ARTICLE IN PRESS

Physiological and Molecular Plant Pathology 68 (2006) 2232

www.elsevier.com/locate/pmpp

Biochemical aspects of reactive oxygen species formation in the

interaction between Lycopersicon spp. and Oidium neolycopersici
Kater ina Tomankovaa,b, Lenka Luhovaa, Marek Petr ivalskya, Pavel Peca, Ales Lebedab,
a

Department of Biochemistry, Faculty of Science, Palacky University in Olomouc, Slechtitelu 11, 783 71 Olomouc-Holice, Czech Republic
b
Department of Botany, Faculty of Science, Palacky University in Olomouc, Slechtitelu 11, 783 71 Olomouc-Holice, Czech Republic
Accepted 26 May 2006

Abstract
Three Lycopersicon spp. accessions differing in the level of resistance to Oidium neolycopersici L. Kiss (tomato powdery mildew) were
studied. Defence reactions occurring in tissue of Lycopersicon esculentum cv. Amateur (susceptible control), Lycopersicon hirsutum f.
glabratum (LA 2128) (highly resistant) and Lycopersicon chmielewskii (LA 2663) (moderately resistant) were investigated during the rst
120 h post-inoculation (hpi). A hypersensitive reaction was detected after 48 hpi in both resistant tomato accessions. Changes in
accumulation of hydrogen peroxide and enzymes involved in its metabolism (catalase, peroxidases, superoxide dismutase) were
monitored. In resistant accessions, intensive H2O2 production correlated with increased activity of cytosolic guaiacol peroxidase,
syringaldazine peroxidase and ascorbate peroxidase. Catalase activity increased especially in moderately resistant L. chmielewskii.
A similar degree of lipid peroxidation occurred in all Lycopersicon accessions. An increase in the concentration of free phenols but
no change in the level of cell-wall-bound phenols were observed during the rst 120 hpi in all Lycopersicon spp. accessions. Spermidine
represented the major part of the total polyamine content. Pathogen-induced lignication was not observed in any of the
studied accessions.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Tomato powdery mildew; Oidium neolycopersici; Wild tomato; Lycopersicon spp.; Defence responses; Resistance; Susceptibility; Hypersensitive
reaction; Catalase; Peroxidase; Hydrogen peroxide; Phenolic compounds; Lignication; Amine oxidase; Polyamines

1. Introduction
The association of reactive oxygen species (ROS)
formation and increased activity of enzymes participating
in their metabolism with the induction of defence responses
has been demonstrated in many plantpathogen interactions [13]. H2O2 generation affects gene expression and
causes inhibition or retardation of fungal development [4].
ROS may act as signalling agents in various plantpathogen interactions [5] and cause reinforcement of plant cell
walls through oxidative cross-linking [6]. Localized production of H2O2 and superoxide is one of the earliest
cytologically and histochemically detectable response
events in plant tissue [7], and is probably involved in
induction of the hypersensitive response (HS) [8]. ROS
Corresponding author. Tel.: +420 58 563 4800; fax: +420 58 563 4824.

E-mail address: lebeda@prfholnt.upol.cz (A. Lebeda).

0885-5765/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pmpp.2006.05.005

have direct antimicrobial activity inhibiting germination of

spores of many fungal pathogens [9]. The pathogen itself
may also generate H2O2 as a pathogenicity factor [10].
Various enzyme systems participate in ROS metabolism
during the pathogen attack [11]. An NADPH-dependent
membrane-bound oxidase (EC 1.6.3.1) releasing extracellular superoxide anion, followed by spontaneous or superoxide dismutase (SOD) (EC 1.15.1.1) catalysed dismutation
of O2 to H2O2 [12] and apoplastic peroxidase (EC 1.11.1.7)
are major enzymes catalysing H2O2 production [13]. It was
suggested that the H2O2 required for the peroxidase (POX)
activity involved in the synthesis of lignin/suberin is derived
from amine oxidase activity (AO) (EC 1.4.3.6) [14,15].
Catalase (CAT) (EC 1.11.1.6) and ascorbate peroxidase
(APX) (EC 1.11.1.11), together with glutathione reductase
(EC 1.8.1.7) and dehydroascorbate reductase (EC 1.6.5.4),
belong to important H2O2-scavenging enzymes, removing
H2O2 through a mechanism known as ascorbate-GSH

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K. Tomankova et al. / Physiological and Molecular Plant Pathology 68 (2006) 2232

pathway (HalliwellAsada pathway) [16]. POXs are included in different physiological processes like cross-linking
of the cell wall proteins [17], pectins by diferulic bridges [18]
and the oxidation of cinnamyl alcohols prior to their
polymerization during lignin and suberin formation [19].
Phenolic compounds (benzoic acid, salicylic acid and other
phenolics) can act as free radical scavengers and protect
cells from their oxidative toxicity [20]. On the other hand,
simple phenols may cause disorders in plant cell membrane
functioning and cause membrane damage, as observed by
e.g. Politycka [21] when the treatment of cucumber roots
with phenolic acids led to membrane damages accompanied
by lipid peroxidation. Lipid peroxides decompose to
produce malondialdehyde, volatile hydrocarbons such as
ethane, pentane and are precursors for the synthesis of
jasmonic acid [22].
Polyamines (PAs) represent an important group of
compounds playing a role in various cell processes (cell
division, protein synthesis, DNA replication and tissue
responses to various stresses) [23]. Exogenous di- and PAs
were shown to stabilize plant cell membranes, protecting
them from damage under stress conditions [24] and
endogenous PAs are also suggested to participate in
sustaining membrane integrity [25]. PAs have been
implicated in molecular signalling events during plant
pathogen interactions [26]. During the HR, PA content has
been reported to increase around and/or in necrotic lesions
and accumulate in the apoplast of infected plants [27].
Inhibitors of PA biosynthesis reduced the rate of HR [28].
Our previous histochemical study, carried out on three
Lycopersicon spp. accessions differing in the response to
Oidium neolycopersici [29,30], pointed out the relationships
between ROS production and plant sensitivity to pathogen
infection [31]. The aim of present study was to continue in
this research and investigate the intensity and timing of the
ROS triggering and the expression of antioxidant enzymes,
lipid peroxidation and possible development of the HR
during defence responses of wild Lycopersicon spp.
accessions against O. neolycopersici.
2. Materials and methods
2.1. Pathogen isolate and its cultivation
O. neolycopersici L. Kiss (isolate C-2) was collected on
tomato cv. Lucy grown in glasshouses of the State
Phytosanitary Administration in Olomouc, Czech Republic
[30]. Susceptible tomato plants (Lycopersicon esculentum cv.
Amateur) placed under plastic covers in a growth chamber
at 18 1C and 12 h photoperiod with a light intensity of
100 mmol/m2 s over day and 15 1C over night were used for
the pathogen maintenance and multiplication [29].
2.2. Plant material and O. neolycopersici inoculation
Seeds of L. esculentum cv. Amateur (highly susceptible
control), Lycopersicon chmielewskii (LA 2663) (moderately

23

resistant, i.e. limited rate of pathogen reproduction) and

Lycopersicon hirsutum f. glabratum (LA 2128) (highly
resistant, i.e. no pathogen reproduction and/or very
limited) [30] were sown in plastic boxes containing Perlite.
Seedlings with fully expanded cotyledons were transferred
to 7 cm diameter plastic pots containing garden soil:peat
(2:1, v/v) and grown in a growth chamber at 20 1C and 12 h
photoperiod. Ten-week-old plants were inoculated with
conidia of O. neolycopersici on the upper side of leaves by
surface contact (dusting/tapping) at the beginning of the
light phase. The second to the fourth true leaves were used
for inoculation. Leaves of tomato cv. Amateur, which
were 80100% covered by fresh (8 days old) sporulating
mycelium of O. neolycopersici, were used as a source of
inoculum. After inoculation, plants were placed under
plastic covers in a growth chamber at 18 1C and 12 h
photoperiod. Control (uninoculated) plants of the same
accessions were cultivated separately under the same
conditions. At certain time intervals (0, 4, 8, 12, 16, 20,
24, 48, 120 h post-inoculation (hpi)), leaves were collected,
frozen under liquid nitrogen and stored at 80 1C. For
detection of cell necrosis, fresh tomato leaves were used.
2.3. Production of hydrogen peroxide and phenolic
compounds
Concentration of hydrogen peroxide in leaf extracts was
measured spectrophotometrically using the reaction of
H2O2 with KI (modied method of Velikova [32]). Leaves
were homogenized in 0.1% TCA in ratio 1:10 (fresh wt/v)
and centrifuged at 12,000g, 15 min at 4 1C. Supernatant
(0.5 mL) was mixed with 1 mL of 1 M KI solution and
incubated 5 min before measuring of the oxidation product
at A390.
Concentration of free and bound phenols was determined using FolinCiocalteau colorimetric reaction [33]
and estimated from the calibration curve for phenol (A750).
Free phenols were extracted in 50% methanol in ratio
1:6 (fresh wt/v) at 90 1C for 90 min. After centrifugation
(14,000g, 15 min), in order to liberate bound phenols, the
pellet was further saponied with 2 mL of 0.5 M NaOH for
24 h at room temperature and neutralized using 0.5 mL of
2 M HCl.
2.4. Cell death and quantification of lignin
Cell death was quantied semiquantitatively using Evans
blue stain capable of binding to necrotic cells [34]. Whole
detached leaves were incubated in 0.05% Evans blue for
15 min. After intensive washing in distilled water, Evans
blue was extracted into 50% (v/v) methanol solution
containing 1% SDS for 30 min at 50 1C. The intensity of
stain was measured against water at 600 nm.
Lignin was quantied by the modied method of Olmos
et al. [35] in extracts prepared by homogenization of plant
tissue in boiling water in ratio 1:10 (fresh wt/v). Supernatant from centrifuged extracts was discarded and pellet

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K. Tomankova et al. / Physiological and Molecular Plant Pathology 68 (2006) 2232

was washed in 100% ethanol, then dried by evaporation at

50 1C in drying oven. The dried pellet was further
incubated in the solution of 18% hydrochloric acid in
ethanol in ratio 1:25 (wt/v) for 3 h and centrifuged. The
supernatant (1 mL) was incubated for 3 h with 10 mL of
20% (w/v) phloroglucinol (dissolved in the solution HCl/
ethanol) and the absorbance was measured at 540 nm.
2.5. Enzyme activity assays
Detached tomato leaves were homogenized on ice in
0.1 M K-phosphate buffer, pH 7.0, containing 1% polyvinyl polypyrrolidone (PVPP) in ratio 1:3 (wt/v). For the
APX assay, the homogenization buffer contained 2 mM
ascorbate. Distribution of syringaldazine peroxidase
(SPOX) and guaiacol peroxidase (GPOX) within the plant
tissue was studied using different extraction buffers
(modied method of Quiroga et al. [36], and Milosevic
and Slusarenko [37]). Cytosolic and weakly ionic-bound
enzymes (phase A) were extracted in 0.1 M K-phosphate
homogenization buffer, pH 7.0, containing 1% PVPP.
After the centrifugation, supernatant was collected for
enzyme activity assay and the rst pellet was washed 3
times with phosphate buffer. In order to liberate membrane-bound and ionic forms of SPOX and GPOX, the
pellet was further extracted (1 h, 4 1C, mixed by inverting
the tubes few times) in the same phosphate buffer
containing 1% Triton X-100 (membrane-bound forms,
phase B). After centrifugation, the pellet was rinsed 3 times
with distilled water and nally incubated (2 h, 4 1C, with
shaking) in homogenization buffer containing in 1 M KCl
(ionically bound cell wall forms, phase C).
GPOX activity was measured by the modied method of
Angelini et al. [14]. Total volume 1.75 mL of the reaction
mixture contained 0.1 M K-phosphate buffer, pH 6, 15 mM
guaiacol and crude extract. The reaction was started by
addition of hydrogen peroxide (nal concentration 5 mM).
Formation of colored product (A436) was followed
continuously for 1 min at 30 1C.
SPOX activity was measured by the modied method of
Quiroga et al. [36] with substrate syringaldazine, an
analogue of lignin monomer. The reaction mixture
contained 0.1 M Na-phosphate buffer, pH 7.0, 0.156 mM
syringaldazine and crude extract. The reaction was started
by the addition of hydrogen peroxide (nal concentration
0.05 mM). Formation of pink oxidation product (A530) was
followed continuously for 1 min at 30 1C.
APX activity was determined according to Klapheck
et al. [38] by following the ascorbate oxidation for 1 min
at 290 nm at 25 1C in a reaction mixture containing 50 mM
K-phosphate buffer pH 7.0, crude extract and 0.5 mM
ascorbate. The reaction was started by the addition of
hydrogen peroxide (nal concentration 0.1 mM).
The activity of CAT was quantied by the modied
method of Goth [39] with ammonium molybdate forming a
stable complex product with unreacted hydrogen peroxide
(A405). Reaction mixture (2.2 mL) contained 45 mM

K-phosphate buffer pH 7.4, 7 mM hydrogen peroxide

and 0.2 mL of the crude extract. Ammonium molybdate
(1 mL of 32.4 mM solution) was added after 4 min of
incubation to stop the consumption of hydrogen peroxide
by CAT present in the extract. Blanks were prepared for
each sample by the addition of the ammonium molybdate
to the reaction mixture immediately, omitting the incubation period.
AO activity was monitored using a coupled reaction with
horseradish peroxidase, 3,5-dichloro-2-hydroxybenzensulphonic acid (DCHBS) and 4-aminoantipyrine, with putrescine as a substrate [40]. The reaction mixture containing
0.1 M K-phosphate buffer pH 7.0, horseradish peroxidase,
1.7 mM 4-aminoantipyrine, 17 mM DCHBS and 25 mM
putrescine was added to start the reaction. The oxidation
product was measured spectrophotometrically (A515).
SOD activity was assayed according to modied method
of Beauchamp and Fridovich [41]. The reaction mixture
contained 50 mM K-phosphate buffer pH 7.8, 10 4 M
ethylene tetraacetic acid (EDTA), 500 mM p-nitrotetrazolium blue (NBT), 100 mM xantine, plant extract and
xantine oxidase, which gave an increase in absorbance at
550 nm at 30 1C. One unit of SOD was dened as the
amount required for inhibition of the NBT reduction by
50% under the assay conditions.
2.6. Lipid peroxidation
Leaves were extracted in 0.1% TCA in ratio 1:5 (fresh
wt/v) and centrifuged at 12,000g for 30 min and 4 1C. One
milliliter of the supernatant was incubated with 4 mL of
20% TCA containing 0.5% thiobarbituric acid (TBA) for
30 min at 95 1C. The reaction was stopped by short cooling
on ice for 10 min and the product was centrifuged at
10,000g for 15 min. The absorbance of the reaction product
was measured at 532 nm.
2.7. Protein estimation
Protein concentration was determined using bovine
serum albumin as a standard according to the method of
Lowry [42].
2.8. PAs in plant tissue
Plant free PAs were detected by HPLC according to the
modied method of Hwang et al. [43]. Plant material was
homogenized on ice in 5% TCA in ratio 1:4 (fresh wt/v)
and centrifuged at 2000g for 5 min. NaOH (2 mL of a 2 M
solution) was added to 1 mL of the supernatant followed
by 10 mL of benzoyl chloride and the mixture was
incubated for 40 min at 25 1C. After addition of 2 mL of
saturated NaCl, benzoylated PAs were extracted with 3 mL
of diethyl ether. After centrifugation (12,000g for 15 min),
the upper organic phase was transferred into Eppendorf
tubes and evaporated to dryness. The residue was dissolved

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K. Tomankova et al. / Physiological and Molecular Plant Pathology 68 (2006) 2232

in 200 mL of the mobile phase (60% methanol, 40% H2O)

and 20 mL aliquots were injected for HPLC analysis.
Standards for the determination of the calibration curve
were prepared by benzoylation of 10 mL of 0.1 M amine
standard solution and injection of 20 mL aliquots. Benzoylated PAs were separated on Supelco C-18 column
(250  4.6 mm ID) by isocratic elution with 60% methanol
at a ow rate of 0.7 mL/min and detected at 254 nm.
2.9. Statistical analysis
Growing, inoculation and sampling of plants were done
in three independent experiments. Leaves of three plants
were considered as 1 sample. Collected plant material was
randomly divided into three parts and analysed. Data
presented in graphs are the means+standard errors of nine
different measurements.
3. Results
3.1. Production of hydrogen peroxide
In plantpathogen interactions, H2O2 has a crucial role
both in plant signalling and defence mechanism during the
time-course of pathogen invasion in the host cell [2,3]. The
level of H2O2 in infected plant tissue was signicantly
higher compared to the control in all tested Lycopersicon
spp. accessions. Differences in the concentration and
timing of H2O2 increase during the rst 120 hpi were
observed between susceptible and resistant accessions. The
concentration of hydrogen peroxide in infected tissue of
susceptible L. esculentum cv. Amateur was lower than
70 mM/g fresh wt, while detected values were 50150 mM/g
fresh wt in moderately resistant accessions L. chmielewskii
and 110380 mM/g fresh wt in highly resistant L. hirsutum.
Concentration of H2O2 in resistant accessions elevated in
two phases: early after inoculation (48 hpi, two-fold
increase) and the second strong increase occurred at
2048 hpi (three-fold increase). In the susceptible accession,
only a late increase of H2O2 content was observed
(2024 hpi) (Fig. 1).

25

3.2. Accumulation of phenolic compounds

In plants, the participation of phenolic compounds and
their metabolites in response to various biotic stress stimuli
is well documented [20]. The level of free phenols increased
dramatically in both resistant and susceptible infected
plants since 2024 hpi. The concentrations of free phenols
after 120 hpi in L. esculentum cv. Amateur reached the
highest values (1600 mmol/g fresh wt), 980 mmol/g fresh
wt in L. hirsutum and only 612 mmmol/g fresh wt in
L. chmielewskii (Fig. 2). No changes in the content of
bound phenols were detected during the rst 120 hpi in any
of the studied Lycopersicon spp. accessions.
3.3. Cell death and lignin deposition
Among plant cellular responses to pathogen invasion,
cell death (hypersensitive reaction) may play a prominent
role, especially in incompatible interactions [28,44]. Spectrophotometric estimation of the extent of cell death
using Evans blue staining of necrotic cells showed the
rst occurrence of cell necrosis in resistant accessions of
L. chmielewskii after 24 hpi and after 48 hpi in L. hirsutum
(Fig. 3). Another plant defence line is generally associated
with increased passive resistance of cell wall, i.e. by
elevated rate of lignin deposition in cells of infected plant
site. No changes in the deposition of lignin were observed
in diseased or healthy plants during the rst 120 hpi (data
not shown).
3.4. Dynamics of POX activities
Correlating with H2O2 accumulation, the activity of
GPOX also increased in infected plants (Fig. 4). GPOX
activity increased steeply during the rst 816 hpi and in
late phases of infection (48120 hpi) in both resistant
accessions. In contrast, the enzyme activity in the
susceptible genotype increased slowly from 24 hpi. At
120 hpi, the activity of cytosolic GPOX reached 160 nkat/g
fresh wt in the case of L. esculentum cv. Amateur, which is
about half the activity observed in both resistant accessions

Hydrogen peroxide (mol/g FW)

600
L. esculentum
cv. Amateur

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum(LA 2128)

400

200

//
0

16

24

//
48 120

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

Fig. 1. Time course of hydrogen peroxide concentration in leaf tissues of Lycopersicon spp. accessions after inoculation by O. neolycopersici. , infected,
n, control plants.

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26

Free phenols (mol/g FW)

2000
L. esculentum
cv. Amateur

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum(LA 2128)

1500

1000

500

//
0

16

24

//
48 120

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

Fig. 2. Time course of the free phenols level in leaf tissues of Lycopersicon spp. accessions after inoculation by O. neolycopersici. , infected, n, control
plants.

A600 (cell death)

2.5
L. esculentum
cv. Amateur

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum
(LA 2128)

1.5
1
0.5
0
0

// //
8 16 24 48 120 0

// //
8 16 24 48 120 0
Time (hpi)

// //
8 16 24 48 120

Fig. 3. Evaluation of cell death by monitoring the uptake of Evans blue

by leaf tissues of Lycopersicon spp. accessions during the infection process
of O. neolycopersici. Uptake of vital dye was quantied by spectrophotometry. , infected, n, control plants.

(360 nkat/g fresh wt in L. chmielewskii and 280 nkat/g fresh

wt in L. hirsutum, respectively). Cytosolic forms of GPOX
comprised the major part of the total enzyme activity,
1.54 times higher than membrane- and ionically bound
forms, depending on genotype. In highly resistant L.
hirsutum, the activity of membrane-bound POX was almost
comparable with the activity of the cytosolic form. In the
case of L. chmielewskii, observed changes of membranebound POX were 4 times lower compared to cytosolic
activity and 2.5 times lower than membrane-bound GPOX
in L. hirsutum. The infection process had no inuence on
ionically bound POX activity in L. esculentum cv.
Amateur. In resistant accessions, the character of changes
of ionically bound and membrane-bound POXs was
similar, but the values of membrane-bound activities were
slightly higher.
Two-to-eleven-fold lower values of SPOX activity were
characteristic for susceptible L. esculentum cv. Amateur in
comparison with healthy resistant accessions. Changes of
SPOX activity during infection process were detected only
in resistant tomato accessions namely after 48 hpi (Fig. 5).
In the case of SPOX, a two-phase increase of the enzyme
activity was not observed. At 120 hpi, the activity of the
enzyme of moderately resistant L. chmielewskii increased

by up to 814% (cytosolic form), 770% (membrane-bound

form) and 357% (ionic-bound form) of the control (the
activity of the control sample was taken as 100%). In
comparison with L. chmielewskii, signicantly lower values
of SPOX activity were detected in highly resistant L.
hirsutum (350% above the control-cytosolic form, 260%
membrane-bound form, 240% ionically bound form).
The activity of cytosolic APX was greatly elevated in
infected tissue of both resistant tomato accessions (Fig. 6),
with the characteristic two-phase increase peak. In
moderately resistant L. chmielewskii, the most dramatic
changes of APX activity were detected from 48 hpi (3 times
higher than in control plants), while in highly resistant
L. hirsutum, the rst substantial increase was observed by
20 hpi with maximum activity at 48 hpi (about 7 times
higher than in control plants).
The extent of cell damage caused by the production of
reactive radicals and oxidative stress related to plant
response to pathogen infection can be estimated by the
determination of the products of membrane lipids peroxidation. Lipid peroxidation occurring during the infection
process was detected in all the studied genotypes. Increased
concentrations of malondialdehyde, the indicator of lipid
peroxidation, were observed from 24 hpi in L. hirsutum and
from 48 hpi in L. chmielewskii and L. esculentum (Fig. 7).
3.5. Dynamics of CAT, SOD and AO activities
The rst increase of CAT activity was found at 412 hpi
in all the studied accessions (early oxidative burst).
A second, more dramatic increase came at 24 hpi in
L. chmielewskii, 48 hpi in L. esculentum and 120 hpi in
L. hirsutum. At 120 hpi, the highest CAT activity was
observed in L. chmielewskii (423 nkat/g fresh wt), while in
L. hirsutum (resp. L esculentum) the detected activity
reached only 194 nkat/g fresh wt (resp. 140 nkat/g fresh wt)
(Fig. 8).
No signicant changes of SOD and AO activities were
detected during the time of our experiment (0120 hpi) in
any of the studied tomato accessions (data not shown).

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27

GPOX (soluble).(nkat/g FW)

400
L. esculentum

L. chmielewskii
(LA 2663)

cv. Amateur

300

200

100

//
0

16

24

//
48 120

(A)
GPOX (membrane-bound).(nkat/g FW)

L. hirsutum
f. glabratum(LA 2128)

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

300
L. esculentum

L. chmielewskii
(LA 2663)

cv. Amateur

L. hirsutum
f. glabratum(LA 2128)

200

100

//
0

16

24

//
48 120

(B)

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

GPOX (ionic-bound).(nkat/g FW)

150
L. chmielewskii
(LA 2663)

L. esculentum
cv. Amateur

L. hirsutum
f. glabratum(LA 2128)

100

50

//
0

16

24

//
48 120

(C)

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

Fig. 4. Time course of the guaiacol peroxidase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici. (A) Soluble,
(B) membrane-bound, (C) ionic-bound GPOX. , infected, n, Control plants.

3.6. PAs in leaf tissue

4. Discussion

Concentrations of free PAs are presented as mg of PA per

g fresh wt of the plant tissue. Different tomato genotypes
contained different amounts of PAs. The level of spermidine was 1.51.9 times higher than the level of putrescine
and 4.56.7 times higher than the level of spermine, thus
making up the major part of total free PA content in each
tomato genotype. The spermidine level increased in two
phases during the experiment: the rst peak appeared
416 hpi and the second at 2448 hpi. This two-phase
increase was characteristic for all Lycopersicon spp.
accessions (Fig. 9).

The interaction of tomato powdery mildew with host

plants have been intensively studied [29,30,45]. These
studies showed a broad range of responses from susceptibility through to resistance and conrmed the HR as an
important and prevailing resistance mechanism in the
defence of Lycopersicon spp. against O. neolycopersici.
However, very little information regarding biochemical
changes accompanying the various responses of tomato to
isolates of O. neolycopersici has been published. We have
aimed to broaden the knowledge in this eld [31] by
carrying out a detailed study of ROS formation, changes in

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28

SPOX (soluble).(nkat/g FW)

500
L. esculentum
cv. Amateur

400

L. chmielewskii
(LA 2663)

300
200
100
0

//
0

16

24

//
48 120

SPOX (membrane-bound).(nkat/g FW)

(A)

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

200
L. esculentum
cv. Amateur

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum (LA 2128)

150

100

50

//
0

16

24

//
48 120

(B)

SPOX (ionic-bound).(nkat/g FW)

L. hirsutum
f. glabratum (LA 2128)

80

L. esculentum
cv. Amateur

//
//
16 24 48 120
Time (hpi)

L. chmielewskii
(LA 2663)

//
0

16

24

//
48 120

L. hirsutum
f. glabratum (LA 2128)

60

40

20

//
0

16

//
48 120

24

(C)

// //
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

Fig. 5. Time course of syringaldazine peroxidase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici. (A) Soluble,
(B) membrane-bound, (C) ionic-bound SPOX. , infected, n, control plants.

400

APX (nkat/g FW)

L. esculentum
cv. Amateur

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum(LA 2128)

300

200

100

//
0

16

24

//
48 120

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

Fig. 6. Time course of ascorbate peroxidase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici. , infected, n control
plants.

ARTICLE IN PRESS
Lipid peroxidation (mol MA/g FW)

K. Tomankova et al. / Physiological and Molecular Plant Pathology 68 (2006) 2232

29

8
L. esculentum
cv. Amateur

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum(LA 2128)

0
0

16

24

//
//
48 120

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48

120

Fig. 7. Dynamics of lipid peroxidation in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici. , infected, n, control plants.

CAT (nkat/g FW)..

500
L. esculentum
cv. Amateur

400

L. chmielewskii
(LA 2663)

L. hirsutum
f. glabratum(LA 2128)

300
200
100
0

//
0

16

24

//
48 120

//
//
16 24 48 120
Time (hpi)

//
0

16

24

//
48 120

Fig. 8. Time course of catalase activity in leaves of Lycopersicon spp. accessions after inoculation by O. neolycopersici. , infected, n, control plants.

Spermidine (g/g FW)

1
0.8
0.6

L. esculentum cv. Amateur

L. chmielewskii (LA 2663)
L. hirsutum f. glabratum (LA 2128)

0.4
0.2
0
0

12

16
20
Time (hpi)

24

48

120

Fig. 9. Time course of spermidine concentration (mg/g fresh wt) in leaves

of Lycopersicon spp. accessions after inoculation by O. neolycopersici.

the activities of enzymes related to ROS metabolism and

the execution of the HR. We compared the resistance
responses of the moderately resistant accession L. chmielewskii (LA 2663), the highly resistant L. hirsutum f.
glabratum (LA 2128) and susceptible L. esculentum cv.
Amateur.
ROS are rapidly produced by plants as a defence
response to pathogen attack [16]. The role of H2O2 as a
signal molecule during HR is well established [46].
Inoculation of tomato leaves with O. neolycopersici caused
marked changes in the level of hydrogen peroxide in plant

tissue accompanied by increase in activity of H2O2scavenging enzymes. In correlation with recently published
results, regarding histochemical study of ROS generation
in the same plantpathogen model [31], rapid mobilization
of defence responses including oxidative burst, demonstrated by a two-phase increase in the level of hydrogen
peroxide, was characteristic for both resistant Lycopersicon
accessions (Fig. 1). It is supposed that the rst increase of
H2O2 concentration within 8 hpi participates in signal
transduction and retardation of the pathogen development.
However, at highly susceptible L. esculentum after rst hpi,
the production of superoxide anion was mostly observed
[31]. In contrast, H2O2 production was not recorded at
susceptible L. esculentum. It is evident that there is a
correlation between the rst observed H2O2 burst [31] and
the time of conidia germination (39 hpi) and the formation appressoria (612 hpi) [30]. The second more extensive
increase of H2O2 concentration in resistant accessions is
connected with various peroxidative reactions leading to
increased defence of infected plants via an HR. Interestingly, the constitutive levels of H2O2 concentration
measured in control plants were 2 orders of magnitude
higher in resistant accession compared to the susceptible.
Thus, both the constitutive level of H2O2 and the plant
capacity to increase H2O2 concentration might contribute
to increased resistance to pathogen attack. The timing of

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30

K. Tomankova et al. / Physiological and Molecular Plant Pathology 68 (2006) 2232

the HR in resistant accessions and its absence in susceptible

plants corresponds to changes of H2O2 concentration and
GPOX activities, respectively [31].
Organisms protect themselves against oxidative stress by
the synthesis of various antioxidant enzymes. The major
Ros-scavenging enzymes of plants includes SOD, GPOX,
APX and CAT. APX might be responsible for the ne
modulation of ROS for signalling, whereas CAT might be
responsible for the removal of the excess of ROS during
stress [16]. In our experiments, CAT activity increased in
two phases in all tomato accessions (Fig. 8). At the time of
the rst increase phase, no changes in H2O2 concentration
were detected except in susceptible L. esculentum cv.
Amateur. It is supposed that the scavenging activity of
CAT corresponds to the rapidity and intensity of H2O2
production in this case. During the second phase, the
highest increase of the CAT activity was found in infected
plants of the moderately resistant accession L. chmielewskii
(LA 2663). In the case of L. hirsutum, the fact that the
activity of CAT was relatively lower may be one of the
factors leading to high H2O2 accumulation. In contrast,
APX activity increased particularly in the second phase in
highly resistant L. hirsutum f. glabratum (LA 2128) (Fig. 6).
These results suggest a complex relationship between the
resistance of the studied Lycopersicon spp. genotypes and
the activity of specic H2O2 scavenging enzymes.
Diversity of the reactions catalysed by plant POXs
accounts for the implication of these proteins in a broad
range of physiological processes such as auxin metabolism,
lignin and suberin formation, cross-linking of cell wall
components, defence against pathogens or cell elongation.
POX activity can be easily detected in the whole lifespan of
various plants: from the early stage of germination to the
nal step of senescence, through the control of cell
elongation, defence mechanisms, and several other roles
[47]. GPOX is one of the important enzymes involved in
defence response of infected plants. A two-phase increase
in GPOX activity was observed, however, only in resistant
accessions. The major increase in activity was in the soluble
cytosolic fraction (Fig. 4). In the susceptible tomato
genotype, very low changes in GPOX activity were
observed in the cytosolic fraction. Considerable changes
of activities of GPOX were found in moderately resistant
tomato. It is supposable that the soluble and bound POXs
may play distinct roles in pathogenesis. Thus, the wallbound enzymes might have a direct role in cell wall
stiffening and lignication, while the cytosolic enzymes
might have an important role in ROS scavenging. The
correlations between intensity of cell death, high content of
H2O2, increasing of soluble and bound GPOX activity, and
high activity of SPOX enzyme (taking part in lignication
process) were observed in genotype with resistant response
(L. hirsutum f. glabratum (LA 2128)) in contrast to
susceptible L. esculentum cv. Amateur.
Phenols and polyphenols are widely distributed among
higher plants and represent an important component of
their defence mechanism [48]. The antioxidant properties

(redox potential) of some polyphenols were shown to be

lower than that of ascorbate [49]. Detailed analysis of
soluble and cell-wall-bound phenols in barley leaf epidermis inoculated with powdery mildews revealed a differential accumulation of the soluble antifungal compound
p-coumaroyl-hydroxyagmatine, especially in resistant
plants [44]. In our case, it is evident that the level of
bound phenols did not signicantly increase during the
120 hpi in any of the studied genotypes; on the contrary,
the level of free phenols became elevated in all genotypes,
most dramatically in the susceptible L. esculentum cv.
Amateur. The relatively lower concentrations of free
phenols in the resistant accessions could not be easily
explained by their consumption by POXs (e.g. SPOX) [49],
as we did not observe increased bound phenols.
Lignin is a phenolic polymer forming structural defence
barriers against pathogen attack and is synthesized from
three phenylpropane alcohols: coniferyl, sinapyl and
coumaryl alcohol. The oxidation of syringaldazine, a lignin
monomer analogue, is involved in the synthesis of lignin
and suberin [50]. In recent experiments (Fig. 5), the activity
of SPOX at 120 hpi was shown to be highly increased, but
only in tomato accessions showing certain degree of
resistance. This result indicates the possible absence of
lignication process, one of the typical plant defence
responses, in susceptible L. esculentum cv. Amateur. On
the basis of the very close relationship between SPOX
activity and the content of free phenols found in resistant
accessions, it might be supposed that lignication will be
intensive mainly in moderately resistant tomato genotypes.
In correlation with our recent results, experiments with
fusaric acid, a non-specic toxin of many pathogenic
Fusarium species, revealed an increase of SPOX on the
second day after the treatment of plant tissue [51]. Changes
in the lignin content during the experiment time (only
120 h) were not detected in any tomato accession either by
biochemical or by histochemical methods [31].
The peroxidation of unsaturated lipids of biological
membranes is the most prominent symptom of oxidative
stress in animals and plants. The production of lipid
peroxides has been proved to be induced by pathogens [22]
and the subsequent products have been shown to possess
antibacterial properties [52] and signalling function [53]. In
a recent experiment, a remarkable increase in lipid
peroxidation measured as production of malondialdehyde
was observed 48 hpi in all accessions (Fig. 7). The highest
changes in lipid peroxidation were demonstrated in
susceptible L. esculentum. It seems that the intensive lipid
peroxidation coupled with the small changes in activities of
antioxidant enzymes during the infection correlate with the
low defence ability of the sensitive accession L. esculentum.
The results of Gobel et al. [22], which focused on the
infection of Solanum tuberosum L. cv. Desiree by Pseudomonas syringae pv. maculicola from the viewpoint of lipid
peroxidation and development of HR, are in agreement
with our observations in resistant tomato. The formation
of lipid hydroperoxides is considered a crucial event in the

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K. Tomankova et al. / Physiological and Molecular Plant Pathology 68 (2006) 2232

development of hypersensitive cell death, appears to be in

major part facilitated by a specic enzyme 9-lipoxygenase
which directly oxidizes fatty acids by molecular oxygen
[22].
Antistress effects and antisenescence actions of diamines
(putrescine, cadaverine), triamine (spermidine) and tetraamine (spermine) have been explained by their ability to
stabilize the intramolecular structure of nucleic acids and
negatively charged plasma membranes by surface phospholipid binding [54]. PAs act as antioxidants by inhibiting
lipid peroxidation: PAs are able to form a ternary complex
with iron and the phospholipid polar heads that may
change the susceptibility to autoxidation and inuence of
Fe2+ and thus change the ability to form free oxygen
radicals [32]. Studies of the PA spectrum under stress
conditions were usually focused to an increase in PA level
[55] but as proved by Politycka and Kubis [56], the level of
PAs under conditions of salt stress or after treatment with
phenol solutions may also decline. Spermidine was proved
to accumulate in intercellular spaces and induce pathogenesis related proteins during HR [57]. In the Lycopersicon
spp.O. neolycopersici interaction, spermidine represented
the major portion of the total free PA content in each
tomato genotype. Its two-phase increase was characteristic
for all Lycopersicon spp. accessions. The content of free
PAs in plants can be decreased through their binding with
other compounds under the inuence of oxidizing enzymes
[58] and one of the forms of PA conjugates are those with
hydroxycinnamic acids. Compared with the concentration
of spermidine, the level of putrescine is lower and because
of its localization in cytoplasm and vacuoles [59],
putrescine has to be transported to the apoplast before it
is oxidized by AO. The participation of AO, enzyme
degrading of PA [58], was not conrmed in defence
response of tomato. In this case, it is supposed that AO
is not a signicant source of the H2O2 required for the POX
activity which is involved in the synthesis of lignin/suberin
during pathogenesis.
In conclusion, recent results proved the participation of
H2O2 and its scavenging enzymes in defence reactions of
this hostpathogen interaction. Variability in the intensity
and timing of the studied events gives the evidence that the
defence mechanisms are linked to different resistance
features of the studied tomato accessions represented by
their ability to trigger ROS production, expression of
antioxidant enzymes, lipid peroxidation and development
of HR. Results on hydrogen peroxide formation, expression of POX activity and execution of HR are in a good
correlation with previously published histochemical experiments [31].
Acknowledgments
The authors thank Prof. A. Slusarenko for critical
reading of the manuscript. This work was supported by the
Grant MSM 6198959215 from the Ministry of Education,
Youth and Sports of the Czech Republic.

31

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