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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.
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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
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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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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.
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
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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
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.
4. Discussion
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500
L. esculentum
cv. Amateur
400
L. chmielewskii
(LA 2663)
300
200
100
0
//
0
16
24
//
48 120
(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)
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
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.
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Lipid peroxidation (mol MA/g FW)
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.
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.
1
0.8
0.6
0.4
0.2
0
0
12
16
20
Time (hpi)
24
48
120
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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