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Plant Physiol. (1998) 118: 617-626
Purification and Characterization of NADP+-Linked
Isocitrate Dehydrogenase from Scots Pine1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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NADP+-isocitrate
dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the
supply of 2-oxoglutarate for ammonia assimilation and glutamate
synthesis in higher plants through the glutamine synthetase/glutamate
synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of
cytosolic localization was detected in green cotyledons of pine
(Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic
efficiency (105 M
1
s1) than the deduced efficiencies for GS and GOGAT in
higher plants. A polyclonal antiserum was raised against pine
NADP+-IDH and used to assess protein expression in the
seedlings. Steady-state levels of NADP+-IDH were
coordinated with GS during seed germination and were associated with
GS/GOGAT enzymes during chloroplast biogenesis, suggesting that
NADP+-IDH is involved in the provision of carbon skeletons
for the synthesis of nitrogen-containing molecules. However, a
noncoordinated pattern of NADP+-IDH and GS/GOGAT was
observed in advanced stages of cotyledon development and in the
hypocotyl. A detailed analysis in hypocotyl sections revealed that
NADP+-IDH abundance was inversely correlated with the
presence of GS, GOGAT, and ribulose-1,5-bisphosphate
carboxylase/oxygenase but was associated with the differentiation of
the organ. These results cannot be explained by the accepted role of
the enzyme in nitrogen assimilation and strongly suggest that
NADP+-IDH may have other, as-yet-unknown, biological
functions.
The synthesis of 2-oxoglutarate represents a connecting point
between carbon and nitrogen metabolism because this ketoacid provides
the carbon skeleton for the assimilation of inorganic nitrogen into
amino acids in higher plants through the GS/GOGAT cycle (Miflin and
Lea, 1980 Traditionally, the Krebs cycle enzyme NAD+-IDH
(EC 1.1.1.41) was considered to be responsible for 2-oxoglutarate
supply in amino acid biosynthesis (Bray, 1983 Cytosolic NADP+-IDH represents 90% to
100% of the activity detected in any plant organ (Chen and Gadal,
1990b We describe the purification and characterization of
NADP+-IDH from a gymnosperm species. The
expression studies during early development in pine suggest that
cytosolic NADP+-IDH is involved in not only the
biosynthesis of nitrogen compounds but also the differentiation of the
hypocotyl. The possible implication of the enzyme in the supply of
ketoacids in reactions other than amino acid biosynthesis is discussed.
Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). In this pathway GS (EC 6.3.1.2) catalyzes the condensation
of the ammonium ion and glutamate to produce Gln. In a second step, the
amido group of Gln is transferred to 2-oxoglutarate by the GOGAT action
(EC 1.4.7.1 and 1.4.1.14) and two glutamate molecules are produced.
These two reactions are of relevance in nitrogen metabolism because Gln
and glutamate are the donors for the synthesis of major nitrogen
compounds in higher plants: amino acids, chlorophyll, polyamines, and
nucleic acids. Therefore, physiological processes that demand a large synthesis of nitrogen-containing molecules also require a net supply of
2-oxoglutarate. Reactions catalyzed by glutamate dehydrogenase (EC
1.4.1.3) and aminotransferases (Givan, 1980) may generate the ketoacid
from glutamate, but when a net synthesis of the amino acid is required,
the 2-oxoglutarate must be supplied by the oxidative decarboxylation of
isocitrate, a reaction catalyzed by two different IDHs differing in
which pyridine nucleotide cosubstrate they use (NAD+ or NADP+).
); however,
NADP+-IDH (EC 1.1.1.42) has recently been
regarded as an alternative pathway when large quantities of the
ketoacid are required (Chen and Gadal, 1990a; Gálvez and Gadal,
1995). This hypothesis was based on the findings that (a) plant
organs exhibit very low NAD+-IDH activity, (b)
NADP+-IDH has a higher affinity for substrates,
and (c) cytosolic aconitase is present in plants and may produce
isocitrate outside of the mitochondria. In angiosperms
NADP+-IDH has been studied in a limited number of
species, including pea (Omran and Dennis, 1971; Randall and Givan,
1981; Chen et al., 1989a), several members of the Solanaceae
(Gálvez et al., 1994; Fieuw et al., 1995; Gallardo et al., 1995),
and cucumber (Canino et al., 1996). It occurs as several isozymes
located in different subcellular compartments, including the cytosol,
chloroplast, mitochondria, and peroxisome (Chen and Gadal, 1990b, and
refs. therein; Rasmusson and Moller, 1990; Gálvez et al., 1994;
Gallardo et al., 1995; Cornu et al., 1996).
; Fieuw et al., 1995; Gallardo et al., 1995; Nieri et al.,
1995), but little is known about its biological role. It has
been suggested that the synthesis of 2-oxoglutarate in the cytosol is
associated with the mitochondrial oxidation of L-malate in
photosynthetic conditions (Hanning and Heldt, 1993), and the
accumulation of cytosolic NADP+-IDH and glutamate
during the ripening of tomato fruit has also been reported (Gallardo et
al., 1995). In addition, it was reported that tobacco mutants with very
low levels of nitrate reductase activity showed a higher expression of
genes involved in nitrogen assimilation, including cytosolic
NADP+-IDH (Scheible et al., 1997). In spite of
the evidence implicating NADP+-IDH in nitrogen assimilation
and glutamate biosynthesis, the role of the enzyme in other plant
metabolic processes consuming cytosolic NADPH and its possible role in
the supply of 2-oxoglutarate to pathways other than the GS/GOGAT cycle
remain to be established. Furthermore, very little information is
available about the provision of the ketoacid for the GS/GOGAT pathway
in trees and especially in gymnosperms.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
). For in vitro experiments water-soaked
maritime pine seeds were sterilized with 10% sodium hypochlorite and
the embryos were detached, placed onto Murashige-Skoog medium
(Murashige and Skoog, 1962), and incubated under continuous white light
(110 µmol photon m2 s1). After the first
evidence of cotyledon development (cotyledon size of 3 mm), the embryos
were harvested, frozen in liquid nitrogen, and stored at 
80°C until
use.
Protein Extraction
The plant material was extracted with a mortar and pestle in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM MgCl2, and 14 mM 2-mercaptoethanol with washed sea sand on ice. The ratio of plant material to buffer to sand was 1.0:4.0:0.5. The extract was filtered through two layers of muslin and clarified by centrifugation at 22,000g for 15 min. The supernatant was used for protein determination, enzymatic analysis, purification of NADP+-IDH, and western analysis. All steps were performed at 4°C.Protein Determination, Enzyme Assays, and Kinetic Studies
Protein was estimated by the method of Bradford (1976) using BSA
as a standard. NADP+-IDH activity was determined by
monitoring the synthesis of NADPH at 340 nm, as described previously
(Gallardo et al., 1995). Kinetic studies were performed with purified
preparations desalted by gel filtration through a Sephacryl S-300
column. Determination of Km values for
NADP+ and 2R,3S-isocitrate
was carried out in 40 mM potassium phosphate, pH 7.5, and 5 mM MgCl2 at different concentrations
of one substrate and at the saturation concentration of the other
substrate (Gallardo et al., 1995). NAD+-IDH
activity was measured as described for NADP+-IDH
but using NAD+ (1 mM) as the pyridine
nucleotide.
Purification of NADP+-IDH from Pine Cotyledons
The major NADP+-IDH form present in Scots pine cotyledons (1-2 cm in size) was purified from 50 g of material following the procedure described by Gallardo et al. (1995)
with modifications. Protein extracts were prepared and fractionated by
precipitation with ammonium sulfate at 45% to 85% saturation. The
precipitates were dissolved and dialyzed against 200 volumes of 10 mM potassium phosphate, pH 7.5, 1 mM
MgCl2, and 14 mM 2-mercaptoethanol
(standard buffer). The dialyzed extracts were loaded onto a
DEAE-cellulose column (21 × 2.5 cm) previously equilibrated in
standard buffer. The column was washed with 200 mL of the same buffer,
and bound proteins were eluted with a linear gradient of 0 to 0.4 M KCl in standard buffer. The fractions with relevant
NADP+-IDH activity were pooled and concentrated
by ammonium sulfate precipitation and extensive dialysis. The enzyme
preparation was further purified by affinity chromatography on a 2-mL
Matrex Red Gel A (Amicon, Danvers, MA) column equilibrated in standard
buffer. After the column was washed with 10 mL of buffer, the proteins were eluted by the application of a linear gradient of 0 to 1 mM NADP+ and 0 to 25 mM
isocitrate in standard buffer. The active fractions were collected and
concentrated by ammonium sulfate precipitation and extensive dialysis.
Estimation of Native Molecular Mass
The molecular mass of pine NADP+-IDH was estimated by gel filtration using a Sephacryl S-300 column (68 × 1.6 cm) previously calibrated with protein markers of known size: urease (483 kD), catalase (240 kD), alcohol dehydrogenase (150 kD), BSA (66 kD), and Cyt c (12.4 kD).Antiserum Production
Polyclonal antibodies against pine NADP+-IDH were raised in New Zealand White rabbits. Immunization of the animals with 200 µg of purified protein was carried out basically as reported previously (Cantón et al., 1996). After blood specimens were
obtained, samples were allowed to clot overnight, and the serum was
recovered by centrifugation for 5 min at 4°C and 5000g.
The final preparation was divided into aliquots and stored at 80°C
until use.
Denaturing Gel Electrophoresis and Western Analysis
Proteins were analyzed by SDS-PAGE using the discontinuous buffer system of Laemmli (1970). After electrophoresis the polypeptides were
electrotransferred to a nitrocellulose filter or visualized by standard
Coomassie blue or silver staining (Morrissey, 1981). Protein blotting,
saturation of blots, incubations with the antisera, and washing steps
were performed as described previously (Gallardo et al., 1995). The
antisera raised against pine NADP+-IDH, cytosolic
GS (Cantón et al., 1996), Fd-GOGAT
(García-Gutiérrez et al., 1995), and the large subunit of
Rubisco (García-Gutiérrez et al., 1993) were diluted
10,000-, 5,000-, 8,000-, and 500-fold, respectively, in 20 mM Tris and 150 mM NaCl, pH 7.5. The
immunocomplexes were detected with a secondary antibody conjugated to
peroxidase with a molar peroxidase to antibody ratio of 3.3 (Vector
Laboratories, Burlingame, CA). Visualization of immunocomplexes was
carried out by assaying peroxidase activity using 3% (v/v)
H2O2 and 0.03 mM 4-chloro-1-naphthol as substrates. Molecular masses were
estimated relative to prestained protein markers (Sigma).
Chlorophyll Determination
Pigments were extracted from hypocotyl sections in 80% (v/v) acetone and total chlorophyll content was determined as reported previously (Graan and Ort, 1984).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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IDH Activities in Scots Pine Tissues
The analysis of IDH activities in pine seedlings revealed that NADP+-IDH activity was notably higher than NAD+-IDH activity in all organs studied (Fig. 1). NADP+-IDH represents approximately 90% of total IDH activity detected in any pine tissue, suggesting that this enzyme has a relevant role in the production of 2-oxoglutarate in pine seedlings. The NADP+-IDH levels were higher in the embryo and cotyledons than in hypocotyls and roots (Fig. 1); however, on a protein-content basis, the activity was higher in hypocotyls and roots (results not shown) and similar (4.5-7.0 nkat/mg protein) to the values described in angiosperm tissues (for review, see Chen and Gadal, 1990b).
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NADP+-IDH Isoenzyme Pattern and Purification from Scots Pine Cotyledons
Although NADP+-IDH exists in higher plants as different isoenzymes located in the cytosol, chloroplast, and mitochondria, most of the NADP+-IDH activity reported so far in plant extracts (90%-100%) corresponds to the cytosolic isoenzyme (Chen and Gadal, 1990a; Gálvez et al., 1994;
Gallardo et al., 1995). To determine the isoenzyme pattern in pine
cotyledons, protein extracts were concentrated by ammonium sulfate
precipitation (0%-90% saturation) and fractionated by ion-exchange
chromatography on a DEAE-cellulose column. The elution profile of
NADP+-IDH showed the presence of a single peak
that eluted from the column at 0.22 M KCl, indicating that
most of the activity detected in the tissue corresponds to the same
isoenzyme (Fig. 2).
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) were used
in western analysis of pine extracts. A 46-kD band corresponding to the
pine NADP+-IDH subunit (as will be shown) was
recognized by an antiserum raised against the cytosolic tobacco
NADP+-IDH, but no cross-reaction was observed
when the antiserum raised against the chloroplast
NADP+-IDH was used (results not shown).
Furthermore, the chloroplast NADP+-IDH isoenzyme,
which is the second major isoenzyme in angiosperm tissues (Randall and
Givan, 1981; Chen and Gadal, 1990b; Gálvez et al., 1994), was not
detected in Percoll-purified chloroplasts from pine cotyledons (not
shown). Together, the above data suggest that cytosolic
NADP+-IDH represents the major
NADP+-IDH isoenzyme in pine cotyledons.
Properties of NADP+-IDH from Pine Cotyledons
85%) that precipitated most of
NADP+-IDH was established in a pilot experiment.
As observed in the ion-exchange chromatography step, the
NADP+-IDH activity was also eluted as a single
peak from the Matrex Red Gel A at 0.27 mM
NADP+ and 6.85 mM isocitrate (not
shown). This purification protocol (summarized in Table
I) renders a homogeneous preparation, as judged by the presence of a single polypeptide in the final preparation (Fig. 3), with an absolute specificity
for NADP+ as the electron acceptor. The enzyme
was purified 250-fold, with a recovery of 37.6%, and the specific
activity of the final preparation was 451.5 nkat/mg protein (Table I).
Based on the data presented in Table I,
NADP+-IDH is a relatively abundant enzyme in pine
cotyledons and represents about 0.4% of total soluble protein.
View this table:
Table I.
Purification of NADP+-IDH from maritime
pine cotyledons
Data are representative of five different purifications from 50 g of
tissue from seedlings with cotyledons 1 to 2 cm in
length.
View larger version (53K):
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Figure 3.
Analysis of polypeptides by SDS-PAGE of the
purification steps of NADP+-IDH. Samples with 20, 10, 5, and 0.5 µg of proteins corresponding to the crude extract, ammonium
sulfate precipitate, DEAE-cellulose, and Matrex Gel Red A,
respectively, were separated by SDS-PAGE and stained with silver. The
positions of molecular mass standards are indicated on the left. The
migration of the NADP+-IDH subunit is marked on the
right.
; Gálvez et al.,
1994; Gallardo et al., 1995). To estimate the native molecular mass of
the pine enzyme, a purified preparation was subjected to gel filtration
through a Sephacryl S-300 column previously calibrated with standard
proteins of known size. The pine enzyme eluted from the column as
a single peak of activity with an apparent molecular mass of 95 kD.
Therefore, pine NADP+-IDH seems to be a dimer
with a subunit size of 46 kD.
) denoted the existence of
noncooperativity of the enzyme for both substrates assayed (not shown).
The Km values of the pine enzyme for
2R,3S-isocitrate and NADP+
were 80 and 10 µM, respectively. The
Kcat of the enzyme was 43 s1 and the ratio
Kcat/Km for
2R,3S-isocitrate and NADP+
were 5 × 105 and 4 × 106 M1
s1, respectively. These kinetic data are in the
value range described or deduced for NADP+-IDH
from angiosperms (Chen and Gadal, 1990b, and the refs. therein; Gálvez et al., 1994; Gallardo et al., 1995).
; Cornu et al., 1996)
and Aspergillus niger (Meixener-Monori et al., 1986) NADP+-IDH and suggest that inhibition of the
enzyme by nucleotides and 2-oxoglutarate is the result of chelating
free Mg2+ and, therefore, of poor regulatory
significance.
Production and Characterization of a Polyclonal Antiserum Raised against Scots Pine NADP+-IDH
A preparation of the purified enzyme was injected into rabbits to raise an antiserum against pine NADP+-IDH. The specificity of the antiserum was assayed by its ability to decrease the NADP+-IDH activity present in a cotyledon extract. As shown in Figure 4A, NADP+-IDH activity decreased when the extract was incubated with increasing volumes of the antiserum obtained, whereas identical volumes of a preimmune antiserum showed little effect on the enzyme activity. The ability of the antiserum to recognize the enzyme was also assessed by western analysis. Total proteins from cotyledons were subjected to SDS-PAGE, transferred onto a nitrocellulose filter, and probed with dilutions of the antiserum ranging from 1,000 to 10,000. In all cases, the 46-kD polypeptide corresponding to the NADP+-IDH subunit was detected in the crude extract. Figure 4B shows a typical experiment in which an aliquot of the purified enzyme was used as a control in the western blot probed with the antiserum diluted 10,000-fold. Because of the high signal-to-noise ratio, a 10,000-fold dilution of the antiserum was used for protein expression studies.
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Accumulation of NADP+-IDH in Germinating Embryos
Seed germination is a complex process that requires the synthesis of specific nitrogen compounds to support the postembryonic differentiation and development of plant organs. To investigate whether NADP+-IDH is associated with germination, we evaluated activity and protein levels in water-soaked seeds and in in vitro germinated maritime pine embryos that were collected upon the first evidence of cotyledon development (cotyledon size of 3 mm). Maritime pine was selected for this study instead of Scots pine because of its larger seed and embryo size, which allowed for easier isolation and better manipulation of the embryos for the in vitro culture.
NADP+-IDH in Developing Scots Pine Seedlings
Analysis of NADP+-IDH in Differentiating Hypocotyl
Characterization of NADP+-IDH Enzyme in Pine Seedlings
NADP+-IDH Expression during Early Development
of Pine
View larger version (43K):
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Figure 5.
Expression of NADP+-IDH and GS in
seeds and in in vitro germinating maritime pine embryos. A,
Protein-soluble extracts were prepared from the megagametophyte
(Megagame.) and embryos of water-soaked seeds (Embryo) and from in
vitro germinating embryos collected upon the first symptoms of
development (Germinat.) and the NADP+-IDH was determined.
The results are the means ± SE of at least three
independent experiments. Proteins (20 µg) of the same samples in
which enzyme activity was determined were separated by SDS-PAGE and
stained with Coomassie blue (B) or blotted onto nitrocellulose filters
and probed with the antibodies raised against pine
NADP+-IDH or against pine cytosolic GS (C) (Cantón et
al., 1996 ). The migration of protein molecular markers is indicated on
the left. The position and size of the immunostained bands are marked
on the right.
; Cánovas
et al., 1993). To investigate whether NADP+-IDH
is associated with the development of the cotyledon, we determined activity and protein level in Scots pine seedlings grown in continuous light and in darkness. In this pine species the chloroplasts are fully
developed when the cotyledon size is 1.0 to 1.5 cm (Palomo, 1997). The
enzymatic activity was similar in cotyledons of light-grown plantlets
at the different stages considered (Fig.
6A). The polypeptide levels of
NADP+-IDH, GS, and Fd-GOGAT were abundant in the
organ during chloroplast development (stages 0.5-1.5 cm; Fig. 6B).
These results are consistent with the view that
NADP+-IDH is involved in the supply of carbon
skeletons for ammonia assimilation and glutamate biosynthesis during
plastid differentiation; however, in later stages of cotyledon
development (stages 2.0-2.5 cm; Fig. 6B)
NADP+-IDH protein content remained abundant,
whereas GS and Fd-GOGAT polypeptide content declined to low levels in
the organ. Similar results were found during cotyledon development in
dark-grown plants (results not shown).
View larger version (29K):
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Figure 6.
Analysis of NADP+-IDH, GS, and
Fd-GOGAT during cotyledon development in Scots pine. A,
NADP+-IDH activity quantified in protein extracts prepared
from embryos of water-soaked seeds (E) and cotyledons of light- and
dark-grown seedlings. The results are the means ± SE
of at least three independent experiments. B, Samples with 20 µg of
proteins corresponding to the same extracts in which enzyme activity
was determined were analyzed by western blotting with the antisera
raised against pine NADP+-IDH, GS (Cantón et al.,
1996 ), and Fd-GOGAT (García-Gutiérrez et al., 1995).
Twenty micrograms of protein was loaded per lane in the gels prepared
for the detection of NADP+-IDH and GS polypeptides, and 40 µg of protein was loaded per lane for the analysis of Fd-GOGAT.
View larger version (25K):
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Figure 7.
NADP+-IDH, GS, and Fd-GOGAT
polypeptides in developing Scots pine hypocotyls. Samples of protein
extracts prepared from the whole hypocotyl of seedlings with cotyledons
0.5, 2.0, and 2.5 cm in length were analyzed by western blotting for
the detection of NADP+-IDH and GS polypeptides. The
migration of protein molecular markers is indicated on the left. The
position and size of the immunostained bands are marked on the right.
Twenty micrograms of protein was loaded per lane in the gels prepared
for the detection of NADP+-IDH and GS polypeptides, and 40 µg of protein was loaded per lane for the analysis of Fd-GOGAT.
). These
characteristics are lost when cells differentiate toward the base of
the hypocotyl during development. To investigate whether the expression
of NADP+-IDH is associated with the
differentiation of this organ, we analyzed the chlorophyll levels and
the NADP+-IDH, GS, Fd-GOGAT, and Rubisco contents
in serial sections of the hypocotyl from the apical meristem (Fig.
8A). The chlorophyll content was high in
the top sections, indicating that chloroplast development takes place
in the young cells formed from the meristem and then decreases toward
the root in a continuous gradient (Fig. 8B). The GS, Fd-GOGAT, and
Rubisco contents matched the pattern of chlorophyll in the organ,
abundant in the sections close to the apical meristem and at very low
levels in the basal sections (Fig. 8C). In contrast to chlorophyll, GS,
Fd-GOGAT, and Rubisco contents, the pattern of
NADP+-IDH polypeptide markedly increased from the
top to the basal sections of the hypocotyl (Fig. 8C).
View larger version (29K):
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Figure 8.
Pattern of NADP+-IDH, GS, Fd-GOGAT,
and Rubisco polypeptides in the hypocotyls of pine seedlings. A, Serial
dissection of the hypocotyl of a Scots pine seedling with cotyledons
2.5 cm in length. Numbers indicate the distance in centimeters from the
apical meristem (the zero value). Total chlorophyll content was
determined in the sections (B) and protein extracts were prepared for
the analysis of NADP+-IDH, GS, and large subunit of Rubisco
(LSU) by western blotting (C). Twenty micrograms of protein was loaded
per lane in the gels prepared for the detection of
NADP+-IDH and GS polypeptides, 40 µg of protein was
loaded per lane for the analysis of Fd-GOGAT, and 5 µg of protein per
lane was loaded for the analysis of Rubisco.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Gálvez and Gadal, 1995). This hypothesis
is supported, among other things, by the fact that
NAD+-IDH activity is 10 to 100 times less
abundant than NADP+-IDH in all plant organs
studied (Chen and Gadal, 1990a). The NADP+-IDH
activity in pine seedlings is also notably higher than
NAD+-IDH and represents about 90% of the total
IDH values detected in any organ. In angiosperms the fractionation of
soluble proteins by ion-exchange chromatography has been used to
resolve the two main NADP+-IDH isoforms reported
in plants, the cytosolic and the chloroplastic isozymes (Chen et al.,
1989a; Gálvez et al., 1994). When such an approach was utilized
to investigate the isoform pattern in pine cotyledons, only one form
was detected, which corresponded to the cytosolic isozyme according to
cross-reactions with antibodies raised against tobacco
NADP+-IDH isozymes. These data are in accordance
with those reported from both photosynthetic and nonphotosynthetic
organs in angiosperm species, in which the cytosolic enzyme represents
90% to 100% of the total NADP+-IDH activity
(Chen and Gadal, 1990a, 1990b; Gallardo et al., 1995; Nieri et al.,
1995).
) and close to that in cultures and tissues with some
photosynthetic capacity, such as mixotrophic tobacco cell cultures and
tomato fruit pericarp (0.1%-0.2%; Gálvez et al., 1994;
Gallardo et al., 1995). The abundance of NADP+-IDH in pine cotyledons may reflect its
participation in several physiological processes that take place in the
organ, since pine cotyledon is both the main photosynthetic organ and
the main storage organ of the seedling. In addition, the synthesis of
2-oxoglutarate and NADPH may be relevant in the organ for the
biosynthesis of nitrogen compounds during chloroplast development and
for the mobilization of the nitrogen reserves required for development of the first true leaves (needles) of the tree.
; Gálvez et al., 1994; Gallardo et al., 1995). Kinetic
studies revealed that the pine enzyme exhibits typical Michaelis
kinetics for both reaction substrates. The
Km values for
2R,3S-isocitrate and NADP+
(80 and 10 µM, respectively) and the
Kcat of the enzyme (43 s1) were also similar to those described or
deduced for the cytosolic enzyme in angiosperms (Gálvez et al.,
1994; Gallardo et al., 1995; Chen and Gadal, 1990b). The catalytic
efficiency of pine NADP+-IDH
(Kcat/Km of
approximately 105
M1 s1 for
2R,3S-isocitrate) is slightly higher than the
efficiency that may be deduced for Fd-GOGAT (104
M1 s1 for
Gln) and cytosolic GS (104
M1 s1 for
glutamate; Lea et al., 1990). Conversely, the molecular and kinetic
data and the fact that the pine protein may be recognized by antibodies
raised against a cytosolic enzyme from angiosperms (Chen et al., 1989b;
this work) suggest that NADP+-IDH is a protein
that was conserved after the evolutive divergence between angiosperm
and gymnosperms and that it could play similar physiological roles in
both.
). Since NADP+-IDH levels are barely
detectable in the megagametophyte of water-soaked pine seeds, most of
the isocitrate produced is probably used by the glyoxalate cycle and,
therefore, a low expression of NADP+-IDH could be
essential in the metabolism of the organ. In contrast, water-soaked
pine embryos contained NADP+-IDH activity and
protein that increased about 4-fold when the postembryonic development
was initiated. A similar pattern was also observed for GS polypeptide,
suggesting that both proteins may be involved in the supply of
precursors for the synthesis of nitrogen compounds associated with the
germination process or in the recycling of nitrogen compounds that
follows the degradation of seed storage proteins during the very early
development of the seedling.
; García-Gutiérrez et al., 1995, 1998). A major difference between pines (conifers) and
angiosperms is the content of GS isozymes, since only cytosolic GS is
expressed in pine during the seedling stage (Cánovas et al.,
1991; Cantón et al., 1993; García-Gutiérrez et al.,
1998). The detection of NADP+-IDH, GS, and
Fd-GOGAT polypeptides during the development of the chloroplast
(cotyledon size of 0.5-1.5 cm) is in agreement with the initial
hypothesis of Chen and Gadal (1990a) and suggests that
NADP+-IDH is associated with the GS/GOGAT cycle
in higher plants. However, when the differentiation of the chloroplast
has been completed (cotyledon size of 2.0-2.5 cm), the expression of
these proteins differ, suggesting that NADP-IDH may play an additional
role not related to the GS/GOGAT cycle.
;
Prescott and John, 1996), and in the hydroxylation of Pro residues of
Hyp-rich glycoproteins (Showalter and Varner, 1989). It is noteworthy
that extensins (Hyp-rich glycoproteins) accumulate in the cell wall of
differentiated cells (Showalter and Varner, 1989) and exhibit a similar
pattern of expression to NADP+-IDH in the
hypocotyl (Bao et al., 1992; Ahn et al., 1996). All of these data
suggest that NADP+-IDH could be involved in the
supply of 2-oxoglutarate for dioxygenase reactions in differentiated
cells and in the decrease of cell wall extensibility associated with
differentiation processes.
).
Nevertheless, our results also suggest that
NADP+-IDH is associated with the differentiation
of the hypocotyl and is not related to the GS/GOGAT cycle and
photosynthetic metabolism in this organ. Therefore, new roles for
NADP+-IDH must be considered to explain the
reported expression.
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FOOTNOTES |
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Received March 23, 1998;
accepted July 10, 1998.
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ABBREVIATIONS |
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Abbreviations: GOGAT, glutamate synthase. GS, Gln synthetase. IDH, isocitrate dehydrogenase.
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ACKNOWLEDGMENTS |
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We thank Prof. P. Gadal (Université Paris Sud) for the antisera against tobacco NADP+-IDHs, Dr. A. García-Gutiérrez for his assistance in the preparation of the antiserum against pine NADP+-IDH, R. Crespillo for technical assistance, and Dr. M.G. Claros for the revision and criticism of the manuscript.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Ahn JH, Choi Y, Kwon YM, Kim S-G, Choi YD, Lee JS (1996) A novel extensin gene encoding a hydroxyproline-rich glycoprotein requires sucrose for its wound-inducible expression in transgenic plants. Plant Cell 8: 1477-1490 [Abstract]
Bao W,
O'Malley DM,
Sederoff RR
(1992)
Wood contains a cell-wall structural protein.
Proc Natl Acad Sci USA
89:
6604-6608
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 [CrossRef][Web of Science][Medline]
Bray CM (1983) Nitrogen Metabolism in Plants. Longman, London
Canino S, Nieri B, Pistelli L, Alpi A, De Bellis L (1996) NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: purification and characterization of a cytosolic isoenzyme. Physiol Plant 98: 13-19 [CrossRef]
Cánovas FM, Cantón FR, Gallardo F, García-Gutiérrez A, De Vicente A (1991) Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings. Planta 185: 372-378
Cánovas FM, McLarney B, Silverthorne J (1993) Light-independent synthesis of LHCIIb polypeptides and assembly of the major pigmented complexes during initial stages of Pinus palustris seedling development. Photosynth Res 38: 89-97
Cantón FR, García-Gutiérrez A, Crespillo R, Cánovas FM (1996) High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinant protein and expression in pine seedlings. FEBS Lett 393: 205-210 [CrossRef][Web of Science][Medline]
Chen RD, Bismuth E, Champigny ML, Gadal P (1989a) Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenase are distinct isoenzymes. Planta 178: 157-163 [CrossRef]
Chen RD,
Bismuth E,
Issakidis E,
Pacot C,
Champigny ML,
Gadal P
(1989b)
Etude immunologique comparée de l
isocitrate déshydrogénase à NADP: une protéine cytosolique bien conservée chez les plantes supérieures.
CR Acad Sci Ser III Sci Vie
308:
459-465
Chen RD, Gadal P (1990a) Do the mitochondria provide the 2-oxoglutarate needed for glutamate synthesis in higher plant chloroplasts? Plant Physiol Biochem 28: 141-145 [Web of Science]
Chen RD, Gadal P (1990b) Structure, functions and regulation of NAD and NADP-dependent isocitrate dehydrogenases in higher plants and in other organisms. Plant Physiol Biochem 28: 411-427
Chen RD, Le Maréchal P, Vidal J, Jacquot JP, Gadal P (1988) Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves. Eur J Biochem 175: 565-572 [Web of Science][Medline]
Cornu S, Pireaux JC, Gerard J, Dizengremel P (1996) NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings. Physiol Plant 96: 312-318 [CrossRef]
Fieuw S, Müller-Röber B, Gálvez S, Willmitzer L (1995) Cloning and expression analysis of the cytosolic NADP+-dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism. Plant Physiol 107: 905-913 [Abstract]
Gallardo F, Gálvez S, Gadal P, Cánovas FM (1995) Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening. Characterization of the predominant cytosolic enzyme from green and ripe pericarp. Planta 196: 148-154
Gálvez S, Bismuth E, Sarda C, Gadal P (1994) Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from mixotrophic tobacco cells. Plant Physiol 105: 593-600 [Abstract]
Gálvez S, Gadal P (1995) On the function of the NADP-dependent isocitrate dehydrogenase isoenzymes in living organisms. Plant Sci 105: 1-14
García-Gutiérrez A, Cantón FR, Gallardo F, Cánovas FM (1993) Immunochemical analysis of chloroplast polypeptides from maritime pine. Phytochemistry 34: 337-341 [CrossRef]
García-Gutiérrez A, Cantón FR, Gallardo F, Sánchez-Jiménez F, Cánovas FM (1995) Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings. Plant Mol Biol 27: 115-128 [CrossRef][Web of Science][Medline]
García-Gutiérrez A, Dubois F, Cantón F, Gallardo F, Sangwan R, Cánovas F (1998) Two different modes of early development and nitrogen assimilation in gymnosperm seedlings. Plant J 13: 187-200 [CrossRef][Web of Science]
Givan CV (1980) Aminotransferases in higher plants. In BJ Miflin, eds, The Biochemistry of Plants, Vol 5. Academic Press, London, pp 329-357
Graan T,
Ort DR
(1984)
Quantitation of the rapid electron donors to P700, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts.
J Biol Chem
259:
14003-14010
Hanning I, Heldt HW (1993) On the function of mitochondrial metabolism during photosynthesis in spinach (Spinacia oleracea L.) leaves. Plant Physiol 103: 1147-1154 [Abstract]
Hedden P (1992) 2-Oxoglutarate-dependent dioxygenases in plants: mechanism and function. Biochem Soc Trans 20: 373-377 [Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 [CrossRef][Medline]
Lea PJ, Blackwell RD, Chen F-L, Hecht U (1990) Enzymes of ammonia assimilation. In PJ Lea, eds, Enzymes of Primary Metabolism, Vol 3. Academic Press, London, pp 257-276
Mariani P, De Carli M, Rascio N, Baldan B, Casadoro G, Gennari G, Bodner M, Larcher W (1990) Synthesis of chlorophyll and photosynthetic competence in etiolated and greening seedlings of Larix decidua as compared with Picea abies. J Plant Physiol 137: 5-14
McQueen-Mason SJ (1996) Expansins and cell wall expansion. J Exp Bot 46: 1639-1650
Meixener-Monori B, Kubicek CP, Harrer W, Schreferl G, Rohr M (1986) NADP-specific isocitrate dehydrogenase from the citric acid-accumulating fungus Aspergillus niger. Biochem J 236: 549-557 [Medline]
Miflin BJ, Lea PJ (1980) Ammonia assimilation. In B Miflin, eds, The Biochemistry of Plants, Vol 5. Academic Press, London, pp 169-202
Morrissey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117: 307-310 [CrossRef][Web of Science][Medline]
Muller RT, Gifford DJ (1995) Purification and characterization of the glyoxysomal enzyme malate synthase following seed germination in Pinus taeda. Plant Physiol Biochem 33: 639-648
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 [CrossRef]
Nieri B, De Bellis L, Biagi PP, Alpi A (1995) NADP+-isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons. Physiol Plant 94: 351-355 [CrossRef]
Omran RG,
Dennis DT
(1971)
Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from a higher plant.
Plant Physiol
47:
43-47
Palomo J (1997) Purificación y caracterización de la isocitrato deshidrogenasa dependiente de NADP+ de pino: análisis cinético-molecular y expresión de la proteína durante el desarrollo. Tesis de Licenciatura, Universidad de Málaga. Málaga, Spain
Prescott AG, John P (1996) Dioxygenases: molecular structure and role in plant metabolism. Annu Rev Plant Physiol Plant Mol Biol 47: 245-272 [CrossRef][Web of Science]
Price NC, Stevens L (1989) Fundamentals of Enzymology. Oxford University Press, Oxford, UK
Randall DD,
Givan CV
(1981)
Subcellular location of NADP+-isocitrate dehydrogenase in Pisum sativum leaves.
Plant Physiol
68:
70-73
Rasmusson AG,
Moller I
(1990)
NADP-utilizing enzymes in the matrix of plant mitochondria.
Plant Physiol
94:
1012-1018
Scheibe W-R, González-Fontes A, Lauerer M, Müller-Röber B, Caboche M, Stitt M (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 9: 783-798 [Abstract]
Showalter AM, Varner JE (1989) Plant hydroxyproline-rich glycoproteins. In A Marcus, eds, Molecular Biology, Vol 15. Academic Press, San Diego, CA, pp 385-519
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) or with a preimmune rabbit serum (
) for 4 h.
The immunocomplexes formed were recovered by centrifugation at
20,000g for 5 min at 4°C, and enzyme activity was
determined in the supernatant. B, Detection of NADP+-IDH by
western analysis. Samples of proteins from a cotyledon extract (20 µg) and the purified preparation (0.5 µg) were subjected to
SDS-PAGE, electrotransferred to a nitrocellulose filter, and probed
with a 10,000-fold dilution of rabbit anti-NADP+-IDH
antibodies. The position and size of the immunoreactive bands are
indicated on the right.