The role of salicylic acid in defense response of tomato to root-knot nematodes

Abstract

Salicylic acid (SA) is involved in hypersensitive reactions of plants to incompatible pathogens and in systemic acquired resistance (SAR) after the attack of necrosis-inducing pests. The possible involvement of SA in defense responses of tomato to root-knot nematodes (Meloidogyne spp., RKNs) was investigated. SA was found not to be responsible for the inhibition of catalase (CAT) detected in the early stages of Meloidogyne-tomato incompatible interactions. CAT extracted from leaves was inhibited only after treatment of the seedlings with SA concentrations as high as 4 mM. Most of the amount of free SA found in plants after SA treatment was detected in the leaves. SA (0.2 mM) was found to cause a competitive inhibition of CAT only at high substrate (H2O2) concentrations. Under different conditions it did not affect, or even enhanced, the enzyme activity. Therefore, it is suggested that SA-mediated CAT inhibition does not operate early in resistance against RKN in tomato, although it might have a role in the consequent lesion formation. Plant uptake of SA was detected by immersion of roots of 1-month-old seedlings in aqueous solutions of SA and SA plus a soil humic acid. Considering the low level of free SA retained by roots, the capacity of exogenously provided SA to act as an elicitor of resistance to root pests is considered unlikely.

Full text

Physiological and Molecular Plant Pathology 68 (2006) 69–78
The role of salicylic acid in defense response
of tomato to root-knot nematodes
Sergio Molinari
a,
, Elisabetta Loffredo
b
a
Institute of Plant Protection, National Research Council, CNR, 70126 Bari, Italy
b
Department of Agro-forestal and Environmental Biology and Chemistry, University of Bari, 70126 Bari, Italy
Accepted 3 July 2006
Abstract
Salicylic acid (SA) is involved in hypersensitive reactions of plants to incompatible pathogens and in systemic acquired resistance
(SAR) after the attack of necrosis-inducing pests. The possible involvement of SA in defense responses of tomato to root-knot nematodes
(Meloidogyne spp., RKNs) was investigated. SA was found not to be responsible for the inhibition of catalase (CAT) detected in the early
stages of Meloidogyne-tomato incompatible interactions. CAT extracted from leaves was inhibited only after treatment of the seedlings
with SA concentrations as high as 4 mM. Most of the amount of free SA found in plants after SA treatment was detected in the leaves.
SA (0.2 mM) was found to cause a competitive inhibition of CAT only at high substrate (H
2
O
2
) concentrations. Under different
conditions it did not affect, or even enhanced, the enzyme activity. Therefore, it is suggested that SA-mediated CAT inhibition does not
operate early in resistance against RKN in tomato, although it might have a role in the consequent lesion formation. Plant uptake of SA
was detected by immersion of roots of 1-month-old seedlings in aqueous solutions of SA and SA plus a soil humic acid. Considering the
low level of free SA retained by roots, the capacity of exogenously provided SA to act as an elicitor of resistance to root pests is
considered unlikely.
r2006 Elsevier Ltd. All rights reserved.
Keywords: Catalase; Humic acid; Hypersensitive reaction; Lycopersicon esculentum;Meloidogyne spp.; R-genes; Salicylic acid
1. Introduction
Tomato (Lycopersicon esculentum Mill.) resistance to
root-knot nematodes (RKNs, Meloidogyne spp.), conferred
by the gene Mi-1 [1], is associated with a localized
hypersensitive response (HR) by the cells at the site of
infection [2]. RKNs enter the roots as motile second stage
juveniles (J2) and migrate intercellularly to the vascular
cylinder where they start to feed on living cells in the zone
of differentiation [3]. In resistant plants, localized cell death
of the root tissue surrounding the nematode occurs, thus
preventing the juvenile developing into the enlarged, egg-
laying adult female [4]. The biochemical events which
follow recognition and lead to HR in incompatible
Meloidogyne-tomato interactions are not well characterized
[5,6].
The involvement of SA in SAR [7] and disease resistance
[8] in plants has been extensively studied. In particular, SA
has been shown to play a crucial role in the induction of
HR [9]. Conversely, SA mediates SAR but not HR in
tobacco treated with the elicitor PB90 from the cotton
blight agent Phytophthora boehmeriae [10]. The mechan-
isms by which SA may induce a defense response against
pathogens have mainly been investigated in Arabidopsis
and rely on NPR1 activation which promotes PR gene
expression [11]. However, enhancement of the SA signal
may also occur through a signal amplification loop
involving reactive oxygen species (ROS) [12]. Rapid
ROS production during the oxidative burst and an HR
are defense responses that are considered hallmarks of
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doi:10.1016/j.pmpp.2006.07.001
Abbreviations: APX, ascorbate peroxidase; CAT, catalase; HA, humic
acid; HR, hypersensitive reaction; PMSF, phenylmethansulfonyl fluoride;
RKNs, root-knot nematodes; SA, salicylic acid; SAR, systemic acquired
resistance

Corresponding author. Present Address: Istituto per la Protezione
delle Piante, CNR, Via G. Amendola 122/D, 70126 Bari, Italy,
Tel.: +39080 5929239; fax: +39080 5580468.
E-mail address: s.molinari@ba.ipp.cnr.it (S. Molinari).

gene-for-gene resistance [11]. SA is able to inhibit the
hydrogen peroxide (H
2
O
2
)-degrading activity of some
catalase (CAT) isoenzmes, thus leading to an increase in
the level of H
2
O
2
, which is generated by the HR-associated
oxidative burst [13].H
2
O
2
has been recognized as a
diffusible signal for gene activation in HR, a trigger for
hypersensitive cell death, a strong antimicrobial molecule
[14], as well as causing induction of PR-1 gene expression
and conditioning SAR [15]. However, many reports
evidenced that H
2
O
2
may not be a second messenger of
SA in SAR signaling [16].
It has already been reported that SA is somehow
involved in the Mi-1-mediated defense responses to RKNs
in tomato [17], although it is still unclear at which stage of
the interaction and by which mechanism it may act. On the
other hand, the ability of exogenously applied SA to induce
resistance to RKNs in tomato is controversial, and, in
some instances, it seems to be linked to the means of
application. Generally, SA treatment seems not to sig-
nificantly limit the degree of J2 infestation, although it may
have an inhibiting effect on the nematode reproduction
index [18–20]. Seedlings soil-drenched with SA solutions at
high concentrations (10 mM) and inoculated with RKNs
did not show any decrease of root galling compared with
untreated inoculated seedlings, whilst reproduction of the
parasites, in terms of number of eggs per root system, was
partially restricted; SA sprayed on seedlings did not have
any effect on nematode infestation [20].
Most of the studies carried out so far on the ability of SA
to elicit SAR come from leaves challenged with necrosis-
forming incompatible pathogens [7] or leaves treated with
exogenous SA [21]. Moreover, it was reported that SA
produced in roots by application of Pseudomonas aerugi-
nosa 7NSK2 could induce systemic resistance to Botrytis
cinerea in tomato [22]. Low amounts of SA applied to
radish roots were able to decrease the percentage of
diseased plants due to Fusarium wilt, although both the
damage of xylem tissue in roots and the yellowing and/or
browning of the leaves were considered as disease
symptoms [23]. Other reports rather indicate the ineffec-
tiveness of SA to elicit SAR in roots [24,25]. Therefore, it is
currently of great interest to establish the role, if any, of SA
in defense responses to root diseases, such as those caused
by nematode attack. Furthermore, it is still unclear why
externally provided SA does not limit the level of RKN
infestation in tomato roots as efficiently as it does against
pests attacking leaves. Moreover, humic acids (HA), which
are ubiquitous in any natural and cultivated soil and are
able to modulate the biological action of SA in young
plants [26], were expected to play a role in SA bioavail-
ability, and its uptake and accumulation in plant tissues.
To address these issues, we compared the capability of
SA to act as inhibitor of CAT in leaves and roots of two
isogenic tomato lines, susceptible and resistant to RKNs,
and determined the amounts and distribution of free SA
between roots and leaves after the treatment of plants with
SA alone or in combination with a soil HA. Our results
show that the level of both SA and H
2
O
2
is crucial for SA
to act as inhibitor of CAT, and, consequently, explicate
one of its possible roles in defense reactions of tomato to
RKNs; moreover, it is suggested that endogenous SA in
roots may be locally involved in lesion formation, which is
part of Mi-mediated resistance against RKNs.
2. Materials and methods
2.1. Plant and parasite material
Seedlings of the tomato near-isogenic lines Motelle,
which carries the gene Mi-1 conferring resistance to RKNs
and potato aphids [27], and the susceptible Moneymaker
were used for inoculation with Meloidogyne incognita.
Plants were cultivated at 24–27 1C in a glasshouse under a
12 h light/12 h dark cycle and watered daily with Hoag-
land’s nutrient solution. The RKNs J2 used for inoculation
were obtained by incubation of egg masses in tap water at
27 1C. One virulent line of M. incognita, which had
previously been selected on Mi-bearing tomato (cv.
VFN8) in a greenhouse and named SM1, was also used
to infest resistant roots. Inoculation was carried out when
seedlings had reached the four true-leaf stage, whereas
older seedlings (one month after sowing, approx. 20 cm
shoot length) were used for SA uptake and CAT
purification experiments.
2.2. Plant treatments and inoculation
Groups of 5 seedlings of cv. Motelle, thoroughly washed
with tap water, were transferred into 4 sets of 8-cm clay
pots each containing: (i) a suspension of active avirulent J2
(500/seedling); (ii) a suspension of virulent SM1 (150/
seedling); (iii) 10 ml of a solution of 1 mM SA; (iv) 10ml of
tap water, this set was used as a control. All the pots were
filled with sterilized quartz sand, watered with Hoagland’s
nutrient solution and kept for 48 h in a growth chamber at
25 1C, with constant illumination. Each set consisted of 6
pots, the experiment was repeated twice.
Groups of 3 resistant tomato seedlings were incubated in
200 ml of 1, 2 and 4 mM SA before the detection of CAT
activity in plant tissues. Incubation consisted in the
immersion of the roots in the test solutions contained in
plastic beakers. Seedlings were kept in a growth chamber at
25 1C under a 12 h light/12 h dark cycle (irradiance
200 mmol photons m
2
s
1
). SA uptake experiments were
carried out with resistant and susceptible seedlings
incubated, as described above, in 1 mM SA and a solution
in which 1 mM SA and 200 ppm HA were allowed to
interact for 16 h under stirring at room temperature
(20721C). Seedlings incubated in distilled water served
as controls. All the test solutions were adjusted to pH 6.5.
The HA used in this work was isolated from an alluvial
German soil according to conventional methods [28] and
stored in homogenized, freeze-dried H
+
-form.
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2.3. Protein extraction for enzyme activity assays
Seedlings from each pot, or plastic beaker, were
thoroughly rinsed with tap water. Roots and leaves,
excised from the shoots, were dried, weighed and collected
on ice. Plant tissues were then cut by a scalpel into very
small pieces to which appropriate volumes of a grinding
buffer consisting of 0.1 M potassium-phosphate buffer, pH
7, 10% glycerol, 10 mM ditiothreitol and the protease
inhibitors PMSF (1 mM), pepstatin (1 mM) and leupeptin
(1 mM) were added. Tissues were homogenized in chilled
glass Potters with pestles connected to a motorized drive.
The coarse homogenates were filtered through 4 layers of
gauze, uniformly distributed into 1.5-ml Eppendorfs and
centrifuged at 10,000 rpm for 3 min in a bench centrifuge.
Supernatants were used directly for enzyme activity assays.
Samples for specific use were dialyzed against 0.1 M KCl
overnight or ultrafiltered at 4 1C through YM-ultrafiltra-
tion membranes (10,000 molecular weigh cut off, Amicon
Co.) in Centricon-10 micro-concentrators.
2.4. SA uptake determination
Aliquots of SA and SA/HA solutions were collected
before and after the incubation of the plants and stored at
about 3 1C until further analysis; the volume of the
solutions before and after the incubation of plants was
also measured. Plants were thoroughly rinsed with distilled
water, then roots and leaves were separated from shoots,
dried and weighed. Tissues were immersed in liquid
nitrogen and ground with a mortar and pestle; samples
were then stored at 80 1C. Before chromatographic
analysis of SA, powdered plant samples were prepared
according to Wu et al. [29]. Briefly, the procedure was
modified as follows. An aliquot of 0.2 g plant tissue was
macerated with 10 ml of 1 mM HCl for 5 min in glass flask
under mechanical shaking at 140 rpm (Universal Table
Shaker mod. 709, Thermo Electron Corporation, San Jose,
CA, USA). Then, the macerate was sonicated at 5 1C for
15 min (Sonics Vibracell mod. VCX 500, Newtown, CT,
USA), and centrifuged at 15,000gat 5 1C for 15 min to
remove the debris (Biofuge Stratos Heraeus, Germany).
The supernatant was collected and extracted twice with
20 ml of ethyl acetate. The ethyl acetate fractions were
combined and evaporated to dryness on a rotary evapora-
tor under reduced pressure at 40 1C. Finally, the residue
was dissolved in 0.5 ml of water/acetonitrile/acetic acid at a
ratio of 67/32/1 (v/v/v) and stored at 4 1C until analysis.
HPLC analysis of SA was conducted according to Loffredo
et al. [26] by using a Spectra System pump (Thermo
Electron Corporation, San Jose, CA, USA) equipped with
a Rheodyne 7125 injection valve fitted with a 20 ml loop. A
Supelcosil
TM
LC-18 chromatographic column (250 mm 
4.5 mm 5mm) and a mobile phase of water/acetonitrile/
acetic acid at a ratio of 67/32/1 (v/v/v) were used. At the
flow rate of 1 ml/min, the retention time for SA was about
11.6 min. The product was quantified with UV detection at
280 nm by using a Spectromonitor 3200 (Thermo Electron
Corporation, San Jose
`, CA, USA) and an external
standard (SA, purityX99%, Sigma-Aldrich, Milano,
Italy). Tests on solution and plant tissue samples were
repeated twice.
2.5. Electrophoresis procedure and CAT activity staining
CAT isozymes were separated by isoelectric focusing on
mini-gels (3.6 cm separation zone) inserted into a Phast-
System
s
equipment (Amersham Bio., Piscataway, NJ,
USA), as already described [30,31]. Mini-gels were loaded
with 4 ml samples of plant tissue extract. Gels were
maintained at 15 1C and runs were stopped at 600 Vh,
which corresponded to approx. 30 min. Pre-programming
of the runs allowed a reliable reproducibility of the band
patterns of the samples. Gels were calibrated by using a
broad pI calibration kit (Amersham Bio., Piscataway, NJ,
USA) containing proteins from 3.5 to 9.3 pI. Relative
electrophoretic mobilities (Rm in cm) were plotted against
pIs and a straight line with a high correlation coefficient
(approx. 0.99) obtained. IEF gels were stained for CAT
activity after the method of Cardy and Beversdorf [32]:
they were soaked in 0.6% H
2
O
2
for 60 s, rinsed with
distilled water and immersed in 0.2% (w/v) potassium
iodide containing 0.1% (v/v) glacial acetic acid until
transparent zones of CAT activity appeared against a dark
yellow background. A second set of gels was incubated in
1 mM SA for 5 min before staining. Since the coloured
background tended to fade rapidly, gels were immediately
drained and scanned by means of a ScanJet II cx (Hewlett
Packard). Digital images were stored in the computer to be
printed.
2.6. Purification of CAT and determination of enzyme
activities
Purification of CAT started from 40 g leaves and 20 g
roots of tomato seedlings resistant or susceptible to RKNs.
Tissues were homogenized in liquid nitrogen and powder
dissolved in 1:5 (w/v) of an extraction buffer consisting of
0.1 M potassium-phosphate buffer, pH 7, 10% glycerol,
10 mM dithiotreitol and the protease inhibitors PMSF
(1 mM), pepstatin (1 mM) and leupeptin (1 mM). Suspen-
sions were ground further by a Polytron
s
PT-10-35
(Kinematica GmbH, Switzerland), filtered through 4 layers
of gauze and centrifuged at 9000gfor 15 min. Ammonium
sulphate 37% (w/v) was mixed to supernatants and the
solutions stirred at 0 1C for 2 h. Protein precipitates were
collected by centrifugation at 14,000gfor 25 min and
suspended in minimal volumes of the extraction buffer
modified with 25% glycerol and 1 mM dithiotreitol. Most
of the starting activity was recovered in these protein
precipitates and improvement of specific enzyme activity
(CAT units mg
1
prot.) was approx. 10-fold in roots and
100-fold in leaves. Aliquots of protein suspensions were
filtered through 0.5 mm Whatman filters and analysed by
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size exclusion HPLC through a 300 7.8 mm BIOSEP
SEC-S3000 column (phenomenex
s
). The column was
equilibrated with 50 mM phosphate buffer, pH 7.6, plus
0.15 M NaCl at a flow rate of 0.5 ml min
1
. The detector
was set at 280 and 407 nm by a dual channel signal mode
and 1 ml-fractions were collected. Two peaks of catalase
activity were recorded and their molecular weights
determined by calibrating the column by a calibration kit
(Sigma Co., USA) consisting of b-amylase, alcohol
dehydrogenase, bovine serum albumin, ovalbumin, carbo-
nic anhydrase, cytochrome c. The molecular weights of the
2 CAT isoforms were calculated as approx. 200,000 and
55,000 daltons; these values are in agreement with those
reported for catalase extracted from chard [33].
CAT activity of tissue extracts was measured as the
initial rate of disappearance of hydrogen peroxide [34],
using 20 mM H
2
O
2
and 25 ml sample in 0.1 M Na-
phosphate, pH 7.0 (0.5 ml final volume); the rate of H
2
O
2
disappearance was followed as decrease in the absorbance
at 240 nm and oxidation of 1 mmole H
2
O
2
min
1
(e¼0.038 mM
1
cm
1
) represented one unit of enzyme.
The spectrophotometric assay for ascorbate peroxidase
(APX) activity was performed after the method of Gerbling
et al. [35]. The reaction mixture contained 0.1 TES, pH 7.0,
0.1 mM EDTA, 1 mM ascorbate from fresh stock solution
(100 mM), 0.13 mM H
2
O
2
,25ml sample extract, in 0.5 ml
final volume. Decrease in absorbance at 298 nm was
followed as ascorbate was oxidized and one unit of enzyme
expressed the ability to oxidize 1 mmole ascorbate min
1
(e¼0.8 mM
1
cm
1
). The sensitivity to SA of the purified
CAT was tested by adding increasing concentrations of SA
(0.1–1 mM) to the above-mentioned reaction mixture for
CAT activity assay. For the calculation of the kinetic
parameters (K
m
,V
max
) of the purified enzyme, CAT
activity was assayed at different H
2
O
2
concentrations
(1–20 mM) in presence or absence of 0.2 mM SA. Catalases
from leaves were not active at the lowest concentrations of
H
2
O
2
used (1–2.5 mM). Lineweaver-Burk plots (double-
reciprocal plots, 1/vagainst 1/s) were made and kinetic
values determined as outlined in [36].
Protein content of the samples was determined according
to Lowry et al. [37].
3. Results
3.1. Changes in SA-sensitive enzymes of tomato tissues after
nematode infestation or SA treatment
Two days after seedling inoculation with an avirulent
population of M. incognita, CAT activity in roots of Mi-
bearing tomato was significantly inhibited (approx. 40%),
whilst no apparent decrease was detected in APX activity
(Fig. 1). Moreover, the specificity of CAT activity
inhibition was evident since inoculation of plants with a
virulent isolate of the parasite, which leads to a compatible
interaction, did not affect root CAT activity. The incuba-
tion of plants in 1 mM SA for 12 h did not result into any
inhibition of CAT activity extracted from excised roots
(Fig. 1A). Dialysis of root extracts impaired CAT activity
(results not shown); however, CAT activity detected in the
extracts after ultra-filtration was 14.674.0 and
9.972.0 units mg
1
protein (n¼37standard deviation),
for uninfested and avirulent M. incognita infested roots,
respectively.
The effect of exogenously supplied SA on CAT of
resistant tomato tissues was further investigated on
seedlings older than those used in the previous set of
experiments. A consistent inhibition of CAT activity in
ARTICLE IN PRESS
Fig. 1. Changes of antioxidant enzymes of roots due to RKN attack. Catalase (CAT) and ascorbate peroxidase (APX) activity, expressed as units mg
1
prot, after inoculation of Mi-bearing tomato seedlings with an avirulent population (inoculated) and a virulent population (Vir-inoculated) of
Meloidogyne incognita, as well as after 12 h incubation of the seedlings in 1 mM salicylic acid (SA-incubated); controls were uninoculated seedlings. Data
are means of two different experiments and each experiment was replicated six times. Values marked by the same letter in each plot do not differ
significantly, according to the least significant difference test (Pp0.05).
S. Molinari, E. Loffredo / Physiological and Molecular Plant Pathology 68 (2006) 69–7872

leaves was obtained only when plants were incubated in
4mM SA (Fig. 2); this treatment also caused marked
symptoms of phytotoxicity on leaves, whilst roots appeared
healthy (data not shown). Accordingly, the incubation of
plants in such high SA concentrations did not cause any
evident inhibition of CAT activity in roots. CAT activity
assays carried out on leaf extracts after dialysis revealed
that dialyzed extracts from control and treated leaves had
the same activity; therefore, it was likely that diffusible and
inhibiting amounts of SA, or other possible dialyzable
inhibitors produced as a result of the toxic action of SA,
were present in the leaf extract of treated plants. To have
direct evidence of transportation of SA from roots to
leaves, free SA was detected in tomato tissues after
immersion of the roots in SA solutions.
3.2. Detection of SA uptake by tomato plants
After 12 h immersion of the roots in 1 mM SA or 1 mM
SA plus 200 ppm HA, plants absorbed, respectively,
approx. 80% and 50% of the SA available in the solutions.
As expected, only a minimal percentage of the total
chemical absorbed was then detected in plant tissues as
free SA (Table 1). In fact, it is well known that SA is
present in plants mostly as its bound-form (b-O-D-glucosyl
SA, GSA) which is the major metabolite of exogenous and
endogenous SA in tobacco [38] and represents the 99.5% of
the total SA in potato [39].Table 1 shows that most of the
free SA accumulated in plants after SA treatment was
detected in leaves. The ratio between free SA detected in
leaves and that detected in roots was particularly high
when plants were treated with SA/HA solutions. Although
the SA absorbed by plants was much lower in the presence
of HA, the amounts detected in leaves were approx. 3-fold
(in susceptible plants) and 2-fold (in resistant plants) higher
than those from plants incubated without HA; conversely,
the amount of free SA retained by roots appeared to be
unchanged by the presence of HA in the medium.
3.3. Inhibition by SA of tomato CAT
The sensitivity of purified tomato CAT to SA inhibition
was then investigated. Generally, CAT extracted from
leaves was found to be less sensitive to SA inhibition than
root CAT (Fig. 3). SA (1 mM) completely inhibited root
CAT, whilst the same concentration caused approx. 60%
inhibition of leaf CAT. In contrast, at a concentration as
low as 0.1 mM, which has been reported as a likely level in
infected tissues [39], SA appeared to have a positive effect
on the activity of CAT extracted from resistant plants,
especially from roots; on the other hand, root and leaf
CAT from susceptible plants were only 20% inhibited.
If proteins from leaf extracts were separated by
isoelectrofocusing according to their net charge, five main
isoforms of catalase were clearly visible (Fig. 4). The span
of the pH gradient used allowed to identify the most
marked band of activity at pH 7.3; a consistent activity was
detected at the border of the separation zone around pH
4.0 (Fig. 4A). If gels were incubated in 1 mM SA before
staining (Fig. 4B), isoforms with the isoelectric point
around the neutrality were considerably inhibited in their
ARTICLE IN PRESS
Fig. 2. Changes of catalase activity due to SA treatment of plants.
Catalase (CAT) activity, expressed as units mg
1
prot. determined in roots,
leaves or dialyzed leaf extract (Dialyzed leaves) of Mi-bearing tomato
seedlings after 12 h incubation in 4 mM salicylic acid (SA-incubated) or in
distilled water (control). Line on each bar indicates standard deviation
(n¼5).
Table 1
Amounts of free salicylic acid (SA) absorbed by tomato plants susceptible and resistant to RKNs and detected in roots and leaves after 12 h incubation in
1 mM SA solution and mixture of 1 mM SA and 200 ppm humic acids (SA/HA)
Sample Free SA absorbed
a
(mgg
1
fresh wt) Free SA in roots (mgg
1
fresh wt) Free SA in leaves (mgg
1
fresh wt)
Susceptible
Control
b
¼¼ 0.4 0.3
SA-incubated 188.0 0.7 1.8
SA/HA-incubated 85.0 0.7 6.2
Resistant
Control ¼¼ 0.4 0.3
SA-incubated 257.2 1.8 3.3
SA/HA-incubated 80.4 2.0 6.7
a
Calculated as the difference between free SA present in solution before and after the incubation time.
b
Plants incubated in distilled water.
S. Molinari, E. Loffredo / Physiological and Molecular Plant Pathology 68 (2006) 69–78 73

activity, whilst activity of more acidic bands was un-
affected.
3.4. Nature of SA inhibition and kinetic parameters of
tomato CAT
As the degree of SA inhibition was previously detected at
saturating concentration of CAT substrate (H
2
O
2
20 mM),
which is far beyond the physiological level of living cells, a
study was undertaken to assay the nature of SA inhibition
on CAT across a larger range of H
2
O
2
concentrations
(from 1 up to 20 mM); the lowest concentrations of this
range better mimic the conditions of tissues undergoing
HR. SA seems to be a modest competitive inhibitor of
CAT from tomato leaves (Fig. 5A,B). It appears to be a
much more effective inhibitor of root CAT at the highest
H
2
O
2
concentrations tested; conversely, at lower concen-
trations, it appears not to inhibit, or even to positively
affect, the enzyme activity (Fig. 5C,D). Table 2 shows that
K
m
of CAT for H
2
O
2
is raised in leaves by the presence of
SA and, consequently, the affinity of the enzyme for its
substrate is reduced, as it occurs in competitive inhibition;
this reduction is much more pronounced for root CAT,
especially that extracted from resistant plants. In resistant
roots, the activity of the enzyme is very slightly lowered by
SA at saturating concentrations (V
max
).
4. Discussion
The data presented in this paper focus on the putative
role of SA in defense response of tomato to RKNs, in
relation to its ability to interact with CAT. SA was found
to inhibit the CAT activity of a SA-binding protein
(SABP1), leading to elevated levels of H
2
O
2
, and to
stimulate the lipase activity of a SABP2 [13,40] in tobacco.
The role of H
2
O
2
as a second messenger of SA in SAR
signaling is a matter of debate [16]. However, at the site of
infection of an incompatible plant–pathogen interaction,
SA levels can be sufficient to cause substantial inhibition of
CAT and ascorbate peroxidase, the other major H
2
O
2
-
scavenging enzyme, although no decrease in CAT activity
could be detected in pathogen-inoculated leaves [8].In
contrast, there has been increasing evidence that CAT of
resistant tomato roots is markedly inhibited in the early
stages of RKN attack [41–43], whilst in compatible
interactions such an inhibition does not occur [18].
Therefore, there was the actual possibility that CAT could
be inhibited by the endogenous SA produced in toma-
to–RKN incompatible interaction [44].
According to the data presented here, inhibition of CAT
in infested root extracts was not altered by ultra-filtration,
ARTICLE IN PRESS
Fig. 3. Inhibition of catalase by SA. Percentage of activity of purified
catalase (CAT) from leaves (A) and roots (B) of Mi-bearing (Res) and the
near-isogenic susceptible (Sus) tomato seedlings at different concentra-
tions (0.1–1 mM) of salicylic acid added in the reaction mixture; 100%
activity is referred to that of purified catalase in the absence of SA. Values
are means of three replicates7standard deviations.
Fig. 4. Inhibition of leaf catalase isozymes by SA. Isoelectrofocusing of
leaf extracts from Mi-bearing tomato seedlings. Mini-gels were stained for
catalase activity which appears as white bands over a dark background.
Before staining, mini-gels were incubated in distilled water (A) or in 1mM
SA solution (B). Gels were calibrated by using standard proteins from 3.5
to 9.3 pI.
S. Molinari, E. Loffredo / Physiological and Molecular Plant Pathology 68 (2006) 69–7874

which presumably releases samples from diffusible phenols,
such as SA. If CAT were inhibited by the endogenous SA
produced during nematode pathogenesis, a corresponding
inhibition of APX should be observed since SA is a known
inhibitor of APX, as well [45]; however, this did not occur.
Moreover, CAT extracted from roots was never inhibited
by SA treatment. It was also shown that when SA was
exogenously provided in large amounts to resistant plants,
the free SA retained by roots was only 1.8 mgg
1
fresh wt,
which means approx. 10 mM tissue average concentration,
assuming a 90% (w/w) tissue water content. Conversely,
root CAT should not be inhibited by SA, up to
concentrations as high as 100 mM, as indicated by the plot
in Fig. 3B. Hence, it is unlikely that the decrease of CAT
activity, detected in the early stages of tomato resistant
reaction to RKNs, may be due to SA inhibition. CAT
inhibition, at the early stages of nematode attack, may
rather result from a direct involvement of the enzyme in the
Mi-1-mediated signal transduction pathway. A rice cDNA
clone, whose product interacted with the cytoplasmic
kinase domain of resistance gene Xa21 in the yeast two-
hybrid system, was found to encode catalase B [46].
Additional CAT inhibition may be due to the high H
2
O
2
levels occurring in roots as an effect of HR, as it is well
established that CAT is inactivated by its own substrate
[47].
After SA feeding of the plants, the amounts of the free
chemical detected in the leaves was approx. 2-fold higher
than those in the roots; in the presence of HA, the
difference was much higher. This means that, under
physiological conditions, free SA is scarcely retained by
roots, moves upward and accumulates into the leaves.
Therefore, the role of SA in tomato resistance to RKNs
may be overestimated when the experimental system
ARTICLE IN PRESS
Fig. 5. Double-reciprocal (Lineweaver-Burk) plots of catalase activity as a function of H
2
O
2
concentration, in presence or absence of SA. Enzyme activity,
expressed as 1/v (units mg
1
prot) 100, was detected at different concentrations of H
2
O
2
(1–20 mM), expressed as 1/[H
2
O
2
]. CAT was purified from: (A)
resistant (Res) leaves; (B) susceptible (Sus) leaves; (C) resistant (Res) roots; (D) susceptible (Sus) roots. Black straight lines refer to CAT in absence of SA,
grey straight lines to catalase in presence of 0.2 mM SA in the reaction mixture. In plots of root CAT in presence of SA, the values refer to marked straight
lines in which concentrations of H
2
O
2
X5 mM were considered. At lower concentrations of substrate, SA effect on CAT activity of roots reflected a
different trend shown by hairlines, whereas leaf catalase was not active.
Table 2
Kinetic parameters of purified catalase extracted from leaves and roots of tomato susceptible (Sus) and resistant (Res) to root-knot nematodes, in absence
or presence of 0.2 mM SA
Leaves Roots
Sus +0.2 mM SA Res +0.2mM SA Sus +0.2 mM SA Res +0.2mM SA
K
m
68 88 95 135 4 6 3 6
V
max
2000 2000 2500 2500 30 19 27 23
K
m
is expressed in mM, V
max
in units mg
1
prot.
S. Molinari, E. Loffredo / Physiological and Molecular Plant Pathology 68 (2006) 69–78 75

chosen is artificial as roots cultured in vitro [17]. Moreover,
this finding casts doubts on the possibility that exogenous
SA may activate defense reactions to root diseases, such as
those caused by nematodes.
It is generally recognized that SA levels at the site of
infection in leaves may be elevated (100–150 mM) [8,38].In
resistant tomato attacked by RKNs most of the free SA
produced in roots was translocated to leaves [44]. This
study showed also that exogenously supplied free SA was
consistently translocated to leaves; roots seem to be able to
retain a low average level of SA. Such a level of SA has
been shown to sustain and not to inhibit CAT activity; on
the other hand, SA has already been reported to protect the
enzyme, depending on different conditions, such as H
2
O
2
concentration [47]. Such findings are in agreement with the
proposed antioxidant role for SA at the site of inflamma-
tion in animals [48], and indicate that it may limit the
spread of toxic peroxidative reactions in tissues not directly
involved in HR.
However, it cannot be ruled out that SA concentration
can rise locally and temporarily at the site of nematode
infection in vivo. Even if it occurred, SA would be able to
inhibit CAT only at elevated concentrations of H
2
O
2
.
Actually, in wheat, accumulation of H
2
O
2
was induced by
SA but not through inhibition of CAT [49]. Therefore, the
biological significance of SA-mediated inhibition of CAT
may be restricted to local lesion formation in infected tissue
[16], where localized H
2
O
2
concentrations may be very
high. Treatment of soybean suspension cells with high
concentrations of H
2
O
2
(6–10 mM) caused cell death,
which could be enhanced by SA or the other CAT inhibitor
3-aminotriazole [12]. Actually, it has been proved here that
the affinity for H
2
O
2
of CAT operating at high substrate
concentrations is drastically lowered by SA in roots
(increase of K
m
). In this way, under these conditions, a
putative localized increase of SA may result into a
consistent inhibition of H
2
O
2
degradation by CAT, thus
lowering the threshold level for H
2
O
2
to trigger cell death
and tissue necrosis, in agreement with the proposed role for
SA to potentiate, or lower the threshold for, specific
resistance responses in R gene-mediated resistance [12].
The fact that SA is scarcely retained by roots may
explain why externally provided SA in tomato roots is not
as efficient as in leaves in inducing defense reactions to pest
challenge. Feeding tomato with relatively low amounts of
SA (0.2 mM) resulted in 83% fewer lesion per leaf in plants
infested by Alternaria solani [50]. In contrast, SA does not
elicit any evident reaction in roots to limit the attack of the
invading nematodes; rather, it somehow causes a partial
restriction on time of nematode development and repro-
duction [18–20]. Considering the data provided here, it is
reasonable to suppose that external SA explicates its
biological functions where it physiologically accumulates.
This was particularly true when SA was allowed to interact
with HA before incubation of tomato plants. The uptake of
SA may be quantitatively and qualitatively different
whether SA is provided to plants as soil-drench or in
hydroponic cultures, for instance. Apparently, plant
uptake of SA is modulated by humic substances which
can raise the threshold concentration of its phytotoxicity
[26].
Separation of leaf CAT isozymes by isoelectrofocusing,
according to their net charge, confirmed that different
isozymes have different sensitivity to SA inhibition and this
may result in different physiological roles, as is the case for
CAT-1 and CAT-2 isozymes of maize [51]. This array of
isozymes characterized by a different SA-sensitivity may
explain why leaf CAT saves approx. 40% of its activity also
in the presence of elevated SA concentrations and the
difficulty to detect CAT inhibition in leaves attacked by
incompatible pests [16]. However, incubation of tomato
seedlings in 4 mM SA for 12 h rapidly led to severe
symptoms of phytotoxicity in leaves, which were associated
with a marked decrease of CAT activity. This finding
suggests that, even if the use of natural or synthetic
inhibitors of key antioxidant enzymes appears profitable in
practical pest management, an accurate investigation on
dosages and their impact on plant metabolism should be
previously carried out in order to avoid uneconomical costs
due to a possible low crop fitness.
Finally, the data shown in this paper indicate that SA is
not responsible for the CAT inhibition observed in the
early stages of Mi-mediated resistance to RKNs. The low
amounts of SA retained by roots as well as the kinetics of
SA–CAT interaction studied in vitro, suggest that SA may
have an anti-inflammatory-like role by limiting the spread
of toxic peroxidative reactions in tissues distant from the
site of infection, although it cannot be ruled out its
involvement in lesion formation at such a site. Further-
more, it resulted unlikely that SA may elicit in susceptible
roots a resistant-like response to restrain RKN invasion,
although it remains to be elucidated the mechanism by
which it is able to limit nematode development and
reproduction.
Acknowledgements
This work was supported by the Italian National
Council of Research (CNR) within the project ‘‘Sustain-
able development of agro-industrial system’’, subproject
AG-PO
4
-IPP-C
1
‘‘Development of innovative control
strategies for plant protection’’.
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