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Plant, Cell and Environment (2000) 23, 853–862

Tolerance of pea (Pisum sativum L.) to
long-term salt stress is associated with induction of
antioxidant defences
J. A. HERNÁNDEZ,1 A. JIMÉNEZ,1 P. MULLINEAUX 2 & F. SEVILLA1
1

Departamento de Nutrición y Fisiología Vegetal, Centro de Edafología y Biología Aplicada del Segura, CSIC, Apartado
4195, E-30100 Murcia, Spain, and 2John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

ABSTRACT
Using two cultivars of Pisum sativum L. with different sensitivity to NaCl, the effect of long-term (15 d) NaCl (70 mM)
treatments on the activity and expression of the foliar
ascorbate–glutathione cycle enzymes, superoxide dismutase isozymes and their mRNAs was evaluated and related
to their ascorbate and glutathione contents. High-speed
supernatant (soluble) fractions, enriched for cytosolic
components of the antioxidant system, were used. In
this fraction from the NaCl-tolerant variety (cv Granada),
the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), Mn-superoxide dismutase (Mn-SOD) and
dehydroascorbate reductase (DHAR) increased, while
CuZn-SOD activity remained constant. In the NaClsensitive plants (cv Challis), salinity did not produce significant changes in APX, MDHAR and GR activities. Only
DHAR activity was induced in cv Challis, whereas soluble
CuZn-SOD activity decreased by about 35%. Total ascorbate and glutathione contents decreased in both cultivars,
but the decline was greater in NaCl-sensitive plants. This
difference between the two cultivars was more pronounced
when the transcript levels of some these enzymes were
examined. Transcript levels for mitochondrial Mn-SOD,
chloroplastic CuZn-SOD and phospholipid hydroperoxide
glutathione peroxidase (PHGPX), cytosolic GR and APX
were strongly induced in the NaCl-tolerant variety but not
in the NaCl-sensitive variety. These data strongly suggest
that induction of antioxidant defences is at least one component of the tolerance mechanism of peas to long-term
salt-stress.
Key-words: Pisum sativum; antioxidant enzymes; ascorbate–glutathione cycle; gene expression; oxidative stress;
salt stress.

INTRODUCTION
One of the most important abiotic factors limiting plant
productivity is water stress brought about by drought or
Correspondence: Dr Francisca Sevilla. Fax: +34 968 396213;
e-mail: fsevilla@natura.cebas.csic.es

© 2000 Blackwell Science Ltd

salinity. Salt stress results in alterations in plant metabolism
including reduced water potential, ion imbalance and toxicity, and reduction of CO2 assimilation (Bohnert & Jensen
1996). The effects of various environmental stresses in
plants are known to be mediated, at least in part, by an
enhanced generation of activated oxygen species (AOS;
O2·-, H2O2 and ·OH) (Hernández et al. 1993, 1995; Mittler
& Zilinskas 1994; Alscher, Donahue & Cramer 1997;
Noctor & Foyer 1998). Although a wide range of genetic
adaptations to saline conditions has been observed and a
number of significant physiological responses have been
associated with tolerance, the underlying mechanisms
of salt-tolerance in plants are still poorly understood.
However, one determinant of salt tolerance could be how
well stressed plants deal with the accompanying oxidative damage to subcellular compartments (Hernández
et al. 1993, 1995; López et al. 1996; Van Camp et al. 1996;
Gueta-Dahan et al. 1997).
To mitigate and repair damage initiated by AOS, plants
have developed a complex antioxidant system. The primary
components of this system include carotenoids, ascorbate,
glutathione and tocopherols, and enzymes such as
superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC
1·11·1.6), glutathione peroxidase (GPX, EC 1.11.1.9), peroxidases and the enzymes involved in the ascorbate–
glutathione cycle (ASC–GSH cycle; Foyer & Halliwell
1976): ascorbate peroxidase (APX, EC 1.11.1.1), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and
glutathione reductase (GR, EC 1.6.4.2) (Noctor & Foyer
1998). Many components of this antioxidant defence system
can be found in different subcellular compartments
(Jiménez et al. 1997, 1998).
Several environmental stresses induce the expression
and/or levels of antioxidative enzymes and their mRNAs
(Edwards et al. 1994; Mittler & Zilinskas 1994; Stevens,
Creissen & Mullineaux 1997). For example, the protein
level of phospholipid hydroperoxide glutathione peroxidase (PHGPX) was shown to be induced in salt-adapted
Citrus sinensis callus and also in leaves from plants grown
on media containing 0·2 m NaCl (Holland et al. 1993; GuetaDahan et al. 1997). However, little is known about the effect
of salt stress on the enzymes of the ASC–GSH cycle in
853

854 J. A. Hernández et al.
plants, and there is not enough information about the
expression of the different SOD isozymes in plants grown
under salt-stress situations. In previous studies with pea
(Pisum sativum L.), we demonstrated that the metabolism
of chloroplasts and mitochondria under NaCl stress
favoured the formation of O2·- radicals and H2O2 in two
cultivars of differing NaCl sensitivity, and that tolerant
plants, but not sensitive ones, responded to NaCl stress with
increased mitochondrial Mn-SOD and chloroplastic CuZnSOD and ascorbate peroxidase activities (Hernández et al.
1993, 1995). The effects of salt stress on the cytosol compartment of pea leaf cells have not been studied, and yet it
is this compartment that might be the most important in the
plant’s response to salt-stress-associated oxidative stress,
such as has been described for the response to paraquat and
to SO2 exposure (Alscher, Donahue & Cramer 1997).
In this paper, we report a more detailed study of the
effect of long-term salt stress on NaCl-tolerant and NaClsensitive pea cultivars. We report here that there are clear
differences between the salt-responsive genotypes in terms
of the activity of antioxidant enzymes and low molecular
weight antioxidants in cytosol-enriched fractions and the
levels of their mRNAs, which might indicate a significant
role for antioxidant defences in conferring NaCl tolerance
in pea plants.

MATERIALS AND METHODS
Growth of plants in salt-stress conditions
Pea (Pisum sativum L.) seeds were surface-sterilized
(ethanol (96% v/v) for 3 min and sodium hypochlorite
(10% v/v) for 5 min), germinated and grown in vermiculite.
Vigorous seedlings were selected for hydroponic culture in
a growth chamber (ASL, Madrid, Spain). First, plants were
cultivated in aerated distilled water for 7 d (Hernández
et al. 1993). Then, plants were transplanted to aerated
optimum nutrient solution for another 7 d. The growth
chamber was set at 24/18 °C, 80% relative humidity and
200 mmol m-2 s-1 of light intensity with a 16 h photoperiod.
After 7 d, 70 mm NaCl were added to the nutrient solution
and plant leaves were sampled at 0, 2, 4, 8 and 24 h and 15 d
after the NaCl was added.

Enzyme extraction and assays
All operations were performed at 0–4 °C. For total extracts,
leaves (1 g) were homogenized with a mortar and pestle in
2 mL of ice-cold 50 mm K-phosphate buffer pH 7·8, 0·1 mm
EDTA containing 5 mm cysteine, 1% w/v PVP, 0·1 mm
PMSF and 0·2% v/v Triton X-100. For APX activity,
20 mm ascorbate was added. The homogenate was centrifuged at 14 000 g for 20 min and the supernatant fraction
was filtered through a column containing 1 mL of Sephadex G-50 equilibrated with the same buffer used for the
homogenization.
Soluble fractions were prepared by homogenizing 2 g
fresh leaf material with a mortar and pestle with 8 mL of a

grinding medium containing 0·35 m mannitol, 30 mm MOPS
buffer (pH 7·5), 4 mm l-cysteine, 1 mm EDTA and 0·2% w/v
BSA (Hernández et al. 1995). For APX activity, 20 mm
ascorbate was added. The homogenate was filtered through
two layers of cheesecloth and centrifuged at 2200 g for 30 s
to pellet the chloroplast fraction. The supernatant was centrifuged at 12 000 g to discard mitochondria and peroxisomes. Then, the 12 000 g supernatant was centrifuged for
20 min at 82 000 g. The supernatant obtained was partially
purified in columns containing Sephadex G-50 equilibrated
with the same buffer used for homogenization, and was
considered as the soluble fraction and used for various
assays.
Marker enzymes were used to measure the contamination of the soluble fractions on both control and NaCltreated plants. CuZn-SOD II activity, estimated by PAGE,
according to Hernández et al. (1999), and glucose-6phosphate dehydrogenase (G6PDH), assayed as described
by Löhr & Waller (1974), were used as chloroplastic
markers. Phosphoenolpyruvate carboxylase (PEPC) was
taken as the cytosolic marker (Winter et al. 1982). Mitochondrial contamination was analysed as the contribution
due to cytochrome c oxidase and Mn-SOD, whereas catalase activity was used as the peroxisomal marker (Jiménez
et al. 1997). Triose phosphate isomerase (TPI) activity,
present in chloroplasts (40%) and cytosol (60%) (Scharrenberger et al. 1985) was also assayed (Feierabend 1975).
APX, DHAR, MDHAR, GR and SOD were assayed
according to Jiménez et al. (1997). The specific activity
values for SOD, APX, DHAR, MDHAR and GR in soluble
fractions, of each cultivar under control and NaCl-stress
conditions, were corrected for cross-contamination using
marker enzymes. These corrections were performed using
the marker activity giving the highest contribution in the
82 000 g supernatant fractions, which under our experimental conditions was due to the chloroplastic CuZnSOD II activity (see below). Therefore, this would give the
most conservative valuations for activities in the supernatant fractions. Protein was estimated according to
Bradford (1976).Ascorbate and dehydroascorbate, reduced
and oxidized glutathione were determined by HPLC as previously described (Jiménez et al. 1997).
For the separation of SOD isozymes, non-denaturing
PAGE was performed on 10% w/v acrylamide gels using a
BioRad mini-protean II dual slab cell (Hernández et al.
1999).

Stomatal conductance measurements
Stomatal conductance was determined on fully expanded
intact leaves with a portable porometer (model Licor 1600,
Li-Cor Inc., Lincoln, Nebraska, USA), using six plants in
each experiment.

RNA extraction and Northern blots
Leaves were ground in liquid nitrogen and RNA preparation was performed by a phenol/LiCl procedure according
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862

Salt tolerance and antioxidant defences in pea 855
to Creissen & Mullineaux (1995). Poly(A+) mRNA was
isolated from total RNA by oligo(dT) cellulose affinity
chromatography (Sambrook, Fricsch & Maniatis 1989). Pea
leaves (30 g) from both cultivars grown in the absence
(control) and in the presence of 70 mm NaCl were used.
Poly(A+) mRNA (up to 5 mg) was separated on 1·5% (w/v)
agarose gels after formaldehyde/formamide denaturation
in MOPS buffer, and transferred to nylon membranes
(Boehringer Mannheim) according to Sambrook et al.
(1989). Membranes were pre-hybridized and hybridized at
65 °C in a 0·9 m NaCl, 10% dextran sulphate and 1% SDS
solution containing 50 mg mL-1 salmon sperm DNA. Prehybridization was carried out for at least 6 h and hybridization was carried out overnight. 32P-labelled DNA probes
were prepared by the method of Feinberg & Vogelstein
(1983). Membranes were washed once in 2¥ SSC, 0·1% SDS
at room temperature for 15 min and twice in 1¥ SSC, 1%
SDS at 65 °C for 15 min. Filters were autoradiographed
using Fuji X-ray film with an intensifying screen at –80 °C.
Signal intensities were estimated from autoradiograms
using a Shimadzu CS-9000 densitometer.
The cDNA probes used were those encoding mitochondrial Mn-SOD (Wong-Vega, Burke & Allen 1991), cytosolic APX (Santos et al. 1996), cytosolic GR (Stevens et al.
1997) and PHGPX (Mullineaux et al. 1998) from pea leaves,
stromatic APX from spinach (Ishikawa et al. 1995), and
cDNA probes encoding cytosolic and chloroplastic CuZnSOD from Pinus sylvestris were obtained from Dr S.
Karpinski (Karpinski et al. 1992).

RESULTS
In previous work, by using different growth parameters
(fresh and dry weight of leaves and leaf area) of plants in
70 mm NaCl-containing grown medium, two pea cultivars
were designated as NaCl-tolerant (cv Granada) and NaClsensitive (cv Challis), respectively (Hernández et al. 1993,
1995). These parameters have now been supplemented with
stomatal conductance data which confirm the designation
of NaCl-tolerant and NaCl-sensitive genotypes. After 15 d
of NaCl treatment, stomatal conductance was reduced
by 13% in the NaCl-tolerant cultivar compared with a
decrease of approximately 88% in the sensitive cultivar
(Fig. 1).
The short-term response to NaCl treatment (up to 24 h
exposure) of the total activities of antioxidant enzymes
and the concentrations and reduced/oxidized ratios of
glutathione and ascorbate, in foliar cell-free extracts or
in the soluble fraction, revealed no statistically significant
responses or differences between the NaCl-tolerant and
NaCl-sensitive cultivars (data not shown). In contrast, longterm (15 d) exposure to 70 mm NaCl did reveal responses
when measured in total leaf extracts (data not shown), and
more so when determined from soluble fractions (Tables 3
and 4).
Cross-contamination of the soluble fraction by other subcellular organelles was estimated, using marker enzymes,
in both control and NaCl-treated plants. When pea leaves
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862

Figure 1. Leaf stomatal conductance (mmol m–2 s–1) of Pisum
sativum plants after a growth period of 15 d in nutrient solutions
containing 0 or 70 mm NaCl. Each histogram is the mean of six
measurements (± SE). Differences from control values were
significant at P < 0·001 (c) according to the Duncan’s multiple
range test.

were subjected to differential centrifugation, the activity of
the marker enzymes indicated a relatively clean separation
of cytosolic fraction (82 000 g supernatant) from organelle
fractions (chloroplasts, mitochondria, and peroxisomes).
In general, the percentage contamination values were quite
similar in both treatments (control and NaCl-treated
plants) (Tables 1 and 2). In NaCl-tolerant plants, the percentage contaminations of the soluble fractions (82 000 g
supernatant) by the chloroplastic CuZn-SOD II were 28
and 21·4% (Tables 1 and 2) in control and NaCl-treated
plants, respectively. In NaCl-sensitive plants, CuZn-SOD II
contamination values increased slightly in soluble fractions
of NaCl-stressed plants in comparison to their values in
control plants (23·75 and 27·7%, respectively) (Tables 1 and
2). This different pattern agrees with the chloroplast
integrity results previously found in both cultivars under
NaCl-stress conditions (Hernández et al. 1995).
Cytosolic PEPC showed up to 200% recovered activity
in the 82 000 g supernatant fractions from control plants,
showing a good correlation with the corresponding fraction
(Table 1). Similar PEPC recovery values were obtained on
NaCl-treated tolerant plants, and these activity values were
slightly lower in NaCl-treated sensitive plants (Table 2).
G6PDH and TPI activities were found at varying degrees
in the 82 000 g supernatant from control plants, up to
23–30% and 68–80%, respectively (Table 1). In NaCltreated plants, the activity values of both enzymes did not
show significant changes in relation to control plants, being
28–30% and 68–78%, respectively (Table 2). It has been
reported that TPI is located in the chloroplasts (about 40%)
and in the cytosol (about 60%) (Scharrenberger et al. 1985),
whereas the bulk of G6PDH has been identified in chloroplasts (80%), and is also present in the cytosol (10%) and
peroxisomes (10%) (Corpas et al. 1998).

856 J. A. Hernández et al.
Table 1. Recovery of marker enzymes in the soluble fraction from control pea leaves
Recovery of total enzyme activity (%)
Fraction

Catalase

Cytochrome c oxidase

TPI

G6PDH

PEPC

MnSOD

CuZn-SOD II

Pisum sativum cv Granada
Crude extract
82 000 g supernatant

100
37·7

100
3·3

100
80·38

100
23·49

100
208

100
17·19

100
28·02

Pisum sativum cv Challis
Crude extract
82 000 g supernatant

100
28·2

100
1·61

100
73·24

100
25·01

100
190

100
14·9

100
23·75

The 82 000 g supernatant was obtained by centrifugation of crude leaf homogenates and partially purified as described in Materials and
methods. Values are means from two independent experiments. Total enzyme activities in the crude extract (nmol min-1) for cv Granada
were: catalase 1·36; cytochrome c oxidase 1·54; TPI, 1064·9; G6PDH, 1494·2; PEPC, 530·2; SOD activity as total units: MnSOD, 237·9; CuZnSOD II, 282·26. Total enzyme activities in the crude extract (nmol min-1) for cv Challis were: catalase 1·70; cytochrome c oxidase 1·74; TPI,
1369·5; G6PDH, 1383·4; PEPC, 660·0; SOD activity as total units: MnSOD, 252·2; CuZn-SOD II, 376·9.

In the 82 000 g supernatant fraction from control plants,
about 15–17% of the mitochondrial and peroxisomal MnSOD activity was found, whereas only 1·6–3·5% of the
cytochrome c oxidase was associated with the 82 000 g
supernatant, and values of about 28–37% for catalase activity were observed (Table 1). Similar results were obtained
in the tolerant NaCl-treated plants (Table 2). However, in
the sensitive NaCl-treated plants, slightly higher recovery
values for catalase and cytochrome c oxidase were obtained
in relation to control plants (Table 2).
Except for catalase activity, for most enzymes, the percentage of activity recovered in the 82 000 g supernatant
was between 13 and 30%. In pea leaves, most of the
ASC–GSH cycle enzymes were found in the chloroplasts
and cytosol (Foyer & Halliwell 1976), whereas in mitochondria and peroxisomes their activities only represents
2·5–3·5% of the total leaf activity (Edwards et al.
1990; Jiménez et al. 1997). According to all these data,
in both control and NaCl-treated plants, the specific
activity values found for all the antioxidant enzymes
in the 82 000 g supernatant were corrected by taking
into account the higher percentage of contamination by

organellar enzymes, which under our experimental conditions was due to the chloroplastic CuZn-SOD II. Its
activity was calculated, in each case, by PAGE of the corresponding soluble fraction and after recording the activity
on the gels (see Materials and methods).
In the 82 000 g supernatant fraction from plants of the
tolerant genotype, NaCl treatment caused an increase in
the activities of the ASC–GSH cycle enzymes (20–35% for
MDHAR, GR and APX and twofold for DHAR; Table 3).
CuZn-SOD I activity did not show any significant change
with salt stress (Table 3). In the 82 000 g supernatant fraction from NaCl-sensitive plants, no changes in the specific
activities of APX, MDHAR and GR were observed,
whereas DHAR increased by 50%. However, in this subcellular fraction from the sensitive genotype, the soluble
CuZn-SOD I had decreased by about 35% of the values
observed in control plants.
A decrease of 50% in the total ascorbate pool of the
soluble fraction was found in the sensitive genotype
exposed to 15 d NaCl stress. This was due to the loss of both
oxidized and reduced forms, but the changes were such that
there was a slight lowering of the ASC/DHA ratio in these

Table 2. Recovery of marker enzymes in the soluble fraction from NaCl-treated pea leaves
Recovery of total enzyme activity (%)
Fraction

Catalase

Cytochrome c oxidase

TPI

G6PDH

PEPC

MnSOD

CuZn-SOD II

Pisum sativum cv Granada
Crude extract
82 000 g supernatant

100
34·3

100
2·01

100
78·53

100
28·68

100
195

100
18·61

100
21·4

Pisum sativum cv Challis
Crude extract
82 000 g supernatant

100
35·7

100
3·24

100
68·2

100
30·1

100
180

100
12·5

100
27·71

The 82 000 g supernatant was obtained by centrifugation of crude leaf homogenates and partially purified as described in Materials and
methods. Values are means from two independent experiments. Total enzyme activities in the crude extract (nmol min-1) for cv Granada
were: catalase 2·09; cytochrome c oxidase 1·55; TPI, 1799·3; G6PDH, 1251·7; PEPC, 484·0; SOD activity as total units: MnSOD, 230·6; CuZnSOD II, 362·7. Total enzyme activities in the crude extract (nmol min-1) for cv Challis were: catalase 1·88; cytochrome c oxidase 1·94; TPI,
1001; G6PDH, 1063; PEPC, 475·0; SOD activity as total units: MnSOD, 229·9; CuZn-SOD II, 193·9.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862

Salt tolerance and antioxidant defences in pea 857
Table 3. Specific activity of antioxidant enzymes in soluble fractions from control and NaCl-treated plants after 15 d of salt stress
CuZn-SOD I

APX

MDHAR

DHAR

GR

Cultivar and treatment

(U mg–1 protein)

(nmol min–1 mg–1 protein)

NaCl-tolerant
Control
70 mm NaCl

9·17 ± 0·94
11·22 ± 0·84

313·3 ± 10·3
420·9 ± 6·4c

49·9 ± 1·2
60·1 ± 0·6c

2·14 ± 0·17
4·29 ± 0·25c

40·81 ± 2·03
50·77 ± 1·9a

NaCl-sensitive
Control
70 mm NaCl

10·70 ± 0·42
6·88 ± 0·47a

264·6 ± 13·8
227·8 ± 13·0

54·9 ± 2·9
52·6 ± 3·3

2·55 ± 0·24
3·85 ± 0·18b

49·48 ± 2·24
46·05 ± 1·47

The activity values were corrected for cross-contamination. Data are the mean ± standard error of at least three different experiments.
Differences from control values were significant at: aP < 0·05; bP < 0·01; cP < 0·001 according to Duncan’s multiple range test.

plants. In NaCl-tolerant plants, both ASC and DHA levels
also fell, but less so and there was no change in the
ASC/DHA ratio (Table 4).
In the soluble fraction of NaCl-treated sensitive plants,
the glutathione pool decreased by 60% compared to the
level in the controls (Table 4). This fall was due to the
decline of both oxidized (GSSG) and reduced (GSH) forms
of glutathione. In the tolerant cultivar, the soluble GSSG
content fell to almost zero after salt treatment, although
more than 50% of the initial soluble GSH remained
(Table 4). Thus, in contrast to ascorbate, there were few
differences observed in the salt-induced changes in glutathione concentrations between sensitive and tolerant
genotypes.
Northern blot analysis of the steady-state levels of
mRNAs encoding the above antioxidant enzymes showed
that tolerant plants responded to long-term NaCl treatment
by increasing mitochondrial Mn-SOD (threefold), cytosolic
APX (threefold), cytosolic GR (fivefold) and chloroplastic
CuZn-SOD II (fivefold) transcript levels (Fig. 2). The levels
of some mRNAs that were screened did not respond to
salt stress in either cultivar, for example stromatic APX and
cytosolic CuZn-SOD (Fig. 2) and chloroplastic GR
(data not shown). In the case of cytosolic CuZn-SOD, its
transcript level even decreased in the NaCl-treated genotype (Fig. 2). Similarly, PHGPX mRNA levels increased

considerably (threefold) in the leaves of tolerant plants but
not in sensitive plants (Fig. 2).

DISCUSSION
The study reported here was an extension of previous
studies aimed at understanding the relationship between
tolerance to salt stress and oxidative stress. A first approach
is to observe which component enzymes of the antioxidant
defences of the plant are regulated in their expression. The
importance of components of the antioxidant defences can
be further assessed using the approach of comparing cultivars differentially responsive to long-term NaCl stress.
The effect of salinity on stomatal conductance has
been shown to be much more dramatic in NaCl-sensitive
than in NaCl-tolerant plants. The data for the NaClsensitive genotype (Fig. 1) are in agreement with stomatal
conductance data from salt-stressed spinach and bean
plants (Robinson, Dowton & Millhouse 1983; Brugnoli &
Lauteri 1991). The increase in AOS production observed in
chloroplasts from NaCl-sensitive plants in relation to
chloroplasts from salt-tolerant ones (Hernández et al. 1995)
may result from stomatal closure causing a decrease in the
CO2 concentration inside the chloroplasts. This, in turn,
might cause a decrease in the concentration of NADP+
available to accept electrons from photosystems I/II and

Table 4. Ascorbate and glutathione concentration in soluble fractions from control and NaCl-treated plants after 15 d of salt stress
ASC

DHA

GSH

(mg g–1 FW)

Cultivar and treatment

GSSG
(nmol g–1 FW)

NaCl-tolerant
Control
70 mm NaCl

159·7 ± 1·6
107·7 ± 3·7b

10·6 ± 0·1
6·9 ± 0·2b

17·5 ± 2·0
9·2 ± 1·1a

0·05 ± 0·003
Not detected

NaCl-sensitive
Control
70 mm NaCl

201·0 ± 2·6
98·1 ± 3·3b

11·7 ± 0·1
5·8 ± 0·2b

20·2 ± 2·7
8·6 ± 0·4a

0·14 ± 0·01
0·022 ± 0·002b

Data are the mean ± standard error of at least three different experiments. Differences from control values were significant at aP < 0·05,
b
P < 0·01 according to Duncan’s multiple range test.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862

858 J. A. Hernández et al.

Figure 2. Northern blot hybridization analysis of mRNA levels
of some antioxidant scavenging enzymes in poly(A+) RNA.
Poly(A+) RNA (5 mg per lane) was isolated from leaves from
pea plants (cv Granada and cv Challis) grown in the absence (C)
or in the presence of 70 mm NaCl (S) for 15 d. The numerical
data above each lane represent the percentage of enzymatic
activity in the 82 000 g supernatant fractions in leaves of the two
cultivars with and without salt treatment (see Table 3). For the
specific activity of mitochondrial Mn-SOD, chloroplastic CuZnSOD and stromatic APX, see Hernández et al. (1993, 1995).
These values are shown here to enable comparison of enzyme
activities with transcript levels for the same enzyme.

thus initiate O2 reduction with the concomitant generation
of AOS (Halliwell 1982).
Data on cytosolic components of the antioxidant
enzymes obtained through differential centrifugation
show considerable agreement (De Gara, de Pinto &
Arrigoni 1997; Gardeström & Edwards 1983; Madamanchi
et al. 1992).
Because most of the antioxidative enzymes measured in
this work (SOD and ASC–GSH cycle enzymes) are located
mainly in the chloroplasts and the cytosol (Foyer &
Halliwell 1976; Madamanchi et al. 1992), our major concern
was the chloroplastic contamination in the 82 000 g supernatant fractions. For that reason, we used, for the activity
corrections, the higher percentage of contamination found,
which, under our experimental conditions was due, as
mentioned above, to the chloroplastic CuZn-SOD II. This
enzyme is a good marker since its localization has been
clearly demonstrated (Palma, Sandalio & del Río 1986;
Hernández et al. 1995, 1999). Results on chloroplastic
CuZn-SOD II activity indicated that the degree of chloroplast contamination in the 82 000 g supernatant fractions
ranged from 21 to 28%. It is important to note that in pea
(cv Challis and cv Granada) chloroplasts, only CuZnSOD II is present, and no Fe-SOD was found (Hernández
et al. 1995). These results contrast with those we have
described in a different pea cultivar (P. sativum cv Puget)
in which two different SOD activity bands, Fe-SOD and
CuZn-SOD II, representing 12–15% and 85–88%, respectively, of the total SOD activity, were identified
(Hernández et al. 1999). In general, the amount of Fe-SOD
activity in higher plants and more specifically in chloroplasts, is very small, compared to that of CuZn-SOD which
is the predominant isozyme (Salin & Bridges 1982; Kurepa
et al. 1997).
Little CuZn-SOD activity has been found in thylakoid
membranes (Ogawa et al. 1985) as the bulk of CuZn-SOD
is localized in the stroma. Thus, we assume that the relative
abundance of chloroplastic CuZn-SOD II is a good marker
for estimating chloroplast breakage.
G6PDH has been extensively used for a long time as a
cytosolic marker (Federico & Angelini 1986; Polle et al.
1990; Ros Barceló 1998; Vanacker et al. 1998a,b). In opposition to the use of G6PDH as a cytosolic marker, a recent
paper by Corpas et al. (1998) reported the presence of
different G6PDH isozymes in pea leaves using different
techniques (differential centrifugations, native PAGE,
SDS–PAGE, Western blot analysis and electron microscopy
immunocytochemistry), and they demonstrated that, at
least in pea leaves, the enzyme is located mainly in chloroplasts (80%), although it is also present in cytosol (10%)
and peroxisomes (10%). So, this enzyme could be useful to
compare the data with those measured using chloroplastic
CuZn-SOD II and to estimate the chloroplast contamination in the soluble fractions. In fact, a percentage of G6PDH
in the soluble fraction greater than 10% could be due
mainly to the major chloroplastic isozyme. On the other
hand, the data for chloroplastic contamination in soluble
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862

Salt tolerance and antioxidant defences in pea 859
fractions calculated according to G6PDH activity were
lower than those obtained using the chloroplastic marker
CuZn-SOD II in both control and salt-treated plants.
Less than 3% of the cytochrome c oxidase activity was in
the 82 000 g supernatant preparations, indicating a low
degree of contamination by mitochondrial membrane
fractions. Cross-contamination by the mitochondrial and
peroxisomal Mn-SOD activity was higher (about 18%).
However, taken together, this suggests a low breakage
of mitochondria. Cross-contamination by peroxisomal
catalase was higher than that by mitochondrial enzymes
(Tables 1 and 2). However, since in pea leaf peroxisomes as
in mitochondria there is only a small amount (2·5–3·5%) of
the ASC–GSH cycle enzymes (Edwards et al. 1990; Jiménez
et al. 1997), the cross-contamination of the 82 000 g supernatant by peroxisomal components cannot be accounted
for the antioxidative enzyme activities detected in it, since
their highest contribution to soluble activities would be
about 1·1%.
Values of about 200% recovered activity of the cytosolic
PEPC were found in the 82 000 g supernatant fractions
after differential centrifugation (Tables 1 and 2), indicating
a high degree of correlation between the supernatant fractions and the cytosolic compartment (Winter et al. 1982;
Stitt et al. 1989; Schinkel, Streller & Winsgle 1998). Taken
together, these results show that the purity of the 82 000 g
supernatant fractions is about 70–80%, which we consider
sufficient and more suitable than crude extracts to make a
meaningful studies of the soluble antioxidant enzymes.
There are other reports in which similar supernatant fractions were taken as the cytosolic fraction (Blinda et al.
1997).
In the soluble fractions from the leaves of NaCl-tolerant
pea plants, long-term exposure (15 d) to NaCl produced
a significant increase in all of the antioxidant enzyme
activities, whereas in salt-stressed sensitive plants, only
DHAR activity increased, while SOD activity significantly
decreased (Table 3). These data should be contrasted with
total leaf extracts which do not show a difference in
response to salt stress between the two genotypes. Thus, the
cytosolic compartment may be important in antioxidant
responses to NaCl. The antioxidant enzymes present in this
compartment may contribute to the protection of other
subcellular compartments in a way similar to the response
of cytosol antioxidant defences to photoinhibitory
light stress in Arabidopsis (Karpinski et al. 1997). Furthermore, in both of these pea cultivars, NaCl stress causes
an increase in O2·- generation in mitochondria together
with an increase in the H2O2 contents in chloroplasts
(Hernández et al. 1993, 1995), and an increased leakage of
H2O2 into the cytosol from mitochondria and peroxisomes
was also suggested (Hernández et al. 1993; Corpas et al.
1993). Thus, the cytosolic compartment may have to scavenge AOS from all other subcellular compartments under
certain stress conditions. This could explain the increase in
antioxidant enzyme activities in the soluble fractions in the
NaCl-tolerant genotype.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862

The up-regulation of DHAR activity in both NaClsensitive and NaCl-tolerant genotypes rather than
MDHAR activity (Table 3), suggests that, in pea subjected
to these stress conditions, ASC is regenerated via glutathione. This is in contrast to the situation described by
Morán et al. (1994) who proposed that MDHAR was the
key detoxifying enzyme under drought stress conditions. It
may be that DHAR activity could participate in ASC
regeneration under conditions of severe stress when
MDHAR activity is limited by the availability of NADH
(Asada & Takahashi 1987). Our data support this view.
In tobacco plants, salinity led to a two- to threefold
increase in the activity of Fe-SOD, cytosolic and chloroplastic Cu-Zn-SOD, APX, DHAR and GR activities (Van
Camp et al. 1996). The increase in cytosolic DHAR activity
has also been observed in sorghum and sunflower plants
grown under drought conditions (Zhang & Kirkham 1996).
It was suggested that the increase in DHAR activity is
induced when cellular ascorbate contents are significantly
decreased (Arrigoni 1994). This increase in DHAR activity
may be required to sustain cycling of oxidized ascorbate
when the flux through the ASC–GSH cycle is increased,
as may occur in transgenic poplar plants with enhanced
expression of Fe-SOD (Arisi et al. 1998). It should be noted
that, neither in the tolerant nor in the sensitive plants did
DHA accumulation take place (Table 4), which could have
been due to the induction of soluble DHAR activity under
salt-stress conditions (Table 3). In NaCl-tolerant plants, the
increase in APX activity, which could contribute to ascorbate oxidation, was paralleled by an increased capacity for
ascorbate regeneration via MDHAR, DHAR and GR
activities (Table 3). Nevertheless, the total soluble ascorbate pool decreased and this was mainly due to the loss of
the reduced form. In NaCl-stressed plants, total chloroplastic ascorbate contents may be higher than in control
plants (Hernández et al. 1995; Meneguzzo et al. 1998). This
could suggest that an enhanced import by chloroplasts of
ascorbate from the cytosol could be induced under salt
stress conditions which could explain the loss of ascorbate
in those soluble fractions.
Like ascorbate, the glutathione content of the soluble
fraction also decreased in NaCl-treated plants from both
cultivars (Table 4). This was despite the increase in GR
activity in NaCl-tolerant plants and the maintenance of this
activity in sensitive ones. The loss of GSH was not attributable to its oxidation to GSSG in spite of the increase in
DHAR activity in both cultivars. The decline in glutathione
contents may be wholly or partly due to depressed rates
of GSH synthesis, increased rates of degradation and/or
GSH transport to other cell compartments or plant organs
(Schneider, Martini & Rennenberg 1992; Jamai et al. 1996;
Herschbach et al. 1998). Glutathione can be synthesized in
both the chloroplastic and cytosolic compartments (Noctor
et al. 1998), but its degradation may be confined to the
cytosol, and under some stress conditions oxidation of GSH
is accompanied by net glutathione degradation (Noctor &
Foyer 1998).Therefore, the loss of glutathione in the soluble

860 J. A. Hernández et al.
fraction (Table 3) may be a consequence of export to other
locations in the cell or the plant.
The increased export of H2O2 from mitochondria,
chloroplasts and peroxisomes to the cytosol under saltstress conditions (Hernández et al. 1993, 1995; Corpas et al.
1993), could also be responsible for the decreased GSH
levels observed in the soluble fractions from both pea
cultivars.
Salt stress is known to result in extensive lipid peroxidation (Hernández et al. 1995; Gosset et al. 1996), and therefore GSH could be used for the conjugation of toxic
membrane lipid peroxidation products, in reactions catalysed by glutathione-S-transferase (GST; Pickett & Lu 1989;
Marrs 1996). This could have contributed to the observed
decrease in glutathione contents in salt-stressed plants of
both genotypes (Table 4). The recent report that salt tolerance in transgenic tobacco seedlings can be enhanced by
over-expressing a GST cDNA adds support to this suggestion (Roxas et al. 1997).
Interestingly, only in the NaCl-tolerant variety was there
a concerted increase in the levels of transcript that encode for some of the enzymes of antioxidant metabolism
(Fig. 2). Collectively, these data and those concerning the
increase in activities of the enzymes and the changes in
antioxidants of the soluble fraction (Tables 3 and 4), point
to an important role for the cytosol antioxidant defences as
one determinant of salt tolerance in peas. However, stressmediated changes in the abundance of a particular transcript do not always correlate with corresponding changes
in antioxidant protein levels and/or enzyme activities
(Edwards et al. 1994; Mittler & Zilinskas 1994; Donahue
et al. 1997; Mullineaux & Creissen 1997; Stevens et al. 1997).
In keeping with this, in the long-term experiments, we
observed that the NaCl-mediated increases in mitochondrial Mn-SOD, cytosolic CuZn-SOD, cytosolic APX,
cytosolic GR and chloroplastic CuZn-SOD mRNA levels
were much greater (Fig. 2) than the increases in the activities of their corresponding enzymes (Table 3). It has been
suggested that changes in the levels of particular isoforms
of such enzymes rather than changes in their total activity
may be more important (Edwards et al. 1994; Stevens et al.
1997).
The PHGPX mRNA level was also significantly induced,
but only in the NaCl-tolerant variety (Fig. 2). This could
explain the low level of lipid peroxidation previously
described in chloroplasts from salt-tolerant plants
(Hernández et al. 1995), since these enzymes reduce lipid
hydroperoxides to their corresponding alcohol using GSH
as the electron donor (Ursini et al. 1995), and this class of
pea PHGPX has been shown to be located in the chloroplast stroma (Mullineaux et al. 1998). PHGPX mRNA
increases in tissues of several plant species undergoing
stress, including salt stress in suspension cells, heavy metal
poisoning, and infection by viral or bacterial pathogens
(Holland et al. 1993; Criqui et al. 1992; Levine et al. 1994;
Gueta-Dahan et al. 1997). Thus, the increase in the level of
PHGPX mRNA in several species subjected to a range
of stresses suggests that common responses occur at the

cellular level, most likely mediated by oxidative damage to
macromolecules.
The increase in transcript levels shown in Fig. 2 could be
due to their de novo synthesis or to their decreased degradation. mRNA stability may be an important factor in regulation of the response to salt stress, as shown in pea plants
subjected to drought and chilling stress (Mittler & Zilinskas
1994; Stevens et al. 1997). Regulation of mRNA stability is
an important point of control of expression of genes in stress
responses (Medhy & Brodl 1998). In pea plants recovering
from drought, although very high levels of APX transcripts
were present in the cell, these transcripts did not associate
with the polysome fraction and therefore did not participate
in protein synthesis (Mittler & Zilinskas 1994). Furthermore, in many plants, abiotic stress can affect translational
processes. For example, in carrot protoplasts, heat shock disrupts the function of both 5¢ cap and 3¢ poly(A) tail structures, and in animals it has been shown that heat shock
affects the initiation of translation (Medhy & Brodl 1998).
In the NaCl-sensitive cultivar, levels of mitochondrial
MnSOD and cytosolic CuZn-SOD transcript were not
affected by salt stress, but activities of mitochondrial
MnSOD (Hernández et al. 1993) and cytosolic CuZn-SOD
decreased by 35%, suggesting an inhibition of these
enzymes by NaCl as previously described in mesophyll
protoplasts (Hernández, del Río & Sevilla 1994).
Stromal APX mRNAs remained at constant levels in both
pea cultivars. In previous work, we have reported that in isolated chloroplasts from tolerant plants NaCl stress produced an increase in APX activity (Hernández et al. 1995).
Similar results were described for cytosolic APX from
radish plants, suggesting that post-translational processes
appear to play an important role in APX expression (López
et al. 1996), although it should be noted that cytosolic APX
in pea did respond to salt stress in the tolerant cultivar
(Fig. 2). These differences between increases or not in
enzyme activities and mRNA levels again emphasize that
control of the expression of plant antioxidant defences is
complex and operates at a several levels.

ACKNOWLEDGEMENTS
This work was supported by grant PB95-0004 from the
DGES (Spain). We would like to acknowledge the Department of Microbiology and Genetics of the University of
Murcia for the facilities to carry out Northern blot analysis. P.M. acknowledges the support of the Biotechnology
and Biological Sciences Research Council through the
Core Strategic Grant to the John Innes Centre. P.M. and F.S.
acknowledge financial support from the British Council,
and J.A.H. acknowledges financial support from The Royal
Society.

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Received 11 February 2000; received in revised form 10 March 2000;
accepted for publication 10 March 2000

© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 853–862


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