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Copyright ©Physiologia Plantarum
2001
PHYSIOLOGIA PLANTARUM 112: 487–494. 2001
Printed in Ireland—all rights reser6ed ISSN
0031-9317
Response of the cultivated tomato and its wild salt-tolerant relative
Lycopersicon pennellii to salt-dependent oxidative stress: The root
antioxidative system
Abed Shalata
a
, Valentina Mittova
b
, Micha Volokita
b
, Micha Guy
b
and Moshe Tal
b,
*
a
Department of Life Sciences,Ben Gurion Uni6ersity of the Nege6,P.O.Box
653
,Beer She6a
84105
,Israel
b
The Blaustein Institute for Desert Research,Ben Gurion Uni6ersity of the Nege6,Sede Boqer Campus
84990
,Israel
*Corresponding author,e-mail
:
motal@bgumail.bgu.ac.il
Received 12 October 2000; revised 16 February 2001
(APX; EC 1.11.1.11) and decreased contents of the antioxidantsThe response of the antioxidant system to salt stress was studied
in the roots of the cultivated tomato Lycopersicon esculentum ascorbate and glutathione and their redox states. In contrast,
Mill. cv. M82 (Lem) and its wild salt-tolerant relative L. increased activities of the SOD, CAT, APX, monodehy-
pennellii (Corr.) D’Arcy accession Atico (Lpa). Roots of control droascorbate reductase (MDHAR; EC 1.6.5.4), and increased
and salt (100 mMNaCl)-stressed plants were sampled at contents of the reduced forms of ascorbate and glutathione and
their redox states were found in salt-stressed roots of Lpa, invarious times after commencement of salinization. A gradual
increase in the membrane lipid peroxidation in salt-stressed root which the level of membrane lipid peroxidation remained
unchanged. It seems that the better protection of Lpa roots fromof Lem was accompanied with decreased activities of the
antioxidant enzymes: superoxide dismutase (SOD; EC salt-induced oxidative damage results, at least partially, from
1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase the increased activity of their antioxidative system.
monodehydroascorbate reductase (MDHAR). However,
two molecules of MDHA can also disproportionate non-en-
zymatically to MDHA and dehydroascorbate (DHA), which
in turn, is reduced to ascorbate via the dehydroascorbate
(DHAR; EC 1.6.5.4) and glutathione reductase (GR; EC
1.6.4.2) route (Asada, 1994). In this route GSH is oxidized
to GSSG by the action of DHAR and GSSG is reduced
back to GSH by the action of GR.
When plants are subjected to environmental stresses oxi-
dative damage may result because the balance between the
production of AOS and their detoxification by the antioxi-
dative system is altered (Hernandez et al. 1993, 1995,
Gomez et al. 1999). The results of numerous studies suggest
that the alleviation of such oxidative damage and increased
resistance to environmental stresses, including salt stress, is
often correlated with a more efficient antioxidative system
(Spychalla and Desborough 1990, Cakmak and Marschner
Introduction
Active oxygen species (AOS) can cause oxidative damage to
many cellular components including membrane lipids,
proteins, and nucleic acids (Halliwell and Gutteridge 1989).
In plants, both enzymatic and non-enzymatic processes par-
ticipate in AOS detoxification. Low molecular mass antioxi-
dants, either hydrophilic such as ascorbic acid (ASC) and
reduced glutathione (GSH) or liphophilic such as h-toco-
pherol and carotenoids, can quench all kinds of AOS (Halli-
well and Gutteridge 1989). Several enzymes are involved in
the detoxification of AOS. Superoxide dismutase (SOD)
converts superoxide to H
2
O
2
. Hydrogen peroxide is scav-
enged by catalase (CAT) and different classes of peroxidases
(Bowler et al. 1992). Ascorbate peroxidase (APX) plays a
key role in the ascorbate-glutathione cycle by reducing H
2
O
2
to water at the expense of oxidizing ascorbate to monodehy-
droascorbate (MDHA) (Asada 1994, Foyer et al. 1994). In
turn, MDHA is reduced to ascorbate by the action of
Abbre6iations – AOS, active oxygen species; APX, ascorbate peroxidase; ASC, ascorbate; CAT, catalase; DHA, dehydroascorbate; DHAR,
dehydroascorbate reductase; GSH, reduced glutathione; GR, glutathione reductase; GSSG, oxidized glutathione; Lem, Lycopersicon
esculentum cv. M82; Lpa, Lycopersicon pennellii acc. Atico; MDA, malondialdehyde; MDHA, monodehydroascorbate; MDHAR, monode-
hydroascorbate reductase; SOD, superoxide dismutase.
Physiol. Plant. 112, 2001 487
1992, Smirnoff 1993, Walker and McKersie 1993, Gossett
et al. 1994, Hernandez et al. 1994, Prasad et al. 1994,
Iturbe-Ormaetxe et al. 1998, Shalata and Tal 1998). The
analyses in these experiments were performed mainly in
leaves or calli derived from them. However, relative to the
leaf where chloroplasts are believed to be the major site of
AOS production (Asada 1996), only scarce information is
available on the root, which is usually the first organ
directly exposed to the salt stress. The salt-dependent in-
crease in activities of mitochondrial Mn-SOD (Kayupova
and Klyshev 1984) and APX (Lopez et al. 1996) was re-
ported in roots of pea and radish, respectively. Meneguzzo
et al. (1999) found that the activities of APX and MDHAR
increased in a salt-tolerant wheat cultivar and decreased in
a salt-sensitive cultivar, suggesting that these enzymes have
a role in salt tolerance in wheat plants. Differences between
the responses of citrus leaves and roots to salinity were
reported by Gueta-Dahan et al. (1997). The activities of
cytosotlic Cu/Zn-SOD and total APX showed a marked
increase in the leaves as compared with minor changes in
the root. In contrast, the activities of phospholipid hy-
droperoxide glutathione peroxidase (EC 1.11.1.9) and cyto-
solic APX isoform increased greatly in the root while
minute changes were found in the leaf (Gueta-Dahan et al.
1997).
In a previous study, Shalata and Tal (1998) suggested
that, as compared with the cultivated tomato (Lem), the
better protection of the leaf of the wild salt-tolerant tomato
species Lpa (Tal and Shannon 1983, Taha et al. 2000) from
salt-depended oxidative stress is because, at least partially,
of the higher inherited activities of SOD and APX and to
higher induced activities of SOD, APX, MDHAR, and
CAT. Here we provide evidence that, similarly to the leaf,
the root of Lpa is also better protected from salt-depended
oxidative stress, which results from the higher efficiency of
their antioxidative system.
Materials and methods
Plant material and growth conditions
Plants of the cultivated tomato, Lycopersicon esculentm
Mill. cv. M82 (Lem), and its wild salt-tolerant (Tal and
Shannon 1983, Taha et al. 2000) relative species, L.pennel-
lii (Corr.) D’Arcy accession Atico (Lpa), were grown in
aerated Hoagland solution in a greenhouse with summer
day/night temperatures of 30/20°C and light of about 1000
mmol m
−2
s
−1
at noon, 8 plants per container of 6 l. Salt
treatment started at the stage of about 4 true leaves and
NaCl concentration was increased by increments of 25 mM
per day until a final concentration of 100 mMwas reached.
Salt-stressed plants were subjected to 100 mMNaCl for 20
days after completing the salt addition. Roots were sam-
pled at various times.
Enzyme extraction and assays
The lower half of the roots was cut, rinsed for 5 min in
cold 0.5 mMCaSO
4
solution and frozen in liquid nitrogen.
The frozen roots were kept at −80°C for further analyses.
Enzymes were extracted from1gofroots using a mortar
and pestle with 5 ml of extraction buffer containing 50 mM
potassium-phosphate buffer (pH 7.8), 0.1 mMEDTA, 1%
(w/v) PVP, 0.1 mMPMSF, 5 mMsodium ascorbate and
0.2% (v/v) Triton X-100. All operations were carried out at
4°C. SOD was determined by monitoring the inhibition of
photochemical reduction of nitro blue tetrazolium accord-
ing to Beyer and Fridovich (1987); APX according to
Jimenez et al. (1997) and corrections were made for low,
non-enzymatic oxidation of ascorbate by H
2
O
2
. MDHAR
was determined by monitoring the decrease in A
340
as a
result of NADH oxidation (MDHA was generated by the
ascorbate/ascorbate oxidase system) (Arrigoni et al. 1981).
GR was determined following Madamanchi and Alscher
(1991), and CAT according to Rao et al. (1996).
Lipid peroxidation
Lipid peroxidation was determined in purified organelles by
measuring the amount of malondialdehyde (MDA, m=155
mM
−1
cm
−1
), a product of lipid peroxidation, by the
thiobarbituric reaction, according to Draper and Hadley
(1990).
Determination of reduced (ASC) and oxidized (DHA)
ascorbate
ASC and DHA were assayed according to Law et al.
(1983). This assay is based on the reduction of Fe
3+
to
Fe
2+
by ASC in acidic solution. The reduced Fe
2+
forms
a pink complex with bipyridil, absorbing at 525 nm. Total
ascorbate (ASC+DHA) was determined by the reduction
of DHA to ASC using dithiothreitol. Aliquots were divided
into equal parts for the determination of total ascorbate
and ASC contents. DHA content was then calculated from
the difference between total ascorbate and ASC.
Determination of reduced (GSH) and oxidized (GSSG)
glutathione
The methods used for analysis of reduced and total glu-
tathione, employed the specificity of GR, as described by
Anderson et al. (1992).
Results
The time course of membrane lipid peroxidation in the
roots of the two tomato species, measured as the content of
MDA, is given in Fig. 1. In Lem roots growing under
normal growth conditions, a small ‘age-dependent’increase
in lipid peroxidation level became apparent after 2 weeks;
however, under salt stress conditions a gradual and large
increase in its lipid peroxidation level was found (Fig. 1A).
In contrast, no change in lipid peroxidation was observed
in either control or salt-stressed roots of Lpa, except for an
indication for a small transient increase during 3 days at
the beginning of salinization (Fig. 1B).
Physiol. Plant. 112, 2001488
In both species, the activities of SOD (Fig. 2), APX (Fig.
3), CAT (Fig. 4), MDHAR (Fig. 5) and GR (Fig. 6)
remained unchanged or slightly decreased with time under
normal growth conditions. In roots of salt-stressed Lem
plants, the activities of two (GR and MDHAR) of these
enzymes remained unchanged and three (SOD, APX and
CAT) of them slightly decreased with time in comparison to
control conditions. In contrast, in the salt-stressed Lpa the
activities of SOD (which was inherently higher in Lpa),
APX, CAT and MDHAR increased with time relative to
control plants, while only that of GR slightly decreased. The
activities of the first 4 enzymes reached a maximum 16 days
after the beginning of salinization and then decreased.
Under normal growth conditions, the contents of the
reduced (ASC) and oxidized (DHA) forms of ascorbate were
fairly constant in roots of both species (Fig. 7). Under salt
stress conditions, however, a continuous decrease (starting 6
days after the initiation of salt stress) in ASC content, and
an abrupt increase in DHA content (4 days after the initia-
tion of salt stress) were detected in Lem. In contrast, in
roots of salt-stressed Lpa, ASC content rapidly increased
(reaching a maximum 16 days after initiation of salinization)
while that of DHA remained unchanged. Consequently, the
ascorbate redox state [ASC/(ASC+DHA)] was decreased
by salinity in Lem and remained unchanged in Lpa (Fig. 7).
Fig. 2. Time course of SOD activity in roots of the cultivated
tomato (Lem) and the wild species (Lpa) under normal and saline
conditions. Additional details as in Fig. 1.
Fig. 1. Time course of lipid peroxidation (represented by MDA) in
roots of the cultivated tomato (Lem) and the wild species (Lpa)
under normal and saline conditions. (A) Lem; (B) Lpa. Open
symbols –control plants, closed symbols –salt-stressed plants.
Values indicate average9
SD
of 12 measurements from two plants
in each of two independent experiments. Time represents days after
initiation (day 0) of salt addition. Plants were subjected to 100 mM
NaCl for 20 days after completing the NaCl addition. The first
samples were taken 12 h after initiation of salinization.
Fig. 3. Time course of APX activity in roots of the cultivated
tomato (Lem) and the wild species (Lpa) under normal and saline
conditions. Additional details as in Fig. 1.
Physiol. Plant. 112, 2001 489
Fig. 4. Time course of CAT activity in roots of the cultivated
tomato (Lem) and the wild species (Lpa) under normal and saline
conditions. Additional details as in Fig. 1.
Fig. 5. Time course of MDHAR activity in roots of the cultivated
tomato (Lem) and the wild species (Lpa) under normal and saline
conditions. Additional details as in Fig. 1.
Fig. 6. Time course of GR activity in roots of the cultivated tomato
(Lem) and the wild species (Lpa) under normal and saline condi-
tions. Additional details as in Fig. 1.
Similarly to ascorbate it was found that: (1) the contents
of the reduced (GSH) and oxidized (GSSG) forms of glu-
tathione were fairly stable with time under the control
conditions (Fig. 8) in both species; (2) GSH content decreased
and that of GSSG increased in the root of Lem, and GSH
increased in Lpa under salt stress; (3) the glutathione redox
state [GSH/(GSH+GSSG)] was decreased by salinity in Lem
and increased in Lpa. Unlike DHA content, which remained
unchanged in Lpa under salinity, GSSG content decreased in
the salinized Lpa roots.
Discussion
Membrane lipid peroxidation is an indicator for an oxidative
damage resulting in the loss of membrane integrity (Smirnoff
1993). The increase of lipid peroxidation in the root of Lem
and its suppression in Lpa under salt stress (Fig. 1) suggest
that, similar to the leaf (Shalata and Tal 1998), the latter is
better protected than the former against salt-dependent oxi-
dative stress. This conclusion is supported by the finding that
H
2
O
2
level increased twofold in salt-stressed Lem but not in
Lpa roots in which H
2
O
2
level was not affected by the salt
treatment (data not shown). The development of salt-depen-
dent oxidative stress in Lem roots probably results from the
lack of salt-dependent up-regulation of its antioxidative
system. The finding that Lpa can up-regulate its antioxidative
system by increasing the activities of SOD, APX, CAT and
MDHAR and of ascorbate and glutathione redox states in
response to salt stress, further supports the above conclusion.
Physiol. Plant. 112, 2001490
Fig. 7. Time course of contents of reduced (ASC) and oxidized (DHA) forms of ascorbate and the ascorbate redox state [ASC/(ASC +
DHA) ratio] in roots of the cultivated tomato (Lem) and the wild species (Lpa) under normal and saline conditions. Open symbols –control
plants, closed symbols –salt-stressed plants. Values indicate average 9
SD
of 12 measurements from two plants in each of two independent
experiments. Time represents days after initiation (day 0) of salt addition. The first samples were taken 12 h after initiation of salinization.
Ascorbate is a major antioxidant in photosynthetic and
non-photosynthetic tissues which reacts directly with AOS,
recycles h-tocopherol and protects enzymes with prosthetic
transition metal ions (Bartoli et al. 2000), and is utilized as
a substrate for APX which catalyses H
2
O
2
detoxification
(Asada 1994, Foyer et al. 1994). As a result of AOS scav-
enging, ASC is oxidized into MDHA, which is sponta-
neously disproportionate to DHA (Bielski 1982). Reduced
ascorbate is regenerated from either MDHA (through the
Mehler-peroxidase cycle (Miyake and Asada 1994) or by the
NADPH-dependent enzyme MDHAR (Buettner and Ju-
rkiewicz 1996)) or DHA (through the ascorbate-glutathione
cycle (Asada 1994, Foyer et al. 1994)). The gradual decrease
of ASC content (Fig. 7) in the salt-stressed Lem, which was
accompanied by a significant increase in DHA and a mea-
surable decrease in MDHAR activity (Fig. 5A), indicates
Physiol. Plant. 112, 2001 491
that the regeneration of ASC from DHA or MDHA under
these conditions is insufficient. It is possible that part of this
failure in Lem is a result of the lack of change in the
activities of DHAR (Mittova et al. unpublished data) and
GR (Fig. 6A). ASC is regenerated either non-enzymatically
by electron transport chain or enzymatically (using MD-
HAR) from MDHA, or from DHA by the ascorbate-glu-
tathione cycle (Asada 1994, Foyer et al. 1994). It was
suggested that, unlike in cotton (Gossett et al. 1994) and pea
(Jimenez et al. 1997), MDHAR plays an important role in
the regeneration of ASC from MDHA in the tomato leaf
(Shalata and Tal 1998, Mittova et al. 2000). The findings
that in the root of salt-stressed Lpa MDHAR activity
increased (Fig. 5B) while DHAR activity failed to change
Fig. 8. Time course of contents of reduced (GSH) and oxidized (GSSG) forms of glutathione and the glutathione redox state [GSH/(GSH+
GSSG) ratio] in roots of the cultivated tomato (Lem) and the wild species (Lpa) under normal and saline conditions. Additional details as
in Fig. 7.
Physiol. Plant. 112, 2001492
(unpublished data), and the activity of GR even decreased
(Fig. 6B) suggest that, similar to the leaf, MDHAR plays a
major role in the regeneration of ASC also in the root.
Increased MDHAR activity and total ASC content in re-
sponse to variety of stresses, which are believed to induce
oxidative stress, have been documented in conifer needles
(Mehlhorn et al. 1986), leaves of wheat (Mishra et al. 1993)
and spinach (Schoner and Krause 1990), and submerged rice
seedlings (Ushimaru et al. 1992).
GSH is involved in ASC regeneration and functions also
as a direct antioxidant of AOS (Noctor and Foyer 1998). It
is also a major regulator of protein thiol-disulfide redox
status and is an important factor in the thiol-disulfide
exchange reactions (Rennenberg 1982, Zhao and Blumwald
1998). Moreover, GSH plays a protective role by increasing
stress tolerance, in particular that of salinity (De Kok and
Oosterhuis 1983) and seems to be an important signal
molecule by acting as a direct link between environmental
stress and key adaptive responses (Rennenberg and Brunold
1994, May et al. 1998, Wingate et al. 1998). The regenera-
tion of GSH from GSSG is mediated by the activity of GR
(Noctor et al. 1998). The increase in GSSG content in
salt-stressed Lem root, apparently at the expense of de-
creased GSH content, suggests that GR activity rate-limits
the regeneration of GSH in this root under salt stress. This
finding supports the suggestion that the combined rates of
ASC regeneration via the DHAR/GR and the MDHAR
routes, in salt-stressed Lem roots, are slower than the rate of
the spontaneous MDHA disproportionation to DHA. The
observation that GSH content increased in salt-stressed Lpa
roots while GR activity, which regenerates reduced glu-
tathione, decreased with time, can be explained by either an
enhanced GSH synthesis and/or its transport to the roots,
or as a result of its decreased consumption by processes such
as degradation, oxidation or chelation (May et al. 1998,
Noctor et al. 1998). The increased GSH content in the
salt-stressed Lpa may reflect, at least partially, its increased
demand as a substrate by enzymes participating in the
detoxification of membrane lipid peroxidation such as glu-
tathione S-transferase (Marrs 1996) and phospholipid hy-
droperoxide glutathione peroxidase (Gueta-Dahan et al.
1997). Another intriguing possibility could be that the in-
creased glutathione redox state in the root cells of salt-
stressed Lpa (Fig. 8) may serve as a signal affecting the
expression of defense genes (Foyer et al. 1997). This possi-
bility may explain the ability of Lpa to up-regulate its
antioxidative genes and the failure of Lem to do so under
salt stress conditions.
The up-regulation of the activities and levels of the an-
tioxidants in response to salt treatment in Lpa roots is
characterized by a rather slow increase of the activities of
SOD, CAT, APX and the levels of ASC and GSH, which
reached their maximum only about 2 weeks after the com-
pletion of salt addition. Foyer et al. (1994) suggested that
the absence of rapid increase in the level of transcripts of the
antioxidant enzymes is related to the role of AOS in signal
transduction, which would be most effective if the oxygen
radical scavenging systems were not drastically increased as
an immediate response to oxidative stress. Whether and how
the slow increase of the antioxidative response is related to
the role of AOS in signal transduction in the salt-stressed
Lpa remains an open question.
Acknowledgements –The first two authors contributed equally.
V.M. is a Jacob Blaustein fellowship incumbent.
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Edited by C. H. Foyer
Physiol. Plant. 112, 2001494
