Plant Physiol. (1999) 119: 1483-1496
Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro
and in Vivo Studies1
Jose Luis Ortega,
Dominique Roche2, and
Champa Sengupta-Gopalan*
Agronomy and Horticulture Department, New Mexico State University,
Las Cruces, New Mexico 88003
 |
ABSTRACT |
Glutamine
synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes
the ATP-dependent condensation of NH3 with glutamate to
produce glutamine. GS in plants is an octameric enzyme. Recent work
from our laboratory suggests that GS activity in plants may be
regulated at the level of protein turnover (S.J. Temple, T.J. Knight,
P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315-325;
S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996]
Plant Physiol 112: 1723-1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular
Approach to Primary Metabolism in Higher Plants. Taylor & Francis,
London, pp 155-177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a
metal-catalyzed oxidation system to produce the ·OH radical, we have
shown that GS is oxidized and that oxidized GS is inactive and more
susceptible to degradation than nonoxidized GS. Histidine and cysteine
protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative
inactivation. The roots of plants fed ammonium nitrate showed a 3-fold
increase in the level of GS polypeptides and activity compared with
plants not fed ammonium nitrate but without a corresponding increase in
the GS transcript level. This would suggest either translational or
posttranslational control of GS levels.
| |
INTRODUCTION |
GS (EC 6.3.1.2) is a key enzyme in nitrogen metabolism. It
catalyzes the biosynthesis of Gln from Glu, ATP, and ammonium. GS from
bacteria consists of 12 identical subunits arranged in two hexamers
stacked face to face; the side-to-side interface of a pair of subunits
constitutes an active site containing two Mn2+
ions (Yamashita et al., 1989
). The two divalent metal ions in the
active site are distinguished by their dissociation constants (Villafranca et al., 1985). Saturation of the high-affinity site, n1, in each subunit by Mn2+
or Mg2+ induces a conformational change,
converting the enzyme from a catalytically inactive to a catalytically
active conformation (Hunt and Ginsburg, 1980). The metal ion at the
n1 site also plays a catalytic role in the
binding of Glu, whereas the second metal ion, n2,
is involved in the binding of ATP (Hunt and Ginsburg, 1980; Liaw et
al., 1993).
In bacteria GS has been shown to be regulated by cumulative feedback
inhibition, covalent modification, and repression/derepression (Stadtman, 1990). Although normally stable, bacterial GS is turned over
when cells are starved for nitrogen (Fulks and Stadtman, 1985),
suggesting that the intracellular level of GS in bacterial cells is
also regulated by proteolysis. The degradation of GS in
Escherichia coli and Klebsiella aerogenes appears
to involve two steps: (a) the enzyme is inactivated by oxidative
modification of a single His residue per subunit (Levine, 1983a; Rivett
and Levine, 1990) and (b) the altered enzyme is then degraded by
endogenous proteases that are capable of degrading the oxidized enzyme
but exhibit little activity on native GS (Roseman and Levine, 1987; Stadtman and Berlett, 1997).
In plants GS is an octamer and has a native molecular mass of
approximately 320 to 380 kD (Stewart et al., 1980). Conservation in the
amino acid sequence in the active site of GS across kingdoms suggests
that plant GS is mechanistically similar to bacterial GS (Shatters and
Kahn, 1989; Sanangelantoni et al., 1990). There are, however,
differences in the ATP-binding site within the active site between the
GS in plants and that in bacterial GS (Kim and Rhee, 1988). It is
generally believed that GS activity in plants is regulated at the
transcriptional level, and most of the research on GS regulation has
focused on this aspect (Hirel et al., 1987; Bennett et al., 1989; Forde
et al., 1989; Walker and Coruzzi, 1989; Edwards et al., 1990; Cock et
al., 1991, 1992; Miao et al., 1991; Roche et al., 1993; Sukanya et al.,
1994; Temple et al., 1995). Very little is known about the regulation
of plant GS at the level of translation, the assembly of holoenzyme,
and enzyme turnover. However, it has been shown that GS in plants is
not regulated by the adenylylation/deadenylylation cascade utilized by
many Gram-negative bacteria (Tate and Meister, 1971). Recent work from
our laboratory suggests that, aside from transcriptional regulation, GS
activity in plants might be regulated at the level of enzyme assembly
or turnover (Temple et al., 1993, 1996; Temple and Sengupta-Gopalan,
1997). To our knowledge, there have been no reports of how GS in plants
is turned over; therefore, in this study we have made the initial step
in understanding the mechanism of turnover of GS in plants by
determining whether regulation by oxidative modification has a role.
The first step in protein oxidation requires the production of oxygen
radicals. This process is mediated by several enzymatic and
nonenzymatic systems. In plants oxygen radicals are generated during
normal physiological processes such as photosynthetic electron transport, mitochondrial respiration, and nitrogen fixation (Allen, 1995; Dalton, 1995). Some enzymatic redox systems can also generate reactive oxygen species (Levine et al., 1981; Stadtman and
Oliver, 1991; Harding et al., 1997; del Rio et al., 1998). The
production of reactive oxygen species increases during physiological
disorders that result from environmental stresses such as temperature
changes, drought stress, and herbicide toxicity (Iturbe-Ormaetxe et
al., 1998), from exposure to high radiance (Landgraf et al., 1997) or
high levels of ozone (Pell et al., 1997), from defense against pathogens (Low and Merida, 1996), and during plant senescence (del Rio
et al., 1998). The injury caused to plant tissues during environmental
stresses is a result of an imbalance between the production of oxygen
radicals and antioxidant defense responses (Foyer et al., 1994).
Nonenzymatic oxidase systems, including the ascorbate/metal/oxygen
system (Levine et al., 1983b) and the mercaptan-mediated MCO system, in
the presence of transition metal ions such as
Fe3+ or Cu2+ (Rhee et al.,
1990; Netto and Stadtman, 1996), are capable of generating ·OH
radicals in vitro. In these systems a series of reactions take place in
which the Fe3+ ion is reduced to
Fe2+ with ascorbate or DTT as the reductants. The
Fe2+ replaces Mn2+ at the
n2 site of the GS. The hydrogen peroxide
generated during the reduction of Fe3+ interacts
with the Fe2+-GS complex and the
Fe2+-peroxide complex and then dissociates into
two reactive species, the ·OH radical and Fe-O (the ferryl ion) (Liaw
et al., 1993; Netto and Stadtman, 1996). Both are extremely reactive
and attack the side chains of amino acid residues proximal to the
n2 metal-binding site (Liaw et al., 1993).
We demonstrate that, like the GS from E. coli, GS from
soybean (Glycine max) roots can be oxidized in vitro with
the MCO system, and the oxidized form is susceptible to degradation
with proteases present in the plant root extract. We also present data
suggesting that turnover of GS in vivo is mediated via an oxidation
step and that in plants grown with exogenous nitrogen the GS is less susceptible to oxidative modification and proteolytic turnover.
| |
MATERIALS AND METHODS |
Plant Material
Soybean (Glycine max L. cv Williams) roots were
obtained from 4-d-old seedlings germinated aseptically in aluminum
trays. Roots were also obtained from 18- to 21-d-old plants that were planted in hydroponic culture vessels (Magenta, Chicago, IL) with vermiculite and grown in nutrient solution without nitrogen for 15 to
17 d; at this stage the nutrient solution was supplemented for 3 to 4 d before harvesting with one of the following: 10 mM KCl, 10 mM ammonium
nitrate, or 0.1% hydrogen peroxide. Root tissues were frozen in liquid
nitrogen and stored at 
80°C until use.
Protein Extraction
All procedures were carried out at 4°C. Roots were ground in
liquid nitrogen with 15% (w/w) insoluble polyvinylpolypyrrolidone and
homogenized with 2 (old tissues) or 5 (young tissues) volumes of
extraction buffer (50 mM Tris-Cl, pH 8.0, 5 mM
EDTA, 5% [v/v] ethylene glycol, 20% [v/v] glycerol, 1 mM magnesium acetate, 1 mM DTT, and a mixture
of protease inhibitors: 50 µg/mL antipain, 1 µg/mL cystatin, 10 µg/mL chymostatin, 2 µg/mL leupeptin, and 1 mM PMSF).
The homogenate was centrifuged for 15 min at 20,000g.
For in vitro oxidation experiments, ammonium sulfate was added to the
4-d-old root extracts to a 30% to 70% saturation level. The protein
precipitate was resuspended in 50 mM imidazole buffer, pH
7.4, and desalted in Sephadex G-25 spin-out columns against 50 mM imidazole, pH 7.4. For in vivo GS oxidation analysis,
root extracts were desalted in Sephadex G-25 columns against a buffer containing 10 mM Tris-Cl, pH 8.0, 1 mM EDTA,
5% (v/v) ethylene glycol, 20% (v/v) glycerol, 1 mM
magnesium acetate, 1 mM DTT, and a mixture of protease
inhibitors: 50 µg/mL antipain, 1 µg/mL cystatin, 10 µg/mL
chymostatin, 2 µg/mL leupeptin, and 1 mM PMSF.
Assay of Enzyme Activity and Protein Determination
Protein concentration was measured by the Bradford (1976) assay,
using BSA as a standard. GS activity was measured
spectrophotometrically at 500 nm by the transferase assay reported by
Ferguson and Sims (1971). Transferase units were calculated from a
standard curve of 
-glutamyl hydroxamate. One unit of transferase
activity is equivalent to 1 µmol -glutamyl hydroxamate
min
1 produced at 30°C. GS activity data
presented are the averages of at least three independent experiments.
Oxidative Inactivation of GS
Inactivation of root GS was carried out by three different MCO
systems, all modifications of the mercaptan-mediated MCO system described by Rhee et al. (1990). Samples of desalted 4-d-old root extract (30%-70% ammonium sulfate fraction) equivalent to 25 µg of
protein were incubated in 50 µL of solution containing 50 mM imidazole, pH 7.4, different concentrations of
FeCl3 (up to 1 mM), and 5 mM DTT, 10 mM GSH, or 20 mM
ascorbate at 4°C. Incubation was performed with and without 1 mM EDTA. Samples were assayed for transferase activity
after incubation. Results are presented as the percentages of activity
compared with the control (no addition). Data are the averages of at
least three independent experiments.
Analysis of the Stability of the GS Protein
Samples (30%-70% ammonium sulfate fraction) of desalted root
extract from 4-d-old soybean plants were incubated in a MCO reaction containing 50 mM imidazole, pH 7.4, 0.125 mM
FeCl3, 5 mM DTT, and 500 µg/mL root
protein for 2 h at 4°C. The root extract for this experiment did
not include any of the protease inhibitors. Incubation was performed
with and without 1 mM EDTA. Samples were then incubated at
30°C to observe the stability of the GS activity and protein for the
next 22 h. Aliquots were taken at several times for estimation of
GS activity and electrophoretic analysis. In one set of experiments, an
oxidized sample after 2 h of incubation at 4°C in the MCO system
was passed through a Sephadex G-25 spin-out column equilibrated with 50 mM imidazole, pH 7.4, 5 mM DTT, and 1 mM EDTA to remove Fe3+ ions. This
sample was then incubated at 30°C for the next 22 h, and the
stability of the GS protein was monitored by one-dimensional SDS-PAGE
and then by western blotting. This experiment was repeated several
times, and the result of a typical experiment is presented here.
Electrophoretic Analysis of GS Polypeptides and Holoenzyme
Protein aliquots were taken at different times during the
incubation in the MCO system. Samples were either boiled in 2% SDS or
brought to 20% glycerol and placed in a 80°C freezer for
electrophoretic analysis in denaturing and native conditions,
respectively. One-dimensional PAGE was performed in a Mini-Protean II
electrophoretic apparatus (Bio-Rad). Analysis of total protein patterns
was performed by SDS-PAGE (Laemmli, 1970) using gradient PAGE gels
from 7% to 15% in which a standard acrylamide:Bis-acrylamide solution
was used. Gels were run at a constant 100 V. Proteins were visualized
by silver staining (Morrissey, 1981).
For analysis of the GS protein the concentration of the crosslinker
used was decreased to 1% of the total acrylamide solution for both
native and SDS-PAGE to increase the separation of the GS1 isoforms in the gels. SDS-PAGE was run in
12% gels at constant voltage. Native gel electrophoresis was run at
4°C using 7.5% gels for 4 h at a constant 100 V.
GS was also analyzed by two-dimensional PAGE (O'Farrell, 1975) with
the modifications previously described (Temple et al., 1996), except
that 10 mM DTT replaced 2-mercaptoethanol in all cases.
Protein samples were boiled in 2% SDS, 1 µg/µL urea was added to
the sample, and the samples were allowed to dissolve at 37°C for 30 min before loading. Volumes equivalent to 0.035 transferase unit
(transferase activity) were loaded in each tube gel, and the tube gels
were extruded from the tubes and equilibrated for 30 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10 mM DTT, and 10% glycerol before being subjected to SDS-PAGE. After
electrophoresis, gels were electroblotted onto nitrocellulose, and the
GS immunoreactive bands or spots were detected with antibodies raised
against nodule GS (Cullimore et al., 1983). Native GS bands were
detected with antibodies against the native enzyme (Lara et al., 1984).
The blots were scanned, and the GS immunoreactive bands were
quantified by using Intelligent Quantifier software (Bio-Image, Ann
Arbor, MI). Experiments were repeated at least three times, and the
results presented here represent a typical experiment. For the analysis of in vivo oxidation of GS, western blots from four different experiments were quantified. To compare between experiments, band intensities were standardized against the total GS intensity in the
control samples (plants fed KCl).
Protection of GS from Oxidative Inactivation
Amino acid and GS substrates were analyzed for their role in
preventing the oxidative inactivation of GS. Protection experiments were performed by the addition of amino acids, ATP, magnesium acetate,
or MnCl2 at a concentration of 5 mM
to desalted 4-d-old ammonium sulfate-precipitated root extracts before
incubation in 100 µL of a MCO reaction (125 µmol FeCl3,
5 mM DTT, 5 mM protectant, and 50 µg of root
protein in 50 mM imidazole, pH 7.4) with or without 1 mM EDTA. GS-transferase activity was assayed after 40 min
of incubation at 4°C. At this time control samples with no protectants were inactivated by 40% to 60%. Inactivation of GS was
calculated as the difference in GS activity between the control sample,
in which 1 mM EDTA was included in the MCO reaction, and the samples incubated in the MCO system containing either the amino
acids or the substrates indicated in Table II. Values are presented as
the percentages of the GS activity lost relative to the control sample,
in which no amino acid or GS substrate was added. Values of
approximately 100% mean no protection, values lower than 100% mean
some protection, and values higher than 100% mean an enhancement in
the inactivation of GS.
View this table:
[in this window]
[in a new window]
|
Table II.
Effect of amino acids and substrates on the
protection of GS inactivation by MCO
A 4-d-old root extract (desalted ammoniun sulfate fraction) was
incubated in 50 mM imidazol (pH 7.4) with 5 mM
amino acids and GS substrates before the addition of a mixture of
FeCl3 and DTT with or without EDTA for 40 min of incubation
at 4°C for partial inactivation (Fig. 1). Samples were assayed for
transferase activity following incubation in the MCO system.
Inactivation was calculated as the difference between each sample and
its respective control in which 1 mM EDTA was added. Values
are the percentages of inactivation compared with the nonprotected
sample. Values below 100% = protection from inactivation; values close
to 100% = no protection. Data are the averages ± SE
of at least three independent experiments (n  3).
|
|
RNA Purification and Northern-Blot Analysis
Total RNA was purified by the LiCl-precipitation procedure (De
Vries et al., 1982). RNA (20 µg) was fractionated on a 1.4% agarose/formaldehyde gel and blotted onto nitrocellulose. Probes were
prepared from inserts isolated from clones: pGS100, coding for the
cytoplasmic "housekeeping" isoform of alfalfa
GS1 (Tischer et al., 1986), the 3
-untranslated
region from the pGSGmD gene for GS1 from soybean
(Roche et al., 1993), and a DNA clone for soybean 28S rRNA (a gift from
Dr. F. Ausubel, Harvard University, Boston, MA). Standard hybridization
conditions, including 50% formamide (Sambrook et al., 1989), were
used. The filter was washed five times with 2× SSC and 0.5% SDS for
20 min at 42°C and exposed. The same filter was used for reprobing
after the DNA probe was stripped by three washes in a boiling solution
of 0.5% SDS. Exposed films were scanned and RNA-hybridization signals
were quantified using Intelligent Quantifier software. Intensities were
standardized for loading against the intensity of the 28S rRNA
hybridization signal.
| |
RESULTS |
Inactivation of Soybean Root GS1 by the Iron/Ascorbate,
Iron/GSH, and Iron/DTT Oxidation System
Our first objective was to demonstrate that GS enzyme from soybean
roots is inactivated in vitro by active oxygen species. We used three
different MCO systems with ascorbate, GSH, or DTT as the reductant and
varying concentrations of FeCl3, and we checked for GS activity (Table I). Incubation of
desalted ammonium sulfate-precipitated extracts of soybean roots was
for 2 h at 4°C. Fe3+ alone at different
concentrations had no effect on GS activity. The reductants by
themselves appeared to promote enzyme activity to a small extent
(20%-25%). DTT with 0.125 mM FeCl3
produced the highest level of inactivation (90%), whereas ascorbate
and GSH required a higher concentration of FeCl3
(0.5-1 mM) for maximal inactivation (75% and 64%,
respectively). Furthermore, of the three reductants used, DTT exerted
the maximum inactivation (90%) after 2 h of incubation. Ascorbate
and GSH, although less effective than DTT in inactivating GS, showed
the maximum inactivation after 1 h of incubation (data not shown).
View this table:
[in this window]
[in a new window]
|
Table I.
In vitro inactivation of soybean root GS by
different MCO systems
A 4-d-old root extract (desalted ammoniun sulfate fraction) was
incubated in 50 mM imidazol (pH 7.4) with FeCl3
(0.25-1 mM) and either DTT, GSH, or ascorbate in the
presence or absence of 1 mM EDTA for 2 h at 4°C, and
samples were assayed for transferase activity. Values are the
averages ± SE of at least three independent
experiments and were calculated as the percentages of the remaining
activity compared with the control with no addition (n 3).
|
|
To demonstrate the involvement of the
Fe3+-catalyzed oxidation reaction in the
oxidation of GS, the desalted ammonium sulfate-precipitated root
extracts were incubated with DTT and 0.125 mM
FeCl3 in the presence or absence of 1 mM EDTA for different times at 4°C. Inactivation of GS in
the absence of EDTA was observed within 30 min, and by 2 h there
was a 90% loss in GS activity (Fig. 1).
In contrast, there was no loss of GS activity in the presence of EDTA
during the first 4 h of incubation at 4°C and only a 40% loss
of GS activity after 24 h of incubation at 30°C.
View larger version (14K):
[in this window]
[in a new window]
| Figure 1.
In vitro inactivation of soybean root GS by a MCO
system. A 4-d-old root extract was incubated with an FeCl3
solution (0.125 mM FeCl3 and 50 mM
imidazol, pH 7.4) and 5 mM DTT in the presence ( ) or
absence ( ) of 1 mM EDTA for the times indicated. The
incubation was at 4°C for the first 2 h and then at 30°C for
the next 22 h. At each time the samples were assayed for
transferase activity. Values are averages ± SE from
at least three independent experiments (n = 3-6).
|
|
Characterization of the Oxidatively Modified GS
Aguirre and Hansberg (1986) showed that oxidized GS subunits from
Neurospora crassa resolved on two-dimensional gels showed a
more acidic pI compared with nonoxidized GS. To determine whether the
oxidized form of GS from roots of soybean exhibit the same phenomenon,
an ammonium sulfate-precipitated extract of 4-d-old soybean roots
(after desalting) was incubated with DTT/FeCl3 (1 and 2 h at 4°C) in the presence or absence of EDTA, and samples were subjected to two-dimensional gel electrophoresis and then analyzed
by immunoblot (Fig. 2). The GS
polypeptides in the EDTA-treated samples resolved into three major
subunits: 
1, 2, and 
1. The samples treated with the MCO system
in the absence of EDTA showed an additional set of three GS
polypeptides with slightly more acidic IEF values than 1, 2, and
1. The 1ox, 2ox,
and 1ox polypeptides represent the oxidized
forms of 1, 2, and 1, respectively. These polypeptides were
also seen in the samples incubated in the MCO system in the presence of
EDTA but at a significantly lower level than in samples that did not
contain EDTA. The spots were all quantified using Intelligent
Quantifier software, and the ratios of the intensity of the oxidized to
the corresponding nonoxidized forms were calculated (Fig. 2B). The
ratio for each form showed an increase in level as the time of
incubation with the MCO system was increased from 1 to 2 h (Fig.
2). The ratio for the 1ox:1 forms showed a
dramatic increase after 2 h of incubation. The data suggest that
the oxidized form of GS subunits can be differentiated from the
nonoxidized form on two-dimensional gels and that the oxidized forms
show an increase with time of incubation in the MCO system.
View larger version (35K):
[in this window]
[in a new window]
| Figure 2.
Two-dimensional PAGE profile of soybean GS
polypeptides from inactive (oxidized) and EDTA-protected GS enzyme. A,
Root extract was incubated in the MCO system with or without EDTA for 1 and 2 h, and the samples were subjected to two-dimensional gel
electrophoresis followed by immunodetection with anti-GS antibodies.
The native GS polypeptides are labeled as 1, 2, and 1, and
their modified versions are labeled as 1ox,
2ox, and 1ox, respectively. B, The spots
were all quantified using Intelligent Quantifier software and the
ratios of the intensity of the oxidized to the corresponding
nonoxidized forms were calculated and plotted on a graph. The figure
represents typical results.
|
|
Fate of Oxidized GS Polypeptides when Incubated with Root Extract
The GS from bacterial systems is known to go through a two-step
turnover mechanism that includes an oxidation step followed by the
degradation of the oxidized GS by endogenous proteases (Roseman and
Levine, 1987). To determine whether the oxidized form of GS from the
roots of soybean is more susceptible to endogenous proteases present in
the roots, the ammonium sulfate-precipitated root extracts after
desalting were incubated in the DTT/FeCl3 system
in the presence or absence of EDTA for different times, and the
extracts were subjected to SDS-PAGE analysis (Fig.
3A). The intensity of immunostained bands
as measured by Intelligent Quantifier software was plotted (Fig. 3C).
The two immunoreactive bands showed only a 15% decrease after 8 h
of incubation and a 50% decrease after 24 h in samples incubated
in the presence of EDTA, whereas in the absence of EDTA the decrease
was almost 95% after 8 h. Concomitantly with the loss of GS
polypeptides, the oxidized samples showed high-molecular-mass
immunoreactive bands. Higher-molecular-mass proteins can be generated
during free-radical reactions via cross-linking of peptides or via
hydrophobic interactions among oxidized proteins (Cervera and Levine,
1988; Stadtman and Berlett, 1997).
View larger version (55K):
[in this window]
[in a new window]
| Figure 3.
Stability of soybean root GS polypeptides when the
oxidized and nonoxidized forms of the enzyme were incubated in the root
extract. A, Four-day-old root extract incubated in the DTT-mediated MCO
system in the presence (DTT/Fe·EDTA) or absence of EDTA
(DTT/FeCl3). Samples were taken at the indicated times, and
the reaction was terminated by the addition of SDS and boiling. The
samples were subjected to SDS-PAGE followed by electroblotting and
immunodetection of GS polypeptides using anti-GS antibodies. B, After
incubation in the MCO system for 2 h, the extract was passed over
a Sephadex G-25 spinout column equilibrated with 50 mM
imidazole, pH 7.4, 10 mM DTT, and 1 mM EDTA to
remove the Fe3+ ion and then incubated for the indicated
times. The samples were then subjected to SDS-PAGE followed by
electroblotting and immunodetection of GS polypeptides using anti-GS
antibodies. C, Bands in A and B were quantified using
Intelligent Quantifier software and plotted. , DTT/Fe·EDTA
treatment; , DTT/FeCl3 treatment;  ,
DTT/FeCl3 system after removal of Fe3+. The
figure represents typical results.
|
|
In another experiment the root extract was incubated in the
DTT/FeCl3 system for 2 h, followed by gel
filtration on Sephadex G-25 to remove all Fe3+,
and then incubated for an additional 22 h. The samples were analyzed by SDS-PAGE and then by immunoblotting; the GS polypeptides were still susceptible to degradation (Fig. 3B), suggesting that the
proteolysis step does not require the presence of
Fe3+ and that oxidative modification and
proteolysis do not have to occur concurrently. Most significant is
that the high-molecular-mass immunoreactive bands seen in the
nondialyzed samples (Fig. 3A) were not detected in the desalted
samples.
Effect of the MCO System on GS Holoenzyme Disassembly
To determine whether proteolysis of subunits was dependent or
independent of holoenzyme disassembly, the extracts used in the
previous experiment (Fig. 3A) were also subjected to native PAGE
followed by activity staining or immunoblot analysis (Fig. 4A). Two immunoreactive bands were
detected: a slower-migrating band and a faster-migrating, more diffused
band. Based on standard molecular-mass markers included in the gel, we
suggest that the faster-migrating band is the tetrameric form of GS,
and the slower-migrating band is the octameric form. Activity staining
of the native gels showed intense activity in the slower-migrating band
in the samples incubated in the DTT/FeCl3 system
in the presence of EDTA and a lower level of activity in the
faster-migrating band. No activity was detected in the samples
incubated in the DTT/FeCl3 system without EDTA
(data not shown). The immunoreactive bands on native gels were
quantified using Intelligent Quantifier software (Fig. 4B).
View larger version (79K):
[in this window]
[in a new window]
| Figure 4.
Stability of soybean root GS holoenzyme in the
native and the oxidized form incubated in root extract. The 4-d-old
root extract was incubated in a DTT-mediated MCO system in the presence
(DTT/Fe-EDTA) or absence of EDTA (DTT/FeCl3), and samples
were removed at the indicated times. The reaction was terminated by the
addition of glycerol to 20%, followed by immediate freezing. A,
Results of native gel electrophoresis (at 4°C) followed by western
blotting and immunodetection using anti-GS antibodies. 8er., Octamer;
4er., tetramer. B, Bands quantified using Intelligent Quantifier
software and plotted. The figure represents typical results.
|
|
The 0-h time for samples incubated in the MCO system does not truly
represent that time because the reaction was terminated only by the
addition of glycerol (up to 20%) and placement of the sample in a
80°C freezer; therefore, there was a time lapse before the reaction
was completely terminated, and the 0-h time more accurately represents
an early time point. At the initial times of the experiment, the GS
forms in the oxidized samples were more intensely stained than the
nonoxidized sample (EDTA protected). This probably resulted from an
increase in the hydrophobicity of the oxidized proteins. Moreover, the
immunoreactive bands for both the octameric and tetrameric forms in the
samples treated with the MCO system without EDTA were more intensely
stained than the corresponding forms in the samples treated with EDTA.
The intensity of immunostaining in the tetrameric and octameric forms was plotted for each time point to determine whether the tetramer was
an intermediate in the turnover of the GS holoenzyme (Fig. 4B).
The tetramer:octamer ratio increased for the samples in the unprotected
MCO system for the first 4 h, after which time the ratio
decreased; in the EDTA-protected system the ratio showed an increase
for the entire 24 h of incubation. A gradual loss of both
immunoreactive bands was detected in the samples incubated in the
DTT/FeCl3 system without EDTA after 2 h, and
by about 24 h of incubation both bands were completely lost. In
contrast, the sample incubated in the DTT/FeCl3
system in the presence of EDTA maintained both immunoreactive bands for
the entire 24-h incubation period. However, the tetrameric form showed
a gradual increase relative to the octameric form as the time of
incubation in the MCO system was increased. This suggests that even in
the protected system there is some loosening of interactions among the
subunits and probably an increase in the hydrophobicity, resulting in
an increase in the immunostaining of the tetramer as the time of
incubation was increased.
Effect of Different Amino Acids and Substrates on MCO-Mediated
Inactivation of GS
Oxidation of GS led to the rapid loss of enzymatic activity,
suggesting that the modified site(s) was near the active site or in a
region that affected the active site. In E. coli GS, the loss of activity was correlated with a loss of a single His and a
single Arg residue per subunit (Farber and Levine, 1986; Climent and
Levine, 1991; Liaw et al., 1993), situated at one of the two metal-binding sites on the enzyme (Yamashita et al., 1989). To determine whether the oxidation sites in the plant GS are similar to
those of bacterial GS, we tested the effect of including some amino
acids on the protection of the GS enzyme in a MCO system (Table
II). Cys, Gln, and His showed the highest
level of protection against inactivation, whereas Arg showed no
protection. We also investigated the oxidative inactivation of GS in
the presence of some of its substrates. As shown in Table II, ATP alone
had a significant protective effect, and ATP plus Glu gave a still higher level of protection. Gln as a product had a higher protective activity, whereas Glu, which is the substrate, had no significant effect. Mg2+ and Mn2+
protect GS from inactivation, an indication of the role of metal ions
in the generation of oxygen radicals. Both Mg2+
and Mn2+ protect the GS enzyme by competing with
Fe2+ for the metal-binding sites on the enzyme.
Mn2+ appeared to be a more potent inhibitor of
inactivation than Mg2+, suggesting that the
oxidative modification site on GS has a higher affinity for
Mn2+ or that Mn2+ can act
as an ·OH scavenger, reducing the amount of
reactive free radicals (Stadtman and Oliver, 1991).
Effect of Hydrogen Peroxide and Nitrate Treatment on GS Activity
and Level of GS Polypeptide and Transcript in Roots of Soybean
To determine whether oxidative modification of GS has any
physiological relevance, the endogenous levels of oxygen radicals were
increased by allowing soybean plants to take up hydrogen peroxide, and
the effect of increased oxygen radicals on GS levels was measured.
Plants (18-21 d old) were grown in hydrogen peroxide (0.1% or 29 mM) for 4 d, at which time visible symptoms in the form of necrotic lesions were seen in the leaves. The fact that Arabidopsis leaves treated with 10 mM hydrogen peroxide for
8 h were shown to have a 367% increase in the in vivo hydrogen
peroxide levels compared with control (Rao et al., 1997), along with
the visible symptoms of necrosis seen in the leaves of our hydrogen peroxide-treated soybean plants, suggests that growing soybean plants
for 4 d in 29 mM hydrogen peroxide must have increased the endogenous hydrogen peroxide levels and therefore the level of
oxygen radicals. GS activity was reduced by almost 50% in the hydrogen
peroxide-treated roots compared with roots from untreated plants.
Furthermore, in vitro inactivation studies have demonstrated that the
GS enzyme from soybean roots is protected from MCO-mediated inactivation if the reaction is performed in the presence of either the
substrate (ATP or Glu) or the product of GS activity (Gln). To
determine whether this phenomenon holds true in vivo, roots of soybean
plants that were treated with or without ammonium nitrate were analyzed
for GS activity. Externally supplied nitrogen would eventually be
converted into ammonia and then into Gln and Glu. Measurement of GS
activity showed that there was an average 2-fold increase in the level
of GS activity in the roots of plants that were fed ammonium nitrate
compared with plants that were not fed nitrogen (Fig.
5A).
View larger version (52K):
[in this window]
[in a new window]
| Figure 5.
Analysis of GS activity and polypeptide profile in
roots of soybean plants treated with hydrogen peroxide and ammonium
nitrate. Soybean plants (15-17 d after planting) were either fed 10 mM KCl (K) or 10 mM ammonium nitrate (N) or
allowed to take up 0.1% hydrogen peroxide (H) for 4 d. The roots
of the 19- to 21-d-old plants were harvested, and the soluble protein
fraction was extracted. A, Average ± SE
(n 4) GS activity (transferase assay) from at
least four independent experiments was calculated and the values were
plotted. UT, Transferase units. B, Typical experiment in
which 1 µg of the root-protein extract from the different treatments
was subjected to SDS-PAGE followed by western blotting using anti-GS
antibodies. The experiment was performed four times, and the results
from a representative experiment are shown here. C, Immunoreactive
bands (GS1 and GS2) were quantified using Intelligent Quantifier
software, and the values were plotted. Values were standardized by
using the total GS band intensity from the control KCl sample as the
standard to compare between experiments. The average values ± SE were plotted (n 4). D, Typical
experiment in which 2.5 µg of the same root extracts were also
subjected to SDS-PAGE in a 7% to 15% PAGE gradient gel, followed by
silver staining. mwm, Standard molecular-mass markers included in the
gel during electrophoresis.
|
|
To determine whether GS polypeptide levels are reflective of the GS
activity levels in the hydrogen peroxide- and ammonium nitrate-treated
plants, protein extracts from control roots, ammonium nitrate-treated
roots, and hydrogen peroxide-treated roots were subjected to SDS-PAGE
and then analyzed by western blotting using GS antibodies (Fig. 5B).
The two immunoreactive bands (GS1 and GS2) were quantified using
Intelligent Quantifier software, and the average values from the
different experiments were plotted (Fig. 5C). In control roots the two
GS1 polypeptide bands (GS1 and GS2) were
found in equal amounts and both forms showed a dramatic increase after
nitrate treatment; the GS1 form, however, showed a greater increase
compared with that of the GS2 form. Both the GS1 and GS2
polypeptides showed decreased levels in the hydrogen peroxide-treated
roots compared with control roots. The GS polypeptide levels in roots
from the various treatments were truly reflective of the enzyme
activity.
Protein extracts from control and treated roots were also subjected to
SDS-PAGE and then silver staining to check for any other changes in the
protein profile resulting from the treatment (Fig. 5D). The overall
profile looked similar except that in the hydrogen peroxide-treated
roots some of the higher-molecular-mass protein bands were not
detectable. The roots fed nitrate also showed the presence of an 85-kD
protein band, probably representing nitrate reductase, that was not
seen in the control or hydrogen peroxide-treated roots.
Total GS1 transcript levels were measured to
determine whether changes in the GS polypeptide level associated with
the uptake of hydrogen peroxide or treatment with ammonium nitrate were
due to changes at the transcript level. Total RNA from the roots of control, hydrogen peroxide-treated, and ammonium nitrate-treated plants
were subjected to RNA analysis using a conserved
GS1 gene probe. The RNA loads were standardized
by probing the blot with a probe for the 28S rRNA. Since the GS1
polypeptide showed a greater increase in roots treated with nitrate,
the RNA blot was also probed with a probe specific for the gene
encoding that polypeptide (pGSGmD3; Roche, 1994). The hybridization
signals were quantified using Intelligent Quantifier software, and the
ratio of the signal with the two GS probes were standardized
individually against the signal obtained with the rRNA probe. As shown
in Figure 6, no
appreciable difference in the level of total GS1
RNA was detected between control and hydrogen peroxide-treated roots.
However, whereas the level of total GS1
transcript showed a 20% increase, the transcript for the specific gene
corresponding to pGSGmD3 showed a 75% increase in the ammonium
nitrate-treated roots compared with the controls.
View larger version (37K):
[in this window]
[in a new window]
| Figure 6.
Analysis of GS1 transcripts in the
roots of soybean plants treated with hydrogen peroxide and ammonium
nitrate. Soybean plants (18 d after planting) were either fed 10 mM KCl (K) or 10 mM ammonium nitrate (N) or
allowed to take up 0.1% hydrogen peroxide (H) for 3 d. The roots
of the 21-d-old plants were harvested, and the total RNA was isolated.
The data from a representative experiment are shown here. A, RNA (20 µg) from each sample was then subjected to electrophoresis in a
formaldehyde-agarose gel. The gel was blotted onto nitrocellulose and
probed with the coding region of pGS100, the 3-untranslated region of
the ammonia-inducible gene (pGSGmD3) or the 28S rRNA gene of soybean.
B, Hybridization signals were quantified using Intelligent Quantifier
software, and the values obtained with the two GS probes were
standardized against the values obtained with the rRNA gene
probe and plotted.
|
|
Taken together, our data suggest that the dramatic increase in
GS1 polypeptides and activity in plants fed
nitrate cannot be accounted for by just transcriptional regulation.
There is specific induction of one gene member or subclass due to
ammonium nitrate treatment at the transcriptional level, but, again,
this was not adequate to account for the increase in the polypeptide level.
Effect of Feeding Hydrogen Peroxide and Ammonium Nitrate on the
Oxidation State of the GS Polypeptides in the Roots of Soybean Plants
To implicate the oxidative modification step in the regulation of
GS activity and enzyme levels associated with the hydrogen peroxide and
ammonium nitrate treatments, root extracts from 21-d-old plants treated
with water, hydrogen peroxide, or ammonium nitrate (see ``Materials and Methods'') were subjected to two-dimensional SDS-PAGE followed by immunoblotting with GS antibodies (Fig.
7). The oxidized and nonoxidized forms of
GS can be distinguished by their two-dimensional gel profile (Fig. 2).
The GS1 subunits resolved into four major spots in all cases: 1, 2, 1ox, and
2ox (the latter two representing the oxidized
forms of 1 and 2, respectively). The 1 spot seen in Figure 2
could not be detected in the control or the ammonium nitrate-treated
samples because 1 is found only in young roots (K. Morey, J.L.
Ortega, and C. Sengupta-Gopalan, unpublished data). However, 1 and
1ox were detectable in the hydrogen
peroxide-treated samples, with a relatively higher level of the
1ox form. The spots were quantified using
Intelligent Quantifier software, and the ratio of the signals in the
1 and 2 spots were compared with those of
1ox and 2ox,
respectively, and the ratios were plotted (Fig. 7B). The level of the
oxidized forms was higher in the hydrogen peroxide-treated roots than
in the control sample. In the ammonium nitrate-treated samples, the
1 protein was more abundant than the 2 form, and, at the same
time, the ratio of the oxidized form to the nonoxidized form for 2
was appreciably lower than in the KCl control sample, whereas there was
a less appreciable difference (24%) in this ratio for the 1 form.
These results suggest that hydrogen peroxide treatment promotes the oxidation of the two GS polypeptides, and ammonium nitrate treatment protects GS polypeptides from oxidative modification.
View larger version (37K):
[in this window]
[in a new window]
| Figure 7.
Two-dimensional PAGE analysis of the GS
polypeptides from soybean roots treated with either hydrogen peroxide
or ammonium nitrate. Soybean plants (17 d after planting) were either
fed 10 mM KCl (K) or 10 mM ammonium nitrate (N)
or allowed to take up 0.1% hydrogen peroxide (H) for 4 d. The
roots of the 21-d-old plants were harvested, and the soluble protein
fraction was extracted. A, Protein equivalent to 0.035 units of GS
activity from each sample was then subjected to two-dimensional PAGE
followed by immunoblotting using the anti-GS antibodies. The
nonoxidized GS polypeptides are labeled 1 and 2 and their
modified versions are labeled 1ox and
2ox, respectively. B, Blots from A were all quantified
using Intelligent Quantifier software and the ratio of the intensity of
the oxidized to the corresponding nonoxidized forms were calculated and
plotted on a graph. Results shown are from a typical experiment.
|
|
| |
DISCUSSION |
The data presented in this paper clearly demonstrate that GS from
plants is subject to MCO in a manner similar to GS from E. coli (Nakamura and Stadtman, 1984), Bacillus subtilis
(Kimura and Sugano, 1992), N. crassa (Aguirre and Hansberg,
1986), Anabaena variabilis (Martin et al., 1997), and
Monoraphidium braunii (Humanes et al., 1995). Furthermore,
as is the case with bacterial GS, the oxidized form of plant GS is more
susceptible to degradation than the nonoxidized form (Roseman and
Levine, 1987). In E. coli GS, the loss of catalytic activity
due to oxidative modification correlated well with the loss of a single
His (His-269) and a single Arg residue (Arg-344) per subunit, and these
residues are situated in one of the metal-binding sites of the enzyme
that is conserved among all GS enzymes from different sources (Farber and Levine, 1986; Climent and Levine, 1991; Liaw et al., 1993).
Whereas we could demonstrate the protection of the soybean root GS from
MCO-mediated oxidation by incubating the enzyme with excess His, Arg
had no effect on the protection of plant GS. Protein-engineering studies have shown that His-269 but not Arg-344 is crucial for bacterial GS activity (Liaw et al., 1993). It is likely that, in spite
of the strong conservation of the Arg-344 residue in all types of GS,
there might be subtle differences in the active site of plant and
bacterial GS. The soybean root GS is also protected from the
MCO-mediated oxidation by Cys. It has been suggested that protection by
Cys is due to a redox effect on the oxidase system (Levine, 1983b), but
this may not be the case because GSH does not show any protective
effect on GS inactivation (Table I). GS sequence alignment has shown
strong conservation of Cys residues among GSs from different kingdoms
(Shatters and Kahn, 1989; Sanangelantoni et al., 1990; Pesole et al.,
1991).
Oxidative modification of Cys residues may prevent the formation of an
active conformation of GS. This would explain the effect of DTT, GSH,
and ascorbate on the activation of GS (Table II). Exogenous His and Cys
might protect against oxidation by simple competition with the
corresponding amino acid residue of the enzyme. It is intriguing that,
although Gln protects the GS from soybean root and B. subtilis from MCO attack, GS from E. coli (Roseman and
Levine, 1987) and N. crassa (Aguirre and Hansberg, 1986) are not protected by Gln, again suggesting some subtle differences in the
active site among the different types of GS. Furthermore, our data also
suggest that, as in bacterial systems (Fulks and Stadtman, 1985), under
low-nitrogen conditions plant GS is subject to turnover and the process
is mediated by oxidative modification of the protein.
Oxidative modification caused a rapid loss of the catalytic activity of
GS within minutes, and by 2 h the inactivation was more than 90%.
In fact, inactivation appeared to be instantaneous, since a 10%
decline in activity could be detected soon after incubation of the
enzyme extract in the MCO system. However, the loss of activity did not
appear to be accompanied by disassembly of the holoprotein, as
evidenced by the results of native gel electrophoresis (Fig. 4). The
two immunoreactive bands on native gels corresponding to the octameric
and the tetrameric forms during the early times of incubation in the
MCO system showed a migration pattern essentially similar to that of
the protected samples. However, the more intense staining of the bands
and the diffused nature of the faster-migrating band in the
nonprotected samples compared with samples that were protected by the
presence of EDTA (Fig. 4) suggest that there were slight conformational
changes that occurred in the octameric and tetrameric form of the
holoprotein as a result of oxidation of the metal-binding sites. The
changes in conformation and surface hydrophobicity probably resulted in
increasing the immunoreactivity of the holoprotein and changing the
migration pattern of the proteins. In GS from B. subtilis
conformational changes in the enzyme were demonstrated by
electrophoresis, crystal diffraction spectral studies, and electron
micrograph studies (Kimura and Sugano, 1992). It is interesting that GS
from E. coli showed no change in the migration pattern in
gels after oxidative inactivation (Kimura and Sugano, 1992). The
presence of oxidized forms of the GS subunits seen in the samples
incubated in the MCO system in the presence of EDTA (Fig. 2) suggests
that GS oxidation/degradation may take place in vivo or during the
extraction procedure.
The oxidative inactivation of GS was not quantitatively related to the
appearance of the oxidized forms of the GS subunits. Therefore, after
2 h of incubation in the MCO system the enzyme activity was
decreased to 10%; at this time only about 50% of the 1 and 2
polypeptides were in the oxidized form (Fig. 2), suggesting that one or
a few oxidized monomers in the oligomer bring about a conformational
change that renders the enzyme inactive. Alternatively, it could
indicate negative cooperation in the oligomer, e.g. one or a few
oxidized monomers in the oligomer bring about a conformational change
that renders the enzyme less active but inaccessible to further
oxidation. It is interesting that the different GS polypeptides showed
different degrees of oxidation when exposed to the MCO system (Fig. 2),
suggesting differences in accessibility to oxidative modification among
the different GS polypeptides. GS polypeptides have been shown to
assemble into holoenzymes with different catalytic activities: the
synthetase:transferase ratio can vary (Bennett et al., 1989), as
can the affinity for Mg2+ (Cullimore et al.,
1983), suggesting that differences in the active sites and the affinity
for divalent cations may result in differences in oxidation of the GS
subunits.
Although inactivation was instantaneous following incubation in the MCO
system, there was only about a 40% loss of the GS polypeptides after
2 h of incubation, suggesting that the quick loss in activity was
due to conformational changes. Liaw et al. (1993) showed that the early
modification in bacterial GS takes place at the
n2 site, eliminating enzyme activity, and the
later modification occurs at the n1 site,
relaxing the GS structure and perhaps enabling proteolytic degradation.
A gradual increase in the tetrameric form relative to the octameric
form is seen during incubation in the MCO system in the presence or
absence of EDTA, suggesting that disassembly of GS also takes place
during the degradation process. The faster rate of loss of the GS
polypeptides in the samples incubated in the MCO system over that
incubated in the presence of EDTA suggests that the oxidized samples
are more susceptible to proteolysis. In bacteria oxidation of GS
holoenzyme increases its hydrophobicity, with a concomitant increase in
its susceptibility to proteolysis (Cervera and Levine, 1988). Oxidized bacterial GS has been shown to be specifically degraded by an E. coli protease (Roseman and Levine, 1987) and by the proteasome complex from mammalian cells (Grune et al., 1997). Whether such a
specific system of proteolysis that recognizes oxidized GS is in place
in plant cells is not known. No immunoreactive peptides smaller than
the authentic size GS peptides could be detected in any of the samples
on SDS-PAGE, as has been detected for GS from E. coli and
B. subtilis. This would suggest that either the proteases in
soybean roots degrade the subunits completely to amino acids or the
antibodies do not have affinity for the GS degradation products.
Our data strongly support the two-step mechanism involving oxidative
modification for GS turnover in plants, and it appears that there are
two modes of turnover: (a) conformational or hydrophobicity changes of
the holoprotein followed by proteolysis and (b) disassembly followed by
proteolysis. However, we have not ruled out the possibility that
oxidative modification is the only route for GS turnover. It is
interesting that, like the samples that had been inactivated by
incubation in the MCO system, GS from samples incubated in the presence
of EDTA also migrated as octamers, tetramers, and probably dimers and
monomers (our gels were overrun). This observation has also been made
with GS from other plant sources (Hopfner et al., 1988; Mack and
Tischner, 1990). This would suggest that in vivo the octameric form of
the GS holoenzyme is in equilibrium with lower-order oligomers in
plants. We also cannot rule out the possibility that the lower-order
oligomers are an artifact of extraction. GS from B. subtilis
and E. coli migrates only as a dodecamer on native gels,
suggesting that there may be some differences in the mechanism by which
GS is turned over in plants compared with bacterial systems.
That oxidative modification is a marking step in the turnover of GS in
plants is further supported by the fact that treatment with hydrogen
peroxide, which would increase the production of ·OH radicals in the
root cells, showed not only a decrease in enzyme activity but also a
decrease in the GS polypeptides. Moreover, our data also showed that
there was a dramatic increase in the level of the oxidized forms of GS
subunits, suggesting that the oxidative modification may be an
intermediate step in the turnover of GS in vivo. The gene for the 1
form was expressed only in the hydrogen peroxide-treated samples. The
gene for the 1 form shows the highest homology to the
gln- gene of bean and therefore may be its ortholog
(Morey, 1997). The gln- gene of bean has been shown to be
expressed in young roots and is induced by both biotic and abiotic
stress (Watson and Cullimore, 1996). This may explain why the soybean
1 gene is expressed in young roots (Fig. 2) and induced in the
stressed roots of hydrogen peroxide-treated plants (Fig. 7).
Furthermore, in accordance with the in vitro studies (Table II)
demonstrating that the GS enzyme could be protected from MCO-mediated turnover by incubating the enzyme with either the substrates or the
product of the reaction, GS activity in vivo could be greatly increased
by feeding the plants ammonium nitrate. This enhancement was not just
at the level of transcription or transcript stability but also at the
level of polypeptide stability. Although there was induction of the
ammonium nitrate-inducible GS1 subclass (Miao et
al., 1991; Roche et al., 1993) at the level of transcription, the
increase in the level of the transcript (approximately 1.5-fold) could
not account for the 2.5-fold increase in the GS polypeptide corresponding to this gene and the more than 2-fold increase in the
overall GS activity level. We attribute this to the protection of the
GS holoenzyme from oxidative modification by the substrates of the
enzyme. Two-dimensional gel analysis of the GS1
polypeptides showed that the oxidized form of the polypeptide 2 was
2- to 3-fold lower in the ammonium nitrate-fed plants than in the
sample from plants that were not fed nitrogen. This suggests that in the presence of the substrate the active site of the enzyme is less
accessible to oxidative modification or the production of oxygen
radicals is enhanced under low-nitrogen conditions. The 1 form,
however, showed only a moderate change in the ratio between the
oxidized and nonoxidized form. It is possible that the increased synthesis of 1 polypeptides resulting from ammonium nitrate
induction of the corresponding genes was accompanied by an increased
turnover of the holoenzyme containing the 1 subunits.
The observations made in this paper may explain the results obtained by
Hoelzle et al. (1992) that feeding nitrogen to nonnodulated soybean
plants resulted in a significant increase in GS activity without
affecting the overall GS1 polypeptide
concentration. The increase in activity in the plants fed nitrogen was
probably due to a lowering in the level of oxidatively modified GS
subunits. On a similar note, we demonstrated that the nodule-specific
GS isozyme is unstable in ineffective soybean nodules, probably because of the absence of fixed nitrogen (in the form of ammonia) in the infected cells of these ineffective nodules (Temple et al., 1996). Our
findings from this study may also explain the discrepancy between GS
activity and polypeptide level in dark-stressed bean nodules (Gogorcena
et al., 1997).
The possibility that MCO modification of enzymes can be used for
selective regulation of enzyme degradation is suggested by the
demonstration that substrates of enzymes can protect them from
oxidative modifications. Such metabolite effects could account for the
differential responses of various enzyme levels to nutritional deficiencies. Thus, nitrogen starvation in bacterial cultures results
in a decrease in the intracellular level of Glu and ATP, substrates
that protect GS against oxidative modification and subsequent
degradation. In the absence of the substrate an enzyme is biologically
inactive and, therefore, its selective degradation can have little
effect on its biological functions. However, by degradation it can
yield amino acids needed for the synthesis of other proteins. In the
same context, it can be argued that in plants the GS enzyme is always
available to metabolize any ammonia that may be produced, thus avoiding
toxic buildup of ammonia. It would follow that in plants the process of
GS turnover and synthesis is a continuous process: When the substrate
is limiting, the enzyme is turned over, and when the substrate is
available, the enzyme is stabilized.
| |
FOOTNOTES |
1
This work was supported by U.S. Department of
Agriculture grant no. 9237305-7941 and by the Agricultural Experiment
Station at New Mexico State University, Las Cruces.
2
Present address: U.S. Department of
Agriculture-Agricultural Research Service, Tifton, GA 31793.
*
Corresponding author; e-mail csgopala{at}nmsu.edu; fax
1-505-646-6041.
Received November 5, 1998;
accepted December 23, 1998.
| |
ABBREVIATIONS |
Abbreviations:
GS, Gln synthetase.
MCO, metal-catalyzed
oxidation.
| |
ACKNOWLEDGMENTS |
We thank Stephen Temple and Tom Knight for critical reading of
the manuscript.
| |
LITERATURE CITED |
Aguirre J,
Hansberg W
(1986)
Oxidation of Neurospora crassa glutamine synthetase.
J Bacteriol
166:
1040-1045
[Abstract/Free Full Text]
Allen RD
(1995)
Dissection of oxidative stress tolerance using transgenic plants.
Plant Physiol
107:
1049-1050
[CrossRef][ISI][Medline]
Bennett MJ,
Lightfoot DA,
Cullimore JV
(1989)
cDNA sequence and differential expression of the gene encoding the glutamine synthetase polypeptide of Phaseolus vulgaris L.
Plant Mol Biol
12:
553-565
Bradford M
(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][ISI][Medline]
Cervera J,
Levine RL
(1988)
Modulation of the hydrophobicity of glutamine synthetase by mixed-function oxidation.
FASEB J
2:
2591-2595
[Abstract]
Climent I,
Levine RL
(1991)
Oxidation of the active site of glutamine synthetase: conversion of arginine-344 to -glutamyl semialdehyde.
Arch Biochem Biophys
289:
371-375
[Medline]
Cock JM,
Brock IW,
Watson AT,
Swarup R,
Morby AP,
Cullimore JV
(1991)
Regulation of glutamine synthetase genes in leaves of Phaseolus vulgaris.
Plant Mol Biol
17:
761-771
[CrossRef][ISI][Medline]
Cock JM,
Hermon P,
Cullimore JV
(1992)
Characterization of a gene encoding the plastid-located glutamine synthetase of Phaseolus vulgaris: regulation of -glucuronidase gene fusions in transgenic tobacco.
Plant Mol Biol
18:
1141-1149
[Medline]
Cullimore JV,
Lara M,
Lea PJ,
Miflin BJ
(1983)
Purification and properties of two forms of glutamine synthetase from the plant fraction of Phaseolus root nodules.
Planta
157:
245-253
[CrossRef][ISI]
Dalton DA
(1995)
Antioxidant defenses of plants and fungi.
In
S Ahmad,
eds, Oxidative Stress and Antioxidant Defenses in Biology.
Chapman & Hall, New York, pp 298-355
De Vries SC,
Springer J,
Wessels JGH
(1982)
Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings.
Planta
156:
120-135
del Rio LA,
Pastori GM,
Palma JM,
Sandalio LM,
Sevilla F,
Corpas FJ,
Jiménez A,
López-Huertas E,
Hernández JA
(1998)
The activated oxygen: role of peroxisomes in senescence.
Plant Physiol
116:
1195-1200
[Free Full Text]
Edwards JW,
Walker EL,
Coruzzi GM
(1990)
Cell-specific expression in transgenic plants reveals nonoverlapping roles for chloroplast and cytosolic glutamine synthetase.
Proc Natl Acad Sci USA
87:
3459-3463
[Abstract/Free Full Text]
Farber JM,
Levine RL
(1986)
Sequence of a peptide susceptible to mixed-function oxidation. Probable cation binding site in glutamine synthetase.
J Biol Chem
261:
4574-4578
[Abstract/Free Full Text]
Ferguson AR,
Sims AP
(1971)
Inactivation in vivo of glutamine synthetase and NAD-specific glutamate dehydrogenase. Its role in the regulation of glutamine synthesis in yeast.
J Gen Microbiol
69:
423-427
[Medline]
Forde BG,
Day HM,
Turton JF,
Shen W-J,
Cullimore JV,
Oliver JE
(1989)
Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.
Plant Cell
1:
391-401
[Abstract/Free Full Text]
Foyer CH,
Descourvieres P,
Kunert KJ
(1994)
Protection against oxygen radicals: an important defense mechanism studied in transgenic plants.
Plant Cell Environ
17:
507-523
[CrossRef]
Top
Abstract
Introduction