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Plant Physiol. (1998) 118: 1327-1335
Role of the Ascorbate-Glutathione Cycle of Mitochondria and
Peroxisomes in the Senescence of Pea Leaves1
Ana Jiménez2,
José A. Hernández,
Gabriela Pastori2,
Luis A. del Río, and
Francisca Sevilla*
Departamento de Nutrición y Fisiología Vegetal,
Centro de Edafología y Biología Aplicada del Segura,
Consejo Superior de Investigaciones Científicas, Apartado 195, E-30080 Murcia, Spain (A.J., J.A.H., F.S.); and Departamento de
Bioquímica, Biología Celular y Molecular de Plantas,
Estación Experimental del Zaidín, Consejo Superior de
Investigaciones Científicas, Apartado 419, E-10080 Granada,
Spain (G.P., L.A.d.R.)
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ABSTRACT |
We investigated the relationship
between H2O2 metabolism and the senescence
process using soluble fractions, mitochondria, and peroxisomes from
senescent pea (Pisum sativum L.) leaves. After 11 d
of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and
monodehydroascorbate reductase (MDHAR) activities localized in the
mitochondrial membrane, were all substantially decreased in
mitochondria. The mitochondrial ascorbate and dehydroascorbate pools
were reduced, whereas the oxidized glutathione levels were maintained.
In senescent leaves the H2O2 content in
isolated mitochondria and the NADH- and succinate-dependent production
of superoxide (O2· ) radicals by
submitochondrial particles increased significantly. However, in
peroxisomes from senescent leaves both membrane-bound APX and MDHAR
activities were reduced. In the matrix the DHAR activity was enhanced
and the GR activity remained unchanged. As a result of senescence, the
reduced and the oxidized glutathione pools were considerably increased
in peroxisomes. A large increase in the glutathione pool and DHAR
activity were also found in soluble fractions of senescent pea leaves,
together with a decrease in GR, APX, and MDHAR activities. The
differential response to senescence of the mitochondrial and
peroxisomal ascorbate-glutathione cycle suggests that mitochondria
could be affected by oxidative damage earlier than peroxisomes, which
may participate in the cellular oxidative mechanism of leaf senescence
longer than mitochondria.
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INTRODUCTION |
In plant cells, chloroplasts, mitochondria, and peroxisomes are
intracellular generators of activated oxygen species such as
H2O2, superoxide
(O2·) radicals, hydroxyl radicals (·OH),
and singlet oxygen (1O2)
(Boveris, 1984 ; Fridovich, 1986; Elstner, 1991; del Río et al.,
1992; del Río and Donaldson, 1995; López-Huertas et al., 1996, 1997).
Some of these activated oxygen species are highly reactive and, in the
absence of protective mechanisms, can produce damage to cell structure
and function (Halliwell and Gutteridge, 1989; Elstner, 1991). Under
nonstressing conditions the antioxidative defense system provides
adequate protection against activated oxygen species (Foyer and
Halliwell, 1976; Fridovich, 1986; Asada and Takahashi, 1987). SOD
catalyzes the dismutation of O2· radicals
to molecular oxygen and
H2O2, thus playing a key
role in this defense mechanism (Fridovich, 1986).
H2O2 scavenging is accomplished by catalase, various peroxidases, and the
ascorbate-glutathione cycle, a series of coupled redox reactions
involving four enzymes, APX, MDHAR, DHAR, and GR (Foyer and Halliwell,
1976; Nakano and Asada, 1981).
Susceptibility to oxidative stress depends on the overall balance
between factors that increase oxidant generation and those cellular
components that exhibit an antioxidant capability (Foyer et al., 1994).
Oxidative damage in plant tissues is especially important during
senescence and is characterized by a notable increase in the metabolism
of activated oxygen species (Kar and Feierabend, 1984; Thompson et al.,
1987; Halliwell and Gutteridge, 1989). In plants senescence symptoms
include chlorophyll and protein loss and increases in lipid
peroxidation and membrane permeability, all of which lead to a
progressive decrease in the photosynthetic capacity (Thompson et al.,
1987).
In a previous study of dark-induced senescence of pea (Pisum
sativum L.) leaves, it was proposed that peroxisomes play an activated-oxygen-mediated role in the oxidative mechanism of this type
of senescence (Pastori and del Río, 1994a). More recently it
was reported that natural senescence of pea leaves causes essentially the same changes in peroxisome-activated oxygen metabolism as dark-induced senescence (Pastori and del Río, 1997).
Little is known about the mechanisms involved in the deterioration of
mitochondrial electron transport during senescence (Thompson et al.,
1987) and the activated-oxygen-related function of mitochondria in leaf
senescence. This contrasts with animal systems, in which a role for the
generation of mitochondria-activated oxygen species in the aging
process has been suggested (Nohl, 1986; Muscari et al., 1990; Yen et
al., 1994; Herrero and Barja, 1997). The importance of mitochondria,
specifically mitochondrial proteins and DNA, as targets of
age-associated free radical attack has been postulated (Yen et al.,
1994) and is receiving much attention. A striking relationship between
mtDNA damage and glutathione oxidation during aging has also been
reported (García de la Asunción et al., 1996).
We recently demonstrated the presence of the ascorbate-glutathione
cycle in mitochondria and peroxisomes of pea leaves, and the
participation of this cycle in the control of
H2O2 concentration in both
cell organelles has been proposed (Jiménez et al., 1996, 1997).
There is considerable evidence supporting the importance of the
intracellular distribution of antioxidative enzymes in the mechanisms
of protection and cell response to oxidative stress conditions (del
Río et al., 1991; Bowler et al., 1992; Hérouart et al.,
1993; Edwards et al., 1994; Foyer et al., 1994; Mullineaux and
Creissen, 1997).
We studied the response of the ASC-GSH cycle components in mitochondria
and peroxisomes during dark-induced senescence of pea leaves with the
aim of establishing a relationship between H2O2 metabolism and the
senescence process. Using soluble fractions, mitochondria, and
peroxisomes purified from senescent pea leaves, we analyzed the level
of antioxidants and antioxidative enzymes, O2· radical production,
H2O2 concentration, and
other oxidative damage parameters.
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MATERIALS AND METHODS |
Plant Material
Pea (Pisum sativum L. cv Lincoln) seeds were obtained
from Royal Sluis (Enkhuizen, The Netherlands). Plants were grown in pots containing aerated nutrient solution in a growth chamber under
optimal conditions for 20 d, as described by Hernández et
al. (1995).
Induction of Senescence
Excised leaves (about 50-60 g fresh weight) from 15- to 17-d-old
pea plants (control) were placed in trays floating in air-saturated distilled water and incubated in permanent darkness at 28°C for 11 d (Pastori and del Río, 1994a). Leaves were then washed
and used for the different assays.
Purification of Cell Organelles
All operations were performed at 0°C to 4°C. Mitochondria and
peroxisomes were isolated from pea leaves after 0 and 11 d in darkness by differential centrifugation, and the washed
12,000g particulate pellet, enriched in mitochondria and
peroxisomes, was centrifuged in self-generated Percoll gradients (28%,
v/v) as previously described (Jiménez et al., 1997). In this
isolation procedure the 12,000g supernatant was considered
to be the soluble fraction. After the sample was centrifuged, fractions
of 1.5 mL were collected dropwise by puncturing the bottom of the tubes using a fractionator (model 185, Isco, Lincoln, NE) equipped with an
optical unit and an absorbance detector. The integrity of the outer
mitochondrial membrane was estimated from the succinate to Cyt
c oxidoreductase activity, as described by Hernández
et al. (1993). The purified mitochondria had intactness between 70% and 90%, and on the basis of the specific activity of HPR, there was a
peroxisome contamination of about 13% (Jiménez et al., 1997).
Similarly, the mitochondrial contamination of peroxisomes was less than
10%, and the intactness of the purified peroxisomes was between 65%
and 70%.
For studies of APX activity, an independent organelle-isolation
procedure was used: 20 mM sodium ascorbate was added to the extraction medium, and all other solutions contained 2 mM
ascorbate to prevent possible inactivation of APX.
Preparation of Submitochondrial Particles
To obtain submitochondrial particles the method described by
Hernández et al. (1993) was followed with some modifications. Particles were prepared by sonication of mitochondria in a disruption medium with a high salt content (20 mM
MgCl2), which produced a high percentage of
inside-out submitochondrial particles (Petit et al., 1991).
Enzyme Assays
Unless otherwise indicated, the activities of all enzymes were
assayed in organelle samples diluted 2- to 5-fold with 50 mM phosphate buffer, pH 7.8, containing 0.1% (v/v) Triton
X-100. APX (EC 1.11.1.11), MDHAR (EC 1.6.5.4), DHAR (EC 1.8.5.1), and GR (EC 1.6.4.2) were assayed as described previously (Jiménez et
al., 1997).
SOD (EC 1.15.1.1) was analyzed by the ferricytochrome c
method using xanthine/xanthine oxidase as the source of superoxide radicals (McCord and Fridovich, 1969). SOD isozymes (Cu,Zn-SOD and
Mn-SOD) were separated by PAGE on 10% gels, as described by Hernández et al. (1994).
The activities of the organelle-marker enzymes catalase (EC 1.11.1.6),
Cyt c oxidase (EC 1.9.3.1), fumarase (EC 4.2.1.2), and HPR
(EC 1.1.1.29) were assayed as described previously (Hernández et
al., 1993; Pastori and del Río, 1994a).
Determination of Total GSH and Total ASC
Total GSH and total ASC were extracted at 0°C from soluble
fractions of pea leaves and from mitochondria and peroxisomes isolated from pea leaves at 0 and 11 d of senescence using a medium without ASC and Cys. For each antioxidant, mitochondria and peroxisomes obtained from two different gradients were combined to obtain a higher
yield of purified organelles. GSH and GSSH were extracted from samples
by mixing with an equal volume of 12% (v/v) perchloric acid containing
2 mM bathophenanthroline disulfonic acid. The resulting
acid extract was frozen, thawed, and centrifuged at 12,000g
for 5 to 10 min. Derivation of the supernatant was carried out as
described previously (Farris and Reed, 1987). HPLC analysis was
conducted following the protocol described by Asensi et al. (1994).
HPLC profiles of these samples were compared with the profiles of
samples treated with the acid solution containing 40 mM
N-ethylmaleimide before its derivation (Asensi et al.,
1994).
Ascorbate was extracted from the soluble fractions and purified
mitochondria and peroxisomes by mixing the samples with an equal volume
of 10% m-phosphoric acid and incubating for 30 min. The
mixture was diluted with distilled water to give a final concentration of 2% m-phosphoric acid and centrifuged at
12,000g for 10 min. ASC and DHA levels in the supernatant
were determined by HPLC as described by Castillo and Greppin (1988).
Other Analytical Methods
The H2O2 concentration
was determined in soluble fractions, mitochondria, and peroxisomes
purified from pea leaves at 0 and 11 d of senescence by a
peroxidase-coupled assay using 4-aminoantipyrine and phenol as donor
substrates (Frew et al., 1983). Soluble fractions (50-100 µL),
intact mitochondria (50-200 µL), and peroxisomes (50-200 µL) were
added to a reaction mixture containing 25 mM phenol, 5 mM 4-aminoantipyrine, 0.1 M potassium phosphate
buffer (pH 6.9), 0.02 µM peroxidase, and 2.5 µM H2O2.
Quinone-imine formation was measured at 505 nm.
Superoxide radical generation by purified submitochondrial particles
was determined by the method of the SOD-inhibitable oxidation of
epinephrine using NADH and succinate as respiratory substrates, according to the method of Boveris (1984). The amount of
O2· radicals produced was calculated using
an  480 of 4.0 mM1 cm1 for
epinephrine (Boveris, 1984). The extent of lipid peroxidation in the
different cell compartments was estimated by determining the
concentration of substances reacting to thiobarbituric acid (Buege and
Aust, 1972). Chlorophyll and proteins were quantified as described by
Hernández et al. (1995).
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RESULTS |
In a previous study two stages in the dark-induced senescence of
detached pea leaves were established by measuring various senescence
parameters (Pastori and del Río, 1994a). Detached pea leaves
were kept in permanent darkness for 14 d, and two clear peaks of
ethylene production and O2 consumption were found
at d 3 and 11. The typical symptoms of leaf senescence developed as the
dark treatment progressed. Chlorophyll loss, lipid peroxidation, and
proteolysis increased throughout the process of dark-induced senescence, especially at d 11, when more than one-half of the chlorophyll had been lost and the formation of MDA and soluble amino
acids had increased about 2- to 3-fold (Pastori and del Río,
1994a). In the present study pea leaf organelles were isolated from
dark-induced senescent leaves at the second well-defined stage of
senescence (11 d of dark treatment), the stage at which severe changes
take place, probably leading to cell death (Pastori and del
Río, 1994a).
A comparison of Percoll-density gradients of young (control) and
dark-induced senescent leaves (11 d of dark treatment) was conducted
using marker enzymes of peroxisomes (HPR and catalase) and mitochondria
(Cyt c oxidase). In the Percoll-density gradients of young
leaves, the main peak of peroxisomes was found in fractions 4 to 8, with a maximum equilibrium density of 1.070 g
cm3 (Jiménez et al., 1997), a value
similar to that previously determined for pea leaf peroxisomes using a
different Percoll gradient (Sandalio et al., 1987). These organelles
were well separated from intact mitochondria, which banded in fractions
10 to 13 and had an equilibrium density of 1.025 g
cm3 (Jiménez et al., 1997). In
dark-induced senescent leaves the pattern of the Percoll-density
gradients showed some minor changes, with the presence of a broad peak
of peroxisomes in fractions 2 to 6, with an equilibrium density of
1.062 g cm3 (Fig.
1). In these leaves, intact mitochondria
were also detected as a broad peak that banded in fractions 8 to 13, with a maximum equilibrium density of 1.026 g
cm3 (Fig. 1). The specific activity of Cyt
c oxidase was lower than 10% in the peroxisomal peak, a
value similar to that found for control leaves, whereas the peroxisomal
contamination of mitochondria was about 15% to 18%, based on the
HPR-specific activity. This mitochondrial contamination by peroxisomes
was slightly higher than that in mitochondria from young leaves.
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| Figure 1.
Purification of mitochondria and peroxisomes from
dark-induced senescent leaves (11 d of dark incubation). Fractions of
1.5 mL were eluted with a gradient fractionator and the activity of
different marker enzymes was assayed. Enzyme activities are expressed
in micromoles per minute per milliliter, protein content as milligrams
per milliliter, and density as grams per cubic centimeter.
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The activities of the enzymes involved in the metabolism of activated
oxygen were measured in soluble fractions, mitochondria, and
peroxisomes from dark-induced senescent pea leaves. After 11 d of
senescence the activities of Mn-SOD, APX, MDHAR, DHAR, and GR had
substantially decreased by about 78%, 50%, 80%, 85%, and 65%,
respectively, in mitochondria (Table I).
These changes were parallel to a significant increase in Cyt
c oxidase-specific activity, which was 3 times higher in
senescent mitochondria than in control mitochondria (from 0.18 ± 0.012-0.50 ± 0.04 µmol min1
mg1 protein).
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Table I.
Specific activities of SOD and ascorbate-glutathione
cycle enzymes in mitochondria isolated from young and dark-induced
senescent pea leaves
Values are expressed as the means ± SE of at least
four independent experiments.
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In submitochondrial particles from young leaves
O2· radicals were generated at slightly
higher rates with NADH than with succinate as the substrate, but
differences were not statistically significant. In the presence of
rotenone the O2· production with NADH was
slightly increased (up to 11%), whereas antimycin enhanced the
O2· generation with succinate by about
25%. After 11 d of senescence both the NADH- and the
succinate-dependent production of O2·
radicals showed a significant increase of up to 2-fold in
submitochondrial particles. In these senescent conditions, rotenone and
antimycin also increased the O2· generation
rates by NADH and succinate, respectively, although the increase
produced by antimycin was higher (about 44%) than that by rotenone
(Table II).
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Table II.
Superoxide production in submitochondrial particles
of young and dark-induced senescent leaves
NADH (50 µM) and succinate (7 mM) were used
as substrates, and rotenone (1 µM) and antimycin (1 µM) were used as inhibitors for the superoxide generation
assays. Values are expressed as the means ± SE of
four different experiments.
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Parallel to all of these changes, the
H2O2 concentration in
isolated mitochondria almost doubled during senescence, although the
peroxidation of lipids in the membranes expressed as MDA production was
apparently unaffected (Table III). In
addition to all of these effects induced by leaf senescence, the
intactness of the external mitochondrial membrane was reduced, from
70% to 90% in mitochondria from young leaves to 55% to 60% in
mitochondria from senescent leaves.
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Table III.
H2O2 concentration and
lipid peroxidation in mitochondria isolated from young and dark-induced
senescent leaves
Values are expressed as the means ± SE of at least
six different experiments.
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The content of the antioxidants ascorbate and glutathione was also
measured in mitochondria after 11 d of leaf senescence, and a
large decrease in total ascorbate was found. The mitochondrial ratio of
ASC to DHA was reduced, mainly due to the loss of ASC, which was
diminished by 70% at 11 d. The GSH pool also decreased after
11 d of dark incubation, to about 60% of the level found in
mitochondria from fresh leaves, whereas the GSSH levels were maintained
and the ratio of GSH to GSSH decreased (Table
IV).
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Table IV.
Ascorbate and glutathione content in mitochondria
isolated from control and dark-induced senescent pea leaves
Antioxidants were extracted and determined as described in ``Materials and Methods''. For each antioxidant, mitochondria obtained from two
density gradients were combined and processed. Values are expressed as
the means ± SE of at least four different
experiments.
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In peroxisomes from senescent pea leaves, the activities of APX and
MDHAR were decreased up to 70% and 83%, respectively, compared with
those from young leaves; GR activity was similar in peroxisomes of
senescent and young leaves; and DHAR activity increased in peroxisomes
of senescent leaves, although not significantly (Table
V). The peroxisomal content of
H2O2 increased
significantly after 11 d of senescence. The rate of lipid
peroxidation, expressed as MDA production, an indicator of oxidative
damage, also significantly increased (about 4-fold) in peroxisomes of
dark-induced senescent leaves (Table VI).
In contrast to mitochondria, in peroxisomes from leaves at d 11 of
senescence, no decreases in the total ascorbate content was found and
no changes in the ratio of ASC to DHA took place. Moreover, in
senescent peroxisomes a large increase in the total glutathione
content, which was mainly GSSH, was observed. This fact was reflected
in a significant decrease in the ratio of GSH to GSSH in senescent
peroxisomes, although the GSH content was also enhanced.
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Table V.
Specific activities of ascorbate-glutathione cycle
enzymes in peroxisomes isolated from young and dark-induced senescent
pea leaves
Values are expressed as the means ± SE of at least
four different experiments.
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Table VI.
Lipid peroxidation, H2O2,
ascorbate, and glutathione content in peroxisomes isolated from young
and dark-induced senescent pea leaves
Antioxidants were extracted and determined as described in ``Materials and Methods''. For each antioxidant, peroxisomes obtained from two
density gradients were combined and processed. Values are expressed as
the means ± SE of at least six (MDA and
H2O2) and four (ascorbate and glutathione)
different experiments.
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Regarding the soluble fractions (12,000g supernatants) at
11 d of senescence, a significant decrease was found in APX (90%) and GR (70%) activities (Table VII),
similar to that occurring in mitochondria. MDHAR activity was slightly
decreased (15%), whereas DHAR activity showed a significant 4-fold
increase in relation to the controls. Nearly a 2-fold increase of the
MDA content in the soluble fraction was observed, and the concentration of H2O2 did not change very
much after 11 d of senescence (Table VIII). In the soluble fraction the
ascorbate content was diminished, with a greater reduction in DHA than
in ASC, which was reflected in an increased ratio of ASC to DHA in
senescent leaves. Unlike ascorbate, the GSSH content was considerably
enhanced by senescence, which was reflected by a decrease in the ratio
of GSH to GSSH. This pattern was very similar to that found in
peroxisomes of senescent leaves (Table VI).
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Table VII.
Specific activities of the SOD isozymes and
ascorbate-glutathione cycle enzymes in the soluble fraction of young
and dark-induced senescent pea leaves
Values are expressed as the means ± SE of at least
three different experiments.
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Table VIII.
Lipid peroxidation, H2O2,
ascorbate, and glutathione content in the soluble fraction of young and
dark-induced senescent pea leaves
Values are expressed as the means ± SE of at least
four different experiments.
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DISCUSSION |
We recently identified the ascorbate-glutathione cycle in
mitochondria and peroxisomes of pea leaves and proposed that this cycle
could represent an important antioxidant protection system against
H2O2 generated in both
plant cell organelles (Jiménez et al., 1997). During senescence
strong oxidative damage takes place, and in this situation the
mitochondrial and peroxisomal ascorbate-glutathione cycle could help to
inhibit any enhancement of activated oxygen species production.
Analysis of self-generated Percoll-density gradients of dark-induced
senescent leaves showed a unique and broad area corresponding to the
mitochondrial fraction, which had an equilibrium density very similar
to that of the mitochondria of young leaves. This agrees with the
results reported by Pastori and del Río (1994a, 1997) in
dark-induced and naturally senescent pea leaves.
Dark-induced senescence produced a significant reduction of Mn-SOD,
DHAR, and GR activities present in the mitochondrial matrix, as well as
APX and MDHAR activities localized in the mitochondrial membrane
(Jiménez et al., 1997). The decrease of these enzyme activities
seems to be a specific response induced by senescence in the
mitochondrial antioxidative enzymes, since Cyt c oxidase activity was significantly increased in senescent leaves.
Both ASC and DHA decreased significantly in mitochondria of
dark-induced senescent leaves, probably because of the
H2O2-mediated oxidation of
ASC and the degradation of DHA. As suggested by Foyer et al. (1994) and
Smirnoff and Palanca (1996), the ASC pool could be reduced by oxidative
stress when the capacity of regenerative systems is exceeded. The
content of GSH was also significantly decreased in pea leaf
mitochondria during senescence, which was probably due to the decrease
in GR activity that took place under the same conditions.
In dark-induced senescent leaves a significant increase in the
mitochondrial H2O2
concentration was found. Similar results have been described by Boveris
et al. (1978) in aged potato tuber submitochondrial particles, in which
O2· generation was also increased. In the
present study rotenone increased the NADH-dependent
O2· generation in submitochondrial
particles from young and senescent pea leaves. At least four NADH
dehydrogenases have been reported in plant mitochondria (Douce, 1985;
Møller, 1997). In the present study the sites where the electrons from
NADH entered the transport chain of the submitochondrial particles were
not determined. Exogenous NADH may have entered at more than one site
(Møller, 1997). Rotenone inhibits strongly but not completely the NADH
oxidation by inside-out submitochondrial particles (Petit et al., 1991)
at the complex I segment of the respiratory chain (Douce, 1985; Siedow
and Umbach, 1995).
Since there was a high proportion of inside-out submitochondrial
particles in the pea leaves in our study, rotenone should have
inhibited a considerable part of the electron transport from the
internal dehydrogenase to oxygen. Therefore, the main
O2· production we found in submitochondrial
particles with NADH and rotenone could have been derived from the
dehydrogenase region. Antimycin A, by blocking the electron flow to Cyt
c oxidase, should enhance O2·
production by ubisemiquinone (Rich and Bonner, 1978). Our results for
O2· radical production by submitochondrial
particles with succinate plus antimycin showed that in senescent
mitochondria the antimycin-inhibited region, i.e. ubiquinone-Cyt
b, is also involved in the increase of
O2· generation.
Although in submitochondrial particles from senescent leaves the
O2· generation with succinate and antimycin
was higher than with NADH and rotenone, differences were not
statistically significant. Similarly, no significant differences were
found for O2· generation with NADH and
succinate. Although some NADH-dependent O2·
production could have taken place after complex I, our results suggest
that both the flavoprotein dehydrogenase and the antimycin-inhibited regions are involved in the increased O2·
generation during senescence. These results disagree with those found
in submitochondrial particles from potato tubers (Boveris et al.,
1978). Taking into account that O2·
radicals are considered the main precursors of mitochondrial H2O2 (Boveris and Cadenas,
1982; Nohl, 1986), we are tempted to propose that the
senescence-induced increase in pea mitochondrial H2O2 might be due to higher
rates of O2· production in mitochondria
from senescent pea leaves.
However, in mitochondria from senescent leaves, the MDA content, an
indicator of lipid peroxidation, showed no significant increase. This
could be explained by the fact that MDA can be readily metabolized by
mitochondria, as was reported by Muscari et al. (1990). Conversely, in
mitochondria from senescent leaves we found a loss in the integrity of
the outer mitochondrial membrane, which has usually been considered to
be an intrinsic feature of oxidative damage in senescent and/or
stressed tissues (Thompson et al., 1987; Buchanan-Wollaston, 1997). The
loss of membrane integrity agrees with previous results from electron
microscopy studies showing that senescence of pea leaves induced a
deterioration in the mitochondrial membrane structure and a slight
disorganization in the matrix and cristae (Pastori and del Río,
1994a). A decrease in mitochondrial membrane integrity could allow the
leakage of H2O2 from the
mitochondria into the cytosol during senescence. This extrusion of
H2O2 could be favored by
the decrease of APX and MDHAR activities in mitochondrial membranes.
In the Percoll-density gradients of dark-induced senescent leaves, a
unique and broad band of peroxisomal fraction was found, which had an
equilibrium density similar to peroxisomes of young and naturally
senescent pea leaves (Pastori and del Río, 1997). In contrast
to mitochondria, ultrastructural membrane alterations in senescent
peroxisomes were not particularly intense.
In peroxisomal matrices of senescent leaves DHAR activity was slightly
enhanced, whereas the GR activity remained unchanged. A slight increase
of the total ASC pool was found in senescent leaf peroxisomes, without
any significant change in the ratio of ASC to DHA. An important
increase in the total GSH was detected, although the ratio of GSH to
GSSH was strongly decreased. These results are consistent with GR and
DHAR acting against ASC oxidation probably produced by
H2O2 and/or
O2· radicals in senescent leaf peroxisomes.
The increase of the H2O2 concentration and MDA content, an indicator of lipid peroxidation, found in peroxisomes of senescent pea leaves is in accordance with
previous results showing increases in O2·
radical production, H2O2
concentration, and lipid peroxidation in peroxisomes of dark-induced
and naturally senescent pea leaves (Pastori and del Río, 1994a,
1997). As a result, a rapid, activated-oxygen-mediated oxidation of GSH
could take place in leaf peroxisomes during senescence. The possibility
of a higher rate of GSH synthesis and/or a more efficient import of GSH
into peroxisomes from chloroplasts or cytosol should also be
considered, since the total GSH pool was considerably increased in
peroxisomes from senescent leaves. The transport of GSH and GSSH
through different cellular membranes has previously been described in
plant systems (Schneider et al., 1992; Tommasini et al., 1993; Jamai et
al., 1996).
In previous reports it was proposed that during pea leaf senescence an
enhancement of the extrusion of peroxisomal membrane-generated O2· radicals into the cytosol could occur,
along with the leakage of overproduced
H2O2 out of peroxisomes
(Pastori and del Río, 1994a, 1997). In our study the decrease
of APX and MDHAR activities, both localized in the peroxisomal
membranes, could have enhanced this potentially serious situation for
all of the cellular compartments because of the possible formation of
the strongly oxidizing ·OH radicals produced by the reaction of
H2O2 with
O2· radicals (Elstner, 1987; Halliwell and
Gutteridge, 1989). Moreover, it is very likely that the peroxisomal
NADH-dependent production of O2· radicals
is intensified by the reverse transition of leaf peroxisomes to
glyoxysomes that occurs during senescence (Landolt and Matile, 1990;
Pistelli et al., 1996; Pastori and del Río, 1997), since more
NADH would be available as a result of the induction of fatty acid
 -oxidation and the glyoxylate cycle.
O2·,
H2O2, and GSH can act as
messenger molecules in cellular signal transduction pathways and also
as factors in plant defense responses (Saran and Bors, 1989;
Hérouart et al., 1993; Levine et al., 1994; Prasad et al., 1994;
del Río et al., 1996; Karpinski et al., 1997). Mitochondria and
peroxisomes could play a role in the oxidative mechanism of senescence
in pea leaves (Pastori and del Río, 1994a, 1994b, 1997) by
favoring the leakage into the cytosol of
O2· and
H2O2, two activated-oxygen
transduction signals that could lead to the expression of specific
genes involved in leaf senescence.
Our results suggest that during senescence oxidative injuries could be
accelerated in mitochondria in relation to peroxisomes because of the
depression of the antioxidant system of mitochondria, resulting in an
enhanced H2O2 production
and membrane damage. Peroxisomes, however, may function longer than
mitochondria in the oxidative mechanism of senescence, because they are
able to respond to increased activated-oxygen production with increased synthesis of antioxidant systems that could partly counteract the
accumulation of activated oxygen species.
| |
FOOTNOTES |
1
This work was supported by the Dirección
General de Investigacion Científica y Technica, Spain (grant
nos. PB92-0492-02 and PB95-0004-02) and by the European Union (grant
no. CHRX-CT94-0605).
2
Present address: Department of Applied Genetics, John
Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
*
Corresponding author; e-mail fsevilla{at}natura.cebas.csic.es; fax
34-68-266613.
Received March 31, 1998;
accepted August 6, 1998.
| |
ABBREVIATIONS |
Abbreviations:
APX, ascorbate peroxidase.
ASC, ascorbate,
reduced form.
DHA, ascorbate, oxidized form (dehydroascorbate).
DHAR, dehydroascorbate reductase.
GR, glutathione reductase.
HPR, hydroxypyruvate reductase.
MDA, malondialdehyde.
MDHAR, monodehydroascorbate reductase.
SOD, superoxide dismutase.
| |
ACKNOWLEDGMENTS |
The authors are grateful to Prof. José Viña
(Departamento de Fisiología, Universidad de Valencia, Spain)
for his valuable help with GSH assays and to Mrs. D. Lapaz for
technical assistance.
| |
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Abstract
Introduction