Journal of Experimental Botany, Vol. 52, No. 359, pp. 1251±1258, June 2001
Tobacco plants that lack expression of functional nitrate
reductase in roots show changes in growth rates and
metabolite accumulation
Robert HaÈnsch1, DesireÂe GoÂmez Fessel1, Christina Witt1, Christine Hesberg1,
Guido Hoffmann1, Pia Walch-Liu2, Christof Engels2, JoÈrg Kruse3, Heinz Rennenberg3,
Werner M. Kaiser4 and Ralf-R. Mendel1,5
1
Botanisches Institut, Technische UniversitaÈt Braunschweig, D-38106 Braunschweig, Germany
Institut fuÈr PflanzenernaÈhrung, UniversitaÈt Hohenheim, D-70593 Stuttgart, Germany
3
Institut fuÈr Forstbotanik und Baumphysiologie, Albert-Ludwigs-UniversitaÈt, D-79110 Freiburg, Germany
4
Julius-von-Sachs-Institut fuÈr Biowissenschaften, UniversitaÈt WuÈrzburg, D-97082 WuÈrzburg, Germany
2
Received 31 August 2000; Accepted 29 December 2000
Abstract
When tobacco is provided with a high nitrate supply,
only a small amount of the nitrate taken up by the
roots is immediately assimilated inside the roots,
while the majority is transported to the leaves where
it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants
have been engineered that showed no detectable
nitrate reductase activity in the roots. These plants
expressed the nitrate reductase structural gene nia2
under control of the leaf-specific potato promoter
ST-LS1 in the nitrate reductase-mutant Nia30 of
Nicotiana tabacum. Homozygous T2-transformants
grown in sand or hydroponics with 5.1 mM nitrate had
approximately 55 ±70% of wild-type nitrate reductase
acivity in leaves, but lacked nitrate reductase acivity
in roots. These plants showed a retarded growth as
compared with wild-type plants. The activation state
of nitrate reductase was unchanged; however, diurnal
variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants
had higher levels of nitrate in the leaves and reduced
amounts of glutamine both in leaves and roots, while
roots showed higher levels of hexoses (3-fold) and
sucrose (10-fold). It may be concluded that the
loss of nitrate reductase acivity in the roots changes
the allocation of reduced nitrogen compounds and
sugars in the plant. These plants will be a useful tool
5
for laboratories studying nitrate assimilation and its
interactions with carbon metabolism.
Key words: Diurnal variation, leaf specific expression, nitrate
reductase, tobacco, roots.
Introduction
The assimilation of nitrate is a highly regulated process
(Crawford, 1995). Nitrate is taken up by the roots, but in
many herbaceous plants only a small amount is reduced
to ammonium within the roots. Plants provided with
a high nitrate supply transport the majority of nitrate
to the leaves where photosynthesis provides energy and
C-skeletons for N-assimilation (Rufty, 1997). N- and
C-assimilation are co-regulated in higher plants by reciprocal molecular and metabolic controls between both
pathways (Kaiser, 1997; Koch, 1997; Ferrario et al.,
1995). In particular, reversible protein phosphorylation
of nitrate reductase (NR, EC 1.6.6.1) plays a key role in
this regulatory network (Glaab and Kaiser, 1995; Huber
et al., 1996; MacKintosh, 1997). The rate of nitrogen
uptake and translocation to the shoot can determine the
rate of shoot growth (see review by Rufty, 1997).
Following uptake into the root cells, nitrate is stored in
vacuoles, reduced by NR, or loaded into the xylem. In the
xylem, nitrate (mainly as KNO3) is transported with the
transpiration stream primarily to source leaves where it
To whom correspondence should be addressed. Fax: q49 531 391 8128. E-mail: [email protected]
ß Society for Experimental Biology 2001
1252
HaÈnsch et al.
can be stored in vacuoles until it is ®nally assimilated. The
products of nitrate assimilation, the amino acids, are
transferred via the phloem to the growth centres of
the shoot, or transported back to the root through the
phloem, for supporting their growth. Root growth and
nitrate uptake are also dependent on a sustained ¯ux of
carbohydrates from the shoot. NR plays a key role in
this interdependent regulatory network although the NR
protein is present in excess in the cells, and only 20±70%
of the NR molecules are active depending on the metabolic state of the cell (Kaiser and Huber, 1994). Studies
with tobacco NR mutants and transgenic plants with
altered expression of NR clearly showed that there is no
direct correlation between plant growth and the nitrate
reduction capacity of the plant, i.e. plants with lowered
NR activity are phenotypically not different from
wild-type plants (Vaucheret et al., 1990; Kleinhofs and
Warner, 1990; Vincentz and Caboche, 1991; Dorbe et al.,
1992; Wilkinson and Crawford, 1993; Foyer et al., 1994;
Gojon et al., 1998). Only when NR activity is lower than
10% of the wild-type level is growth affected (MuÈller and
Mendel, 1989; Foyer et al., 1994; Scheible et al., 1997a).
The role of nitrate reduction in the roots of herbaceous
plants is not fully understood. It may constitute only
10% of the total nitrate reduction capacity of the plant,
depending on plant type and N-nutrition (Gojon et al.,
1998). Recently it was suggested that nitrate might serve
not only as nutrient but also as signal for regulating
N- and C-metabolism (Scheible et al., 1997b). In order to
elucidate the role of root nitrate reduction, the consequences of a complete loss of this enzymatic activity
in the roots on metabolism and plant development were
studied. For this purpose tobacco plants were constructed
without detectable NR activity in the roots. In this report,
the ®rst analyses of these transformed plants are
presented and it is shown that the loss of nitrate reductase
activity in the roots changes the allocation of reduced
nitrogen compounds and sugars when NR is present only
in the leaves.
Materials and methods
Plasmid construction
All restriction endonuclease and ligase reactions (Sambrook
et al., 1989) were performed using the buffer conditions recommended by their respective manufactureres. The intermediate
plasmid constructions were made in the pRT-series (Toepfer
et al., 1988). Two binary vectors were constructed using the
HindIII site of the pGA472 (An et al., 1987). The ®rst vector
contained the CaMV-35S promoter driving the nitrate reductase
cDNA (nia2) of Nicotiana tabacum (Vaucheret et al., 1989;
Vincentz and Caboche, 1991) followed by the polyA-tail
from CaMV-35S. In the second vector, the 35S-promoter was
replaced by the ST-LS1 promoter from Solanum tuberosum
(Stockhaus et al., 1987) kindly provided by Dr Uwe Sonnewald,
Gatersleben. For transformation, the Agrobacterium tumefaciens
strain C58C1upGV2260 was used.
Plant material and transformation
Experiments were performed with Nicotiana tabacum cv.
Gatersleben (GAT). The nitrate reductase-de®cient mutant
Nia30 (MuÈller, 1983) derived from this wild type was used as
recipient in transformation studies. The plants were grown
in vitro on MS medium (Murashige and Skoog, 1962) supplemented with ammonium succinate in the case of the Nia30
mutants (Vaucheret et al., 1990). Leaf disc transformation and
shoot regeneration were performed as described previously
(Horsch et al., 1985). Regenerated plantlets were maintained
on medium containing 50 mg l 1 kanamycin and screened by
Southern blotting of genomic DNA to verify the integration of
the foreign genes. The primary transformants were transferred
into soil in controlled environment chambers (Hereaus-VoÈtsch,
HPS 1500, Balingen, Germany) with 14 h daily light periods to
allow self-fertilization.
Cultivation of plants and determination of relative
growth rate (RGR)
Seeds of wild-type N. tabacum cv. GAT and the transformants
were germinated on soil in a dayunight-regime of 14u10 h,
25u20 8C, a relative humidity of 80% and 350±400 mE m 2s 1
PAR. After 3 weeks the plants were transferred to sand-®lled
0.5 l pots or to 3.0 l pots containing a well-aerated nutrient
solution of the following composition: 2.0 mM Kq, 2.5 mM
Ca2q, 1.2 mM Mg2q, 0.5 mM H2PO4 , 1.2 mM SO24 , 10 mM
FeEDTA, 10 mM H3BO3, 0.5 mM Mn2q, 0.5 mM Zn2q, 0.1 mM
Cu2q, and 0.07 mM MoO24 . Nutrient solutions were changed
three times a week. Sand-cultured plants were supplied daily
with the complete nutrient solution containing 5 mM ammonium nitrate. During the ®rst week of hydroponic growth, NO3
(3.4 mM) plus NHq
4 (1.7 mM) were added to the nutrient
solution. Subsequently, NO3 (5.1 mM) was the sole nitrogen
source of the plants for both the hydroponically and the
sand-grown plants.
Growth was measured as the increase in fresh or dry weigth
(DW) between ®ve harvest occasions between 4±8 weeks after
sowing. Dry weight was determined after oven-drying at 80 8C
for 14 h. RGR for single plants was calculated according to:
RGR ˆ ln (DW ®nal plantuDW seedling)udays of growth.
Extraction and detection of mRNA and protein
Plants were harvested, shoot and root biomass were determined,
and the tissues were frozen in liquid nitrogen and stored at
80 8C. Tissues were extracted by grinding to a ®ne powder in
a small mortar and pestle cooled in liquid nitrogen.
For RNA-analysis, 1 g powder was transferred to a precooled
15 ml centrifuge tube and total RNA was extracted (Verwoerd
et al., 1989). Gel electrophoresis was performed in a formamideformaldehyde-agarose-gel loaded with 40 mg of total RNA.
The RNA was blotted to a charged membrane (Hybond-Nq,
Amersham-Pharmacia-Biotech, Freiburg, Germany) and ®xed
by UV-crosslinking followed by hybridization with a ¯uorescein-dUTP-labelled-DNA-probe. Bands of NR mRNA were
visualized with an anti-¯uorescin antibody labelled with alkaline
phosphatase. Hybridization and detection were performed with
CDP-Star Detection System according to the manufacturer
(Amersham-Pharmacia-Biotech).
Protein from 20 mg tissue was extracted and loaded (Scheible
et al., 1997b) on SDS-PAGE consisting of 7.5% acrylamide
(Laemmli, 1970). The proteins were electroblotted to HybondPVDF (Amersham-Pharmacia-Biotech) and immuno-detected
with NR-peptide antibodies (Scheible et al., 1997b) kindly
Plants without root nitrate reductase
provided by Dr Mark Stitt (Heidelberg). The NR bands were
visualized using the ECL-Plus-System (Amersham-PharmaciaBiotech).
Determination of NR activity
Nitrate reductase activity was determined (Scheible et al.,
1997a). The activation state of NR is given by the ratio of
its activity in extraction and reaction buffers with either 10 mM
EDTA or with 10 mM MgCl2 (Kaiser and Spill, 1991).
Analysis of amino-compounds and tissue nitrate concentration
Amino compounds were extracted from plant material by
a modi®cation of the method described earlier (Gessler et al.,
1998a). Roots and the ®rst two adult plus the youngest leaves
were ground with mortar and pestel, respectively. Aliquots of
0.1 g of the frozen powder were homogenized in 0.2 ml of
buffer containing 20 mM HEPES (pH 7.0), 5 mM EGTA,
10 mM NaF, and 1.2 ml of chloroform:methanol (1.5:3.5 (vuv)).
The homogenate was incubated for 30 min at 4 8C. Subsequently, water-soluble metabolites were extracted twice with
0.6 ml double-distilled water. The aqueous phases were combined and freeze-dried (Alpha 2±4, Christ, Osterode, Germany).
The dried material was dissolved in 1 ml lithium citrate buffer
(0.2 mM, pH 2.2) for amino acid analysis. Amino compounds
were separated and subsequently detected after post-column
derivatization with ninhydrin by an automated amino acid
analyser (Biochrom, Pharmacia LKB, Freiburg, Germany).
Analyses of amino compounds and nitrate in rootsushoots of the
wild type and the transformant at day and night were performed
in 5±6 independent replicates each. For statistical analysis
data were subjected to ANOVA (Microcal Origin, Version 3.5,
Microcal Software, Inc.)
Tissue nitrate was determined according to the method
described previously (Gessler et al., 1998b) using a conductivity
detector module (CDM, Dionex).
Sugar determination
Leaf samples (0.5 g FW) were ground in small mortars in liquid
nitrogen. The frozen powder was suspended in 5 ml deionized
water, and grinding continued until thawing. Subsequently,
samples were boiled for 3 min in a heating block and cooled
immediately on ice. After removal of insoluble material by
centrifugation (16 000 g, 20 min, 2 8C), the supernatant was
used after suitable dilution to separate and quantify sugars by
isocratic ion chromotography with PAD (pulsed amerometric
detection) (4500 i, Carbopac P1 column plus precolumn, 0.12 M
NaOH as eluent, Dionex, Idstein, Germany). Mixtures containing sorbitol, glucose, fructose, sucrose, and maltose (0.1 mM
each) were used as standards every ®fth sample. Occasionally,
extracts were passed through mixed-bed ion exchange columns
before sugar analysis in order to check for ionic contamination
which might give a signal on the PAD. However, in all cases,
analysed contaminations were negligible.
Statistics
Biomass and nitrate reductase activity values were obtained
from 3 leaves per plant using 3±5 plants per line. The results
are givenp
as the mean values for each population with the
SE ˆ snu n 1, where sn is the SD. If not pointed out
otherwise, for biochemical analyses the youngest fully expanded
three leaves (7 and 15 cm in length) were pooled. The values
given for these analyses represent the means of three plants, and
1253
each experiment was repeated independently three times.
Northern and Western analyses were done with a mix of ground
powder from these plants.
Results
Construction of tobacco plants without NR activity
in the roots
For engineering tobacco plants without detectable NR
activity in the roots, tobacco NR-cDNA was introduced
into the widely used tobacco NR-mutant Nia30. This
mutant is a homozygous double mutant in the NR loci
nia1 nia2 that was puri®ed by backcrossing to the wild
type (MuÈller, 1983). It lacks detectable NR activity in
leaves and roots and is unable to grow in soil. Cultured
in vitro, Nia30 plants are able to use ammonium-succinate
as the nitrogen source but cannot grow on nitrate (MuÈller,
1983). For transformation, the nia2-cDNA (Vaucheret
et al., 1989) was under the control of the potato ST-LS1
promoter that drives expression of the genes only in
green tissues (Stockhaus et al., 1987). As a control,
Nia30 mutants were transformed with NR-cDNA under
the control of the constitutive CaMV-35S promoter.
T0-plants that exhibited NR activity in the leaves were
subjected to Southern blot analysis of genomic DNA
using a nptII- and a nia2-(Moco-domain only) radiolabelled probe, respectively. Three plants (H, P and U) with
a one copy-integration of the transgene were chosen to
produce homozygous T2-seeds.
Untransformed Nia30-mutants were unable to grow
in hydroponic culture or sand with nitrate as the sole
nitrogen source and died. Among the 35S-NR transformants, plants were identi®ed that showed NR activities
similar to the wild type both in leaves and roots.
Compared to the wild type, the three LNR plants
(LNR ˆ leaf nitrate reductase) had NR activities between
25% and 70% in the leaves, and under no circumstances
were NR activity detectable in the roots.
LNR plants show retarded growth
LNR plants were considerably smaller than the wild type
of the same age while 35S-NR plants grew as fast
as wild-type plants. Figure 1A shows the phenotype of
homozygous T2-plants of the LNR type, i.e. retarded
growth with smaller leaves as compared with a wild-type
plant of the same age. Eight weeks after germination,
LNR plants had accumulated less than half the biomass
compared with the wild type (Fig. 1B). Between 4 and 8
weeks of cultivation the RGR were not different (LNR:
0.112 and wild type: 0.119). Given enough time LNR
plants developed into plants of similar size as the wild
type (not shown) with a ¯owering time of 83.2"5.3 d
(69.4"4.1 d in wild type). The retarded growth of LNR
plants made it necessary to measure NR activities not
1254
HaÈnsch et al.
only in leaves of the same plant age but also in leaves of
a given developmental stage and size when comparing
LNR plants with wild-type plants. Seven-week-old wildtype plants correspond in developmental stage and size
to LNR plants of an age between 8 and 9 weeks. NR
activities were therefore determined separately for the ®rst
four fully expanded leaves at regular intervals between
7 and 10 weeks after sowing. Figure 2 shows that NR
activities stayed comparable over a period of 7±9 weeks
for both wild-type and LNR plants. The slower growth of
the LNR plants was surprising because it was previously
shown that tobacco plants with considerably reduced NR
activity exhibited no growth retardation (Foyer et al.,
1994). When Nia30 mutants were transformed with
35S-NR cDNA they showed NR activity in both leaves
and roots and grew as fast as wild-type plants. Thus the
slower growth of the LNR plants might be attributable to
the loss of root NR activity. Therefore LNR lines H and U
were chosen for further analyses and were compared with
wild-type plants and, where indicated, also with Nia30
transformed with 35S-NR cDNA as the positive control.
Correlation of nitrate reductase protein-level and
enzyme-activity
Homozygous T2-generation plants of the LNR type and
the wild type were analysed for NR activity and for the
amount of NR protein expressed in the leaves and roots.
Figure 3A shows that NR activity correlates with the
amount of NR-protein detected by NR-speci®c antibodies. LNR plants had approximately half the NR
activity of the wild type in their leaves which directly
correlated with the amount of NR protein detected
immunologically. Further, the activation state of NR
was analysed according to Kaiser and Spill (Kaiser and
Spill, 1991). Figure 3B shows that the percentage of
active NR in the leaves of both wild-type and LNR plants
ranged from 60% to 80%, whereas the activation state of
the root NR was much lower in the wild type.
Diurnal variation of NR expression
Fig. 1. (A) Phenotype of sand-grown plants of homozygous
T2-generation, 7-week-old: N. tabacum cv. GAT wild-type plant (left),
35S-U-plant (middle) and LNR H- and LNR U-plant (right).
(B) Accumulation of biomass in leaves (closed symbols) and roots
(open symbols) of N. tabacum wild type (m) and homozygous
T2-generation LNR H-plants (j). Values presented are for LNR H
plants, LNR U plants gave similar values. The increase of fresh and
dry weight of hydroponically grown plants was measured over a period
of 8 weeks starting from seed germination.
Fig. 2. NR activities in single leaves of wild-type and LNR H plants
measured in weekly intervals between 7 and 10 weeks after sowing.
NR activities were determined 3 h after beginning of the light period
separately for the ®rst (three) four fully expanded leaves. Seven-week-old
wild-type plants correspond in developmental stage and size to LNR
plants of an age between 8 and 9 weeks. Nitrogen source: 5.1 mM KNO3
as described in Experimental procedures.
Expression of NR was characterized by a typical diurnal
variation of the level of mRNA, protein and enzyme
activity (Fig. 4). For generating LNR plants the potato
ST-LS1 promoter was used that is expressed only in
photosynthetically active, green tissues (Stockhaus et al.,
1989). This promoter is known to increase transcription
Fig. 3. Comparison of NR-activity with the protein-level (A), and
activation state of NR (B) in leaves (top) and roots (bottom) of 7-weekold N. tabacum cv. GAT wild-type and LNR H plants. In (A) the NR
activities of wild-type plants were set to 100%, NR-protein was
visualized by Western blotting (20 mg protein loaded per lane) of
the same sample that was used for determining of NR-activity. In (B)
the total amount of NR activity (determined in Mg-free buffer) was
compared with the fraction of enzyme active in the presence of 10 mM
Mg2q (striped columns) (as determined by Kaiser and Spill, 1991).
Plants without root nitrate reductase
about 2-fold when the green tissues are exposed to light
(Stockhaus et al., 1987), but nothing is known about its
behaviour during a complete day±night cycle. Therefore,
NR expression was analysed in LNR plants and it was
compared to the wild type. Figure 4 also shows that the
LNR type also exhibited diurnal variation of NR
expression on the level of enzyme activity, re¯ecting the
strength of the ST-LS1 promoter in dark and light;
however, the amplitude of this variation was not so
pronounced as in the wild type. The level of NR-mRNA
in wild-type leaves showed the expected diurnal variation
with a maximum during the night, while the amount of
NR protein was decreased at night. Also in wild-type
roots, the amount of NR protein was lower at the end of
the day and during the night. In LNR leaves, however,
constitutive NR-mRNA levels were measured over the
whole period. LNR roots showed a NR-mRNA pattern
similar to the wild type. The trace amounts of NR protein
in LNR roots originated from the mutated and unstable
NR protein of mutant line Nia30 that was used as
recipient for the transformation experiments.
Soluble nitrogen compounds in the shoot and roots
In the leaves, total soluble non-protein nitrogen (TSNN)
contents were substantially higher in the LNR transformants as compared to the wild type and the 35S-NR
plants (Table 1). Enhanced TSNN levels in the leaves of
1255
LNR plants were attributed to an enhanced NO3 content
that overcompensated the strongly reduced levels of
amino-N. Most remarkably, glutamine levels in the
leaves of the LNR transformants were over 95% reduced
as compared to the wild type and to the 35S-NR plants
(Table 1). NHq
and glutamate levels of the leaves
4
were higher in the wild type and the 35S-NR plants
as compared to the LNR type.
In the roots, TSNN contents were slightly lower as
compared to the leaves (Table 1). Both, TSNN and NO3
contents were similar in the roots of the wild type,
35S-NR plants and the LNR plants. Still, the glutamine
levels in the roots of the LNR transformants were over
90% reduced as compared to wild type and 35S-NR
plants, which is also re¯ected in the lower levels of aminoN in LNR roots. Total protein contents in wild type
and LNR were also examined. They were very similar
in leaves and roots of wild-type, LNR and 35S-NR
plants, with a trend towards higher values in LNR roots
(data not shown).
Hexoses and sucrose in the shoot and roots
at day and night
The contents of glucose, fructose and sucrose of the leaves
did not show differences between LNR plants and the
wild type (Table 2). As expected, the sugar content of
the leaves increased during the day. In the roots of LNR
plants, however, a strong accumulation of sucrose did
occur with levels 8±10 times higher than in the wild type
(Table 2). Hexose levels in the LNR roots were 2±3 times
higher as compared to the wild type, but did not increase
during the day. Malic acid concentrations in roots of LNR
were 5±10 times higher than in the wild type, and cation
concentrations were also increased (data not shown).
Discussion
Fig. 4. Diurnal variation of mRNA, protein-level and activity of nitrate
reductase in leaves (top) and roots (bottom) of 7-week-old N. tabacum
cv. GAT wild-type plants (left) and LNR H plants (right) grown
in hydroponics. NR activity was determined as total amount of NR
(total height of the columns) and as active enzyme (dark grey part of the
column). The levels of mRNA (40 mg loaded per lane) and protein (from
20 mg tissue loaded per lane according to Scheible et al., 1997b) were
determined using Northern and Western blot analysis, respectively.
Nitrate reduction in the roots of herbacious plants
like tobacco constitutes approximately one-tenth of the
total nitrate reduction capacity of the plant (Gojon et al.,
1998). To study the consequences of the loss of this small
percentage of NR in the roots for plant metabolism and
development, tobacco plants were constructed without
detectable NR activity in the roots. This might principally
be achievable by an antisense approach using a strong
and tightly expressing root-speci®c promoter. Antisense
approaches are a good means for lowering the expression
of a given gene, but only rarely reduce its expression
tightly to zero. This problem was circumvented by using
a completely NR-de®cient mutant as recipient for the NR
cDNA in sense under control of a leaf-speci®c promoter.
The potato ST-LS1 promoter chosen for these experiments is known to increase transcription about 2-fold
when exposed to light and it is active only in green
1256
HaÈnsch et al.
Table 1. Total soluble non-protein nitrogen (TSNN) contents (mmol N g 1FW;"SD) in leaves and roots of N. tabacum cv. GAT wild
type, 35S-NR plants, and LNR-lines H and U
Samples were taken 3 h after beginning of the light period. Data shown are means of 5±6 plants each. Amino compounds not shown here did not
exhibit signi®cant differences.
Leaves
TSNN
Amino-N
NO3
NH4
Ser
Glu
Gln
Roots
TSNN
Amino-N
NO3
NH4
Ser
Glu
Gln
Wild type
35S-U
LNR-H
LNR-U
54.10"0.85
15.44"6.04
36.64"5.06
2.01"0.13
0.64"0.14
1.69"0.22
2.75"1.08
34.62"0.88
19.28"1.87
14.16"1.04
1.18"0.05
0.62"0.08
2.35"0.17
3.73"0.33
89.03"5.08
2.81"0.20
85.89"5.27
0.33"0.01
0.31"0.02
0.98"0.13
0.12"0.01
80.56"32.24
2.91"0.22
77.26"32.44
0.38"0.02
0.38"0.01
0.95"0.05
0.17"0.01
42.12"2.09
8.23"2.13
33.48"0.02
0.41"0.02
0.26"0.01
1.64"0.52
1.92"0.56
48.26"2.01
10.64"0.42
37.06"2.50
0.56"0.08
0.43"0.02
2.52"0.08
2.24"0.22
39.14"4.89
3.16"0.30
35.82"4.56
0.16"0.02
0.29"0.04
0.73"0.06
0.25"0.01
24.87"1.58
2.33"0.09
22.40"1.49
0.13"0.01
0.25"0.02
0.56"0.09
0.14"0.02
Table 2. Hexoses and sucrose in leaves and roots on N. tabacum cv. GAT wild type and LNR-plants
Values presented are for LNR-H plants. LNR-U plants gave similar values. Samples were taken at the end of the night, 30 min before the light period,
and 3 h before the end of the photosynthetic period. Concentrations are given in mmol g 1 FW ("SD).
Night
Leaves
Roots
Glucose
Fructose
Sucrose
Hex:Suc
Glucose
Fructose
Sucrose
Hex:Suc
Day
Wild type
LNR-H
Wild type
LNR-H
1.58"0.88
1.27"0.79
1.16"1.04
2.46
1.27"0.49
1.77"0.95
0.53"0.42
5.74
1.81"0.62
1.51"0.53
1.71"1.93
1.94
3.72"1.78
4.87"2.71
5.30"3.79
1.62
3.38"1.09
2.34"1.40
3.97"1.81
1.44
2.01"1.03
2.86"1.61
0.50"0.25
9.74
2.68"0.79
1.93"0.41
3.29"2.92
1.40
3.64"2.15
4.88"2.71
4.33"3.58
1.97
tissues containing photosynthetically active chloroplasts
(Stockhaus et al., 1987). In the leaf, it is expressed only in
the mesophyll cells and the stomata guard cells, but not
in the epidermis (Stockhaus et al., 1989). The leaf mesophyll is also the main site of nitrate assimilation so that
the ST-LS1-driven NR expression is appropriately placed.
The ST-LS1 promoter turned out to be very tight in
its organ speci®city, i.e. no residual NR activity was
ever detected in the roots of the LNR transformants. It
permitted a diurnal expression pattern of NR activity,
albeit not with the same amplitude as observed for the
nia-promoter (Fig. 4). The ST-LS1 promoter, however is
not nitrate inducible. As in wild-type plants, the amount
of NR protein in LNR plants is decreased at night,
and also the activation state of NR in wild-type and
LNR plants was very similar (Fig. 4). NR-mRNA levels,
however, stayed constant over the whole period in LNR
plants. Here it has to be taken into account that mutant
line Nia30 used as a recipient for the transformations
experiments has point mutations in the two nia genes
rendering its NR protein inactive and unstable, but the
genes are still transcribed. Therefore the NR-mRNA
expression pattern observed in LNR leaves is the sum
of two transcriptions: one driven by the residual nia
promoters and the other driven by the introduced ST-LS1
promoter.
The LNR transformants showed a slower growth than
the wild type. This was surprising for several reasons: (1)
In wild-type tobacco, root NR activity is approximately
10 times lower than leaf NR activity (Gojon et al., 1998)
and hence its loss should not cause such a delay in
growth. (2) Tobacco plants with lowered NR activity did
not show growth retardations. Only when NR activity
was lower than 10% of the wild-type level was growth
affected (MuÈller and Mendel, 1989; Foyer et al., 1994;
Scheible et al., 1997a; Gojon et al., 1998). (3) The latter
plants with less than 10% of wild-type NR activity
showed, however, actual 15 NO3 reduction rates between
30±70% of those measured in wild-type plants (Gojon
et al., 1998). These high rates, however, were also
Plants without root nitrate reductase
observed in detached leaves so that Gojon et al. came to
the conclusion that in tobacco the contribution of
the roots to total nitrate reduction is not high (Gojon
et al., 1998). Further, the data of these authors demonstrate that there is a discrepancy between the potential
nitrate reduction (as measured by NR activity) and the
actual nitrate reduction, and this discrepancy has not
been solved yet. LNR plants had approximately 50% of
wild-type NR activity in the leaves which corresponded
with the amount of NR protein detected immunologically. Taking into account the results of other authors
(Gojon et al., 1998; Foyer et al., 1994), neither the amount
of NR protein expressed in leaves nor the activity and
the activation state of NR could account for the slower
growth of LNR plants.
The loss NR activity in the roots of LNR plants had an
effect on their development. One could argue that this
phenotype is unrelated to NR but rather might be due to
secondary mutations in the recipient plant Nia30. This
possible intrepretation, however, can be ruled out because
the control 35S-NR transformants that regained NR
activity both in leaves and roots showed normal growth
and metabolite values. The determination of NR activity
in leaves over a period of 7±9 weeks after sowing gave
rather steady values for single leaves of wild-type and
LNR plants. Thus, it rules out the possibility that strongly
changed NR activities that might occur during different
developmental stages were the reason for the observed
growth retardation.
More detailed biochemical analyses of the LNR
plants showed that they had striking changes in nitrogen
and sugar content. A strong difference in total soluble
nitrogen contents between wild-type Nicotiana tabacum
cv. `Gatersleben' and the transformants was observed.
The glutamine content of the LNR plants was more than
90% reduced as compared to wild-type plants while
35S-NR transformants that do express root NR showed
normal levels of glutamine. The glutamate contents in
LNR plants were only moderately lowered (2±3-fold), in
both leaves and roots. The decreased levels of glutamine
and glutamate in the leaves of LNR plants could be
explained with the reduced NR activity in the leaves of
LNR plants. However, the following explanation should
also be considered: because root NR activity was not
measurable in the LNR transformants and NO3 was
the sole source of nitrogen of the plants, the amino-N
accumulated in the roots cannot originate from NO3
reduction and assimilation in the roots. Thus, the roots of
the LNR plants constitute a larger sink for amino
compounds than the roots of the wild type because
down-regulation of NR activity in LNR roots creates an
additional demand for reduced nitrogen and seems to
interact with the cycling pool of amino compounds in
the plants. Glutamine, as the major amino compound
allocated from the leaves to the roots in the phloem of
1257
several species (Gessler et al., 1998a), has apparently
to satisfy this additional demand which might explain
the sharp decline of glutamine levels in the leaves of
LNR plants that was not seen in the 35S-NR control
transformants. Roots need reduced nitrogen in order
to maintain an appropriate rate of growth, therefore
glutamine as the major source for LNR roots is readily
metabolized.
The lack of NR activity in the roots of the LNR
transformants resulted in enhanced accumulation of
NO3 in the leaves but not in the roots. This might be
explained in two ways: either the lower NR activities in
the leaves are the reason for the higher NO3 contents
oruand NO3 accumulation in leaves is under control
of root NR activity. 35S-NR control transformants
expressing root NR do not accumulate high NO3 in
the leaves. Scheible et al. suggested that higher concentration of nitrate in the shoot acts as a signal to regulate
shoot±root allocation of nitrogen in tobacco (Scheible
et al., 1997b).
In LNR roots, reduced nitrogen cannot be gained by
any nitrate reduction in the root. As a consequence, all
reduced nitrogen has to be allocated by phloem transport
from the leaves to the root. Winter et al. showed that
phloem transport of amino acids is mediated by a mass
¯ow of sucrose driven by active phloem loading of sucrose
(Winter et al., 1992). Thus, enhanced phloem loading of
sucrose may be required to achieve an allocation of
amino compounds suf®cient to maintain appropriate root
growth. This view is supported by the observation that
not only the levels of nitrogen metabolites were changed
in transformants lacking root NR activity, but also
sugar metabolism was affected. Roots of LNR plants had
sucrose levels 6±10 times higher than in the wild type.
Also the hexose levels in the roots of the LNR transformants were 2±3 times higher than in the wild type,
while the sugar levels in the leaves of LNR plants and
the wild type were very similar. Roots of LNR grew
smaller than roots of wild type with respect to biomass
production. Apparently, the lack of nitrate reduction
produces signals controlling root growth in a yet unknown
manner. Accordingly, sugar accumulation in LNR roots
may be considered as a general consequence of impaired
growth. In addition, the high sucrose accumulation
suggests, that sucrose translocation or unloading was
a major point of direct or indirect control by nitrate
reduction. Detailed analyses (J Kruse, unpublished
results) of metabolite allocation in Nia30-transformants
will further elucidate the interactions between nitrate
reduction, carbon metabolism, allocation, and growth.
Acknowledgements
We gratefully acknowledge the ®nancial support of the Deutsche
Forschungsgemeinschaft.
1258
HaÈnsch et al.
References
An G, Ebert PR, Mitra A, Ha SB. 1987. Binary vectors. Plant
Molecuar Biology Manual, Sec A3u2.
Crawford NM. 1995. Nitrate: nutrient and signal for plant
growth. The Plant Cell 7, 859±868.
Dorbe M-F, Caboche M, Daniel-Vedele F. 1992. The tomato
nia gene complements a Nicotiana plumbaginifolia nitrate
reductase-de®cient mutant and is properly regulated. Plant
Molecular Biology 18, 363±376.
Ferrario S, Valadier MH, Morot GJF, Foyer C-H. 1995. Effects
of constitutive expression of nitrate reductase in transgenic
Nicotiana plumbaginifolia L. in response to varying nitrogen.
Planta 196, 288±294.
Foyer C-H, Lescure JC, Lefebvre C, Morot GJF, Vincentz M,
Vaucheret H. 1994. Adaptations of photosynthetic electron
transport, carbon assimilation and carbon partitioning
in transgenic Nicotiana plumbaginifolia plants to changes in
nitrate reductase activity. Plant Physiology 104, 171±178.
Gessler A, Schultz M, Schrempp S, Rennenberg H. 1998a.
Interaction of phloem-translocated amino compounds with
nitrate net uptake by the roots of beech (Fagus sylvatica)
seedlings. Journal of Experimental Botany 49, 1529±1537.
Gessler A, Schneider S, Weber P, Hanemann U, Rennenberg H.
1998b. Soluble N compounds in trees exposed to high loads
of N: a comparison between the roots of Norway spruce
(Picea abies) and beech (Fagus sylvatica) trees grown under
®eld conditions. New Phytologist 138, 385±399.
Glaab J, Kaiser WM. 1995. Inactivation of nitrate reductase
involves NR-protein phosphorylation and subsequent
`binding' of an inhibitor protein. Planta 195, 514±518.
Gojon A, Dapoigny L, Lejay L, Tillard P, Rufty TW. 1998.
Effects of genetic modi®cations of nitrate reductase expression on 15NO3 uptake and reduction in Nicotiana plants.
Plant, Cell and Environments 21, 43±53.
Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholz DA,
Rogers SG, Fraley RT. 1985. A simple and general method
to transferring genes into plants. Science 227, 1229±1231.
Huber SC, Bachmann M, Huber JL. 1996. Post-translational
regulation of nitrate reductase activity: a role for Ca2q and
14-3-3 proteins. Trends in Plant Science 1, 432±438.
Kaiser WM. 1997. Regulatory interaction of carbon and nitrogen
metabolism. Progress in Botany 58, 159±163.
Kaiser WM, Huber SC. 1994. Post-translational regulation
of nitrate reductase in higher plants. Plant Physiology 106,
817±821.
Kaiser WM, Spill D. 1991. Rapid modulation of spinach leaf
nitrate reductase by photosynthesis: in vitro modulation by
ATP and AMP. Plant Physiology 96, 368±375.
Kleinhofs A, Warner RL. 1990. Advances in nitrate assimilation.
In: Mi¯in BJ, Lea PJ, eds. The biochemistry of plants, Vol. 16.
Intermediary nitrogen metabolism. New York: Academic
Press, 89±120.
Koch KE. 1997. Molecular crosstalk and the regulation of C
and N-responsive genes. In: Foyer CH, Quick WP, eds.
A molecular approach to primary metabolism in higher plants.
Taylor & Francis Ltd., 105±124.
Laemmli UK. 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227,
680±685.
MacKintosh C. 1997. Regulation of CuN metabolism by reversible protein phosphorylation. In: Foyer CH, Quick WP, eds.
A molecular approach to primary metabolism in higher plants.
Taylor & Francis Ltd., 179±201.
MuÈller AJ. 1983. Genetic analysis of nitrate reductase-de®cient
tabacco plants regenerated from mutant cells: evidence for
duplicate structural genes. Molecular and General Genetics
192, 275±281.
MuÈller AJ, Mendel RR. 1989. Biochemical and somatic cell
genetics of nitrate reduction in Nicotiana. In: Wray JL,
Kinghorn JL, eds. Molecular and genetic aspects of nitrate
assimilation. Oxford: Oxford University Press, 166±185.
Murashige T, Skoog F. 1962. A revised medium for rapid
growth and bioassay with tobacco tissue cultures. Physiology
Plantarum 15, 473±497.
Rufty TW. 1997. Probing the carbon and nirogen interaction:
a whole plant perspective. In: Foyer CH, Quick WP, eds.
A molecular approach to primary metabolism in higher plants.
Taylor & Francis Ltd., 255±273.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning:
a laboratory manual. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory.
Scheible W-R, GonzaÂlez-Fontes A, Morcuende R, Lauerer M,
Geiger M, Glaab J, Gojon A, Schulze E-D, Stitt M. 1997a.
Tobacco mutants with a decreased number of functional nia
genes compensate by modifying the diurnal regulation of
transcription, post-translational modi®cation and turnover
of nitrate reductase. Planta 203, 304±319.
Scheible W-R, Lauerer M, Schulze E-D, Caboche M, Stitt M.
1997b. Accumulation of nitrate in the shoot acts as a signal to
regulate shoot-root allocation in tobacco. The Plant Journal
11, 671±691.
Stockhaus J, Eckes P, Rocha-Sosa M, Schell J, Willmitzer L.
1987. Analysis of cis active sequences involved in the leafspeci®c expression of a potato gene in transgenic plants.
Proceedings of the National Academy of Sciences, USA 84,
7943±7947.
Stockhaus J, Schell J, Willmitzer L. 1989. Correlation of
the expression of the nuclear photosynthetic gene ST-LS1
with the presence of chloroplasts. EMBO Journal 8,
2445±2451.
Toepfer R, Schell J, Steinbiss HH. 1988. Versatile cloning
vectors for gene expression and direct gene transfer in plant
cells. Nucleic Acids Research 16, 8725.
Vaucheret H, Kornenberger J, Rouze P, Caboche M. 1989.
Complete nucleotide sequence of the two homeologous
tobacco nitrate reductase genes. Plant Molecular Biolology
12, 597±600.
Vaucheret H, Chabaud M, Kornenberger J, Caboche M. 1990.
Functional complementation of tobacco and Nicotiana
plumbaginifolia nitrate reductase mutants by transformation
with the wild-type allele of the tobacco structural genes.
Molecular and General Genetics 220, 468±474.
Verwoerd TC, Dekker BMM, Hoekema A. 1989. A small-scale
procedure for the rapid isolation of plant RNAs. Nucleic
Acids Research 17, 2362.
Vincentz M, Caboche M. 1991. Constitutive expression
of nitrate reductase allows normal growth and development
of Nicotiana plumbaginifolia plants. EMBO Journal 10,
1027±1035.
Wilkinson JQ, Crawford NM. 1993. Identi®cation and characterization of a chlorate-resistant mutant of Arabidopsis
thaliana with mutations in both nitrate reductase structural
genes NIA1 and NIA2. Molecular and General Genetics 239,
289±297.
Winter H, Lohaus G, Heldt H. 1992. Phloem transport of amino
acids in relation to their cytosolic levels in barley leaves. Plant
Physiology 99, 996±1004.