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Plant Physiol. (1998) 117: 293-302
Overexpression of Nitrate Reductase in Tobacco Delays
Drought-Induced Decreases in Nitrate Reductase
Activity and
mRNA1
Sylvie Ferrario-Méry*,
Marie-Hélène Valadier, and
Christine H. Foyer
Laboratoire du Metabolisme, Institut National de la Recherche
Agronomique, Route de Saint-Cyr, F-78026 Versailles, France (S.F.-M.,
M.-H.V.); and Department of Environmental Biology, Institute of
Grassland and Environmental Research, Plas Gogerddan, Aberystwyth,
Ceredigion SY23 3EB, United Kingdom (C.H.F.)
 |
ABSTRACT |
Transformed (cauliflower mosaic virus
35S promoter [35S]) tobacco (Nicotiana plumbaginifolia
L.) plants constitutively expressing nitrate reductase (NR) and
untransformed controls were subjected to drought for 5 d.
Drought-induced changes in biomass accumulation and photosynthesis were
comparable in both lines of plants. After 4 d of water
deprivation, a large increase in the ratio of shoot dry weight to fresh
weight was observed, together with a decrease in the rate of
photosynthetic CO2 assimilation. Foliar sucrose increased
in both lines during water stress, but hexoses increased only in leaves
from untransformed controls. Foliar NO3
decreased rapidly in both lines and was halved within 2 d of the
onset of water deprivation. Total foliar amino acids decreased in
leaves of both lines following water deprivation. After 4 d of
water deprivation no NR activity could be detected in leaves of
untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much
more stable than NR activity. NR mRNA abundance increased in the leaves
of the 35S-NR plants and remained constant in controls for the first
3 d of drought. On the 4th d, however, NR mRNA suddenly decreased
in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls.
The phosphorylation state of the protein was unchanged by long-term
drought. There was a strong correlation between maximal extractable NR
activity and ambient photosynthesis in both lines. We conclude that
drought first causes increased NR protein turnover and then accelerates
NR mRNA turnover. Constitutive NR expression temporarily delayed
drought-induced losses in NR activity. 35S-NR expression may therefore
allow more rapid recovery of N assimilation following short-term water
deficit.
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INTRODUCTION |
C and N metabolism are co-regulated in higher plants. Energy and C
skeletons required for N assimilation are provided either directly or
indirectly (via Suc) by photosynthesis. A high rate of
CO2 assimilation favors a high rate of N
assimilation and vice versa (Ferrario et al., 1995 ). Molecular and
metabolic controls are implicated in the C to N interaction, involving
reciprocal regulation between the pathways of C and N assimilation
(Champigny and Foyer, 1992). The present study concerns the regulation
of NR, the first enzyme of primary N assimilation in plants. This enzyme is regulated at the transcriptional level by the availability of
the substrate NO3 and by the
end product of the N assimilation pathway, Gln. NR activity is also
regulated posttranscriptionally by a phosphorylation-dephosphorylation mechanism. The dephosphorylated and phosphorylated NR proteins are
equally active, but phosphorylation sensitizes the enzyme to inhibition
by an inhibitory 14-3-3 protein (NIP) in the presence of
Mg2+ (Glaab and Kaiser, 1995; MacKintosh et al.,
1995). Both types of NR regulation respond to the changes in C
metabolism, since transcription is stimulated by Suc (Cheng et al.,
1992; Vincentz et al., 1993) and NR inhibition by protein
phosphorylation is stimulated by low rates of C fixation (Kaiser and
Förster, 1989).
Nutrient deficiencies are an intrinsic feature of water deficits in
natural and controlled environments (Talouizite and Champigny, 1988;
Larsson et al., 1989; Larsson, 1992; Pugnaire and Chapin, 1992;
Beyrouty et al., 1994; Brewitz et al., 1996). The loss of transpiration
and turgor causes a decrease in
NO3 absorption by the roots
and in transport from the roots to the leaves (Shaner and Boyer, 1976;
Larsson, 1992). NO3
availability then limits NO3
assimilation. NR can be inhibited soon after the onset of water deprivation (Plaut, 1974), but Gln synthetase and other related enzymes
are relatively unaffected (Becker and Fock, 1986a, 1986b; Foyer et al.,
1998). Drought-induced decreases in foliar N have been shown to
specifically limit the capacity for recovery from water deficits in
prairie grasses (Heckathorn and De Lucia, 1994, 1995). In such species
decreases in foliar N of up to 40% induced by water stress persisted
long after water had been restored to the plants (Heckathorn and De
Lucia, 1994, 1995). As a direct result of this N deficit,
photosynthesis was impaired, but photosynthesis and leaf N recovered in
parallel once water was restored to the plants (Heckathorn et al.,
1997).
Many posttranscriptional control mechanisms respond to water stress,
including mRNA processing, transcript stability, translation efficiency, and protein turnover (Ingram and Bartels, 1996). Protein kinases involved in the transcriptional regulation of protein synthesis
are induced by water stress in Arabidopsis thaliana (Urao et
al., 1994). Proteolytic activity increases during drought, and enhanced
protease activity is implicated in the acceleration of the protein
turnover observed under these conditions. Consequently, typical
proteinogenic amino acids and Pro accumulate in water-stressed plants
(Fukutoku and Yamada, 1984).
An important question arises concerning the molecular basis for
drought-induced decreases in NR activity (Foyer et al., 1998). Decreased NO3
availability will inhibit NR gene transcription and decrease the
stability of NR mRNAs. It could also affect other factors such as
posttranscriptional controls. In maize leaves NR gene transcription is
specifically and rapidly inhibited by water stress (Foyer et al.,
1998). It was therefore of interest to study the responses of NR in
water-stressed tobacco (Nicotiana plumbaginifolia) plants in which the native NR gene had been replaced by a
35S-NR cDNA construct (Vincentz and Caboche, 1991). In these
transformants NR gene expression is constitutive and should not respond
to water deficits via metabolite-mediated changes in gene expression.
Other effects of metabolites such as
NO3 on NR mRNA stability or
protein turnover are still operative and the NR protein remains posttranscriptionally regulated by phosphorylation and by proteolysis in these plants (Vincentz and Caboche, 1991; Vincentz et al., 1993;
Ferrario et al., 1995, 1996; Nussaume et al., 1995). The present study
involved the application of water stress to 35S-NR transformants, which
are well characterized and provide an unparalleled opportunity to
advance the understanding of the regulation of NR activity in the
drought response in plants.
| |
MATERIALS AND METHODS |
Untransformed and transformed (35S-NR) tobacco (Nicotiana
plumbaginifolia; Vincentz and Caboche, 1991) plants were grown in pots in a growth chamber with a 16-h photoperiod at a temperature of
23°C day/18°C night at 170 µmol m2
s1 irradiance. The plants were supplied daily
with a complete nutrient solution containing 10 mm
NO3 and 2 mm
NH4+ (Coïc and Lesaint,
1975). When the plants reached 7 weeks of age, irrigation was
discontinued for a period of 5 d for 12 plants of each type
(water-stressed plants). Six plants of each type continued to receive
irrigation (control plants). Three days after ceasing irrigation, six
water-stressed plants of each line were rewatered with the complete
nutrient solution (rehydrated plants). Each day during water
stress, the fourth leaf from the apex was harvested (the length of the
first leaf from the apex was  1 cm).
For each treatment, leaves from one-half of the plants (three plants)
were harvested and pooled to study leaves at similar developmental
states. Leaves were harvested 3 h after the beginning of the
photoperiod and immediately frozen in liquid N and then reduced to a
fine powder and stored at  80°C until they were used for biochemical
analyses. An aliquot of this fine powder was then lyophilized for the
extraction of amino acids.
All experiments were carried out three times. In the first two
experiments leaves were used for biochemical analyses as well as
photosynthesis and biomass measurements.
Statistics
Values given for biomass and photosynthesis measurements were
obtained from a minimum of three leaves per plant from between 3 and 10 plants per line depending on the experiment (see tables and figure
legends). The results are given as the mean values for each population
with the se =  n/
 1, where n is the
sd. For biochemical analyses all of the leaves of 3 plants
were pooled. The values given for these analyses represent the means of
the leaves of 3 pooled plants.
Biochemical Analyses
NR Activity
NR was extracted from an aliquot of the leaf powder stored at
80°C. The extraction buffer, which consisted of 50 mm
Mops-KOH, pH 7.8, 5 mm NaF, 1 µm
Na2MoO4, 10 µm FAD, 1 µm leupeptin, 1 µm microcystin, 0.2 g/g fresh weight PVP, 2 mm
 -mercaptoethanol, and 5 mm EDTA, was added to the leaf
powder. A 50-µL aliquot of the uncentrifuged crude extract was
retained for chlorophyll determination. The crude homogenate was then
centrifuged for 5 min at 12,000g and 4°C. The NR activity
and the NO3 content in the
supernatant were assayed immediately. The maximal NR activity
(unphosphorylated form) was measured in the presence of 5 mm EDTA. The activity of the unphosphorylated form was
determined in 10 mm MgCl2. The
reaction mixture consisted of 50 mm Mops-KOH buffer, pH
7.5, containing 1 mm NaF, 10 mm
KNO3, 0.17 mm NADH, and either 10 mm MgCl2 or 5 mm EDTA.
The reaction was stopped after 8 or 16 min by the addition of an equal
volume of sulfanilamide (1%, w/v in 3 n HCl) followed by
n-napthylethylenediamine
dihydrochloride (0.02%, w/v), and the A540
was measured. The activation state of NR is defined as the activity
measured in the presence of 10 mm
MgCl2 divided by the activity measured in the
presence of 5 mm EDTA (expressed as a percentage).
RNA Extraction
Total RNA was extracted from frozen material. The extraction
medium consisted of phenol/100 mm Tris-HCl, pH 8.0, 0.1 m LiCl, 10 mm EDTA, 1% SDS/chloroform-isoamyl
alcohol (24:1, v/v) at a ratio of 1:1:1 (v/v/v). Extracts were
incubated at 80°C as described by Verwoerd et al. (1989). The aqueous
phases, collected by centrifugation at 20,000g for 5 min,
were incubated with an equal volume of 4 m LiCl overnight
at 0°C. Total precipitated RNA was then collected by centrifugation
at 20,000g for 30 min and dissolved in an aqueous solution
of 1% diethylpyrocarbonate. RNA was precipitated by incubation with
0.3 m sodium acetate, pH 5.6, overnight at 20°C. The
precipitated RNA was collected by centrifugation at 20,000g
for 20 min and resuspended in 1% diethylpyrocarbonate. Concentrations
of RNA were estimated spectrophotometrically at 260 nm.
Northern Analysis
The extracted RNA was separated by electrophoresis in 1.3%
agarose gels containing 17% formaldehyde (Maniatis et al., 1982) and
transferred to nylon hybridization-transfer membranes (Genescreen Biotechnology Systems, NEN Research Products, Boston, MA) and cross-linked at 80°C for 2 h. Hybridization with
32P-labeled NR and -ATPase cDNA probes was
performed in 50% formamide, 0.1% SDS, 0.9 m NaCl, 0.9 m Na3PO4, 5 mm EDTA (pH 7.4), 5× Denhardt's solution (0.1% Ficoll
[type 400, Pharmacia], 0.1% PVP, and 0.1% BSA), and 1 mg/100 mL
denatured salmon-sperm DNA. The membranes were incubated overnight at
42°C and then washed twice in 2× SSC (1× SSC = 0.15 m NaCl and 15 mm sodium citrate) and 0.1% SDS. They were then incubated with 0.2× SSC and 1% SDS at 65°C for 5 min
as described by Maniatis et al. (1982).
For the second hybridization with an ATPase probe, the membranes were
initially washed in 0.1× SSC and 0.1% SDS for 3 h. Relative mRNA
amounts were determined by densitometric scanning of the autoradiograms
(Power Look II scanner, UMAX Data Systems, Taiwan) and an advanced
quantifier (J-D Match, BioImage Systems Corp., Ann Arbor, MI). The NR
probe consisted of a 1.6-kb internal EcoRI tobacco
nia2 cDNA fragment as described by Vaucheret et al. (1989). The probe used for detection of the nuclear-encoded -subunit of the
mitochondrial ATPase was obtained from N. plumbaginifolia as
described by Bountry and Chua (1985).
Carbohydrate Analysis
Carbohydrates were extracted in 1 m
HClO4 from the leaf powder that had been stored
at 80°C. The uncentrifuged crude extract was retained for assay of
pheophytin and the rest was centrifuged for 5 min at 12,000g
and 4°C. The pellet was used for starch determination. The
supernatants (500 µL) were neutralized with 200 µL of 0.5 m Tris-HCl, pH 7.5, and 60 µL of 5 m
K2CO3. The precipitate was removed by centrifugation for 5 min at 12,000g, and Suc,
Glc, and Fru were analyzed enzymatically in the supernatant for 5 min at 12,000g (Galtier et al., 1995).
For starch determination, the pellet was resuspended in water and
incubated at 100°C for 2 h following hydrolysis by  -amylase and amyloglucosidase in 20 mm sodium acetate, pH 4.6, for
3 h at 50°C. The Glc formed was assayed as above (Galtier et
al., 1995).
Amino Acid Analysis
Total amino acids were extracted as described for the
carbohydrates and determined by the Rosen colorimetric method (Rosen, 1957).
For determination of amino acid composition, amino acids were extracted
from the lyophilized powder with 2% 5-sulfosalicylic acid (10 mg dry
weight mL1). The crude extracts were
centrifuged at 12,000g for 5 min, and an aliquot of the
supernatant was analyzed by ion-exchange chromatography (model LC5001
analyzer, Biotronics, Lowell, MA; Rochat and Boutin, 1989);
physiological program run with lithium citrate buffers and detection at
A570 and
A440 after postcolumn derivatization with
ninhydrin (Rochat and Boutin, 1989).
Determinations of NO3 and
Chlorophyll
NO3 content was analyzed
in the supernatant from the leaf extracts for NR activity according to
the method of Cataldo et al. (1975). Chlorophyll (from the same
extracts) and pheophytin (extracts for carbohydrates) were assayed as
described by Arnon (1949).
Photosynthesis
The rate of net CO2 assimilation, the
stomatal resistance, and the transpiration of attached tobacco leaves
were measured using an IR gas analyzer (model LCA4, Analytical
Development Co., Hoddesdon, UK).
| |
RESULTS |
Biomass
After 5 d of water deprivation, the fresh weight accumulation
in the shoot was decreased (75%) relative to that of the plants that
were continuously irrigated (Fig. 1A).
This decrease in biomass accumulation included water loss from the
plant tissues as demonstrated by the large increase in the ratio of dry
weight to fresh weight (Fig. 1C). When determined as dry weight,
biomass accumulation was decreased in the shoot by approximately 30%
compared with continuously irrigated plants (Fig. 1B). Since 5 d
of drought caused such severe water loss from the plants that they were
unable to recover following restoration of the water supply (data not shown), plants deprived of water for 3 or 4 d were used in the following experiments. When plants deprived of water for 3 d were rehydrated, biomass was increased after 2 d compared with those deprived of water for the whole experimental period (Fig. 1), but
no differences were observed between the two plant lines under these
conditions. No differences in biomass were found between the two lines
during drought or following rehydration. Leaf samples were selected at
random from the plant populations for the following measurements.
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| Figure 1.
Biomass accumulation in well-irrigated control
plants (stippled bars), in plants deprived of water for 5 d (black
bars), and in plants deprived of water for 3 d and subsequently
rehydrated for 2 d (gray bars). Effects on shoot biomass (A),
shoot dry weight (B), and shoot dry weight to fresh weight ratio (C)
were measured in untransformed N. plumbaginifolia (WT)
and 35S-NR transformants (C1).
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Photosynthesis and Foliar Carbohydrate Contents
Photosynthetic rates (Fig. 2)
decreased from the 2nd d of water deprivation, reaching a minimum (30%
of the initial value) on the 4th d in both tobacco lines. Rehydration
at d 3 allowed recovery of photosynthetic activity to control rates
within 2 d, suggesting that the dehydrated state was not
irreversible at this stage (Fig. 2). Foliar carbohydrate contents were
relatively constant over the first 3 d of the experiment (Fig.
3). On d 4 of water stress, however, Suc
was increased and starch was decreased in both lines of plants (Fig.
3A). On d 4 of water deprivation, foliar hexose contents also increased
but only in the untransformed plants (Fig. 3, B and C). Rehydration at
d 3 caused a rapid but transient increase in leaf hexoses in both
lines, whereas Suc and starch contents decreased.
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| Figure 2.
The effect of water deprivation ( ,  )
and rehydration (shaded symbols) compared with well-watered conditions
( ,  ) on ambient photosynthesis in untransformed (circles and
dotted lines) N. plumbaginifolia and 35S-NR
transformants (triangles and bold lines).
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| Figure 3.
The effect of water deprivation on the
carbohydrate contents of leaves of untransformed N. plumbaginifolia (stippled bars) and of 35S-NR transformants
(shaded bars). Foliar Suc (A), Fru (B), Glc (C), and starch (D) were
measured in plants deprived of water for 4 d. The effect of
rehydration on d 3 untransformed N. plumbaginifolia
(white bars) and 35S-NR transformants (black bars) is also shown. Chl,
Chlorophyll.
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Foliar NO3 Content
In the absence of water deficit, the foliar
NO3 content was higher in the
leaves of untransformed plants than in those of the 35S-NR
transformants, which is consistent with previously published
observations (Ferrario et al., 1996).
NO3 decreased on the 2nd d of
drought in the leaves of both lines as a result of water stress (to a
value of 50% of the irrigated controls in less than 2 d).
Rehydration at d 3 induced a rapid increase in the
NO3 content of the leaves
(Fig. 4A).
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| Figure 4.
The effect of water deprivation on the foliar
NO3 content (A) and on foliar amino acid
accumulation (B) in untransformed N. plumbaginifolia
(stippled bars) and in 35S-NR transformants (shaded bars). The effect
of rehydration after 3 d of water stress in untransformed
N. plumbaginifolia (white bars) and in 35S-NR
transformants (black bars) is also shown. Chl, Chlorophyll.
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Total Foliar Amino Acids
The amino acid and Gln contents of the leaves of the well-watered
35S-NR plants were higher than those of the untransformed controls
(Fig. 4; Table I), as has been reported
previously (Quilleré et al., 1994). Water deprivation caused a
decrease in total foliar leaf amino acid contents of both the
untransformed and 35S-NR plants (Fig. 4B). During the 1st d of water
deprivation, however, the amino acid content of the leaves of the
35S-NR plants was much higher than that of the untransformed controls
(Fig. 4B). As the duration of water stress increased, the foliar amino
acid content of the 35S-NR plants dramatically decreased such that similar values were obtained in both lines by d 2 (Fig. 4B).
Rehydration after 3 d caused an increase in foliar amino acids
(within 24 h), but there were no longer differences between the
two lines. Water deficit induced a decrease in all amino acids (Table
I). The high Gln content observed in the leaves of the well-watered 35S-NR plants was maintained on the 1st d of water stress (Table I),
whereas the Gln pool decreased in the leaves of the untransformed controls. Rehydration increased Gln and other amino acids in both lines
(Table I).
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|
Table I.
Foliar amino acid composition in the 35S-NR
(C1) and untransformed tobacco (WT) lines during water deprivation
and rehydration
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Foliar NR Activity, NR Activation State, and NR mRNA Content
Maximal extractable NR activity was higher in the leaves of the
35S-NR transformants than in those of the untransformed controls in
well-watered conditions (Fig. 5A). Foliar
NR activity decreased from d 2 of water stress in both lines, but the
decrease was more pronounced in the leaves of the untransformed
controls than in those of the 35S-NR transformants. After 4 d of
water deficit no NR activity could be detected in the leaves of the
untransformed plants, whereas more than 50% of the original NR
activity remained in the leaves of the transformed line (Fig. 5, A and
B). Rehydration induced an increase in NR activity in the untransformed
line; NR activity approached control values within 2 d of
restoring the water supply, and there was no longer any difference in
NR activity between the two lines. The NR activation state was not modified by the water stress (Fig. 5C).
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| Figure 5.
The effect of water deprivation on maximal
extractable NR activity (A), on NR activity extracted and assayed in
the presence of Mg2+ (B), and on the NR activation state
(C). A and B, Leaves from untransformed N. plumbaginifolia (stippled bars) and in 35S-NR transformants
(shaded bars) were compared. The effects of rehydration after 3 d
of water stress on untransformed N. plumbaginifolia (white bars) and in 35S-NR transformants (black bars) were measured on
d 4. C, Untransformed controls ( ) and 35S-NR transformants ( )
were subjected to water stress for 3 d and then water was restored
for a further 2 d ( , ). Chl, Chlorophyll.
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Twice the amount of NR mRNA (expressed as a percentage of
-ATPase) was present in the leaves of the 35S-NR line compared with
those of the untransformed controls (Fig.
6). The difference in NR mRNA content was
most marked after 3 d of water deficit, when NR mRNA abundance in
the transformed line was about 7 times that of the untransformed
controls (Fig. 6). This difference was due entirely to an increase in
NR mRNA in the transformed line. Steady-state transcript abundance was
not changed in the untransformed controls at d 3, whereas NR activity
had decreased in the leaves of the untransformed controls (Figs. 5A and
6). This suggests that the stability of the NR protein, but not NR
mRNA, was affected by water stress at this time. On d 4 of water
stress, NR mRNA decreased suddenly in the 35S-NR transformants,
decreasing to values similar to those found in the untransformed
controls grown in similar conditions (Fig. 6). Rehydration did not
restore NR mRNA abundance in the untransformed controls, which
decreased even further, whereas in the 35S-NR leaves recovery was
rapid. In the untransformed controls NR mRNA abundance had not
recovered after 24 h of rehydration (Fig. 6), whereas it was
nearly four times that measured at the beginning of the experiment in
the 35S-NR transformants.
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| Figure 6.
The effect of water deprivation on NR mRNA
accumulation in leaves of untransformed N. plumbaginifolia (WT) and 35S-NR transformants (C1) expressed as
a percentage of ATPase mRNA. Plants were deprived of water
immediately after the first measurement on day t0. mRNA abundance was then measured at the same point in the photoperiod on
consecutive days of water stress (t1, t2,
t3, and t4) and after 1 d of rehydration
following 3 d of water deprivation (t4R).
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Relationships between Maximal Extractable NR Activity and
Photosynthetic Activity
A correlation between maximal extractable NR activity and net
photosynthesis was observed in both lines. Decreases in photosynthetic activity following water deprivation were accompanied by comparable decreases in NR activity (Fig. 7). NR
activity was always higher in the leaves of the 35S-NR line than in
untransformed controls at similar photosynthetic activities (Fig. 7).
When photosynthesis was maximally inhibited as a result of water
deprivation, NR activity was undetectable in the untransformed plants
(Fig. 7). In the 35S-NR transformants, however, NR activity was always
detectable even in severe water stress. Therefore, the relationship
between maximal extractable NR activity and CO2
assimilation rate was shifted in the transformed plants compared with
untransformed controls, but the two parameters always decreased in
parallel in both lines of plants (Fig. 7).
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| Figure 7.
The relationship between maximal extractable NR
activity and ambient photosynthesis in leaves of untransformed ()
N. plumbaginifolia and of 35S-NR () transformants
during water stress. Chl, Chlorophyll.
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| |
DISCUSSION |
Drought induced a rapid decrease in NR activity in untransformed
tobacco leaves similar to that observed in maize (Foyer et al., 1998)
and in other species (Plaut, 1974; Heuer et al., 1979). During the
first 3 d of drought this was caused by a decrease in NR protein.
On d 4 of water deprivation NR transcripts also decreased in both
lines. Drought-induced changes in NR gene expression caused by
differences in foliar NO3 and
sugar content should only largely affect the native NR promoter, but
drought-induced effects on the expression of the 35S promoter are also
possible. In the present study differences between the 35S-NR
transformants and the untransformed lines were evident in NR
transcripts. NR mRNA abundance greatly increased in the 35S
transformants over the first 3 d of drought but was stable in the
leaves of untransformed controls during this period.
Only at an advanced stage of dehydration (4 d) was NR mRNA decreased in
both lines. In the 35S-NR line NR mRNA abundance was restored within
24 h of rehydration but in the transformed controls it did not
recover during this period. Therefore, 35S-NR expression appears to be
less inhibited by drought than expression of the native NR promoter.
Figure 6 clearly demonstrates that NR transcript abundance is increased
as a result of water deficit in the transformants. NR mRNA stability
may be affected by water stress. Effects on stability would be
comparable if the concentrations of metabolic factors affecting
stability were similar in both lines. The decrease in NR mRNA abundance
after 4 d of drought might be a response to changes in metabolite
concentrations. NO3
concentration affects NR mRNA stability and NR gene transcription (Galangau et al., 1988). Gln may exert a negative influence on NR mRNA
stability, since NR transcript abundance increased following drought as
the foliar Gln pool decreased.
Changes in NR activity were observed in both lines during water stress,
but NR activity persisted in the leaves of transformants for much
longer than in the untransformed control leaves. Water stress induces
proteases that increase protein turnover (Ingram and Bartels, 1996).
Cys proteases were induced within 10 h of the onset of water
stress in A. thaliana (Koizumi et al., 1993). In
addition, a thiol proteinase has been identified as an NR-inactivating factor in barley leaves (Hamano et al., 1984). Proteases may be induced
by drought in more or less the same manner in both tobacco lines used
in this study. The induction of an NR-specific protease may explain the
observed decrease of NR activity, since loss of NR protein occurred in
the absence of changes in NR mRNA abundance.
In the 35S-NR line, NR activity was always higher than that of the
untransformed controls and remained present even when the photosynthetic activity was decreased to a minimum value. Relatively high rates of transcription or deregulation of transcription in the
35S-NR line could compensate for losses in NR protein incurred as a
result of increased protease activity. The high level of NR activity
found in the leaves of the 35S-NR line during drought could also result
from the more or less ubiquitous expression of the 35S promoter, which
allows expression of the NR gene in all plant tissues, unlike the
native NR promoter, which is expressed only in leaf mesophyll cells.
Consequently, if proteolytic degradation of the NR protein is tissue
specific, it would be less efficient in the 35S-NR line than in the
untransformed controls.
Phosphorylation of the NR protein has been shown to occur rapidly
(within hours) as a result of water deficit (Kaiser and Förster,
1989; Brewitz et al., 1996). In addition, the phosphorylated form of
the NR protein has been suggested to be less stable than the
unphosphorylated protein and, therefore, perhaps more sensitive to
proteolytic degradation (Lejay et al., 1997). In the present study only
the longer-term effects of water stress were studied. NR activation
state was not modified as a result of drought (Fig. 5), suggesting that
phosphorylation of NR protein may be an early but transitory response
to water stress. The only report of long-term increases in the
phosphorylation state of NR during drought concerns maize, a
C4 plant (Foyer et al., 1998). Furthermore,
water-deficit-induced changes in phosphorylation state may differ
between species and, therefore, would be different in tobacco compared
with spinach (Kaiser and Förster, 1989), tomato (Brewitz et al.,
1996), or maize (Foyer et al., 1998), in which this type of regulation
has been observed.
The results presented here suggest that water stress initially causes a
decrease in the stability of the NR protein. The effects of this change
were observed much earlier in the untransformed controls than in the
35S-NR transformants. This might be interpreted as a decrease in the
sensitivity of the 35S transformants to drought-induced effects on N
metabolism. The total foliar amino acid pool was higher in the 35S-NR
leaves at the beginning of the experiment and remained higher on the
1st d of drought. This suggests that more efficient N assimilation can
occur in the 35S-NR transformants than in the untransformed controls
during short-term (24-h) water deficits. As long as
NO3 did not limit NR
assimilation, the amino acid contents of the leaves of the 35S-NR line
did not decrease. Substantial decreases in foliar
NO3 have been reported in
droughted leaves (Heckathorn and De Lucia, 1994, 1995), whereas total
amino acid levels may increase in the advanced stages of drought
because of proteolysis (Fukutoku and Yamada, 1984) and perturbations in
the translocation of amino acids from shoots to roots (Larsson, 1992).
Primary metabolism must maintain the supply of C skeletons, ATP, and
reducing power to drive N assimilation during water stress. In the
early states of drought, dehydration causes stomatal closure and
CO2 fixation is limited by
CO2 availability. Photosynthetic electron
transport, mitochondrial respiration, and photorespiration are still
active and can even increase at this stage (Krampitz and Fock, 1984).
The NADH required for NO3
reduction in leaves can be provided by several different sources, such
as oxidation of glyceraldehyde 3-P or substrate oxidation in the
tricarboxylic acid cycle or Gly oxidation (Kumar et al., 1988).
However, shoot biomass production was comparable in the two lines over
the first 5 d of water stress. Similarly, photosynthesis was
decreased by water deficits to a comparable degree in both lines.
Therefore, constitutive NR expression did not facilitate higher
photosynthetic rates in the 35S-NR transformants than in the
untransformed controls, as was already observed with varying NO3 supply (Ferrario et al.,
1995). However, correlations between maximal extractable NR activity
and net photosynthesis were observed in both lines regardless of the
foliar NR activity prior to water deprivation.
These findings demonstrate coordinate regulation of photosynthetic
CO2 assimilation and NR activity in tobacco
leaves. In C4 prairie grasses drought-induced
losses in photosynthetic capacity were shown to result largely from
decreases in shoot N; recovery of photosynthesis following drought was
only possible when shoot N contents were restored (Heckathorn and De
Lucia, 1994, 1995; Heckathorn et al., 1997). The present study
demonstrates that coordinate control of C and N assimilation can also
be observed in tobacco. In this case, the regulatory relationship
involves total extractable NR activity and net photosynthesis.
Metabolic cross-talk between C and N metabolism involves multiple steps of coordinate control in which many metabolic signals such as NO3, Gln, Suc, and reductants
participate.
The molecular and metabolic basis for the correlations presented in
Figure 7 must therefore be highly complex and also indicate that the
precise regulatory coordination is perturbed, at least in the short
term, by constitutive NR expression. This not only suggests that at
least part of the coordinate regulation involves regulation of NR gene
transcription in the untransformed plants but also demonstrates that
the other mechanisms of NR regulation cannot compensate for the absence
of normal transcriptional controls (at least in the short term). Only
after 4 d of water stress were NR mRNA levels decreased in both
lines. At this point NR activity was still present in the leaves of the
transformants. Although other factors such as
NO3 availability interact to
limit the flux through the pathway of N assimilation, there is no doubt
that the transformants are better equipped in terms of available NR
protein to rapidly restore N assimilation, when favorable conditions
return, than the untransformed controls. No immediate benefit was
observed in terms of biomass accumulation in the short term, but under
field conditions of fluctuating water availability constitutive NR
expression may confer a physiological advantage by providing a
preemptive modification, preventing slowly reversible losses in
N-assimilation capacity.
| |
FOOTNOTES |
1
This work was funded by European Economic
Community Biotechnology (contract no. BIO2 CT93 0400) and was a project
of the Technical Priority Network D Nitrogen Utilization and
Efficiency.
*
Corresponding author; e-mail christine.foyer{at}bbsrc.ac.uk; fax
44-1970-828357.
Received August 20, 1997;
accepted February 2, 1998.
| |
ABBREVIATIONS |
Abbreviations:
NR, nitrate reductase.
35S, 35S promoter from
cauliflower mosaic virus.
| |
ACKNOWLEDGMENT |
We are indebted to Yvette Roux for assistance with amino acid
analyses.
| |
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Top
Abstract
Introduction