Journal of Horticultural Science & Biotechnology (2004) 79 (4) 560564

Oxidative metabolism in tomato plants subjected to heat stress

By R. M. RIVERO, J. M. RUIZ and L. ROMERO*
Department of Plant Biology, Faculty of Science, University of Granada, E-18071, Granada, Spain
(e-mail: lromero@ugr.es)
(Accepted 23 February 2004)
SUMMARY
Tomato plants (Lycopersicon esculentum L. cultivar Tmknvf2) were grown for 30 d at two temperatures (25C, optimal
temperature, and 35C) with the objective of determining the effect of heat stress on oxidative metabolism. The leaf
concentrations of the antioxidant compounds ascorbate (AsA), dehydroascorbate (DHA), reduced glutathione
(GSH), oxidized glutathione (GSSG) and the activities of the enzymes superoxide dismutase (SOD), catalase (CAT),
guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione
reductase (GR), as well as total hydrogen peroxide (H2O2) concentration and shoot dry weight, were determined. High
temperature stress caused: (1) decreased shoot weight, (2) accumulation of H2O2, (3) increased SOD activity,
(4) decreased activities of CAT, GPX, APX, DHAR, GR (associated with detoxifying H2O2), and (5) increased levels
of the antioxidant compounds AsA, DHA, GSSG, and GSH. In addition, our data demonstrated that heat stress
occurred in tomato plants at 35C, and this temperature may have initially inhibited the ascorbate/glutathione cycle
and then provoked an oxidative burst, indicated by foliar H2O2 accumulation.

any crops are cultivated in areas where climatic

conditions are not always ideal and where
temperatures may periodically fall far below, or rise
substantially above optimum levels. Such conditions can
trigger oxidative metabolism in plants, brought on by an
overproduction of active oxygen species (AOS) such as
superoxide radicals (O2), hydroxyl (OH), singlet
oxygen (1O2), and hydrogen peroxide (H2O2) (Elstner
and Oswald, 1994; Prasad et al., 1994; Queiroz et al.,
1998). The AOS are highly reactive and can damage
proteins, chlorophylls, membrane lipids and nucleic acids
ultimately, upsetting the homeostasis of the organism
(Shaaltiel and Gressel, 1986; Scandalios, 1993). To
prevent or alleviate these damages, plants have
developed various mechanisms based on the production
of defence antioxidants (Jahnke et al., 1991; Walker and
McKersie, 1993; Hodges et al., 1997).
The electrons generated by the activity of
photosystem II (PSII) give rise to the formation of
different AOS, the radical O2 being one of the most
important (Salin, 1989; Bowler et al., 1992; Scebba et al.,
1998). The enzyme superoxide dismutase (SOD) brings
about the dismutation of the radical O2 to H2O2, and is
commonly considered as the first line of cell defence
(Halliwell and Gutteridge, 1989). H2O2 can be detoxified
in different cell compartments by different enzymes,
either, on the one hand, by the activities of guaiacol
peroxidase (GPX) or catalase (CAT), which bring about
the catalysis of H2O2 to H2O (Peters et al., 1989;
Takahama and Oniki, 1992), or, on the other hand, by an
oxidation-reduction system of antioxidant metabolites
(ascorbate and glutathione) which involves other
enzymes, such as ascorbate peroxidase (APX),
dehydroascorbate peroxidase (DHAR) and glutathione
*Author for correspondence.

reductase (GR) called the ascorbate/glutathione cycle

or route of Halliwell-Asada (Asada and Takahashi,
1987; Halliwell and Gutteridge, 1989; Salin, 1989;
Ushimaru et al., 2000).
The exposure of plants to stress situations generally
triggers antioxidant defence systems, since the
regulation of the coding genes for these enzymes
appears to be highly sensitive to the rise in cellular
levels of AOS produced under these conditions (del Ro
et al., 1991; Palma et al., 1991; Scandalios, 1993; Allen,
1995; Rao et al., 1996). For example, higher cellular
levels of H2O2 under thermal stress indicate a clear rise
in AOS production (Lafuente and Martinez-Tllez,
1997; Paolacci et al., 1997). Both high as well as low
temperatures can prompt H2O2 accumulation in
different plant tissues resulting from stronger SOD
activity and by a partial inhibition of the enzymes in
charge of detoxification (Yamakawa, 1983; Campa,
1991; Gaspar et al., 1991). In this way, H 2O2
accumulation in plants subjected to thermal stress is
considered the prime cause for the reduction in plant
biomass (Queiroz et al., 1998).
The tomato is a plant that requires optimum
temperatures of 2226C for growth and development
(Brinen 1979; Maroto 1995), and exposure to higher
temperatures can notably alter its metabolism. In light of
the above facts, the aim of the present work was to
examine the enzymatic activities as well as the
concentration of the antioxidant compounds involved in
the development of the oxidative metabolism in tomato
plants grown at 25C and 35C, to determine the capacity
of these plants to acclimatize and develop under high
temperatures. When these plants are grown at high
temperatures (35C), H2O2 accumulates and cannot be
eliminated even by the glutathione/ascorbate cycle, as its
main enzymes are possibly inactivated by the same high

R. M. RIVERO, J. M. RUIZ and L. ROMERO

temperatures. This H2O2 accumulation would, first,
reduce foliar biomass and, afterwards, kill the plant. On
the contrary, tomato plants grown at optimum
temperatures (25C) develop well, indicating good
detoxification of AOS and therefore a correct
functioning of the antioxidant systems of the plant.

MATERIAL AND METHODS

Plant growth
Seeds of tomato (Lycopersicon esculentum L. cultivar.
Tmknvf2) were germinated and grown for 30 d in a growth
chamber at a growth temperature of 25C (Brinen, 1979;
Maroto, 1995). Afterwards, 12 plants were transferred to a
cultivation chamber set at 35C (day/night). The
experiments were carried out at constant temperatures
(day/night) because the aim of this work was to investigate
the influence of a specific temperature on antioxidant
enzymes and compounds. Thereafter, 12 plants were
grown at 25C (control plants) and another 12 plants were
grown at 35C (heat stress). Both experiments (25C and
35C) were conducted for 30 d (3060 d after sowing). The
growth chamber was maintained at a relative humidity of
6080% and 16 h of photoperiod at a photosynthetic
photon flux density (PPFD) of 350 mol m2 s1 (measured
at the top of the plants).
During all experiments, the seedlings were grown in
individual pots (25 cm upper diameter, 17 cm lower
diameter, 25 cm in height) filled with vermiculite, and the
plants received a nutrient solution (pH 6.06.1) of: 2 mM
KNO3, 4 mM (NO3)2Ca, 1.5 mM NaH2PO4, 2 mM CaCl2,
3 mM SO4K2, 1.25 mM MgSO4, 5 M Fe-EDTA, 2 M
MnSO4, 1 M ZnSO4, 0.25 M CuSO4, 0.05 M
(NH4)6Mo7O24 and 2.5 M H3BO3 (van Zinderen, 1986).
The nutrient solution was contained in a 50 l tank, which
was subsequently distributed to plants by a localized drip
irrigation system.
Plant sampling
Leaves of these plants were sampled on day 60 after
sowing. The material was rinsed three times in H2O
following disinfection with 1% non-ionic detergent
(Decon 90) (Wolf, 1982), and then blotted on filter paper.
Half of the plants of each treatment were used for the
analysis of SOD, CAT, GPX, APX, DHAR, GR, H2O2,
AsA, DHA, total ascorbate, GSH, GSSG and total
glutathione (triplicate assays for each extraction). The
other half of the plants were used to determine shoot dry
weight. Leaves were dried in a forced-air oven at 70C
for 24 h. Dry weight (DW) was recorded and expressed
as g dw shoot1.
Metabolite assays
The methods used for extraction of H2O2 were those
of McNervin and Uron (1953) and Brennan and Frenkel
(1977). Hydroperoxides form a specific complex with
titanium (Ti+4), which can be measured by colorimetry at
415 nm. The concentration of peroxide in the extracts
was determined by comparing the absorbance against a
standard curve representing a titanium-H2O2 complex
from 0.1 to 1 mM. The hydroperoxides represent the
total peroxides.
Reduced ascorbate (AsA), dehydroascorbate
(DHA), and total ascorbate (AsA + DHA) were

561

determined spectrophotometrically following Gossett

et al. (1994). Tomato tissue (11.5 g FW) was
homogenized over ice with 0.5 g inert sand and 10 ml of
ice-cold freshly prepared 5% (w/v) m-phosphoric acid
with a mortar and pestle. The homogenate was
centrifuged at 10000 g for 15 min at 2C. Total ascorbate
was determined by incubating at 25C for 30 min with
100 l supernatant, 110 mM KH2PO4, 3.6 mM EDTA,
and 1.5 mM DTT to reduce all DHA to AsA in 700 l
total volume. After incubation, 100 l of 0.5 % (w/v) nethylmaleimide was added to remove excess DTT.
Measurements of AsA were treated in a similar manner
except that 200 l of deionized H2O was substituted for
DTT and n-ethylmaleimide. Colour was developed in
both series of reaction mixtures (total and reduced
ascorbate) by the addition of 400 l 10% (w/v)
trichloroacetic acid (TCA), 400 l 44% o-phosphoric
acid, 400 l of 65 mM
-
1-dipyridyl in 70% ethanol,
and 200 l of 110 mM FeCl3. The reaction mixtures were
then incubated at 37C for 45 min in a water bath and
quantified at 525 nm. From the same extract, AsA
and total ascorbate were assayed. Ascorbate standards
of between 0.001 and 0.5 mol ml1 ascorbate in
m-phosphoric acid were analysed in the same manner as
extracts. For each sample, DHA was estimated from the
difference of total ascorbate and AsA.
The oxidized glutathione (GSSG), reduced
glutathione (GSH) and total glutathione (GSSG + GSH)
amounts were determined spectrophotometrically
following Gossett et al. (1994). Tomato tissue (0.51 g FW)
was homogenized in a mortar and pestle over ice along
with 0.5 g inert sand and 10 ml of ice-cold freshly made
5% (w/v) 5-sulfosalicylic acid. The homogenate was
centrifuged at 10000 g at 2C for 15 min. Two solutions
were then prepared. Solution A (pH 7.2) consisted of
100 mM Na2HPO4 40 mM NaH2PO4H2O, 15 mM EDTA,
1.8 mM 5,5-dithiobis(2-nitrobenzoic acid), and
0.04% (w/v) bovine serum albumin (BSA). Solution B
(pH 7.2) consisted of 1.0 mM EDTA, 50 mM imidazole,
0.2% (w/v) BSA, and 2 units per ml of glutathione
reductase. Total glutathione was measured in a reaction
mixture consisting of 400 l solution A, 320 l of solution
B, 400 l of a 1:25 dilution of supernatant in 0.5 M
KH2PO4 (pH 7.0), and 80 l of 3 mM NADPH. GSSG
was analysed in a similar manner except that 1 ml of 1:10
diluted supernatant in 0.5 M KH2PO4 (pH 6.5) was first
incubated with 20 l 2-vinylpyridine at 25C for 45 min.
From the same extract, GSSG and total glutathione were
assayed. A standard curve was developed by preparing
solutions of 0.002 to 0.0001 g ml1 GSH in 60 ml1
m-phosphoric acid (pH 2.8) containing 1 mM EDTA,
diluting 1:2000 with the 50 ml l1 Na2PO4 at 412 nm, and
analysing in the same manner as the extracts. Levels of
GSH were estimated as the difference between total
glutathione and GSSG.
Enzyme assays
SOD activity was assayed by monitoring the inhibition
of nitroblue tetrazolium (NBT) reduction, according of
the methods of Giannopolitis and Ries (1977) and Beyer
and Fridovitch (1987), with some modifications (Yu et al.,
1998). A 5 ml reaction volume were used, containing
50 mM HEPES (pH 7.6), 0.1 mM EDTA, 50 mM Na2CO3
(pH 10.0), 13 mM methionine, 0.025% (v/v) Triton

562

Heat and oxidative metabolism

TABLE I
Effect of temperature on SOD activity, dry weight (D.W.) and H2O2 concentration. SOD activity expressed as units SOD (mg prot)1 (min)1.
D.W. expressed as g per plant H2O2 expressed as mmol (g F.W.). Data
are means SE (n=6). Levels of significance are represented by ***
at P<0.001
Temperature
25C
35C
Signif.

SOD Activity

H2O2

D.W.

1.27 0.12
7.12 0.22
***

2l.6l 1.66
76.46 2.4l
***

363 2.21
175 1.03
***

X-100, 63 M NTB, 1.3 M riboflavin and appropriate

aliquot of enzyme extract. The reaction mixture was
illuminated for 15 min; PPFD was 380 mol m2 s1.
Identical reaction mixtures that were not illuminated
were used to correct for background absorbance. One
unit of SOD activity was defined as the amount of
enzyme required to cause 50% inhibition of the
reduction of NTB as monitored at 560 nm.
CAT activity was determined as described by Badiani
et al. (1990) by following the consumption of H2O2
(extinction coefficient, 39.4 mM1 cm1) at 240 nm for
3 min.
GPX activity was determined as described by Kalir
et al. (1984) and Ruiz et al. (1998) by the oxidation of
guaiacol in presence of H2O2 (extinction coefficient,
26.6 mM1 cm1) at 470 nm.
APX activity was determined according to Gossett
et al. (1994) by following the decrease in A290 of an assay
mixture containing 0.5 mM ascorbate (extinction
coefficient, 2.8 mM1 cm1).
DHAR activity was determined following Ushimaru
et al. (2000).
GR activity was assayed as described by Ushimaru
et al. (1992).
Activities rates were determined at substrate
saturation in our enzyme assays. All activities were
expressed as a function of the oxidized or reduced
substrate per milligram of protein per minute. The
protein concentration was determined by the method of
Bradford (1976) using BSA as standard.
Statistical analysis
Analysis of variance was used to assess the
significance of treatments. Results represent mean
values SE. A correlation analysis was also conducted
to determine the relations between the different
variables. Levels of significance are represented by * at
P<0.05, ** at P<0.01, *** at P<0.001 and n.s.: not
significant by ANOVA at P=0.05 tested differences
between the two temperatures and separated ANOVAs
for each variable and we did t-test for each variable and
made a Bonferroni correction. Each experiment was
replicated three times.

RESULTS AND DISCUSSION

The tomato, which requires optimum temperatures of
2226C for growth and development, undergoes
metabolic alterations at supraoptimal temperatures
(Brinen, 1979; Maroto, 1995). Under our experimental
conditions, the highest values for shoot dry weight were
registered at 25C (optimum temperature for this
species) and the lowest at 35C (Table I), representing a
52% difference. Under stress conditions caused by high
temperatures, in addition to metabolic alteration,
overproduction of AOS can trigger oxidative damage
(Paolacci et al., 1997). Proper functioning of the
scavenging system is essential to maintain the
concentration of AOS (Scebba et al.. 1998). SOD
catalyses the dismutation of O2 to H2O2 and O2 (McCord
and Fridovitch, 1969; Ushimaru et al., 2000). Increased
SOD activity in spinach plants was found during
exposure to high temperatures (Schner and Krause,
1990), and high SOD activity has been associated with
heat in plants where overproduction of O2 is involved
(Bowler et al., 1992; Rao et al., 1996). In our research,
SOD activity was significantly higher (P<0.001) at 35C
than at 25 C (seven-fold higher) (Table I).
In our experiments, H2O2 concentration rose
significantly at 35C (P<0.001), to almost 3.5-fold higher
than that found at 25C (Table I). This appears to suggest
that greater H2O2 accumulation may be due largely to
the rise in SOD activity at 35C, given the positive
relation found between these two parameters (SOD
activity-H2O2 r = 0.912***).
The above results could explain the dry-weight
reduction in tomato plants subjected to 35C, since one
of the initial symptoms of H2O2 accumulation is reduced
foliar biomass (Table I; Willenkens et al. 1997). The lower
concentration of H2O2 at 25C, coinciding with the
highest foliar biomass (Biomass-H2O2 r = 0.823***),
appear to corroborate an inverse relationship between
the amount of biomass of a plant and its foliar H2O2
concentration.
Many studies have shown that CAT and GPX
activities increase during exposure to high temperatures
(Pinhero et al., 1997). However, in our experiments, CAT
and GPX diminished at 35C compared with 25C by
75% and 54%, respectively (Table II). The lower CAT
and GPX activities in our tomato plants subjected to
35C could also account for the demonstrated high
accumulation of foliar H2O2 (CAT activity-H2O2
r = 0.935***; GPX activity-H2O2 r = 0.907***). Finally,
the reduction of the CAT and GPX activities at 35C
could be due to an inactivation of these enzymes by the
higher temperature.
The activities of APX, DHAR and GR (Table II) in
all cases had behaviour similar to that found for the
CAT and GPX activities (Table II). At 35C, we found a

TABLE II
Effect of temperature on antioxidant enzymes in tomato plants. GPX activity expressed as mol guaiacol oxidized (mg prot)1 (min)1; CAT activity
expressed as mol H2O2 oxidized (mg prot)1 (min)1; APX activity expressed as mol AsA oxidized (mg prot)1 (min)1; DHAR activity expressed as
mol DHA oxidized (mg prot)1 (min)1; GR activity expressed as mol GSH oxidized (mg prot)1 (min)1. Data are means SE (n=6). Levels of
significance are represented by *** at P<0.001
Temp

GPX

CAT

APX

DHAR

GR

25C
35C
Signif.

39.17 2.23
18.32 1.48
***

17.21 1.36
2.24 0.19
***

98.24 4.96
21.33 1.94
***

119.2 5.85
38.65 2.19
***

125.2 6.39
26.37 2.01
***

R. M. RIVERO, J. M. RUIZ and L. ROMERO

563

TABLE III
Effect of temperature on ascorbate and glutathione concentrations. AsA, DHA and total ascorbate expressed as mol ascorbate (g f.w.)1 GSH, GSSG
1
and total glutathione expressed as mol glutathione (g F.W.) . Shoot dry weight expressed as g per plant. Data are means SE (n=6). Levels of
significance are represented by*** at P<0.001
Temperature
25C
35C
Signif.

AsA
2.75 0.13
14.75 0.45
***

DHA

Total Ascorbate

GSSG

GSH

0.44 0.03
2.41 0.12
***

3.19 0.22
17.17 0.35
***

0.157 0.01
0.64 0.011
***

0.563 0.053
2.166 0.064
***

reduction of these activities of 77%, 67% and 77%,

respectively compared with 25C. As with CAT and
GPX, the relationships between the enzymatic activities
of the glutathione-ascorbate cycle and the H2O2;
concentration (APX activity-H2O2 r = 0.754***;
DHAR activity-H2O2 r = 0.567*; GR activity-H2O2
r = 0.789***) could also explain the accumulation of
this compound in tomato plants subjected to 35C.
These results are contrary to those reported by Paolacci
et al. (1997), who found increased activities only in
plants treated with high temperatures but able to
develop heat tolerance.
In our experiments, the statistically significant
decrease (P<0.001) in enzymatic activities at 35C
appears to imply an enzymatic inactivation by the high
temperatures and a lack of heat tolerance in tomato
plants. In plants grown at 25C, we found significant
higher activities of the enzymes in charge of
eliminating H2O2, confirming that the antioxidant
defence system of the plant functions correctly under
optimum growth conditions, impeding the massive
accumulation of H 2O 2 and maintaining correct
functioning of its cell metabolism.
The concentration of the antioxidant compounds that
participate in the glutathione/ascorbate cycle (AsA,
DHA, total ascorbate, GSH, GSSG and total
glutathione) at 35C and 25C are shown in Table II.
Both substrates behaved similarly at the different
temperatures, with concentrations proving consistently
lower at 25C than at 35C. Under heat stress, the plant
boosts synthesis of different forms of glutathione and
ascorbate in the cell, for these to be used as necessary
substrates for the glutathione/ascorbate cycle, and in this
way aid in the detoxification of different AOS produced,

Total Glutathione
3.71 0.706
16.157 0.706
***

thereby prolonging plant survival as long as the stress

conditions persist. However, our results indicate that
possibly, with the treatment at 35C, there is an
inactivation of the principal enzymes of this pathway due
to the high temperatures. This may explain the higher
concentrations of glutathione and ascorbate at 35C,
both in its total as well as in its oxidized and reduced
forms, since these substrates were not being used by the
APX, DHAR or GR. Therefore, H2O2 would not be
detoxified and, in accord with our experimental results,
showing the highest H2O2 concentrations at 35C. At
25C the glutathione and ascorbate concentrations were
lower, supporting the hypothesis that under normal
growth conditions the antioxidant defence systems
continue to function (Salin, 1989), although at a slower
pace that needed under heat stress. This fact supports the
idea that high temperatures inhibit these activities.
On the other hand, we found that the concentrations
of the oxidized forms both of ascorbate (DBA) and
glutathione (GSSG) were less than the concentrations
of their reduced forms (AsA and GSH) (Table III). This
is normal, the initial substrate is AsA for the
detoxification of H2O2, beginning with the
ascorbate/glutathione cycle. In addition, for efficient
detoxification, this ascorbate must be regenerated by
DHAR activity, which is possible only if there are high
GSH concentrations in the medium. As indicated above,
the high temperatures could inhibit the antioxidant
enzymes of the ascorbate/glutathione cycle, and thus it is
logical that the AsA and GSH concentrations should be
higher than those of the oxidised forms (DHA and
GSSG), since these substrates are not being used by
these enzymes to detoxify the H2O2, so this compound
accumulates in the cells of these plants.

REFERENCES
ALLEN, R. (1995). Dissection of oxidative stress tolerance using
transgenic plants. Plant Physiology, 107, 104954.
ASADA, K. and TAKAHASHI, M. (1987). Production and scavenging
of active oxygen in photosynthesis. In: Photoinhibition. (Kyle,
D. J., Osmond, C. B. and Amtzen, C .J., Eds). Elsevier,
Amsterdam, The Netherlands, 2278.
BADIANI, M., DE BASI, M .G. and FELICI, M. (1990). Soluble peroxidase from winter wheat seedlings with phenoloxidase-like
activity. Plant Physiology, 93, 48994.
BEYER, W .F. and FRIDOVITCH, I. (1987). Assaying for superoxide
dismutase activity: some large consequences of minor changes
in conditions. Analytical Biochemistry, 161, 55966.
BOWLER, C., VAN MONTAGU, M. V. and INZ, D. (1992). Superoxide
dismutase and stress tolerance. Annual Review of Plant
Physiology and Plant Molecular Biology, 43, 83116.
BRADFORD, M. M. (1976). A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing the
principle of protein binding. Analytical Biochemistry, 72, 24854.
BRENNAN, T. and FRENKEL, C. (1977). Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiology,
59, 4116.

BRINEN, G. H. (1979). Plant and row spacing, mulch and fertilizer

rate effects on watermelon production. Journal of the American
Society for Horticultural Science, 104, 7246.
CAMPA, A. (1991). Biological roles of plant peroxidases: Known and
essential function. In: Peroxidases in chemistry and biology,
Vol II. (Everse, J., Everse, K. and Grishman, M. B., Eds). CRC
Press, Boca Raton, Florida, USA, 2550.
DEL RO, L. A., SEVILLA, F., SANDALIO, L. M. and PALMA, J. M.
(1991). Nutritional effect and expression of SODs: induction
and gene expression; diagnostics; prospective protection
against oxygen toxicity. Free Radical Research Communications,
1213, 81927.
ELSTNER, E. F. and OSWALD, W. (1994). Mechanism of oxygen activation during plant stress. Proceedings of the Royal Society of
Edinburgh, Section B-Biological Sciences, 102 B, 13154.
GASPAR, T. H., PENEL, C., HAGEGA, D. and GREPPIN, H. (1991).
Peroxidases in plant growth, differentiation and development
processes. In: Biochemical, molecular and physiological aspect
of plant peroxidases (Lobarzewsky, J., Greppin, H., Penel., C.
and Gaspar, T. H., Eds). University of Geneva, Switzerland,
24980.

564

Heat and oxidative metabolism

GIANNOPOLITIS, C. N. and RIES, S. K. (1977) Superoxide dismutase

occurrence in higher plants. Plant Physiology, 59, 30914.
GOSSETT, D. R., MILLHOLLON, E. P. and LUCAS, M. C. (1994)
Antioxidant responses to NaCl stress in salt-tolerant and saltsensitive cultivars of cotton. Crop Science, 34, 70614
HALLIWELL, B. and GUTTERIDGE, J. M. C. (1989). Free radicals in
biology and medicine. Second Edition. Clarendon Press,
Oxford, UK.
HODGES, D. M, ANDREWS, C. J., JOHNSON, D. A. and HAMILTON, R. I.
(1997). Antioxidant enzyme and compound responses to chilling stress and their combining abilities in differentially sensitive
maize hybrids. Crop Science, 37, 85763.
JAHNKE, L. S., HULL, M. R. and LONG, S. P. (1991). Chilling stress and
oxygen metabolizing enzymes in Zea mays and Zea diploperennis. Plant, Cell & Environment, 14, 97104.
KALIR, A., OMRI, G. and POLJAKOFF-MAYBER, A. (1984). Peroxidase
and catalase activities in leaves of Halimione portulacoides
exposed to salinity. Physiologia Plantarum, 62, 23844.
LAFUENTE, M. T. and MARTNEZ-TLLEZ, M. A. (1997). Effect of the
high temperature conditioning on ethylene, phenylalanine
ammonia-lyase, peroxidase and polyphenol oxidase activities in
flavedo of chilled Fortune mandarin fruit. Journal of Plant
Physiology, 150, 6748.
MAROTO, J. V. (1995). Desairollo ptimo para las plantas de tomate.
In: Horticultura herbcea especial. (Maroto, J.V., Ed).
Mundiprensa, Madrid, Spain, 71475.
MCCORD, J. M. and FRIDOVITCH, I. (1969). Superoxide dismutase.
An enzymatic function for erythrocuprein (hemocuprein).
Journal of Biological Chemistry, 244, 604955.
MCNERVIN, W. M. and URON, P. F. (1953). Separation of hydrogen
peroxide from organic hydroperoxides. Analytical Chemistry,
25, 17601.
PALIYATH, G. and FLETCHER, R. A. (1995). Paclobutrazol treatment
alters peroxidase and catalase activities in heat-stressed maize
coleoptiles. Physiology and Molecular Biology of Plants, 1, 1718.
PALMA, J. M., GARRIDO, M, RODRGUEZ-GARCA, M. I. and
DEL RO, L. A. (1991). Peroxisome proliferation and oxidative
stress mediated by activated oxygen species in plant peroxisomes. Archives of Biochemistry and Biophysics, 287, 6874.
PAOLACCI, A. R., BADIANI, M., DANNIBALE, A., FUSARI, A. and
MATEUCCI, G. (1997). Antioxidant and photosynthesis in leaves
of Triticum durum Desf. seedlings acclimated to non-stressing
high temperature. Journal of Plant Physiology, 150, 3817.
PETERS, J. L., CASTILLO, F. J. and HEATH, R. L. (1989). Alteration
of extracellular enzymes in pinto beans leaves upon exposure
to air pollutants, ozone and sulphur dioxide. Plant Physiology,
89, 15964.
PINHERO, R. G., RAO, M. V., PALIYATH, G., MURR, D. .P. and
FLETCHER, R. A. (1997). Changes in activities of antioxidant
enzymes and their relationship to genetic and paclobutrazolinduces chilling tolerance of maize seedlings. Plant Physiology,
114, 695704.
PRASAD, T. K, ANDERSON, M. D., MARTIN, B. A. and STEWARD, C. R.
(1994). Evidence for chilling induced oxidative stress in maize
seedlings and regulatory role for hydrogen peroxide. The Plant
Cell, 6, 6574.

QUEIROZ, C. G. S., ALONSO, A., MARES-GUIA M. and

MAGALHAES, A. C. (1998). Chilling-induces changes in membrane fluidity and antioxidant enzyme activities in Coffea
arabica L. roots. Biologia Plantarum, 41, 40313.
RAO, M. V., PALIYATH, G. and ORMROD, D. P. (1996). Ultraviolet-B
and ozone-induced biochemical changes in antioxidant
enzymes of Arabidopsis thaliana. Plant Physiology,
110, 12536.
RUIZ, J. M., BRETONES, G, BAGHOUR, M., BELAKBIR, A. and
ROMERO, L. (1998). Relationship between boron and phenolic
metabolism in tobacco leaves. Phytochemistry, 48, 26972.
SALIN, M. L. (1989). Toxic oxygen species and protective system of
the chloroplast. Physiologia Plantarum, 72, 6819.
SCANDALIOS, J. G. (1993). Oxygen stress and superoxide dismutases.
Plant Physiology, 101, 712.
SCEBBA, F., SEBASTIANI, L. and VITAGLIANO, C. (1998). Changes in
activity of anti oxidative enzymes in wheat (Triticum aestivum
L.) seedlings under cold acclimation. Physiologia Plantarum,
104, 74752.
SCHNER, S. and KRAUSE, G. H. (1990). Protective systems against
active oxygen species in spinach: response to cold acclimation
in excess light. Planta, 180, 3839.
SHAALTIEL, Y. and GRESSEL, J. (1986). Multienzyme oxygen radical
detoxification system correlated with paraquat resistance in
Coniza bonariensis. Pesticide Biochemistry and Physiology,
26, 228.
TAKAHAMA, U. and ONIKI, T. (1992). Regulation of peroxidasedependent oxidation of phenolics in the apoplast of spinach
leaves by ascorbate. Plant and Cell Physiology, 33, 37987
USHlMARU, T., KANAZAWA, S., SANO, S. and KOSHTOA, T. (2000).
Changes in antioxidative enzymes in cucumber cotyledons
during natural senescence: Comparison with those during darkinduced senescence. Physiologia Plantarum, 109, 2116.
USHIMARU T., SHIBASAKA, M. and TSUJI, H. (1992). Development of
O2 detoxification system during air adaptation of submerged
rice seedlings. Plant and Cell Physiology, 33, 106571.
VAN ZINDEREN, E. M. (1986). Development of hydroponic system
and look into the future. Annual Conference on Hydroponics.
Hydroponics Society of America. Concord, CA,USA, 3712.
WALKER, M. A. and MCKERSIE, B. D. (1993). Role of the ascorbateglutathione antioxidant system in chilling resistance of tomato.
Journal of Plant Physiology, 141, 2349.
WILLENKENS, H., CHAMNONGPOL, S., DAVEY, M., SCHRAUDNER, M.,
LANGEBARTELS, C., VAN MONTAGU, M., INZ, D. and
VAN CAMP, W. (1997). Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. The EMBO Journal,
16, 480616.
WOLF, B. (1982). A comprehensive system of leaf analysis and the
use for diagnosis of crop nutrients stress. Communications in
Soil Science and Plant Analysis, 13, 103559.
YAMAKAWA, B. (1983). Grafting. Vegetable handbook (Nishi,
Yokendo Book Co., Ed.). Tokyo, (in Japanese).
YU, Q., OSBORNE, L. and RENGEL, Z. (1998). Micronutrient deficiency changes activities of superoxide dismutase and ascorbate
peroxidase in tobacco plants. Journal of Plant Nutrition,
21, 142737.