Download Nitrate reductase activity in chicory roots

Journal of Experimental Botany, Vol. 48, No. 306, pp. 59-65, January 1997
Journal of
Experimental
Botany
Nitrate reductase activity in chicory roots
following excision
Christophe Vuylsteker, Brigitte Huss and Serge Rambour1
Laboratoire de Physiologie et Genetique Moteculaire V6g4tales, Universite des Sciences et Technologies de
Lille, F-59655 Villeneuve d'Ascq Cedex, France
Received 15 April 1996; Accepted 24 July 1996
Abstract
In young chicory plantlets [Cichorium intybus L. Witloof
cv. Flash), nitrate assimilation takes place mainly in
the roots. Nitrate reductase activity (NRA) was measured in roots deprived of shoot control by excision and
transferred into a sucrose-containing medium. Such a
treatment resulted in a drop of about 6 0 % of NRA
within 3 h. The level of NR protein decreased after
12 h and the level of NR-mRNA after several days. This
adaptation of nitrate assimilation to excision was
affected by a phosphorylation-dephosphorylation
mechanism as shown by increased sensitivity to magnesium of in vitro NRA. Okadaic acid, a serinethreonine protein phosphatases inhibitor, enhanced
the decrease of NRA. Conversely, staurosporine, a
serine-threonine protein kinases inhibitor, antagonized the inhibition of NRA. This suggests that excision
caused a rapid inactivation of NRA in roots of chicory
by modifying the phosphorylation balance towards a
phosphorylated NR form which could enter an inactive
complex.
Key words: Chicory, nitrate reductase, staurosporine.
Introduction
As much as 25% of the energy of photosynthesis is
consumed by the nitrate assimilation pathway
(Solomonson and Barber, 1990). As a consequence, most
of the fast growing plants reduce nitrate in their leaves
where the main part of the reducing power arises directly
from light via ferredoxin (Beevers and Hageman, 1980).
Thus nitrate reduction does not compete with the photoreduction of CO2 (Robinson, 1988). Because of such a high
energy requirement, regulation of NR is often viewed as
an energy balance between nitrate assimilation and CO2
reduction (Oaks, 1985). This scheme is supported by data
from Cheng et al. (1992) and Vincentz et al. (1993) who
showed that expression of reporter genes directed by 5'
flanking regions of NR genes is regulated by sucrose in
the absence of light. Conversely glutamine inhibits the
induction of NR in tobacco and Nicotiana plumbaginifolia
(Deng et al., 1991; Vincentz et al., 1993).
When the nitrate assimilatory pathway occurs in the
roots, high amounts of photosynthates must be imported
and oxidized to provide the required reductants, energy
and carbon skeletons. It has been assumed that the
mechanisms involved in the control of nitrate reduction
were similar in leaves and roots. Since the heterotrophic
status of the roots imposes a total dependence of nitrate
reduction on photosynthates supplied by the leaves, the
relationship between NRA and light or photosynthesis
remains indirect compared to leaves (Huppe and Turpin,
1994). This implies some differences in the control mechanisms. However, fewer studies have been done on the
metabolic regulation of nitrate reduction and assimilation
in roots, particularly in the carbohydrate storing ones.
Chicory which is a biennial Asteraceae, develops a
rosette of leaves and a tuberous root at the end of the
first year of its growth cycle. It provides an interesting
model for studying spatial and temporal regulation of
nitrate assimilation. In chicory, in vivo NRA remains
higher in roots than in leaves whether the plants are
grown in vitro or in fields until they become tuberous
(Dorchies and Rambour, 1985). The nitrate content is
high in roots during this period and accounts for almost
10% of the total nitrogen (Limami et al., 1993). During
tuberization, which starts at approximately the third
month, NRA decreases in roots and leaves supply the
1
To whom correspondence should be addressed. Fax: +33 20 43 68 49. E-mail: rambour©univ-Lille1 .fr
Abbreviations: ATPase, ATP synthase; GS, glutamine synthetase; NR, nitrate reductase; NRA, nitrate reductase activity; TUB, tubulin.
© Oxford University Press 1997
60
Vuylsteker et al.
plant with reduced nitrogen (Dorchies and Rambour,
1985). Other enzymatic activities involved in the nitrate
assimilation pathway such as glutamate synthase or glutamine synthetase show similar seasonal modifications
(Sechley et al., 1991). At that later stage, roots accumulate
inulin and reduced nitrogen, predominantly as amino
acids whereas their nitrate content becomes insignificant
(Limami et al., 1993). Thus, nitrate reduction in chicory
is controlled by the nitrate flux and is tightly linked to
modifications between sink and source strength during
the developmental cycle.
In roots, NRA is controlled by NR synthesis in
response to nitrate (Oaks, 1985), cytokinins (Hanisch
Ten Cate and Breteler, 1982), or by NR proteolysis
(Poulle et al., 1987). In maize roots, glutamine exerts a
particularly strong inhibition on NR at the transcriptional
level (Li et al., 1993). Recent evidence shows that NR is
under metabolite control that is exerted by a reversible
phosphorylation/dephosphorylation reaction. In leaves,
dark decreases NRA by inhibiting dephosphorylation of
the NR protein (Kaiser and Huber, 1994) whereas in
roots maintained in the dark, the decrease of NRA is
related rather to slow protein turnover than to slow
dephosphorylation (Glaab and Kaiser, 1993). Conversely,
phosphorylation of root NR occurs in response to anaerobiosis (Glaab and Kaiser, 1993; Kaiser et al., 1993).
Recent data demonstrated that NR inactivation is
achieved by the formation of a complex comprising
phosphorylated NR and an inhibitor protein (Kaiser and
Huber, 1994; MacKintosh et al., 1995; Bachmann et al.,
1995). Dephosphorylation releases active NR and the
inhibitor protein.
Detopping plantlets abolishes the shoot-to-root relationships. In the absence of sucrose, nitrate reduction is
rapidly decreased (Brouquissee/o/., 1992). This resembles
senescence in leaves. The decrease of enzymatic activities
related to nitrogen assimilation and the subsequent
increases of protein and amino acid degrading activities
are metabolic adaptations to maintain respiratory activity
(Brouquisse et al., 1991; Saglio and Pradet, 1980). Root
excision and their transfer into a liquid medium containing sucrose should abolish the shoot control on nitrate
reduction without creating a carbohydrate starvation. It
could be inferred that excised chicory roots supplied with
sucrose constitute a system in which nitrate reduction is
only determined by the nitrogen demand of the root.
A rapid inactivation of NR by a phosphorylation
reaction immediately after the roots were detopped is
reported here. Thereafter, the levels of both NR protein
and NR-mRNA were strongly decreased.
Materials and methods
Plant material
Chicory seeds {Cichorium intybus L. var. Witloof, cv. Flash)
were surface-sterilized and germinated on solid growth medium
H15, containing 15 mM sucrose, salts of Heller (1953) and
7 g P ' agar. The growth chamber was maintained at 22±1 °C
with a photoperiod of 16/8 h (light/dark) and a light irradiance
of 14 ^M m " 2 s " ' . After 18 d, plants which developed two
cotyledons and four leaves were decapitated, and twelve uniform
roots were transferred, in aseptic conditions, into flasks
containing 50 ml H15 liquid medium.
Inhibitors
Staurosporine was dissolved in DMSO at a concentration of
50/xgml" 1 , stored at — 20 °C, and used at a final concentration
of 50ngml"'. Okadaic acid was dissolved in a solution made
of ethanol and liquid medium (HI5) (1 : 1, v/v) at a concentration of 25/i.gml" 1 , and used at a final concentration of
0.8/igmr 1 .
All chemicals were from Sigma Chemical Co., St Louis, MO.
In vivo nitrate reductase activity
The roots were harvested at different times, weighed and
assayed for NRA according to Jaworski (1971). Individual
roots were introduced in 2 ml of the incubation mixture
comprising 62.5 mM KNO 3 (5 vols), 37.5 mM K-phosphate
buffer pH 7.5 and 1.2% 1-propanol (v/v). Measurements were
made on five independent samples. Experiments were repeated
at least twice. Nitrite was revealed by adding 0.5 ml (11 mM in
3 M HC1) and 0.5 ml of aqueous 10 mM jV-1 naphthyl ethylene
diamine dichloride. NRA was expressed as nmol nitrite produced min" 1 g" 1 FW.
In vitro nitrate reductase activity
In vitro assays are derived from Merlo et al. (1995). Roots were
frozen and ground in a chilled mortar. Extraction buffer
contained 50 mM HEPES-K.OH pH 7.5, 5 mM MgCl2) 0.5 mM
EDTA, 14 mM 2-mercaptoethanol, 0.1% (v/v) Triton XI00,
10% (v/v) glycerol, 50,xM leupeptin, 0.5 mM PMSF, and
polyvinylpyrrolidone 10% (w/v). Extracts were desalted on G25
Sephadex columns equilibrated with the same buffer without
EDTA and MgCl2. The incubation medium contained 100 fA
extract and 400 ^1 of a mixture comprising 50 mM
HEPES-KOH pH7.5, 10 mM KN0 3 , 0.2 mM NADH, and
10 ^M FAD. Modulation of the activation status of NR in
vitro, was performed by adding either 2 mM EDTA or 5 mM
MgCl2 to desalted extracts. Incubation was performed at 30 °C
for 5 min, and the reaction was then stopped by adding 50 [A
0.5 M zinc acetate. Excess NADH was oxidized with phenazine
methosulphate (final concentration 10 ftM). Nitrite was revealed
as above and NRA was expressed as nmol of nitrite min"' mg~'
protein. The protein content was measured according to
Bradford (1976) with bovine serum albumin as a standard.
ELISA immunoquantification of NR proteins
The NR level was quantified by the two sites ELISA procedure
according to Cherel et al. (1986) using monoclonal anti-NR
maize 96925 and S6 polyclonal anti-NR maize antibodies. These
antibodies were first tested against chicory root NR by Western
blot analysis and immunoprecipitation assays.
Total RNA extraction
Total RNA was extracted from the root tissues according to a
procedure derived from Chirgwin et al. (1979). One gram tissue
was ground in liquid nitrogen to a fine powder which was
suspended in 5 vols of 4 M guanidium thiocyanate containing
0.1 M TRIS-HCI and 1% (v/v) 2-mercaptoethanol. Nucleic
acids were then extracted by phenol'chloroform coupled with
Reversible phosphorylation of nitrate reductase
ethanol precipitation (0.75 vol. ethanol and 0.08 vol. 1 M acetic
acid). Nucleic acids were pelleted and dissolved in 10 mM
TRIS-HC1 pH 7.5. RNAs were selectively precipitated with
2 M lithium chloride. RNA were finally dissolved in diethyl
pyrocarbonate-treated sterile water.
61
ATPase, cDNA from a beta subunit of Nicotiana plumbaginifolia
(Boutry and Chua, 1985); TUB, cDNA from alpha tubulin of
Daucus carota (Borkid and Sung, 1985).
Results
Northern analysis
20 /xg total RNA were run in a 1.5% (w/v) agarose formaldehyde
gel (Sambrook et al., 1989). Subsequently, blotting was achieved
on Hybond-N + (Amersham) membranes. DNA probes were
labelled with [a-P32] dCTP (111 TBq mM " ' ICN) using random
priming (T 7 Quickprime Pharmacia). Hybridizations were
performed according to Church and Gilbert (1984); membranes
were then exposed to X-ray films (Kodak X-Omat AR) at
— 80 °C using intensifying screens.
Intensity of the bands was estimated after scanning and
digitization using a Microtek Color/Gray scanner (Biorad)
connected to a Macintosh LCIII (Apple) computer. The
software used was the free ware NIH-1.56.
The probes were: NR, a partial cDNA from nitrate reductase
of Cichorium intybus (X 84102 EMBL Data Library; Palms
et al., 1996); GS1, a complete cDNA from cytosolic glutamine
synthetase of Nicotiana tabacum (gift of B. Hirel, unpublished);
In vivo NRA in isolated roots after their transfer into a liquid
medium
Roots were excised and immediately transferred into
liquid medium. In vivo NRA decreased about 63% within
the first 3 h following the transfer. Thereafter, NRA
diminished slowly until the 3rd day and remained stable
from the 3rd day onwards (Fig. 1). The level of NR
protein, measured by ELISA analysis, remained stable
during the first hours, but severely decreased 12 h after
the transfer (Fig. 1). The level of total soluble proteins
remained stable during the first 2 d and subsequently
decreased about 20% (data not shown). Thus, a decrease
of the soluble protein level could not explain the decrease
of NRA during the first 2 d.
In vitro NRA
a
1
3
96
120
Tune (hours)
Fig. 1. Time-course of in vivo NRA and NR protein levels in detopped
chicory roots. Roots of 18-d-old plantlets were detopped and transferred
into liquid medium. 100% activity was referred as NRA at the onset of
the transfer (T = 0). Means±SD (n=10) The NR-protein level was
measured by the two sites ELISA method. One representative ELISA
measurement among three repeats was figured.
In vitro NRA was measured in roots before they were
excised. In the presence of EDTA in the reaction mixture,
NRA reached 12 nmol NO2~ min" 1 mg" 1 protein.
Without EDTA, but in the presence of 5 mM magnesium,
it was decreased by about 60% (Table 1). Sensitivity of
in vitro NRA towards magnesium is currently referred as
an estimation of the level of inactivated NR. According
to data from MacKintosh et al. (1995), Mg 2 + ions
promote the linkage between active phosphorylated NR
and NIP (nitrate reductase inhibitor protein). EDTA
chelates divalent cations and thus prevents the formation
of the inactive complex. Thus, measures with EDTA
reflect potential NR activity depending on the level of
NR protein. With Mg 2 + ions, NRA depends on the level
of active NR under either a dephosphorylated or phosphorylated form. Consequently, the ratio between NRA
measured with Mg 2 + ions and NRA measured with
EDTA, reflects the ratio between active NR and total NR.
Three hours after the roots were transferred, the level
of NR protein was not much modified (Fig. 1). Similarly,
Table 1. In vitro NRA: effects of excision and addition of 0.1 nM staurosporine or I JXM okadaic acid
Inhibitors were added aseptically into the liquid medium prior to the transfer of excised roots. In vitro NRA was assayed with either EDTA or
Mg 2 * ions. One significant experiment is figured among three repeats. Activities were expressed as nmol NOf min" 1 mg" 1 total soluble protein.
The percentage of activation corresponds to the ratio of NRA with Mg 2 + /NRA with EDTA x 100.
Before excision
After excision
5d
3h
In vitro NRA with EDTA
In vitro NRA with Mg^ +
Percentage of activation
12±l 6
4.9 ±1.4
40
Control
Staurosporine
Okadaic acid
Control
10.8 ±1.22
<1.6
<15
9.3 ±1.5
4.8±1.1
52
6.5± 1.5
nd
nd
3.40 ±1.6
nd
nd
62
Vuylsteker et al.
NRA assayed in the presence of EDTA was not significantly reduced: it reached 10.8 nmol min~' mg" 1 protein
(versus 12 nmol min" 1 mg" 1 protein before the roots were
detopped). Conversely, in the presence of Mg 2 + ions
NRA was inactivated over 85% (Table 1). Thus inhibition
of NRA during the first hours was probably related to
increased phosphorylation of NR.
Five days after the transfer, NR assayed with EDTA
only reached 3.40 nmol min" 1 mg" 1 protein. The level of
NR protein which dropped about 66% 12 h after the
transfer, remained low on day 5 (Fig. 1). Thus, whereas
initial inactivation of NR during the first 3 h, was related
to increased phosphorylation which was probably followed by the formation of an inactive complex. Loss of
NRA on day 5 would be related to lowered levels of
NR protein.
Northern blot analysis
Northern blot analysis did not reveal any modification of
the mRNAs of either NR, GSl or jSATPase during the
first hours following the transfer of the roots into the
liquid medium (Fig. 2). Thereafter, theNR, GSl, tubulin,
and /SATPase mRNA levels decreased by about 60%
between the 3rd and the 4th days (Fig. 2) and decreased
NRA may be accounted for by lowered transcription of
the NR gene. However, decreased transcription was not
specific to NR.
Effect of inhibitors of protein phosphorylation
It was decided to verify the hypothesis of a phosphorylation effect by using okadaic acid which is a specific
inhibitor of protein phosphatases of the 1 and 2A types
Time (h)
NR
72
of vertebrates, yeasts and plants (Cohen et al., 1990) and
staurosporine which inhibits various serine and threonine
protein kinases by competing with ATP (MacKintosh
and MacKintosh, 1994).
Addition of 1 fiM okadaic acid to the liquid medium,
strongly emphasized the in vivo NRA drop (Fig. 3).
Conversely, addition of 0.1 nM staurosporine increased
NRA which reached a level exceeding the initial one.
Moreover, this stimulatory effect was maintained for at
least 24 h (Fig. 3).
For in vitro NRA assays, roots were excised and
transferred into liquid medium containing either okadaic
acid or staurosporine; NRA was measured 3 h later.
Okadaic acid dramatically decreased NRA assayed in the
presence of EDTA. As NRA was severely lowered in the
presence of okadaic acid, accurate measurements of NRA
could not be carried out in the presence of Mg 2 + ions.
In the presence of staurosporine, NRA was more
modified when assayed with Mg 2 + ions than with EDTA
(Table 1). Indeed, NRA in the presence of EDTA was
lowered by 17%. In the presence of Mg 2 + ions, activities
were three times higher than in control excised roots
attaining values measured in roots of intact plantlets.
Sequential delivery of staurosporine
In a first set of experiments, roots were excised, transferred
for 3 h into media with or without staurosporine and
assayed for in vivo NRA. It was 3.5-fold higher with
staurosporine than in controls (Fig. 4a). When staurosporine was added 3 h after the roots were transferred, NRA
160
1 uM Okadaic acid
Control
0,1 nM Staurosporine
96
120
••III
80
ATPsynthase
TUB
GS 1
28SrRNA —
HHHHH
Time (hours)
Fig. 2. Northern blot analysis of total RNA from excised chicory roots.
The time-scale corresponds to different stages of the culture in liquid
medium. Probes were cDNAs of nitrate reductase (NR), f) subunit of
ATP synthase (ATP synthase), a-tubuline (TUB), and cytosolic
glutamine synthetase (GSl). The lower panel corresponds to 28S rRNA
coloured with acridine orange. It allows control of the loaded amounts
of RNA.
Fig. 3. Effect of 0.1 nM staurosporine and 1 ^M okadaic acid on in
vivo NRA. Staurosporine and okadaic acid were added 3 h after the
roots were transferred. NRA was expressed as nmol NOf min' 1 g"1
FW. Values are means±SD (n = 5) of one significant experiment.
Assays were performed three times and the data varied within the same
order of magnitude.
Reversible phosphorylation of nitrate reductase
4.a
4.b
4.c
Fig. 4. In vivo NRA in relation to sequential delivery of 0.1 nM
staurosporine. (a) Roots were excised and transferred for 3 h into liquid
medium with or without staurosporine (D) NRA in intact roots. NRA
in excised roots grown for 3 h with ( • ) or without ( • ) staurosporine.
(b) Excised roots harvested on control plantlets were first grown in
liquid medium for 3 h. Staurosporine was then added and NRA assayed
3h latter. ( • ) NRA in roots grown with staurosporine. NRA in
controls grown for 3 h ( • ) and 6 h (D) without staurosporine. (c)
Roots were grown for 48 h in liquid medium before staurosporine was
added and NRA was assayed 3 h later Symbols are as in (b). (d) ( • )
NRA in roots of intact plantlets incubated during 4.5 h with 0.1 nM
staurosporine ( • ) controls. Roots were then harvested on plantlets
that were treated for 4.5 h with staurosporine, transferred for 3 h in
liquid medium and assayed for NRA. ( • ) NRA with staurosporine
(GO) NRA without staurosporine. This figure represents one significant
experiment from three similar repeats.
assayed 3 h later was increased about 250% and recovered
NR activities of undetopped roots (Fig. 4b). When the
supply of staurosporine occurred 2 d after the roots were
transferred, NRA was only increased by 60% and never
recovered NR activities measured in intact roots (Fig. 4c).
Decrease of the NR protein level (Fig. 1) probably
accounts for this result.
In a second set of experiments, staurosporine was
supplied for 4.5 h to intact plantlets. Roots were then
excised and immediately assayed for in vivo NRA which
was not significantly modified compared to controls
(Fig. 4d). Roots of pretreated plantlets were then excised,
transferred for 3 h into liquid media with or without
staurosporine and assayed for in vivo NRA. NR activities
were maintained in both conditions (Fig. 4d) indicating
that pretreatment with staurosporine suppressed the
excision-induced drop of NRA.
Discussion
When excised roots of young chicory plantlets were
transferred into liquid medium, NRA decreased about
60% within the first 3 h. Then, NRA decreased slowly
and was stabilized from the 3rd day on.
63
NRA decreased well before the level of the NR protein
which decreased about 50% 12 h after the transfer of the
roots. Although the level of NRA does not always match
NR protein (Oaks, 1994), the data could be explained in
terms of post-translational regulations, such as reversible
phosphorylation-dephosphorylation reactions of the NR
protein. According to Kaiser, in vitro reactivation of
phosphorylated NR is prevented by divalent cations such
as magnesium and increasing phosphorylation is associated with inhibition of NRA (Kaiser and Huber, 1994).
In excised roots of chicory, increased sensitivity of in vitro
NRA towards Mg 2 + ions during the first hours following
decapitation, is in good agreement with the assumption
of the formation of a phosphorylated inactive complex.
However, in vitro NRA in roots of intact plantlets was
also highly sensitive to magnesium. NRA of maize roots
was more susceptible to magnesium than NRA in the
leaves (Merlo et al., 1995).
In order to assess whether a reversible phosphorylation-dephosphorylation reaction contributed to the
regulation of NRA in excised roots, inhibitors of either
phosphatases or protein kinases were used. Okadaic acid,
an inhibitor of PP1 and PP2A protein phosphatases, was
known to prevent the reactivation of inactivated NR in
leaves and roots, indicating that reactivation requires an
active protein phosphatase (MacKintosh, 1992; Glaab
and Kaiser, 1993; Huber et al., 1992). In detopped chicory
roots, okadaic acid emphasized the loss of in vivo and in
vitro NR activities. This is in agreement with both models
of Kaiser and Huber (1994) and of MacKintosh et al.
(1995): inhibition of phosphatases locks the reactivation
process of NR and maintains the inactive NR-NIP complex, the formation of which is activated by Mg 2 + ions,
at a high level. In vitro NR activities in the presence of
EDTA, were assayed in extracts made from roots which
grew for 3 h with okadaic acid. Even if EDTA permits
dissociation of the inactive NR-NIP complex, a 3 h
treatment with okadaic acid probably favoured accumulation of the inactive NIP-NR complex and may explain
why NRA remained lower than in controls (Table 1).
Conversely, staurosporine, an inhibitor of serinethreonine-dependent protein kinases prevented the
decrease of NRA in excised roots immediately after their
transfer. Delayed additions exerted poor effect, seemingly
because the level of NR protein was reduced (Fig. 1).
NRA in intact roots was not modified with staurosporine,
indicating that the bulk NR might be in an active
dephosphorylated form. Maintaining excised roots for
several days in liquid media, resulted in lowering both
NR and NR-mRNA levels. In chicory, such a long-term
effect might progressively exhaust the pool of NR reactivable by staurosporine. Decay of the NR protein level
before NR-mRNA could be accounted for by the hypothesis that phosphorylated NR is a preferential target for
protein degradation (Kaiser and Huber, 1994). In addi-
64
Vuylsteker et al.
tion to NR-mRNA, the levels of GS1 and jSATPase
mRNAs equally decreased. The absence of growth in
excised roots and suppression of shoot to root correlations
probably reduced sink strength and several biosynthetic
pathways such as nitrogen assimilation and respiration.
Several factors may be modified by excision. First,
sucrose starvation could hinder the nitrogen assimilation
pathway (James et al., 1993). Secondly, suppression of
the aerial part of the plants reduced the pool of available
nitrate in barley roots within a few hours (Zhen et al.,
1992). In detopped roots of chicory these factors probably
did not account for decreased NRA, since sucrose was
added to both solid and liquid media. Moreover the
nitrate content was not decreased several hours after
excision (data not shown) indicating that nitrate was
probably not a limiting factor of NRA in excised roots.
In conclusion, in young chicory plantlets, NRA, which
mainly occurs in roots, is probably under metabolite
control. Excision of roots which affect the metabolite
balance induced a rapid inhibition of NRA. This regulation involves the phosphorylation state of the NR protein.
Phosphorylation of NR can also be considered as the
result of a general activation of phosphorylation reactions
in response to a stress. Activation of protein kinase
activities by wounding has indeed been reported (Usami
et al., 1995).
Acknowledgements
We thank Dr G Conejero (INRA, Montpellier) for the gift of
a maize anti-NR polyclonal antibody; Dr M Caboche for the
gift of the maize monoclonal antibody and Dr T Moureaux
(INRA, Versailles) for her helpful assistance in ELISA determination of NR contents, Dr M Boutry (University of Leuven)
and Dr B Hirel (INRA, Versailles) for the generous gift of the
N. plumbaginifolia j3 ATP synthase-cDNA and the N. tabacum
glutamine synthetase GSl-cDNA, respectively. This work was
supported by grants from Conseil Regional Nord-Pas de Calais.
Christophe Vuylsteker was supported by a MERS fellowship.
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