-h light L, 5 mM NO
3 −
lanes 3, 8 and 13; 72-h dark D, 5 mM NH
4 +
lanes 4, 9 and 14; 72-h dark D, 5 mM NO
3 −
lanes 5, 10 and 15. An arrow head indicates DNA – protein complexes of fragment IIB that are specific for extracts from leaves of plants kept in the
dark lanes 9 and 10.
Fig. 3. Competition of NiR promoter fragment IIB binding activity. Lane 1: 0.2 ng
32
P-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 H
2
O 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
