Journal of Experimental Botany, Vol. 57, No. 8, pp. 1747–1758, 2006
Oxygen Metabolism, ROS and Redox Signalling in Plants Special Issue
doi:10.1093/jxb/erj191 Advance Access publication 12 May, 2006
Antioxidative enzymes from chloroplasts, mitochondria,
and peroxisomes during leaf senescence of nodulated
pea plants
José M. Palma1,*, Ana Jiménez2, Luisa M. Sandalio1, Francisco J. Corpas1, Marianne Lundqvist2,
Manuel Gómez3, Francisca Sevilla2 and Luis A. del Rı́o1
1
Departamento de Bioquı´mica, Biologı´a Celular y Molecular de Plantas, Estación Experimental del Zaidı´n, CSIC,
Apartado 419, 18080 Granada, Spain
2
Departamento de Biologı´a del Estrés y Patologı´a Vegetal, Centro de Edafologı´a y Biologı´a Aplicada del Segura,
CSIC, Apartado 195, 30080 Murcia, Spain
3
Departamento de Agroecologı´a y Protección Vegetal, Estación Experimental del Zaidı´n, CSIC, Apartado 419,
18080 Granada, Spain
Received 24 January 2006; Accepted 13 March 2006
Abstract
In this work the influence of the nodulation of pea
(Pisum sativum L.) plants on the oxidative metabolism
of different leaf organelles from young and senescent
plants was studied. Chloroplasts, mitochondria, and
peroxisomes were purified from leaves of nitrate-fed
and Rhizobium leguminosarum-nodulated pea plants
at two developmental stages (young and senescent
plants). In these cell organelles, the activity of the
ascorbate–glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase
(MDHAR), dehydroascorbate reductase (DHAR), and
glutathione reductase (GR), and the ascorbate and
glutathione contents were determined. In addition, the
total superoxide dismutase (SOD) activity, the pattern
of mitochondrial and peroxisomal NADPH-generating
dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative
metabolism enzymes were also analysed in these
organelles. Results obtained on the metabolism of
cell organelles indicate that nodulation with Rhizobium
accelerates senescence in pea leaves. A considerable
decrease of the ascorbate content of chloroplasts,
mitochondria, and peroxisomes was found, and in
these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.
Key words: Ascorbate–glutathione cycle, chloroplasts, mitochondria, peroxisomes, root nodules, senescence.
Introduction
Leaf senescence is a genetically regulated process characterized by oxidative stress and chlorophyll and protein breakdown. During senescence, an enhancement of
reactive oxygen species (ROS) is produced along with an
increase in proteolytic activity (del Rı́o et al., 1998; Palma
et al., 2002). ROS are maintained under certain levels by
a battery of molecules with antioxidant capacity. The most
important antioxidative enzymes are catalase, peroxidases,
superoxide dismutase (SOD), and those of the ascorbate–
glutathione cycle, a series of coupled redox reactions involving four enzymes: ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate
reductase (DHAR), and glutathione reductase (GR). The
main non-enzymatic antioxidant molecules are ascorbate
and glutathione, which are also integrated in the cycle mentioned above, and a-tocopherol, b-carotene, and flavonoids
(Noctor and Foyer, 1998; Halliwell and Gutteridge, 2000).
The ascorbate–glutathione cycle is an important antioxidant protection system against H2O2 generated in different cell compartments. Its occurrence has been reported
in chloroplasts, mitochondria, peroxisomes, and cytoplasm
(Dalton et al., 1993; Jiménez et al., 1997; Noctor and
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1748 Palma et al.
Foyer, 1998; Asada, 2000). SOD is also present in these
four compartments. Chloroplasts contain basically the
CuZn-SOD and Fe-SOD isoforms, whereas in mitochondria only the Mn-SOD isozyme has been clearly localized
(Alscher et al., 2002; del Rı́o et al., 2003a). In peroxisomes, both CuZn-SOD and Mn-SOD have been found
either as soluble proteins in the organelle matrix, or bound
to membranes in the case of Mn-SOD (del Rı́o et al.,
2003a, b). Catalase is localized in peroxisomes and is the
most characteristic marker enzyme of these organelles
(Baker and Graham, 2002).
In chloroplasts, leaf senescence produces the disorientation of the grana, dilation of thylakoids, and a considerable
loss of photosynthetic pigments with a concomitant formation of plastoglobuli (Smart, 1994; del Rı́o et al.,
1998). However, mitochondria remain relatively intact
until the last stages of senescence (Quirino et al., 2000).
In peroxisomes, the photorespiratory pathway is diminished during leaf senescence whereas the glyoxylate cycle
enzymes are induced (Buchanan-Wollaston, 1997), thus
allowing the energy provision by a combined action of
the b-oxidation and glyoxylate cycle (Pistelli et al., 1996;
del Rı́o et al., 1998). In this process, leaf peroxisomes
are metabolically converted into glyoxysome-type peroxisomes (Nishimura et al., 1996), and this is accompanied
by a proliferation of the peroxisomal population (Droillard
and Paulin, 1990; Pastori et al., 1997).
In leguminous plants, nodules are able to fix atmospheric
nitrogen by the action of nitrogenase, but in the absence
of oxygen which is a strong ROS-mediated inhibitor of
this enzyme (Gage and Margolin, 2000; Santos et al.,
2000). The nodule bacteria and plant metabolism interact
in a complex process where a number of genes and signal
molecules belonging to both counterparts are involved
(Schultze and Kondorosi, 1996; Hirsch, 1999; Gage and
Margolin, 2000). Finally, nitrogen fixed in root nodules is
exported to the aerial part of the plant and used for plant
growth and development (Gage and Margolin, 2000;
Schultze and Kondorosi, 1996; Puppo et al., 2005).
Different studies have focused on the alterations of nodules during senescence, which are associated to oxidative
stress and proteolytic processes and have some similarities
to the process of leaf senescence (Pladys and Vance, 1993;
Kardailsky and Brewin, 1996; Matamoros et al., 2003;
Sheokand and Brewin, 2003; Puppo et al., 2005). Nevertheless, the influence of nodulation on leaf senescence and
its associated oxidative metabolism has not been studied
in nodulated plants at the subcellular level.
In this work, the influence of nodulation on leaf senescence was investigated in different cell compartments
of pea plants. For this purpose, a battery of antioxidative
enzymes and the non-enzymatic antioxidants ascorbate and
glutathione, distributed in chloroplasts, mitochondria, and
peroxisomes, were analysed in young and senescent leaves
from both nitrate-fed and Rhizobium-nodulated pea plants.
Materials and methods
Plant material and growth conditions
Pea seeds (Pisum sativum L., cv. Phoenix) were kindly supplied
by Dr Peter Römer from Südwestdeutsche Saatzucht (Rastatt,
Germany). Seeds were surface-sterilized with ethanol for 3 min and
were germinated in sterilized vermiculite for 14 d in controlledenvironment chambers. Healthy and vigorous seedlings were then
selected and planted in two sets of vermiculite pots. One set was
grown with nitrogen-free Hoagland’s nutrient solution (Olivares
et al., 1980) and these seedlings were inoculated with 60 ml of a
suspension of Rhizobium leguminosarum biovar. viciae (c. 106 cells
per plant) obtained from Microbio Rhizogen Corporation, UK. The
second set of plants was grown with the same solution, supplemented with 5 mM KNO3, and was not inoculated (controls). Plants
were grown in a controlled environmental chamber with a day/
night temperature cycle of 25/19 8C, 60/85% relative humidity,
600 lmol mÿ2 sÿ1 (incandescent 40 W Philips; fluorescent: Sylvania
24-F96T12VHO/CW) and 14 h photoperiod. Plants were grown for
12 weeks until an advanced fructification stage when both nodules
and leaves had fully senesced. Nine-week-old plants, which were
initiating the flowering stage, were considered as young plants.
Purification of cell organelles
All operations were performed at 0–4 8C. Chloroplasts, mitochondria,
and peroxisomes were purified by differential and density-gradient
centrifugations. For each organelle, the whole purification procedure
was carried out both in the absence and the presence of ascorbate.
Samples for the determination of ascorbate peroxidase activity were
prepared in the presence of ascorbate. Thus, extraction media of
leaves contained either 0 or 20 mM ascorbate, whereas all the other
media used in each purification procedure, including the gradient
layers, contained either 0 or 2 mM ascorbate.
Chloroplasts
For the purification of chloroplast from pea leaves, the method based
on the centrifugation in Percoll density-gradients (21–60%, p/v) was
used (Palma et al., 1986). This method results in two chloroplast
bands in the Percoll gradients with different densities but sharing
similar intactness (78–85%) (Palma et al., 1986). To remove Percoll
from the gradient samples, the chloroplast bands were diluted
five times with a medium containing 20 mM MOPS-KOH, pH 7.2,
0.3 M mannitol, 1 mM EDTA, and either 0 or 2 mM ascorbate,
and they then were centrifuged at 12 000 g for 20 min. The pellets
were resuspended in the same medium and used for further assays.
Mitochondria
Mitochondria were purified from pea leaves by a method which
involves a self-generated Percoll gradient, as described by Jiménez
et al. (1997). This method supplies mitochondrial fractions with
less than 10% cross-contamination by peroxisomes and intactness
percentages between 70–90%.
Peroxisomes
For the purification of peroxisomes from pea leaves two different
methods were used. To determine the activity of different characteristic peroxisomal enzymes (catalase, hydroxypyruvate reductase,
glyclolate oxidase, and malate synthase), organelles were purified
by sucrose density-gradient centrifugation (López-Huertas et al.,
1995). Peroxisomes purified by this method showed less than 2%
contamination by other organelles and intactness percentages of
about 90%. However, for the analysis of superoxide dismutase activity,
H2O2 content, and levels of ascorbate and glutathione, a method
based on Percoll density-gradient centrifugation was used which
Antioxidative enzymes in nodulated peas
supplies 70% intact peroxisomes (Sandalio et al., 1987). Percoll was
removed from gradient samples as indicated above for chloroplasts.
Enzyme assays
Ascorbate peroxidase (APX; EC 1.11.1.11) was determined by
monitoring the initial ascorbate oxidation by H2O2 at 290 nm
(Hossain and Asada, 1984). To test the feasibility of the assay,
20 mM p-chloromercuriphenylsulphonic acid (pCMS), a specific
inhibitor of APX, was used (Mittler and Zilinskas, 1991). Monodehydroascorbate reductase (MDHAR; 1.6.5.4) was assayed by measuring
the monodehydroascorbate-dependent NADH oxidation, and monodehydroascorbate was generated by the ascorbate/ascorbate oxidase
system (Arrigoni et al., 1981). The rate of monodehydroascorbateindependent NADH oxidation (without ascorbate and ascorbate
oxidase) was subtracted from the monodehydroascorbate-dependent
reaction.
Dehydroascorbate reductase (DHAR; EC 1.8.5.1) was determined
according to the method of Dalton et al. (1993) by following the
increase of ascorbate formation at 265 nm using N2-saturated buffer.
The reaction rate was corrected by the non-enzymatic reduction of
dehydroascorbate by glutathione (GSH). A factor of 0.98, to account
for the small contribution to the absorbance by GSSG, was also
considered. Glutathione reductase (GR; EC 1.6.4.2) was assayed
by monitoring the NADPH oxidation coupled to the reduction of
GSH (Edwards et al., 1990). The reaction rate was corrected for the
small, non-enzymatic oxidation of NADPH by GSSG. Superoxide
dismutase (SOD; EC 1.15.1.1) was determined by the ferricytochrome c reduction method using xanthine/xanthine oxidase as the
source of superoxide radicals (McCord and Fridovich, 1969).
NADP-dehydrogenases were determined by monitoring the
NADPH formation at 340 nm, but using their specific substrates,
and following basically the methods previously described for
these enzymes (Corpas et al., 1998, 1999). The substrates used
were: oxaloacetate for NADP-malate dehydrogenase (NADPMDH; EC 1.1.1.82), malate for the malic enzyme (ME; EC 1.1.140),
glucose-6-phosphate for glucose-6-phosphate dehydrogenase
(G6PDH; EC 1.1.1.49), 6-phosphogluconate for 6-phosphogluconate
dehydrogenase (6PGDH; EC 1.1.1.44), and isocitrate for the NADPisocitrate dehydrogenase (NADP-ICDH; EC 1.1.1.42). The total
NADPH rate formation in purified organelles was calculated by
the sum of their respective dehydrogenase activities, NADP-MDH,
ME, and NADP-ICDH in mitochondria and G6PDH, 6PGDH and
NADP-ICDH in peroxisomes.
Hydroxypyruvate reductase (HPR; EC 1.1.1.29) was assayed by
following the oxidation of NADH (Schwitzguébel and Siegenthaler,
1984), and glycolate oxidase (GOX; EC 1.1.3.1) was determined by
monitoring the formation of the complex glyoxylate–phenylhydrazone at 324 nm (Kerr and Groves, 1975). Malate synthase (MS;
EC 4.1.3.2) activity was analysed by measuring the absorbance at
412 nm of the mercaptide group formed by 5-59-dithiobis-(2nitrobenzoic) acid and the free acetyl-CoA generated by the enzyme
(Hock and Beevers, 1966). The determination of catalase activity was
performed by the method of Aebi (1984). Protein concentration was
determined according to Bradford (1976) using BSA as standard.
Determination of total ascorbate and glutathione in
organelles
The antioxidants ascorbate and glutathione were determined in
chloroplasts, mitochondria, and peroxisomes immediately after the
purification of these organelles by Percoll density-gradient centrifugation. All media used for these determinations were free of ascorbate
and cysteine. Basically, the methods described by Jiménez et al.
(1997) were followed.
Total ascorbate was extracted with 5% (v/v) phosphoric acid,
and the levels of reducted (ASC) and oxidized (DHA) ascorbate in
1749
organelles were determined by HPLC (Castillo and Greppin, 1988).
GSH and GSSG were extracted with 12% perchloric acid containing
2 mM bathophenanthroline disulphonic acid. Then samples were
frozen, centrifuged and derivatized (Farris and Reed, 1987), and an
internal c-Glu-Glu standard was used. HPLC analysis was performed
following the protocol described by Asensi et al. (1994).
Results
In recent studies carried out in our laboratory in pea plants,
the effect of nodulation with Rhizobium leguminosarum
on the relationship between leaf senescence and oxidative
stress was studied (Vanacker et al., 2006). In nitrate-fed
(controls) and nodulated pea plants different physiological
parameters were investigated during plant growth, and
in leaf crude extracts the level of H2O2, and the extent of
lipid peroxidation and protein oxidation were also determined. The results obtained established that plants grown
for 9 weeks were at the initial flowering stage, and after
12 weeks they were at advanced fructification and were
clearly senescent. Accordingly, in this work for comparative purposes 9-week-old plants were considered as young
plants, whereas 12-week-old plants were regarded as
senescent plants.
Chloroplasts
The activity of the ascorbate–glutathione cycle and other
organelle-specific enzymes was determined in chloroplasts,
mitochondria, and peroxisomes isolated from pea leaves
of nitrate-fed and nodulated young and senescent plants.
In choroplasts from nitrate-fed plants, APX, MDHAR
and DHAR activities notably decreased in senescent plants
compared with young plants (Fig. 1). However, in nodulated plants only DHAR was clearly affected by senescence, and, to a lower extent, APX. GR activity did
not change with time in both plant treatments. In general,
all enzyme activities were higher in nitrate-fed than in
nodulated young plants, especially APX and MDHAR.
In chloroplasts, the content of reduced ascorbate (ASC)
did not vary in nitrate-fed and nodulated plants of the same
age (Table 1). However, in both treatments a considerable
decline of ASC was observed in senescent plants. The
oxidized form of ascorbate (dehydroascorbate, DHA) was
only detected in senescent plants, and the ratio ASC/DHA
was similar in both sets of plants (Table 1). In senescent
plants the content of reduced glutathione (GSH) was
slightly higher in nodulated than in nitrate-fed plants. The
content of oxidized glutathione (GSSG) considerably decreased with time in nitrate-fed plants, but in nodulated
plants this parameter was not modified. Comparison among
treatments revealed that the GSSG content was negatively
affected by nodulation (Table 1). In all types of plants,
reduced glutathione (GSH) was the predominant form of
this non-enzymatic antioxidant. The GSH/GSSG ratio
was considerably higher in nodulated than in nitrated-fed
1750 Palma et al.
Fig. 1. Activities of the ascorbate–glutathione cycle enzymes in chloroplasts from nitrate-fed and nodulated pea plants at two different growth times.
Values are expressed as the mean 6SEM of at least three independent experiments. APX, ascorbate peroxidase; MDHAR, monodehydroascorbate
reductase; DHAR, dehydroascorbate redcutase; GR, glutathione reductase.
Table 1. Ascorbate and glutathione contents in chloroplasts from nitrate-fed and nodulated pea plants at two different growth times
Values are expressed as the mean6SEM of at least three independent experiments. ND, not detected.
DHA
ASC/DHA GSH
GSSG
GSH+GSSG
GSH/GSSG
ASC
(ng 100ÿ1 g leaves) (ng 100ÿ1 g leaves) (ng 100ÿ1 g leaves)
(lg 100ÿ1 g leaves) (lg 100ÿ1 g leaves)
Nitrate-fed
9 weeks 48.3862.25
12 weeks 7.1360.38
Nodulated
9 weeks 48.0062.25
12 weeks 6.7560.38
ND
1.7160.43
–
4.17
1.5260.40
1.4260.09
0.07560.002
0.01360.001
1.595
1.433
20.3
109.2
ND
1.7160.43
–
3.95
1.4960.07
1.8860.11
0.00460.001
0.00460.001
1.526
1.923
413.9
437.2
plants, and this ratio was strongly enhanced by senescence
in nitrate-fed plants.
Mitochondria
In mitochondria isolated from pea leaves, most of the
ascorbate–glutathione cycle enzyme activities increased
with time in both sets of plants, the most apparent response
being observed in nodulated plants (Fig. 2). Nevertheless,
APX activity diminished with senescence in the nitratefed treatment. Comparisons between both plant treatments
showed that in senescent nodulated plants MDHAR, DHAR,
and GR activities were much higher than in nitrate-fed
ones. In mitochondria from young plants the ascorbate
content increased with nodulation, but this parameter declined in senescent nodulated plants (Table 2). No DHA
could be detected in mitochondria of both nitrate-fed and
nodulated plants. GSH was the predominant form in mitochondria from the two treatments and decreased with time
in nitrate-fed plants. The GSH/GSSG ratio increased with
senescence in nitrate-fed plants but under the same conditions diminished in nodulated plants.
In purified mitochondria, the activities of NADPdependent malate dehydrogenase (NADP-MDH) and
NADP-dependent malic enzyme (NADP-ME) notably decreased in senescent leaves of both nitrate-fed and nodulated plants, while an opposite pattern was observed for
the NADP-isocitrate dehydrogenase (NADP-ICDH) which
increased in senescent plants (Fig. 3). The comparison
between both plant treatments showed that only NADPMDH activity was negatively affected by nodulation,
especially in young plants. As indicated in Fig. 3, the theoretical total amount of NADPH generated in leaf mitochondria by these three dehydrogenases did not vary significantly
as a consequence of either the plant nitrogen regime or age.
Peroxisomes
The ascorbate–glutathione cycle was also studied in peroxisomes isolated from leaves of nitrate-fed and nodulated
plants. APX and MDHAR activities were reduced in
Antioxidative enzymes in nodulated peas
1751
Fig. 2. Activities of the ascorbate–-glutathione cycle enzymes in mitochondria from nitrate-fed and nodulated pea plants at two different growth times.
Values are expressed as the mean 6SEM of at least three independent experiments. APX, ascorbate peroxidase; MDHAR, monodehydroascorbate
reductase; DHAR, dehydroascorbate redcutase; GR, glutathione reductase.
Table 2. Ascorbate and glutathione content in mitochondria from nitrate-fed and nodulated pea plants at two different growth times
Values are expressed as the mean 6SEM of, at least, three independent experiments. ND, not detected.
DHA
(ng 100ÿ1 g leaves)
GSH
(ng 100ÿ1 g leaves)
GSSG
(ng 100ÿ1 g leaves)
GSH+GSSG
(ng 100ÿ1 g leaves)
GSH/GSSG
92.5612.3
97.9621.4
ND
ND
0.8360.13
0.4560.05
0.01860.006
0.00260.001
0.848
0.452
46.1
225
235.5629.6
109.5625.1
ND
ND
0.7560.03
0.8060.08
0.01960.004
0.07360.019
0.769
0.873
39.5
11.0
ASC
(ng 100ÿ1 g leaves)
Nitrate-fed
9 weeks
12 weeks
Nodulated
9 weeks
12 weeks
senescent plants of both treatments (Fig. 4). On the contrary,
DHAR and GR increased with time in both treatments. The
effect of nodulation on these enzymes was very clear since
both DHAR and, to a lower extent, GR were reduced in
comparison with nitrate-fed plants. In peroxisomes only
the reduced ascorbate form (ASC) was detected by HPLC
(Table 3). The ASC content was higher in nitrate-fed plants
that in nodulated ones. As reported for mitochondria, no
DHA could be detected in leaf peroxisomes from both
sets of plants. No changes were observed in the reduced
form of glutathione (GSH) as a result of nodulation or
age. The GSH/GSSG ratio was enhanced at the senescence stage in both nodulated and nitrate-fed plants.
Some of the enzymes involved in the main metabolic
pathways of peroxisomes were analysed. The photorespiratory enzymes glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR) diminished in senescent plants of
both treatments (Fig. 5). Comparison between nitrate-fed
and nodulated plants showed that the activity of these
two photorespiratory enzymes was negatively affected by
nodulation. The activity of the glyoxylate cycle enzyme
malate synthase (MS) behaved in an opposite way to that
of the photorespiratory enzymes. Considerable increases
were observed in senescent plants, and this effect was more
dramatic in nodulated plants. With respect to catalase, a
characteristic enzyme of the oxidative metabolism of
peroxisomes, its activity pattern was similar to that of
GOX and HPR, with notable decreases as a result of plant
senescence and nodulation.
The activity of the NADPH-generating dehydrogenase
from leaf peroxisomes, glucose-6-phosphate dehydrogenase (G6PDH) decreased with time but not with plant
nodulation (Fig. 6). 6-Phosphogluconate dehydrogenase
(6PGDH) was not modified by senescence in both treatments, although this enzyme activity was much lower in
nodulated plants compared with the nitrate-fed ones. The
NADP-dependent isocitrate dehydrogenase activity
(NADP-ICDH) was higher in nitrate-fed plants and was
1752 Palma et al.
Fig. 3. Mitochondrial NADPH-generating enzymes from nitrate-fed and nodulated pea plants at two different growth times. Values are expressed as the
mean 6SEM of, at least three independent experiments. NADP-MDH, NADP-dependent malate dehydrogenase; NADP-EM, NADP-dependent malic
enzyme; NADP-ICDH, NADP-dependent isocitrate dehydrogenase.
Fig. 4. Activities of the ascorbate–glutathione cycle enzymes in peroxisomes from nitrate-fed and nodulated pea plants at two different growth times.
Values are expressed as the mean 6SEM of at least three independent experiments. APX, ascorbate peroxidase; MDHAR, monodehydroascorbate
reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase.
slightly lowered by senescence in both plant treatments.
As a consequence of all these changes, in peroxisomes,
the theoretical amount of NADPH generated by the endogenous NADP-dehydrogenases slightly decreased with
senescence in both treatments, and was negatively affected
by nodulation (Fig. 6).
Finally, considering the localization of superoxide dismutase isozymes in different cell compartments, the effect
of nodulation on superoxide dismutase (SOD) activity was
analysed in chloroplasts, mitochondria, and peroxisomes
purified from the two sets of pea plants. In chloroplasts,
SOD decreased both with time and with nodulation, but
the opposite pattern was observed in peroxisomes
(Table 4). In these organelles, SOD showed a considerable increase in senescent plants from both treatments,
and this activity was also enhanced by nodulation. By contrast, no significant differences were observed in the mitochondrial SOD activity of nitrate-fed and nodulated plants.
Antioxidative enzymes in nodulated peas
1753
Table 3. Ascorbate and glutathione content in peroxisomes from nitrate-fed and nodulated pea plants at two different growth times
Values are expressed as the mean6SEM of, at least, three independent experiments. ND, not detected.
Nitrate-fed
9 weeks
12 weeks
Nodulated
9 weeks
12 weeks
ASC
(ng 100ÿ1 g leaves)
DHA
(ng 100ÿ1 g leaves)
GSH
(ng 100ÿ1 g leaves)
GSSG
(ng 100ÿ1 g leaves)
GSH+GSSG
(ng 100ÿ1 g leaves)
GSH/GSSG
186.8611.6
218.6611.9
ND
ND
0.6560.08
0.7060.04
0.01260.009
0.00960.004
0.662
0.709
54.2
77.8
140.363.6
115.768.0
ND
ND
0.8260.14
0.6960.04
0.02360.008
0.01460.005
0.843
0.704
35.7
49.3
Fig. 5. Enzyme activities of photorespiration, the glyoxylate cycle, and oxidative metabolism in peroxisomes from nitrate-fed and nodulated pea plants
at two different growth. Values are expressed as the mean 6SEM of at least three independent experiments. HPR, hydroxypyruvate reductase; GOX,
glycolate oxidase; MS, malate synthase; CAT, catalase.
Discussion
The effect of soil nitrogen limitation on some foliar antioxidants distributed in chloroplasts and mitochondria from
spinach leaves was studied by Logan et al. (1999), and
there are some reports on antioxidants of legume nodule
organelles and their role in nitrogen fixation (Dalton et al.,
1993; Iturbe-Ormaetxe et al., 2001). However, as far as
is known, this is the first report on the effect of nodulation
on the relationship between leaf antioxidants and senescence in different cell compartments.
Results obtained on the metabolism of leaf chloroplasts,
mitochondria, and peroxisomes indicate that nodulation
of pea plants with Rhizobium accelerates its progression
towards senescence. Chloroplasts of mesophyll cells are
dismantled in the early phases of senescence, when degradation pathways for chlorophyll and proteins are interconnected (Inada et al., 1998; Quirino et al., 2000;
Hortensteiner and Feller, 2002). The rate of senescence
and the remobilization of leaf nitrogen are related to the
nitrogen nutrition status of the plant and the source/
sink relationships (Crafts-Brandner et al., 1998; Masclaux
et al., 2000; Hörtensteiner and Feller, 2002). Therefore,
in nodulated plants, chloroplasts gain special relevance
since in mesophyll cells of C3 plants these organelles accumulate as much as 75% of the total cell nitrogen as part
of the Rubisco and other stromal proteins (Hörtensteiner
and Feller, 2002).
Chloroplasts are one of the target organelles of ageassociated oxidative stress, and some of their antioxidant
barriers have been studied (Munné-Bosch and Alegre,
2002). Our results indicate that the loss of ascorbate in
chloroplasts from both nitrate-fed and nodulated young
plants might contribute to the age-promoted oxidative stress
response. Ascorbate is one of the main antioxidants present in chloroplasts, which can regenerate the thylakoidal
a-tocopherol and directly scavenge the excess superoxide
.
1
(Oÿ
2 ) and hydroxyl ( OH) radicals and singlet oxygen ( O2)
1754 Palma et al.
Fig. 6. Enzyme activities of NADPH-generating dehydrogenases in peroxisomes from nitrate-fed and nodulated pea plants at two different growth
times. Values are expressed as the mean 6SEM of at least three independent experiments. G6PDH, glucose-6-phosphate dehydrogenase; 6PGDH,
6-phosphogluconate dehydrogenase; NADP-ICDH, NADP-dependent isocitrate dehydrogenase.
Table 4. Superoxide dismutase (SOD) activity in chloroplasts,
mitochondria and peroxisomes from nitrate-fed and nodulated
pea plants at two different growth times
Values are expressed as the mean 6SEM of, at least, three independent
experiments.
SOD (U mgÿ1)
Chloroplasts
Nitrate-fed
9 weeks
12 weeks
Nodulated
9 weeks
12 weeks
Mitochondria
Nitrate-fed
9 weeks
12 weeks
Nodulated
9 weeks
12 weeks
Peroxisomes
Nitrate-fed
9 weeks
12 weeks
Nodulated
9 weeks
12 weeks
5.5160.77
2.4060.21
2.6460.83
1.9360.99
10.3562.80
8.1461.31
7.5460.3
11.5862.65
21.4162.00
87.7866.41
35.4463.22
73.7867.40
during senescence (Smirnoff, 2000). Our results suggest
that, in senescent plants, ascorbate cannot perhaps be used
to remove H2O2 by the choroplastic ascorbate–glutathione
cycle since all enzyme activities involved in this pathway
are diminished. Moreover, in these organelles nodulation
seems to produce a similar effect to senescence in all components of the ascorbate–glutathione cycle. In addition, the
decrease in SOD activity suggests that chloroplasts from
senescent nodulated and nitrogen-fed plants are less efficient than those from young plants to remove the Oÿ
2
radicals generated in the photosynthetic electron transport
chain. The decrease in SOD activity, together with that
observed in the enzymes of the ascorbate–glutathione
cycle, suggests that an overproduction of Oÿ
2 and H2O2
probably takes place in chloroplasts. Recently, an increase
in the H2O2 content, associated with a reduction in the ascorbate level was reported in chloroplasts from pea plants
subjected to salt stress (Gómez et al., 2004). However, the
pattern of both SOD and APX activities described by these
authors was different from that described in this work.
Glutathione distribution among the organelles was more
homogeneous than ascorbate, although in all types of
plants chloroplastic GSH+GSSG represents about 50% of
the total cell content. Mitochondria and peroxisomes
contributed in a similar proportion to the overall glutathione bulk. Contrary to what happened with ASC, total
glutathione increased with senescence in chloroplasts from
nitrate-fed and nodulated plants (Table 5). In mitochondria,
glutathione percentages only varied with time in nitratefed plants, showing a similar pattern to that reported for
glutathione in mitochondria from senescent Cucurbita
pepo leaves (Zechmann et al., 2005). The level of peroxisomal glutathione was differently affected by senescence
in the two treatments (Table 5). Thus, the contribution
of peroxisomes to the cellular glutathione pool increased in
nitrate-fed plants, whereas in nodulated plants these organelles contained less glutathione.
The values of ascorbate and glutathione contents determined in this work in cloroplasts, mitochondria, and
Antioxidative enzymes in nodulated peas
1755
Table 5. Relative ascorbate and glutathione content in chloroplasts, mitochondria and peroxisomes from nitrate-fed and nodulated
pea plants
ASC (%)
Nitrate-fed
9 weeks
12 weeks
Nodulated
9 weeks
12 weeks
GSH+GSSG (%)
Chloroplasts
Mitochondria
Peroxisomes
Chloroplasts
Mitochondria
Peroxisomes
99.4
95.5
0.2
1.3
0.4
2.9
51.4
55.2
23.7
17.4
21.3
27.3
99.4
96.8
0.3
1.6
0.3
1.7
48.6
54.9
24.5
24.9
26.9
20.1
peroxisomes differ substantially from the data obtained
for these antioxidants in crude extracts of leaves from
nitrate-fed and nodulated plants (Vanacker et al., 2006)
which are about three orders of magnitude higher. However, there are several reason to explain these differences:
(i) the relatively low purification yield of cell organelles,
as a result of their lability during the isolation procedure,
particularly in the case of peroxisomes (Palma et al.,
1986; López-Huertas et al., 1995; Jiménez et al., 1997);
(ii) the permeability of chloroplastic, mitochondrial, and
peroxisomal membranes to both ascorbate and glutathione
can produce losses to the medium of these metabolites
during their purification procedures; and (iii) the data
obtained in crude extracts, besides ascorbate and glutathione from the three cell organelles analysed in this work,
also include the contents of other cell compartments
such as the nucleus, cytoplasm, and apoplast. Therefore,
the ascorbate and glutathione data reported in this work,
although do not strictly represent real values, for the
reasons mentioned above they can be useful from a comparative viewpoint to obtain information about change
tendencies in the antioxidant levels of cell organelles from
the different plant treatments.
In root nodules, the mitochondrial ascorbate–glutathione
cycle functions as a detoxifying mechanism involved in
the protection against oxidative processes which interfere
with nitrogen fixation (Iturbe-Ormaetxe et al., 2001). It
has been also found that this pathway, together with other
antioxidative systems from root and foliar mitochondria
are up-regulated by salinity in salt-tolerant Lycopersicon
plants (Mittova et al., 2003, 2004). In the experimental
conditions in this study, the capability of scavenging hydrogen peroxide by mitochondrial APX was limited in
nodulated and senescent plants. Under these conditions, a
possible increase in H2O2 concentration might occur in
mitochondria.
In plant systems, mitochondrial MDH, ME, and ICDH
have been reported to regenerate NADPH for use in organelle metabolism (Møller and Rasmusson, 1998). Thus, the
NADPH necessary for GR activity is provided by the
NADPH-generating enzymes malate dehydrogenase, malic
enzyme, and isocitrate dehydrogenase. In pea plants, the
activity of these enzymes varied greatly with nodulation
and time. However, in the overall balance the total
NADPH generation rate did not show variations with
treatments.
In animal mitochondria, the role of senescence-associated
oxidative stress has been thoroughly reviewed (Sastre
et al., 2003; Huang and Manton, 2004). In plants, mitochondria remain intact until the last stages of senescence (Inada et al., 1998; Quirino et al., 2000), and in pea
leaves a role for the mitochondrial ascorbate–glutathione
cycle and SOD during this physiological process was
reported (Jiménez et al., 1998). These authors found that
dark-induced leaf senescence was characterized by a decrease of SOD, APX, MDHAR, DHAR, and GR mitochondrial activities as well as a reduction in the ascorbate
and glutathione pools of these organelles. However, in the
experimental conditions used here, natural senescence was
studied, which might explain the slight but not dramatic
changes that were observed in the antioxidant systems.
Peroxisomes are cell organelles bound by a single membrane and characterized by an essentially oxidative type of
metabolism (del Rı́o et al., 2003a, b). Leaf peroxisomes
house many of the photorespiratory enzymes and, during
leaf senescence, these organelles undergo a metabolic
conversion into glyoxysome-type peroxisomes (Nishimura
et al., 1996; del Rı́o et al., 1998). Glyoxysomes are also
present in cotyledons from oilseeds and contain the glyoxylate cycle enzymes. This pathway, along with the boxidation enzymes, converts lipids into carbohydrates,
useful for germination and late stages of senescence. Thus,
during leaf senescence, a decline in photorespiration and
an enhancement of the glyoxylate cycle occurs. The decrease
in peroxisomal GOX activity in N2-fixing plants compared
with nitrate-fed plants is in agreement with previous results
obtained by Frechilla et al. (1999) in crude extracts from
pea leaves. These results showed that pea leaf peroxisomes
developed senescent symptoms in nodulated and senescent
plants, as indicated by the activity pattern of the photorespiratory enzymes HPR and GOX and by the glyoxylate
cycle enzyme MS. Malate synthase is an enzyme whose
activity has been exclusively associated to glyoxysomes in
germinating oilseeds and to peroxisomes in senescent
leaves. Thus, the strongly enhanced MS activity found in
nodulated plants clearly indicates that these plants are
1756 Palma et al.
senescent. Catalase (CAT) and SOD results also confirm
these symptoms, since a decreasing CAT activity but an
enhanced SOD activity were found during both natural and
induced senescence of pea plants (Pastori and del Rı́o, 1994,
1997; Jiménez et al., 1998).
The activity pattern of peroxisomal APX, and MDHAR
on one hand, and DHAR and GR on the other, correlates
well with the pattern reported in pea leaves during induced
senescence (Jiménez et al., 1998), and also reinforces the
functional link of these enzymes: APX and MDHAR
associated with the peroxisomal membrane, and DHAR
and GR soluble in the organellar matrix (Jiménez et al.,
1997). Overall, the ascorbate–glutathione cycle seems to
be less affected by senescence than by nodulation. The
presence of the ascorbate–glutathione cycle in mitochondria and chloroplasts has led to the proposal that, in
Arabidopsis, an integrated ascorbate–glutathione antioxidant defence linked at the level of the genome may take
place (Chew et al., 2003). The contribution of peroxisomal
ascorbate and glutathione relative percentages to the total
of these antioxidants in the three cell organelles, together
with the high specific activity of the ascorbate–glutathione
cycle enzymes, give peroxisomes a special relevance in
the ascorbate–glutathione cycle of the whole cell.
Considering the relative ascorbate content in the three
organelles analysed, the level of vitamin C was reduced
in chloroplasts from senescent nitrate-fed and nodulated
plants compared with young plants, but no specific changes
due to nodulation were observed (Table 5). By contrast,
the contribution of mitochondria and peroxisomes to the
total ASC content was notably enhanced with time in the
two treatments. In nitrate-fed plants, the ascorbate level of
peroxisomes was twice that found in mitochondria, which
shows the importance of peroxisomes in the ascorbate
metabolism of the three cell organelles studied. Chloroplastic ascorbate represents about 95–99% of the total cell
ascorbate analysed, which is consistent with chloroplasts
as the main ascorbate store in leaves where this antioxidant plays an important role in photosynthesis (Noctor
and Foyer, 1998; Asada, 2000; Smirnoff, 2000).
In conclusion, considering the oxidative metabolism of
chloroplasts, mitochondria, and peroxisomes, it might be
postulated that, in these experimental conditions, 12-weekold pea plants develop senescence symptoms in leaves
at a subcellular level, which is in agreement with the
physiological and biochemical analyses previously described in whole plants and leaf crude extracts, respectively
(Vanacker et al., 2006). This time-dependent evolution is
accelerated by nodulation with Rhizobium and is basically
characterized by a decline in the leaf antioxidant capacity.
The role of ROS metabolism as a source of redox signals
in chloroplasts, mitochondria and peroxisomes (Corpas
et al., 2001; Foyer and Noctor, 2003), has a special relevance in the symbiotic system reported in this work,
and suggests that the physiological efficiency of the
plant–Rhizobium association might perhaps be improved
by enhancing the antioxidant capacity of the aerial parts of
the plant.
Acknowledgements
This work was supported by an RTN grant of the European
Commission (HPRN-CT-2000-00094). Authors are indebted to
Dr Peter Römer (Südwestdeutsche Saatzucht, Rastatt, Germany)
for the provision of pea seeds cv. Phoenix. The valuable technical
help of Ms Elena Sánchez-Romero and Mr Carmelo Ruı́z-Torres is
also acknowledged.
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