Plant Physiol. (1999) 119: 219-230
A Conserved Acidic Motif in the N-Terminal Domain of Nitrate
Reductase Is Necessary for the Inactivation of the Enzyme in the Dark
by Phosphorylation and 14-3-3 Binding1
Emmanuelle Pigaglio2,
Nathalie Durand, and
Christian Meyer*
Laboratoire de Biologie Cellulaire, Institut National de la
Recherche Agronomique, Centre de Versailles, F-78026 Versailles
cedex, France
 |
ABSTRACT |
It has
previously been shown that the N-terminal domain of tobacco
(Nicotiana tabacum) nitrate reductase (NR) is
involved in the inactivation of the enzyme by phosphorylation, which
occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a
mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic
virus 35S RNA promoter and introduced in NR-deficient mutants of the
closely related species Nicotiana plumbaginifolia. We
found that the deletion of a conserved stretch of acidic residues
led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic
stretch seems to have the same effects on NR activation state as the
deletion of the N-terminal domain. A hypothetical explanation for these
observations is that a specific factor that impedes inactivation
remains bound to the truncated enzyme. A synthetic peptide derived from
this acidic protein motif was also found to be a good substrate for
casein kinase II.
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INTRODUCTION |
Most higher plants obtain the nitrogen metabolites needed for
growth and development by taking up and assimilating nitrate. After
active transport into the cell, nitrate is reduced to nitrite by NR (EC
1.6.6.1-2), a cytosolic enzyme. Subsequently, nitrite is reduced to
ammonium by nitrite reductase, which is localized in the chloroplast.
The expression of the NR gene is highly regulated at the
transcriptional level by many endogenous and environmental factors, including hormones, light, nitrogen source, and carbohydrates (for
review, see Hoff et al., 1994
; Crawford, 1995). These transcriptional regulations probably determine the long-term fluctuations in the NR
protein level. On the other hand, a reversible posttranslational regulation of the NR protein involving protein phosphorylation allows
short-term modulation of the enzyme activity in response to light-dark
transitions, variations in photosynthetic activity, CO2 level, intracellular pH, or oxygen
availability (Kaiser and Förster, 1989; Kaiser and
Brendle-Behnisch, 1991; Huber et al., 1992; Kaiser et al., 1992, 1993;
MacKintosh, 1992; Kaiser and Huber, 1994a). Inactivation of NR is
linked with phosphorylation both in vivo and in vitro.
Proteins involved in the phosphorylation/dephosphorylation mechanism of
spinach NR have been purified and characterized, and a two-step
regulation model was proposed (Spill and Kaiser, 1994; Glaab and
Kaiser, 1995; MacKintosh et al., 1995). According to this model,
spinach NR is first phosphorylated on Ser-543, which is conserved among
higher-plant NRs (Douglas et al., 1995; Bachmann et al., 1996b) and
then becomes inactivated upon binding of a factor called NR inactivator
protein, which was recently identified as a mixture of 14-3-3 proteins
suggested to interact with the regulatory phosphorylation site of NR
(Bachmann et al., 1996a; Moorhead et al., 1996). Moreover,
inactivation of the enzyme can be evidenced only when NR activity is
measured in the presence of magnesium in the millimolar range. The
magnesium ion plays an important role in the mechanism of NR
inactivation: it is needed for both the NR phosphorylation step and for
the maintenance of NR in its inactive form (Kaiser and Huber, 1994b).
After chromatography, three distinct peaks of NR kinase activities were
identified in spinach extracts (Bachmann et al., 1996b; Douglas et al.,
1997).
Evidence for inactivation of NR from other plant sources by the same
mechanism has been obtained for squash (Lillo, 1993), cabbage (Kojima
et al., 1995), maize (Huber et al., 1994; Li and Oaks, 1994), barley
(Decires et al., 1993), Nicotiana plumbaginifolia (Nussaume
et al., 1995), pea (Glaab and Kaiser, 1993), and Arabidopsis (Labrie and Crawford, 1994; Su et al., 1996). The
precise mechanism of NR inactivation is as yet poorly understood
because a complete structural model of the NR molecule is still
lacking.
What is known is that NR is a homodimeric enzyme composed of 100- to
115-kD monomers. Each monomer is organized in three main domains
housing the three prosthetic groups of NR, MoCo, heme, and FAD.
Electrons from NADH travel successively through the FAD, heme, and MoCo
domains before reaching the nitrate molecule for its reduction (for
review, see Hoff et al., 1994; Campbell, 1996). These domains are
linked by protease-sensitive hinges, and the regulatory phosphorylation
site of NR is located on the first hinge, which connects the MoCo and
the heme domains (Douglas et al., 1995; Bachmann et al., 1996b). The NR
protein sequences are well conserved among higher plants and when
compared with fungi or algal NR sequences (apart from the N-terminal
region, which varies both in sequence and length; Nussaume et al.
[1995]).
This observation prompted us to investigate the possible role of this
N-terminal region by expressing in transgenic N. plumbaginifolia an NR-coding sequence carrying a 56-amino acid
deletion in this region (
NR protein, Nussaume et al., 1995). It was
found that the NR enzyme was still active and that, apart from a
higher thermosensitivity, showed the same enzymatic properties as the wild-type enzyme. However, the NR protein activity was no longer regulated by darkness and phosphorylation; indeed, the NR activation state (the percentage of active enzyme) was higher than in the wild
type and was unaffected by light-dark transitions. Moreover, the NR
protein was insensitive to MgATP inactivation in vitro. A relationship
between NR inactivation and degradation was also confirmed, because the
NR protein was more stable in the dark than the complete NR protein
(Nussaume et al., 1995).
A similar result was obtained for spinach, in which it was shown that,
when the NR protein had lost its first 45 amino acids by proteolysis,
the NR activity could no longer be inhibited by 14-3-3 binding,
although the degraded enzyme was still phosphorylated (Douglas et al.,
1995). These results strongly suggest that the N-terminal region of NR
is somehow involved in and is required for the inactivation of the
enzyme by phosphorylation. So far, the question of how this N-terminal
region of NR participates in the inactivation process remains largely
unanswered, mainly because the deletion that was made originally does
not allow us to determine which residues are involved in this process.
To determine which segments of the NR N-terminal sequence are necessary
for the posttranslational inactivation of the protein, we introduced
smaller deletions within the N-terminal region of the tobacco
(Nicotiana tabacum) NR to express the resulting
deleted NR proteins in the closely related species N. plumbaginifolia. The deleted NR proteins were expressed in an
NR-deficient mutant background. The thermosensitivity, inactivation,
and degradation of NR after light-dark transitions were compared in
wild-type and transgenic plants expressing the complete NR-coding
sequence, the NR protein, or smaller deletions in the N-terminal
region.
| |
MATERIALS AND METHODS |
Plant Material and Growth Conditions
Plants of Nicotiana plumbaginifolia var Viviani (line
pbH1D) were used for all experiments. The C1 (Vincentz and Caboche, 1991) transgenic line was obtained by transformation of the N. plumbaginifolia E23 nia mutant with a full-length
coding sequence from the tobacco Nia2 gene linked to a CaMV
35S RNA promoter. The del8 (Nussaume et al., 1995) transgenic line was
obtained in the same way except that the NR-coding sequence carried a
56-amino acid deletion in the N-terminal region. Seeds were sown in
vitro on solid B-N medium (Gabard et al., 1987) supplemented
with 10 mM KNO3. Germination was
performed under the following photoperiod conditions: 8 h at a
light intensity of 75 µE m
2
s1 (1 h at 20°C, 6 h at 25°C, and
1 h at 20°C) and 16 h in darkness at 17°C. Plants were
grown either in the greenhouse or in a growth cabinet. In the latter
case, the photoperiod was 16 h of light (2 h at 20°C, 12 h
at 25°C, and 2 h at 20°C; 225 µE m2
s1) followed by 8 h of darkness at 17°C.
Plants in continuous darkness were kept at 17°C. In all cases plants
were grown until the rosette stage (about 3 weeks) and watered daily
with a nutritive solution (Coïc and Lesaint, 1975).
NR Protein Sequence Analysis
NR protein sequence alignment was performed using the Wisconsin
package (Genetics Computer Group, Madison, WI). The structure predictions were made using the PHD neural network (Rost and
Sander, 1994) and were performed at the EMBL (Heidelberg, Germany).
Construction of the Chimeric A-NR and S-NR Genes
Standard procedures were used for recombinant DNA manipulations
(Maniatis et al., 1982). Enzymes were used according to the supplier's
recommendations.
Two internal deletions (A and S, which correspond, respectively,
to the deletion of an acidic domain and of a conserved Ser residue in
the NR N-terminal region) were introduced in the complete NR-coding
sequence corresponding to the tobacco Nia2 gene (carried by
plasmid pCS22, Vincentz and Caboche, 1991) using a site-directed
mutagenesis method based on PCR (Chen and Przybyla, 1994). This method
uses oligonucleotides whose sequences contain the expected deletion to
synthesize megaprimers in a first PCR round. The different steps of the
construction are summarized in Figure 1.
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| Figure 1.
Construction of the plasmid vectors expressing the
deleted NR proteins A-NR and S-NR. We first amplified by PCR a
198-bp fragment containing the A deletion and a 110-bp fragment
containing the S deletion using, respectively, the primers 26 and 27 or the primers 26 and 28 (A). The amplified fragments called,
respectively, megaprimers A and S, were then used as primers for
a second PCR with primer 6 (B). The resulting DNA fragments were cloned
between the SstI and Spel sites of the
plasmid pBluescript (C). These new plasmids were digested by
SpeI and PstI and an
SpeI-PstI fragment was then introduced
downstream of the deleted sequence to recover the complete NR-coding
sequence (D). These constructs were called pA-NR and pS-NR. The
dark bars represent the NR-coding sequence. S, SstI; Sp,
SpeI; P, PstI.
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First, a 198-bp fragment containing the A deletion (the A
megaprimer) was amplified by PCR (Fig. 1A) using the plasmid pCS22 as template with oligonucleotide 26 (5
-ATCGAGCTCTTTTAGAATAATCCA-3) and oligonucleotide 27 (5-ATTTGAAGGTACTCATTATCAAGGTAAATGGTGGAA-3). In parallel, a 110-bp
fragment containing the S deletion (the S megaprimer)
was obtained using oligonucleotide 26 and oligonucleotide 28 (5-GTTGCAGCCACGAACCCGGGGCTTGAAAGA-3). The sequence of
primer 26 was derived from the sequence just upstream of
the NR-coding sequence in plasmid pCS22, the last base of primer 26 being the first base of the NR-coding sequence. Primer 27 (reverse)
corresponds to nucleotides 279 to 297 and 337 to 353 of the tobacco
Nia2 genomic sequence (Vaucheret et al., 1989),
which introduces a 39-bp deletion in the NR-coding sequence. Primer
28 (reverse) corresponds to nucleotides 196 to 210 and 223 to 237 of the tobacco Nia2 genomic sequence, which introduces a
12-bp deletion.
The A megaprimer was then used for a second PCR round (Fig. 2B) with
oligonucleotide 6 (Meyer et al., 1995) using the plasmid pCS22 as a
template. This produced a fragment of the NR-coding sequence (exon 1 and the beginning of exon 2) containing the A deletion. A DNA
fragment containing the S deletion was synthesized in the same way,
using the megaprimer S and oligonucleotide 6 as primers.
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| Figure 2.
Alignment of the N-terminal domain protein
sequences from plant, fungal, and algal NRs. Sequences were first
aligned using the PileUp program (Genetics Computer Group), and the
resulting alignment was refined by hand. Sequences are all from the
SwissProt database, the algal NR sequence (nia volca) is
from V. carterii, and the fungal NR
sequence (nia ustma) is from U. maydis.
Conserved residues are boxed. Acidic residues are shaded dark gray, and
basic residues are shaded light gray. The 56-amino acid sequence
deleted in the NR protein is indicated (dashed box), as are the
sequences deleted in the A-NR and S-NR proteins (bold boxes).
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The two PCR products were gel purified, digested by SstI and
SpeI, and cloned in the same sites in plasmid pBluescript
(Stratagene). The presence and accuracy of the deletions were verified
in the resulting positive clones by DNA sequencing.
The two recombinant plasmids were digested by SpeI and
PstI, and an SpeI-PstI fragment from
pCS22 containing the end of the NR-coding sequence was cloned
downstream of the previous inserts (Fig. 1D). This reconstituted a
complete NR-coding sequence and created the pA-NR and pS-NR
plasmids.
To construct binary vectors expressing the A-NR and the S-NR
proteins, the complete A-NR and S-NR chimeric sequences from pA-NR and pS-NR were isolated as an
SstI-PstI fragment, blunt-ended, and cloned into
the blunt-ended KpnI site of the binary plant transformation
vector pBinDH51 (Vincentz and Caboche, 1991). This put the A- and
S-NR-coding sequences under the control of the CaMV 35S RNA promoter
and terminator and created the binary plasmids pBA-NR and pBS-NR.
Plant Transformation and Regeneration
The recombinant vectors pBA-NR and pBS-NR in the
Escherichia coli XL1-Blue strain (Bullock et al., 1987) were
mobilized into Agrobacterium tumefaciens strain LB4404 as
described by Bevan (1984). The NR-deficient nia mutant E23
of N. plumbaginifolia, which is unable to use nitrate as
a nitrogen source, was transformed as described previously by
infecting leaf discs with an A. tumefaciens culture
(Vaucheret et al., 1990). The resulting calli were selected on a
medium containing 100 mg L1 kanamycin and 10 mM ammonium succinate as the sole nitrogen source and
regenerated into plantlets as described previously (Vaucheret et
al., 1990). The transformed calli were halved, and each half was placed
on a regenerating medium containing either succinate ammonium or 10 mM potassium nitrate as the sole nitrogen source. Regenerated plantlets that were restored for the ability to utilize nitrate for growth were then transferred to the greenhouse and are
referred to as primary transformants. Genetic analysis of the progeny
obtained from selfing primary transformants (the
R1 generation) was performed on B-N medium
containing 100 mg L1 kanamycin and 10 mM
potassium nitrate.
Extraction and Analysis of RNA
Total RNAs were extracted from frozen leaf material and analyzed
by northern blots as described by Crété et al. (1997). Hybridization was performed using the 1.6-kb
EcoRI-EcoRI cDNA fragment corresponding to the
tobacco Nia2 gene (Vaucheret et al., 1990). Ethidium bromide
staining of rRNAs after gel migration was used to ensure homogenous
loading of the RNA samples.
NR Extraction and Activity Measurement
Leaves were harvested 2 h after the beginning of the day
period from plants grown in the greenhouse. For plants that were kept
in the dark, leaves were harvested from two plants under a green light
at each sampling time and were immediately frozen in liquid nitrogen
and stored at 
80°C.
Frozen leaves were ground in liquid nitrogen and extracted in 4 mL
g1 fresh weight buffer A (50 mM
Hepes-KOH, pH 7.6, 10 mM magnesium chloride, 1 mM DTT, 5 µM FAD, and 1 µM
leupeptin). The whole procedure was carried out at 4°C. The mixture
was incubated for 15 min on ice, and the homogenate was centrifuged at
12,000g for 10 min. The supernatant (crude extract) was
either used immediately for NR activity assays or was subjected to
further steps of purification.
For ammonium-sulfate precipitation, powdered ammonium sulfate was
gradually added to the cool crude extract until 40% saturation at
4°C. The solution was slowly stirred for 1 h at 4°C and
centrifuged at 12,000g for 30 min. The pellet was dissolved
in buffer A in one-tenth of the original volume.
NR activity measurements were carried out in buffer B (50 mM Hepes-KOH, pH 7.6, 10 mM magnesium chloride,
140 µM NADH, and 5 mM potassium nitrate). In
some experiments EDTA (15 mM) was added to the reaction
buffer. The reaction was initiated by adding 200 to 300 µL of extract
and was carried out for 5 to 12 min at 27°C in a total volume of 1 mL. The nitrites formed during the assay were revealed as previously
described (Meyer et al., 1995).
The activation state of NR is defined as the ratio of the NR activity
measured in the presence of free magnesium ions to the NR activity
measured in the presence of EDTA. This activation state is expressed as
a percentage and reflects the amount of active NR in an extract.
In Vitro CKII Assays on an Acidic Synthetic Peptide
CKII activity was assayed by measuring the incorporation of
32P from
[
-32P]ATP into a synthetic peptide, as
detailed by Davies et al. (1989). A recombinant human CKII was obtained
from New England Biolabs and was used according to the supplier's
recommendation. A synthetic peptide (RRREEET*EEE; New England Biolabs),
the substrate for human CKII, was used as a positive control. The
synthetic peptide derived from the tobacco acidic domain corresponds to
residues 55 to 69 of the NIA2 tobacco protein sequence
(SSSEDDDDDDEKNEG, acidic peptide). The labeling reaction (final volume
30 µL) consisted of 100 units of human CKII (0.2 µL), 1 µL of
CKII substrate peptide (final concentration 245 µM), or 6 or 12 µL of acidic peptide (300 and 600 µM,
respectively); 0.2 mM [-32P]ATP
(200 µCi/µmol); 20 mM Tris-hydrochloride, pH 7.5; 50 mM potassium chloride; and 10 mM magnesium
chloride. After incubation for 30 min at room temperature, a 20-µL
aliquot was removed and spotted onto a 2- × 2-cm square of
phosphocellulose paper. The papers were then washed in orthophosphoric
acid (75 mM), with a final wash in acetone, dried, and
placed in 1.5-mL plastic tubes, and the bound radioactivity was
measured by direct counting (Cerenkov counts).
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RESULTS |
Construction of Plant Vectors Expressing NR Proteins Deleted in the
N-Terminal Region
The N-terminal extensions of higher-plant NR protein sequences
were aligned and compared with an algal sequence from Volvox carterii and with a fungal NR sequence from Ustilago
maydis (Fig. 2). As noted previously (Nussaume et al., 1995),
these sequences are poorly conserved even among higher plants.
However, in the plant sequences two regions showing more conserved
features were identified within the NR N-terminal extensions: a
consensus sequence, RXDSPVR, which contained a conserved Ser residue
(amino acids 24-30 of the NIA2 tobacco protein sequence) found in most
higher-plant sequences (Fig. 2), and an
acidic domain rich in Asp and Glu residues (amino acids 53-68 of the
NIA2 tobacco protein sequence), which also contained a stretch of
conserved Ser residues.
The first motif (RXDSPVR) is absent in the NR N-terminal sequences from
Leguminosae (soybean, bean, and lotus) and less conserved in
Cruciferae, whereas it is found in the NADH:NR sequences from monocots
(rice and Nia1 in barley). Conversely, an acidic stretch preceded by one to four Ser residues appeared to be present in all
higher-plant sequences examined (Fig. 2). One may then wonder whether
these protein sequences play any role in the inactivation of NR by
phosphorylation, because they are contained in the previous 56-amino
acid deletion that was shown to abolish inactivation of NR in the dark.
To investigate this, these two motifs were removed from the tobacco
NR-coding sequence using a site-directed mutagenesis method based on
PCR (Fig. 1). The resulting pA-NR construct carries a 13-amino acid
deletion (amino acids 54-66 of the NIA2 tobacco protein sequence)
corresponding to the acidic domain, whereas in the pS-NR construct,
the 4 amino acids surrounding the conserved Ser were removed (amino
acids 25-28 of the NIA2 tobacco protein sequence). The truncated
NR-coding sequences A-NR (2.715 kb) and S-NR (2.742 kb) were
placed under the control of the CaMV 35S RNA promoter and terminator in
the plant-transformation vector pBinDH51 (Vincentz and Caboche, 1991),
which produced the binary plasmids pBA-NR and pBS-NR.
Complementation of the E23 NR-Deficient Mutant of N. plumbaginifolia by the Chimeric Genes
A-NR and S-NR
The E23 nia mutant of N. plumbaginifolia is
deficient for NR activity and is unable to grow with nitrate as the
sole nitrogen source. Indeed, the NR structural gene is disrupted by an
insertion of the Tnp2 retrotransposon in the first exon (C. Meyer, unpublished results). As a consequence, the E23 mutant does not
produce any full-length NR mRNA and is devoid of NR protein. The E23
mutant was transformed with either the pBA-NR construct or the
pBS-NR construct by agroinoculation of leaf discs with A. tumefaciens. To avoid any selection pressure on the recovery of
transgenic plants expressing a functional NR protein,
kanamycin-resistant calli were regenerated on a medium containing
ammonium as the nitrogen source. The selected calli were then cut in
half and grown on either ammonium or nitrate. Finally, the regenerated plantlets that were able to grow on a medium containing nitrate as the
only nitrogen source were retained and then transferred to the
greenhouse. Two independent primary transformants
(R0) carrying the A-NR gene (A1 and A2) and
four independent primary transformants carrying the S-NR gene (S2,
S3, S4, and S9) were obtained. For each primary transformant, the
NR sequence surrounding the introduced deletion was amplified by
PCR and verified by DNA sequencing (data not shown).
The transformants were phenotypically similar to the wild type or to
the transgenic N. plumbaginifolia overexpressing either the
NR (C1 plants) or the NR protein (del8 plants). They grew vigorously
both in vitro and in the greenhouse with 10 mM nitrate as
the sole nitrogen supply.
The selfed progeny (R1) of the primary
transformants were studied for the transmission of the
kanamycin-resistance marker and for their ability to grow on nitrate.
Three primary transformants (A2, S2, and S4) were found to carry a
single functional kanamycin-resistance locus. Three other primary
transformants (A1, S3, and S9) showed an aberrant Mendelian segregation
(data not shown). The A1, A2, S2, S4, and S9 lines were retained for
further studies.
NR activities were measured for each transformant in the
R1 plants and compared with the NR activity of
the wild-type, C1, and del8 plants (Fig.
3). All of the transformants had NR
activity lower than the wild type. The same range of NR activity was
observed for transgenic plants expressing the NR protein (Nussaume
et al., 1995).
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| Figure 3.
NR activities in wild-type (WT) and
transgenic N. plumbaginifolia plants ectopically
expressing NR. Plants were grown in the greenhouse and harvested at the
beginning of the light period. Results are from two independent
experiments. C1 and del8, Transgenic plants expressing, respectively, a
wild-type NR protein (35S-NR) and the NR protein (35S-NR). A1 and
A2, Transgenic plants expressing the A-NR protein. S2, S4, and S9,
Transgenic plants expressing the S-NR protein.
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Thermosensitivity of the A-NR and S-NR Proteins
A difference in thermosensitivity between the NR and NR
proteins was previously observed when NR activity was assayed in vitro
using ammonium sulfate-precipitated extracts. The activity of the NR
protein was found to be more sensitive to temperature than the activity
of the wild-type NR (Nussaume et al., 1995).
The thermosensitivity of the A-NR and S-NR proteins was compared
with that of NR and NR proteins by measuring NR activity in
ammonium sulfate-precipitated extracts (Fig.
4). In the wild type the NR activity was
higher at 30°C than at 20°C, whereas after 30 min the NR activity
was lower at 35°C than at 20°C. For the NR protein expressed in
the del8 transgenic plant, NR activity dramatically decreased after 10 min at 30°C or 35°C. The increase in temperature had the same
effect on the S-NR protein and wild-type NR protein activities (Fig.
4). Conversely, the thermosensitivity of the A-NR protein was
intermediate between that of the wild type and that of the NR
proteins. In the A1 and A2 transgenic plant extracts the nitrite
accumulation measured when the enzyme was incubated at 30°C was lower
than that measured at 20°C, but higher than that in del8 extracts at
the same temperature (Fig. 4). At 35°C the nitrite accumulation was
comparable in A1, A2, and del8 plant extracts. These results
suggest that the S-NR protein is as stable as the wild-type NR,
whereas the A-NR protein is more thermosensitive (although less so
than the NR protein).
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| Figure 4.
Thermosensitivity of the A-NR and S-NR
proteins. NR activity (nitrite accumulation) was assayed in vitro at
different temperatures (20°C, 30°C, and 35°C) on ammonium
sulfate-precipitated extracts obtained from the wild-type (wt) or from
different transgenic lines. Abbreviations of the genotypes of the
transgenic lines are as defined in the legend of Figure 3.
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Reversible Inactivation of NR by Darkness
Spinach NR is known to be inactivated in the dark, and this
inactivation was best revealed when NR activity was assayed in a
magnesium-containing buffer. The inactivation was reversible in vitro
by adding EDTA to the reaction buffer (Kaiser and Brendle-Behnisch, 1991). Therefore, the NR activation state, or percentage of active NR,
can be estimated by calculating the ratio between NR activity assayed without EDTA (which corresponds to the activity of
the dephosphorylated NR in the extract) and NR activity assayed
with EDTA (reactivated NR). It was previously shown that the enzyme from N. plumbaginifolia was also inactivated in the dark,
but at that time the kinetics of NR inactivation had not been
investigated (Nussaume et al., 1995).
Figure 5 shows the inactivation time
course of NR in wild-type N. plumbaginifolia plants exposed
to darkness. The light-dark transition induces a decrease in NR
activity of about 65%. The half-time of the inactivation reaction was
about 15 min, which is in agreement with previous results in spinach
leaves (Huber et al., 1992; Kaiser et al., 1992). Until 30 min of
darkness this inactivation is fully reversible by EDTA. After that
point NR inactivation becomes irreversible, probably because of the
degradation of the protein. Such results are in agreement with previous
reports of species such as spinach (Huber et al., 1992; Kaiser et al., 1992; MacKintosh, 1992), barley (Decires et al., 1993), cabbage (Kojima
et al., 1995), maize (Li and Oaks, 1994), pea (Glaab and Kaiser,
1993), and squash (Lillo, 1993).
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| Figure 5.
Inactivation kinetics of extractable NR from
N. plumbaginifolia leaves in response to a light-dark
transition. Plants grown in the greenhouse with light (L) were put in a
dark room (t = 0) and kept in darkness for various times. Leaves
were harvested from two different plants and NR was extracted in a
magnesium-containing buffer. NR activity was measured in the crude
extracts with 15 mM EDTA ( ) or without EDTA ( ).
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Effect of Light on the Expression of the A-NR and S-NR
Chimeric Genes and on the Activity of the A-NR- and S-NR-Deleted
Proteins
The posttranscriptional regulation by light of the expression of a
35S-NR gene was demonstrated previously (Vincentz and Caboche, 1991).
To study the effect of light on the expression of the A-NR and
S-NR genes, wild-type and control transgenic plants (C1 and del8)
were kept in the dark for 72 h, along with A1, A2, S2, S4, and S9
transgenic plants (Fig. 6A).
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| Figure 6.
Effect of light on the expression of the A-NR
and S-NR chimeric genes. A, Plants grown in the greenhouse were
placed in the dark and leaves were harvested at the beginning of a
normal day/night cycle (I) and after 72 h of darkness (II). B,
Northern analysis of total RNA (5 µg) using as a probe the 1.6-kb
EcoRI fragment of the tobacco Nia2 NR cDNA. Ethidium
bromide staining of rRNA (total RNA) is shown as a control for
homogenous loading. Abbreviations of the genotypes of the transgenic
lines are as defined in the legend to Figure 3 (A2 is another sample
from the A2 line).
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In wild-type plants the NR mRNA level was lower in the light than in
the transgenic plants and decreased to undetectable levels after
72 h of darkness (Fig. 6A). In the transgenic plants C1, del8, A1,
A2, S2, S4, and S9, in which the transcription of the NR gene is under
the control of the CaMV 35S promoter, the NR mRNA accumulation was
unaffected by the dark treatment (Fig. 6B), as was observed previously
for the C1 and del8 transgenic plants (Vincentz and Caboche, 1991;
Nussaume et al., 1995). In the transgenic plants the NR mRNA remained
constitutively overexpressed throughout the dark treatment (data not
shown). Since the transcriptional regulation by light of NR expression
was absent in these transgenic plants, the modulation of NR expression
by light should only be the result of posttranscriptional regulation.
To study the effect of darkness on the activity of the deleted proteins
A-NR and S-NR, leaf samples were harvested from the same plants
at the beginning of the light period and at various times after the
onset of darkness (30 min and 2, 4, 6, 24, 48, and 72 h). The
effect of darkness on the activation state of the NR proteins expressed
in wild-type and transgenic plants was investigated first (Fig.
7). In illuminated leaves about 40% to
50% of NR was active in the wild-type, C1, and S plants, whereas the
amount of active NR was higher in the del8 and A plants (approximately 60%, Fig. 7). Upon transfer of the plants to darkness, NR was further
inactivated in the wild-type, C1, and S plants (approximately 25% of
active NR after 30 min), and this low activation state remained more or
less constant during the dark period (Fig. 7). On the contrary, the
activation state of NR in the del and A plants seemed to be unaffected
by darkness (approximately 60%, Fig. 7). The results with the C1 and
del8 plants agree with those previously obtained by Nussaume et al.
(1995). Taken together, these results suggest that, although the
S-NR protein behaves like the wild-type protein in response to a
light-dark transition, the deletion of the acidic motif (A-NR)
abolishes the NR inactivation by darkness and, therefore, has the same
effect as the 56-amino acid deletion of the NR protein.
View larger version (67K):
[in this window]
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| Figure 7.
NR activation state in leaves of wild-type and
transgenic N. plumbaginifolia plants during prolonged
darkness. Plants were grown in the greenhouse. Leaves were first
collected at the beginning of a normal day/night cycle (L), the plants
were subsequently placed in the dark, and leaves were harvested at the
indicated times from four different plants of the same genotype. NR
activity was assayed in crude extracts for 10 min with or without EDTA
(15 mM). The NR activation state is the percentage of
active NR, which corresponds to the ratio between NR activity assayed
without EDTA and NR activity assayed with EDTA. Abbreviations of the
genotypes of the transgenic lines are as defined in the legend to
Figure 3.
|
|
We have also represented the maximal NR activity (assayed in the
presence of EDTA) measured in the above experiment, which should
reflect the total amount of functional NR protein (Fig. 8). After 30 min of darkness the maximal
NR activity for all genotypes tested showed little change compared with
the maximal NR activity measured in the light (data not shown).
Conversely, after 2 h of darkness the maximal NR activity
decreased in the plants to about 25% to 35% of the NR activity
measured in the illuminated leaves (Fig. 8), which indicates a probable
degradation or irreversible inactivation of the NR protein. The NR
activity then decreased rapidly in the wild-type, C1, and S plants and
was almost undetectable after 48 h in the dark. In the del8 and A
plants, the maximal NR activity also decreased but less rapidly (Fig.
8). After 4 h in the dark the NR activity measured with EDTA was
always 3- to 4-times higher in the del8 and A plants than in the
wild-type, C1, or S plants. Again, these results suggest that the
characteristics of the A-NR protein are similar to those of the
NR protein and that the A-NR protein, although finally degraded
after an extended period of darkness, was less sensitive to degradation
than the wild-type NR protein or the S-NR protein.
View larger version (55K):
[in this window]
[in a new window]
| Figure 8.
NR total activity in leaves of wild-type and
transgenic N. plumbaginifolia plants during prolonged
darkness. Leaves were harvested as described in the legend to Figure 7.
NR activity was then assayed in the crude extracts for 10 min in the
presence of 15 mM EDTA. The NR activities shown correspond
to the NR activities measured with EDTA in the experiment described in
Figure 7. NR activity is given as a percentage of the NR activity
measured in the light (L, 100%). Values for specific NR activities in
the light (in nanomoles of nitrite produced per minute per milligram of
protein) were 24 for wild type, 23 for C1, 11 for del8, 15 for A1, 14 for A2, 12 for S2, 13 for S4, and 11 for S9.
|
|
Phosphorylation of the NR N-Terminal Acidic Cluster by CKII
The 13-amino acid sequence that was deleted in the A-NR protein
contains three consecutive Ser residues followed by one Glu and six Asp
residues (Fig. 2). Such a sequence is reminiscent of a consensus CKII
phosphorylation site (S/TXXE/DX; Pearson and Kemp, 1991). The consensus
sequence for CKII phosphorylation includes a cluster of acidic residues
on the C-terminal side of the target Ser or Thr, with Asp successfully
replacing Glu (Pearson and Kemp, 1991). A synthetic peptide was derived
from the deleted protein sequence in the A-NR protein and then used
as a substrate for a kinase assay with human CKII (Table
I). It appears that this peptide (amino
acids 55-69 of the NIA2 tobacco protein sequence) can be efficiently
phosphorylated in vitro by human CKII.
View this table:
[in this window]
[in a new window]
|
Table I.
Phosphorylation by CKII of a synthetic peptide
derived from the tobacco NR N-terminal acidic cluster
The in vitro phosphorylation by human CKII of a substrate peptide and
of the peptide derived from the sequence of the tobacco NR N-terminal
acidic cluster (acidic peptide) were compared. For details, see
``Materials and Methods''. The possible phosphorylation sites for
CKII are in bold type and are underlined.
|
|
| |
DISCUSSION |
Complementation of NR Deficiency by the A-NR- and
S-NR-Deleted Genes
The transformation of the nia E23 mutant of N. plumbaginifolia with the A-NR and S-NR deleted genes allowed
wild-type NR activity and a wild-type phenotype to be restored. These
observations confirm previous results obtained with transgenic E23
plants expressing either the full-length tobacco NR cDNA (C1 plants,
Vincentz and Caboche, 1991) or the truncated NR protein (del plants,
Nussaume et al., 1995) under the control of the CaMV 35S RNA promoter. However, the A and S transformants did not seem to be less fertile than
the wild type, unlike the del transformants (Nussaume et al., 1995).
The 4 (S deletion) or 13 (A deletion) amino acids that were
removed from the tobacco NR sequence were not required for the
functionality of the protein. Such a result was expected, because these
two deletions are included in a larger region of 56 amino acids that
was previously shown to have no effect (except on thermosensitivity) on
the functionality of the NR protein (Nussaume et al., 1995).
Role of the Two Deleted Regions in the in Vitro Stability of
the NR Protein
The NR protein was previously found to be less stable than the
NR protein in vitro when the NADH:NR activity was assayed on an
ammonium sulfate-precipitated extract, whereas no difference was
detected when assayed on a crude extract (Nussaume et al., 1995). Since
the NADH:Cyt c reductase activity was not affected in the
NR protein, it was concluded that this instability concerned only
the MoCo domain. We then postulated that the 56-amino acid deletion
might alter MoCo binding and that the instability of the NR protein
observed after precipitation could be due to the removal of plant
factors ensuring MoCo binding or stabilization (Nussaume et al., 1995).
The A-NR and S-NR proteins were found to behave differently in
response to temperature by exhibiting different thermosensitivity profiles (Fig. 4). The S-NR protein was as stable as the wild-type NR, whereas the A-NR was more thermosensitive (although less so than
the NR protein). These results suggest that the sequence deleted in
the A-NR protein was the structural feature involved in the loss of
MoCo and, therefore, in the thermosensitivity of the protein. The
fact that the A-NR protein was more stable than the NR protein
could then be explained by a larger destabilization of the NR
protein due to the size of the deletion. In both the A-NR and the
NR proteins, the thermosensitivity of the NR protein was linked to a
higher activation state of the enzyme and to the insensitivity to dark
inactivation in N. plumbaginifolia (compare Figs. 4 and 7).
The actual relationship between the NR N-terminal acidic motif and the
stability of MoCo binding is as yet undetermined, since structural data
concerning the MoCo domain are relatively scarce.
The S-NR Protein Exhibits the Same Properties as the Tobacco
Wild-Type NR in N. plumbaginifolia
The alignment of the NR N-terminal domain protein sequences
revealed two main conserved motifs among higher plants (Fig. 2). One of
these motifs (RXDSP) was centered around Ser-27 in the tobacco NIA2
sequence and was therefore included in the sequence that was removed in
the NR protein. Since several Ser residues were found to be
phosphorylated in spinach (Huber et al., 1992), Arabidopsis (Labrie and
Crawford, 1994), and maize (Huber et al., 1994) NR, we questioned
whether this conserved Ser was involved in the NR-inactivation process
by introducing a four-amino acid deletion (residues 25 to 28 of the
NIA2 tobacco protein sequence) into the tobacco NR-coding
sequence and expressing the resulting S-NR protein in transgenic
N. plumbaginifolia plants. The S-NR protein was found to
be inactivated and degraded in the dark to the same extent as the
wild-type NR protein.
This suggests that the conserved RXDSP motif in the NR N-terminal
domain is not directly involved in the inactivation of the enzyme by
phosphorylation, which is in agreement with this motif being absent in
the NR N-terminal sequences from Leguminosae (Fig. 2). Nevertheless,
the modulation of the S-NR activity has been investigated only in
response to light modifications; therefore, this motif could be
involved in the regulation of NR activity in response to other external
or internal changes.
Identification of a 13-Amino Acid Sequence Involved in the
Posttranslational Inactivation of NR
The second conserved motif that was identified in the NR
N-terminal domain was a stretch of acidic amino acids preceded by several Ser residues (Fig. 2). An NR-coding sequence deleted from this
motif was then obtained and expressed in transgenic N. plumbaginifolia plants under the control of the 35S promoter. It
was found that the A-NR activation state was higher than for the
wild-type NR protein and was unaffected by the light-dark transition,
even after prolonged darkness. This suggests that, like the NR
protein (Nussaume et al., 1995), A-NR is less sensitive to
inactivation by phosphorylation. Therefore, the absence of the above
motif in the NR protein is probably the reason for the loss of
posttranslational regulation. This acidic motif seems to be necessary
for the inactivation of NR but may not be sufficient. A spinach NR that
had been partially proteolyzed at the N terminus could not be
completely inactivated by phosphorylation and 14-3-3 binding, even
though it still contained the N-terminal acidic motif (Douglas et al.,
1995).
After an extended period of darkness, the A-NR and NR activities
measured in the presence of EDTA were found to disappear less quickly
than wild-type NR activity. These activities should reflect the total
amount of reactivatable NR protein. This suggests that the A-NR and
NR proteins are more stable than the wild-type NR in the dark.
Indeed, 48 h of darkness led to a 10- to 12-fold decrease in NR
and A-NR total activity, whereas the decrease was about 50- to
100-fold in wild-type NR. After 72 h of darkness the NR and
A-NR activities were still higher than for the wild-type NR (data
not shown). The finding that the NR and A-NR proteins were still
present after extended periods of darkness is in agreement with our
previous results (Nussaume et al., 1995). However, the total amount of
NR protein measured in the dark was lower in the present study,
possibly because of the methods that were used to measure the amount of
NR protein. In the present study, NR activity was assayed as NADH:NR
activity after reactivation by EDTA. We now believe that this assay is
more representative of the amount of functional NR in the plant. In a
previous study, Nussaume et al. (1995) determined the amount of NR
protein by using an ELISA or a partial NR activity measurement
(NADH:Cyt c reductase activity), in which the NR protein can
be recognized by the antibody or exhibit a partial activity even if
partially degraded. These results point to a close relationship between NR inactivation by phosphorylation and degradation, as has been suggested in previous reports (Nussaume et al., 1995; Kaiser and Huber,
1997).
It has been shown that NR is phosphorylated on a conserved Ser residue
located in the hinge separating the MoCo and the heme domain (Douglas
et al., 1995; Bachmann et al., 1996b), and it has been suggested that
the inactivating 14-3-3 proteins bind this phosphorylated Ser. The
question that remains is the actual role of the acidic motif within the
N terminus. Secondary structure prediction by the PHD neural network
(Rost and Sander, 1994), which is based on multiple alignment,
suggested with a high probability that the whole N-terminal domain
forms an exposed loop ending with a short 
-helix beginning right
after the acidic stretch. The sequence that was removed in the A-NR
protein is somewhat reminiscent of a sequence rich in P, E/D, S, and T
residues (the "PEST" motif; Rechsteiner and Rogers, 1996), which
has been shown to serve as a proteolytic signal. For example, it was
proposed that phytochrome is rapidly degraded upon light absorption
because of the exposure of a potential PEST sequence (Rechsteiner and Rogers, 1996).
It has also been shown that constitutive phosphorylation by CKII of
I
B, a cytoplasmic protein sequestering the animal transcription factor NF-B, is required for its degradation (Lin et al., 1996; Schwarz et al., 1996). Interestingly, phosphorylation by CKII is
located in a C-terminal PEST motif that is required for degradation of
the IB protein. Therefore, it seems that phosphorylation by CKII
of PEST sequences, which often contain consensus sites for CKII
phosphorylation, could be required for their role in proteolysis. The
fact that a peptide derived from the NR N-terminal acidic motif is a
good substrate for CKII further supports the involvement of this
sequence in NR degradation. Indeed, the A-NR and NR proteins, in
which the acidic motif is absent, seem to be more stable than the
wild-type enzyme. As a hypothetical model for NR degradation, we
propose that upon phosphorylation of the regulatory Ser residue in the
first hinge domain the NR N-terminal acidic motif phosphorylated by
CKII could serve as a signal for NR proteolysis.
This model does not solve the problem of the role of the acidic motif
in modulating NR inactivation in the dark. It was previously shown that
the NR protein could be inactivated by yeast 14-3-3 proteins when
purified from dark-exposed leaves of del plants in the presence of
phosphatase inhibitors (Lillo et al., 1997). When purified in the
absence of phosphatase inhibitors the NR protein was less inhibited,
suggesting that the deletion of the N-terminal domain does not hinder
phosphorylation of the NR enzyme in planta or its inactivation in
vitro by yeast 14-3-3 proteins. This is true only when the NR
protein is purified, because this inactivation was not observed in
crude extracts (Lillo et al., 1997).
We propose the following hypothetical model to explain our
observations: A putative NR activation factor binds to the
dephosphorylated form of NR. Upon phosphorylation of the regulatory Ser
residue, the acidic motif itself or the whole N-terminal domain
triggers the release of this factor by becoming exposed to the outside. This would then allow binding of 14-3-3 proteins and subsequent inactivation of the NR enzyme as well as degradation of the protein by
CKII phosphorylation. This model could explain the previous set of
data: In NR or A-NR proteins expressed in N. plumbaginifolia, the NR activation factor would remain bound to
NR, and therefore the binding of 14-3-3 proteins would either not take
place or would be very reduced (Nussaume et al., 1995; this work). Upon purification of NR the putative NR activation factor would be lost and
inhibition of the NR protein could take place (Lillo et al., 1997).
However, we do not know yet whether such an activating factor exists.
Experiments are currently under way to characterize the putative NR
activation factor and to confirm the above hypothetical model. The in
vivo phosphorylation of NR by CKII also remains to be confirmed, as
well as its role in the NR protein degradation observed upon
inactivation.
| |
FOOTNOTES |
1
This work was supported by a grant from the
Ministère de l'Education Nationale et de la Recherche
Scientifique to E.P.
2
Present address: Institute of Botany, University
of Heidelberg, Im Neuenheimer Feld 360, DE-69120 Heidelberg, Germany.
*
Corresponding author; e-mail meyer{at}versailles.inra.fr; fax
33-1-30-83-30-99.
Received June 8, 1998;
accepted September 25, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
CKII, casein
kinase II.
MoCo, molybdenum cofactor.
NR, nitrate reductase.
| |
ACKNOWLEDGMENTS |
We thank Jacques Goujaud and Krystyna Gofron for care of the
plants, Marie-France Dorbe for help with plant transformation, Marie-Thérèse Leydecker and Thérèse Moureaux
for assistance with activity measurements, and Pierre Rouzé, who
first identified the acidic domain in the NR protein sequence. We thank
Carol McKintosh for bringing the CKII phosphorylation site to our
attention. We are also indebted to Françoise Vedele, Michel
Caboche, Helen North, and Hoai-Nam Truong for critical and careful
reading of the manuscript and for stimulating discussions and advice.
| |
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