Plant Biology ISSN 1435-8603

RESEARCH PAPER

Salicylic acid treatment via the rooting medium interferes with

stomatal response, CO2 fixation rate and carbohydrate
metabolism in tomato, and decreases harmful effects of
subsequent salt stress
. Szepesi1, M. L. Simon2 & I. Tari1
P. Poor1, K. Gemes1, F. Horvath1, A
1 Department of Plant Biology, University of Szeged, Szeged, Hungary
2 Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary

Keywords
Hexokinase; photosynthetic rate; Solanum
lycopersicum; soluble sugars; stomatal
conductance.
Correspondence
I. Tari, Department of Plant Biology,
University of Szeged, H-6701 Szeged,
Kozepfasor 52, P.O. Box 654, Hungary.
E-mail: tari@bio.u-szeged.hu
Editor
R. Leegood
Received: 18 February 2010; Accepted: 20
February 2010
doi:10.1111/j.1438-8677.2010.00344.x
ABSTRACT
Salicylic acid (SA) applied at 10)3 M in hydroponic culture decreased stomatal con-
ductance (gs), maximal CO2 fixation rate (Amax) and initial slopes of the CO2 (A Ci)
and light response (A PPFD) curves, carboxylation efficiency of Rubisco (CE) and
photosynthetic quantum efficiency (Q), resulting in the death of tomato plants.
However, plants could acclimate to lower concentrations of SA (10)7)10)4 M) and,
after 3 weeks, returned to control levels of gs, photosynthetic performance and solu-
ble sugar content. In response to high salinity (100 mM NaCl), the pre-treated
plants exhibited higher Amax as a function of internal CO2 concentration (Ci) or
photosynthetic photon flux density (PPFD), and higher CE and Q values than salt-
treated controls, suggesting more effective photosynthesis after SA treatment.
Growth in 10)7 or 10)4 M SA-containing solution led to accumulation of soluble
sugars in both leaf and root tissues, which remained higher in both plant parts dur-
ing salt stress at 10)4 M SA. The activity of hexokinase (HXK) with glucose, but not
fructose, as substrate was reduced by SA treatment in leaf and root samples, leading
to accumulation of glucose and fructose in leaf tissues. HXK activity decreased fur-
ther under high salinity in both plant organs. The accumulation of soluble sugars
and sucrose in roots of plants growing in the presence of 10)4 M SA contributed to
osmotic adjustment and improved tolerance to subsequent salt stress. Apart from
its putative role in delaying senescence, decreased HXK activity may divert hexoses
from catabolic reactions to osmotic adaptation.

heavy metal (Mishra & Choudhury 1999) or salt (Tari et al.

INTRODUCTION
The phytohormone salicylic acid (SA) has been associated
with various physiological processes in higher plants and is
an important signalling molecule of biotic and abiotic stres-
sors. SA has been identified in exudates of soil-borne micro-
organisms and plant roots (Chou & Patrick 1976). Pathogen
infections that elicit SA accumulation and the SA signalling
pathway have been reported (Stout et al. 1999; Catinot et al.
2008), and the SA content of tissues may also increase in
response to abiotic stressors (Freeman et al. 2005; Pal et al.
2005). The interactions of SA with other abiotic or biotic sig-
nalling pathways, e.g. with jasmonic acid or ethylene signal-
ling, are time-dependent (Koornneef & Pieterse 2008;
Koornneef et al. 2008), suggesting that there may be an
interaction window between the hormonal effects (van Dam
2009). This means that the response timing is an important
aspect of the cross-talk between various abiotic or biotic
interactions.
Treatment with exogenous SA has been shown to decrease
the harmful effects of different abiotic stressors, such as UV-B
and ozone (Yalpani et al. 1994), chilling (Janda et al. 1999),
2002) and water stress (Bezrukova et al. 2001). This is related
to the generation of sublethal oxidative stress induced by SA
and enhanced expression and activity of redox-controlled
antioxidant enzymes.
SA induces rapid stomatal closure in several plant species
(Raskin 1992) and may therefore be expected to affect the
rate of photosynthesis. The various investigations of the
effects of SA on the photosynthetic process have resulted in
contradictory data. Pancheva et al. (1996) found that
100 lm SA decreased leaf growth, chlorophyll content, maxi-
mal rate of CO2 fixation and activity of ribulose-1,5-bisphos-
phate carboxylase-oxygenase (Rubisco) in barley. In contrast,
SA elevated photosynthetic electron transport rate in wheat
chloroplasts (Sahu et al. 2002), decreased harmful effects of
cadmium on photosynthesis in maize plants (Krantev et al.
2008), counteracted salt stress-induced growth inhibition
and enhanced net photosynthesis in a salt-tolerant wheat
genotype (Arfan et al. 2007) and in tomato (Gemes et al.
2008). It was considered that these effects of SA were not
due to stomatal limitation but were associated with meta-
bolic factors.

Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 105
Effects of salicylic acid on photosynthesis
SA has been found to inhibit the activity of catalase
(CAT), an enzyme participating in photorespiration of C3
plants (Chen et al. 1993; Dat et al. 1998), and consequently,
increases H2O2 content of the tissues. This effect is isoen-
zyme-specific in maize (Horvath et al. 2002) or transient in
cereals (Janda et al. 2003). Activation of antioxidative defence
systems by SA pre-treatment through elevation of the H2O2
level is involved in alleviation of paraquat-generated oxidative
stress in tobacco and cucumber (Strobel & Kuc 1995), and
SA also relieves paraquat-induced inhibition of CO2 assimila-
tion in barley (Ananieva et al. 2002).
Photosynthetic activity may change in time and may deter-
mine the substrate availability for carbohydrate metabolism.
Phosphorylation is the first step in regulation of the supply
of hexoses for biosynthesis and metabolism. Hexokinases (EC
2.7.1.1) (HXK) catalyse the conversion of glucose and fruc-
tose into hexose monophosphates, thereby permitting entry
of carbon skeletons into catabolism, e.g. glycolysis or anabolic
processes such as biosynthesis or storage of sucrose. More-
over, HXK is known to function as a glucose sensor. Among
plant HXKs, HXK1 in Arabidopsis is the best-characterised
glucose sensor; it can be detected at least partially in the
nucleus of isolated protoplasts (Yanagisawa et al. 2003) or is
attached continuously to mitochondria (Balasubramanian
et al. 2007). It has been suggested that the HXKs from Oryza
sativa, OsHXK5 and OsHXK6, have the dual ability to target
mitochondria and nuclei and to participate in repression of
sugar-regulated genes, such as the small subunit of Rubisco
(Cho et al. 2009). It is also possible that HXK acts at the
translation level on the glucose-dependent repression of the
Rubisco promoter-luciferase (RBCS-LUC) fusion construct
(Balasubramanian et al. 2007). An increase in levels of free
hexoses may serve as a signal for repression of photosynthetic
genes (Koch 1996), but can also trigger pathogenesis-related
gene expression in the tissues, resulting in more successful
acclimation to stressors (Herbers et al. 1996).
We earlier found that pre-treatment with 10)7 or 10)4 m
SA resulted in stomatal closure and a water potential decrease
in tomato plants in short-term experiments, but, after
osmotic volumetric adaptation, water potential of the tissues
was restored (Szepesi et al. 2005, 2009). This osmotic adapta-
tion, in concert with the time- and tissue-specific priming of
antioxidant defence mechanisms, contributed to decreased
sensitivity of plants pre-treated with 10)4 m SA, but not with
10)7 m SA, to subsequent salt stress induced by 100 mm
NaCl (Szepesi et al. 2009).
The present study was conducted to assess whether appli-
cation of SA in hydroponic culture medium of tomato could
time-dependently modify stomatal conductance, CO2 assimi-
lation rate and activity of HXKs, enzymes diverting carbohy-
drates to glycolysis, and whether the time-dependent changes
in photosynthetic activity and carbohydrate metabolism could
affect acclimation of tomato plants to subsequent salt stress.
MATERIAL AND METHODS
Plant material
Seeds of tomato (Solanum lycopersicum cv. Rio Fuego) were
germinated at 26 C for 3 days in the dark, and seedlings
were subsequently transferred to perlite for 2 weeks. The
106 Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands
Poor, Gemes, Horvath, Szepesi, Simon & Tari
plants were then placed in hydroponic culture containing
2 mm Ca(NO3)2, 1 mm MgSO4, 0.5 mm KCl, 0.5 mm
KH2PO4 and 0.5 mm Na2HPO4, pH 6.0. The concentrations
of micronutrients were 0.001 mm MnSO4, 0.005 mm ZnSO4,
0.0001 mm (NH4)6Mo7O24, 0.01 mm H3BO4 and 0.02 mm
Fe(III)-EDTA. The plants were grown in a controlled
environment under 300 lmol m)2 s)1 light intensity
(F36W GRO lamps, Sylvania, Germany), with 12-h light
12-h dark period, a day night temperatures of 24 22 C and
relative humidity of 5560%. The nutrient solution was chan-
ged every second day. Prior to being subjected to salinity
stress, the plants were pre-treated for 3 weeks with 10)7
10)4 m SA. The SA was present in the culture medium
throughout the whole period of the experiments. Samples
were prepared in three replicates at 10:00 h from the second,
fully expanded young leaf 24 h or 1, 2 and 3 weeks after SA
exposure. The experiments were conducted from 10:00 to
4:00 h and were repeated three to five times. Salt stress was
then imposed by transferring the plants for 710 days into
hydroponic culture containing 100 mm NaCl.
Measurement of light and CO2 response curves, chlorophyll a
fluorescence and stomatal conductance
The net photosynthetic rate (A, lmol CO2 m)2 s)1), sub-sto-
matal concentration of CO2 (Ci, lmol mol)1) and chloro-
phyll a (Chl a) fluorescence were measured on the second,
fully expanded leaf with a portable photosynthesis system
(LI-6400, LI-COR, Inc., Lincoln, NE, USA), as described by
Guoth et al. (2009). Light response curves were recorded
under constant conditions (25 C, 65 10% relative humid-
ity, and controlled CO2 supply of 360 lmol mol)1) while
increasing photosynthetic photon flux density (PPFD) from 0
to 1200 lmol m)2 s)1. The A versus PPFD curves were fitted
using the equation A Amax 1  eAqPPFDLcp , where Amax
is maximum photosynthesis at light saturation, Aq is photo-
synthetic quantum efficiency (the initial slope of the photo-
synthetic increment at low light levels) and Lcp is the light
compensation point (PPFD at A = 0) (Peek et al. 2002;
Brodersen et al. 2008).
The CO2 response curves were measured at ambient O2
concentration (21%) and saturating PPFD (500 lmol m)2
s)1), while the external CO2 concentration was raised from
0 to 1200 lmol mol)1. A Ci curves were fitted according to
Pfanz et al. (2007) with the exponential equation
y Y0 a1  ebx , using the Sigma Plot 8.0 program,
where a is Amax, and b is carboxylation efficiency, CE (mol
CO2 m)2 s)1), which corresponds to the initial slope of the
A versus Ci curve (von Caemmerer & Farquhar 1981).
Leaves were dark-adapted for 20 min before measurement
of F0, the minimal fluorescence, using weak measuring light.
Maximum fluorescence (Fm) was measured by applying a
0.2-s saturating pulse of 3000 lmol m)2 s)1. The leaves were
then illuminated continuously with white actinic light of
700 lmol m)2 s)1. After 20 min, steady-state fluorescence
(Fs) was recorded, and maximum fluorescence level (Fm) in
the light-adapted state was determined with saturating pulses.
The actinic light was then turned off and minimum fluores-
cence level in the light-adapted state (F0) determined by illu-
minating the leaf with a 3-s far-red pulse (5 lmol m)2 s)1).
The maximum quantum yield of PSII photochemistry, Fv Fm
Poor, Gemes, Horvath, Szepesi, Simon & Tari
(Fm F0 Fm), the photochemical quenching coefficient,
qP = (Fm Fs) (Fm F0) (Bilger & Schreiber 1986), non-
photochemical quenching (NPQ = Fm Fm 1) (Bilger &
Bjorkman 1990) and actual quantum yield of PSII electron
transport in the light-adapted state (FPSII = (Fm Fs) Fm)
(Genty et al. 1989) were determined.
Stomatal conductance to water vapour (gs) was also mea-
sured in the middle of apical leaflets of the second expanded
leaf, using a steady-state porometer (PMR-2; PP Systems,
UK) under greenhouse conditions at a light intensity of
300 lmol m)2 s)1.
Measurement of total sugar, glucose, fructose and sucrose content
For determination of total sugar content, fresh plant material
was frozen in liquid N2, and homogenised in 5 cm3 of 2 mm
HEPES buffer (pH 7.5) in 80% ethanol. The samples were
incubated in an 80 C water bath for 1 h, and then centri-
fuged. The pellet was extracted again with 5 cm3 of the same
buffer. The combined extract was evaporated to dryness
under vacuum at 45 C. The dried samples were dissolved in
3 cm3 distilled water in the presence of 0.3 g polyvinylpyrr-
olidone, and centrifuged after 30 min. Total sugar content
was determined by the phenol-sulphuric acid method
(Dubois et al. 1956). Sucrose, glucose and fructose content
was assessed with the Boehringer Mannheim R Biopharm
0716 260 sugar analysis kit according to the manufacturers
instructions. The glucose concentration was determined by
converting D-glucose with HXK to glucose-6-phosphate
(G-6-P) in the presence of G-6-P dehydrogenase, and cor-
rected for a blank sample. The NADPH formed was
measured at 340 nm. D-fructose, which was converted to
fructose-6-phosphate (Fru-6-P) by HXK, was determined
subsequently after conversion by phosphoglucose isomerase
to G-6-P. Sucrose was hydrolysed by invertase and D-glucose
was measured after inversion (total D-glucose) according to
the principle outlined above. The amount of NADPH formed
was stoichiometric in relation to the amounts of D-glucose,
D-fructose and sucrose.
Determination of hexokinase (EC 2.7.1.1) activity
The HXK activities of plant samples were determined with
glucose or fructose as substrate according to Whittaker et al.
(2001) with some modifications. A 1.5 cm3 reaction mixture
contained 100 mm KH2PO4 buffer (pH 7.5), 10 mm glucose
or 5 mm fructose, 1 mm ATP, 2 mm MgCl2, 1 mm EDTA,
0.5 mm NADP+, 1 U G-6-P dehydrogenase and 1 U phos-
phoglucose isomerase from bakers yeast and 100 ll plant
extract. Activity measurements were taken by following the
increase in absorbance at 340 nm for 5 min at 25 C. G-6-P
dehydrogenase (240 U mg)1 protein) and phosphoglucose
isomerase from bakers yeast (400600 U mg)1 protein) were
purchased from Sigma-Aldrich.
Statistical analysis
Statistical analysis was carried out with SigmaStat 3.1 statisti-
cal software. After analysis of variance (anova), Duncans
multiple comparisons were performed. Differences were con-
sidered significant if P 0.05.
Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands
Effects of salicylic acid on photosynthesis
RESULTS
Plant short-term responses
To clarify whether tomato plants growing in the presence of
various SA concentrations differ in photosynthetic response,
short- and long-term experiments were conducted to deter-
mine changes in stomatal conductance and photosynthetic
activity. As expected, stomata closed after 3 h of treatment
with SA (data not shown), and there was a very significant
decrease at SA concentrations as low as 10)7 m after 1 day
(Fig. 1).
As closure of stomata may limit CO2 diffusion into chlo-
roplasts, the photosynthetic activity of plants was assessed
through CO2 and light response curves (Fig. 2A and B). Amax
in leaves treated with 10)3 m SA was significantly reduced,
and the initial slopes of both the A Ci and A PPFD curves,
which allow estimation of carboxylation efficiency of Rubisco
and photosynthetic quantum efficiency, respectively, were less
steep than in control samples. The reduction in photosynthe-
sis was accompanied by initiation of tissue degeneration and
subsequent death of the plants. In contrast, Amax of the A Ci
curves increased after 10)7 and 10)4 m SA exposure, and that
of A PPFD curves was significantly higher after 10)7 m SA
treatment, suggesting that SA pre-treatment could improve
photosynthetic performance of plants even after only 1 day
of exposure.
Since SA was found to be photosynthetically active,
changes in soluble sugar content and activity of HXK,
which catalyse the first step of glucose catabolism in gly-
colysis, were determined. There were no significant changes
in enzyme activity in the roots, or in the fructose phos-
phorylating activity in leaf and root tissues (Fig. 3A), but
activity of HXK decreased with glucose as substrate in leaf
samples at 10)410)3 m SA, suggesting activation of a car-
bohydrate-saving mechanism (Fig. 3B). In samples taken
1 day after 10)4 m SA treatment, the total soluble sugar
content of leaf tissues increased significantly (Fig. 4B,
day 1).
Fig. 1. Changes in stomatal conductance (gs) on abaxial surfaces of the
second fully expanded leaf of tomato plants treated with 10)710)3 M sali-
cylic acid for 1 day. Mean SE, n = 15. Bars with different letters indicate
significant differences at the 0.05 level (Duncans multiple range test).
107
Effects of salicylic acid on photosynthesis
Fig. 2. Responses of CO2 assimilation rate to intercellular CO2 concentra-
tion (Ci) (A), and photosynthetic light intensity (PPFD) (B) in leaves of
tomato plants grown in hydroponic culture containing 10)710)3 M sali-
cylic acid for 1 day. Mean SE, n = 3.
Plant long-term responses
Changes in photosynthesis
In the long-term experiments, plants could clearly acclimate
to SA, and control levels of stomatal conductivity (Fig. 4A)
and photosynthetic activity (Fig. 5A and C) were restored;
moreover, the plants accumulated significantly more soluble
sugars in leaf and root tissues after 4 weeks of growth in SA-
containing solution (Fig. 4B, week 4). The decrease in gs was
considerable, relative to the untreated controls, after exposure
to 100 mM NaCl, but the decline was stronger in salt-stressed
controls and in plants pre-treated with 10)7 m SA than in
those pre-treated with 10)4 m SA (Fig. 4A, week 4). The max-
imum CO2 assimilation rate was significantly reduced as a
function of both Ci and PPFD in salt-stressed plants, but the
response was less harmful in leaves pre-treated with SA
(Fig. 5). Analysis of A Ci curves revealed that SA at both con-
centrations increased Amax and carboxylation efficiency of Ru-
bisco and, compared to salt-stressed controls, reduced CO2
compensation points under salinity stress (Table 1). The Amax
in response to increasing light intensity was elevated during
salt stress in SA-treated plants. These plants may convert light
energy more effectively in photochemical reactions and had
significantly higher quantum yield (Q) under salt stress. Light
compensation points were also significantly higher in pre-
treated plants than in the salt-stressed controls (Table 2).
108 Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands
Poor, Gemes, Horvath, Szepesi, Simon & Tari
Fig. 3. Fructose (A) and glucose (B) phosphorylation activity of crude pro-
tein extracts prepared from leaves and roots of tomato plants after 24 h
of 10)710)3 M salicylic acid treatment. Mean SE, n = 3. Bars with dif-
ferent letters are significantly different at the 0.05 level (Duncans multiple
range test; n.s.: not significant). The tests were performed separately for
leaf and root samples and are indicated by normal and italic letters,
respectively.
The values of Fv Fm revealed that the various treatments
did not cause irreversible damage to PSII reaction centres,
and effective quantum yield (FPSII) was significantly higher
in leaves pre-treated with SA than in salt-treated controls. At
700 lmol m)2 s)1, SA pre-treatment increased photochemical
quenching at the expense of heat dissipation (NPQ) under
salt stress, which suggests that pre-treated plants subjected to
high salinity maintained photochemical activity and electron
transport rate comparable to that of untreated controls
(Table 3). The fluorescence induction parameters and preser-
vation of chlorophyll content under salt stress (Szepesi et al.
2009) suggest that SA pre-treatment improved functioning of
the photosynthetic apparatus.
Carbohydrate content and hexokinase activity
In order to reveal whether the increase in photosynthetic
activity under salt stress in plants pre-treated with SA can
lead to changes in carbohydrate assimilation and metabolism,
total soluble sugar, glucose, fructose and sucrose content was
determined, and activities of HXKs measured. The content of
soluble sugars increased as compared with salt-stressed con-
trols in leaves of plants grown in SA under high salinity, but in
root this was only observed at 10)4 m SA (Fig. 4B, week 4).
The increase in soluble sugars relative to the untreated con-
trols occurred in parallel with accumulation of the hexoses,
glucose and or fructose in leaves of plants adapted to SA.
Poor, Gemes, Horvath, Szepesi, Simon & Tari Effects of salicylic acid on photosynthesis

A B

Fig. 4. Changes in stomatal conductance (gs) measured on the abaxial surface of the second, fully expanded leaf of tomato (A) and in total sugar content
of samples harvested from the second leaf and from roots of tomato plants (B) treated with 10)710)4 M salicylic acid and then exposed to 100 mM NaCl
for 1 week. Mean SE, n = 15 (stomatal conductance) or 3 (total sugar content). Bars with different letters indicate significant differences at the 0.05
level (Duncans multiple range test; n.s.: not significant). The tests were performed separately for leaf and root samples and are indicated by normal and
italic letters, respectively.

In roots, the hexose content significantly decreased at 10)7 m nied by a decrease in sucrose content in leaves, which
SA and hexose accumulation declined further under salt declined further under high salinity in control plants and at
stress (Fig. 6B and C). The increase in hexoses was accompa- 10)4 m SA. In roots, the opposite tendency was observed.

Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 109
Effects of salicylic acid on photosynthesis Poor, Gemes, Horvath, Szepesi, Simon & Tari

Fig. 5. Responses of CO2 assimilation rate to intercellular CO2 concentration (Ci) (A) and photosynthetic light intensity (PPFD) (C) in leaves of tomato
plants grown in hydroponic culture containing 10)710)4 M salicylic acid. Pre-treated plants were then exposed to 100 mM NaCl for 1 week (B and D).
Mean SE, n = 5.

Table 1. The parameters of the A Ci curves in leaves of tomato plants grown in hydroponic culture containing 10)7 or 10)4 M salicylic acid. Pre-treated
plants were then exposed to 100 mM NaCl for 1 week.

treatments Amax (lmol CO2 m)2 s)1) CE (mol CO2 m)2 s)1) Ccp (lmol CO2 mol)1)

control 15.18 0.32a 0.047 0.000a 69.06 2.45b

control + 100 mM NaCl 9.01 1.06b 0.026 0.006b 106.69 5.83a
10)7 M SA 16.44 1.07a 0.048 0.002a 68.98 6.43b
10)7 M SA + 100 mM NaCl 10.89 2.29ab 0.044 0.005a 76.26 6.30b
10)4 M SA 15.97 0.40a 0.048 0.002a 78.31 3.40b
10)4 M SA + 100 mM NaCl 11.59 1.98a 0.043 0.005a 85.23 7.43b

Amax = maximal assimilation (lmol CO2 m)2 s)1); CE = carboxylation efficiency (mol CO2 m)2 s)1); Ccp = CO2 compensation point (lmol CO2 mol)1).
Mean SE, n = 5. Means denoted by different letters are significantly different at P 0.05 as determined by Duncans multiple range test.

The roots of plants grown in the SA-containing culture solu-
tion accumulated more sucrose than controls, both in the
absence and in the presence of 100 mm NaCl (Fig. 6A).
In response to SA, there was a significant decrease in glu-
cose phosphorylation activity of HXKs prepared from leaf
and root tissues (Fig. 7A and B). This decreased further to
very low levels following salt treatment. Fructose phosphory-
lation activity was more sensitive and displayed greatly
decreased activity in the presence of 100 mm NaCl in SA pre-
treated tissues.
DISCUSSION
The beneficial effect of SA on photosynthesis may be
manifested in a wide array of metabolic and physiological
processes. Chlorophyll and carotenoid content was enhanced
in many plant species at low SA concentrations (Hayat et al.
2005), and SA treatment also proved to be effective in
increasing pigment content of tomato leaves under salt stress
(Tari et al. 2002; Szepesi et al. 2009). However, the results of
Hayat et al. (2008) revealed significant declines in photosyn-
thetic parameters, leaf water potential, chlorophyll and rela-
tive water content in response to exogenously applied SA in
tomato plants exposed to water stress.
Our results demonstrated, that the effects of SA on photo-
synthesis depended on the applied SA concentration and
duration of treatment. The harmful effects of salt stress in
tomato were alleviated by sublethal concentrations of SA,
which trigger osmotic adaptation in parallel with antioxidant
defence mechanisms (Szepesi et al. 2009). In this, the
response of roots was as significant as that of leaves.
It was found that SA at 10)3 m decreased the efficiency of
CO2 fixation as a function of both PPFD and Ci, but this
treatment proved to be lethal in 1 to 2 weeks. The sensitivity,
and hence response of a plant to SA, depends on the geno-
type (Arfan et al. 2007) and also on the developmental phase

110 Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands
Poor, Gemes, Horvath, Szepesi, Simon & Tari Effects of salicylic acid on photosynthesis

Table 2. Parameters of photosynthetic light (A PPFD) response curves in leaves of tomato plants grown in hydroponic culture containing 10)7 or 10)4 M

salicylic acid. Pre-treated plants were then exposed to 100 mM NaCl for 1 week.

treatments Amax (lmol CO2 m)2 s)1) Q (mol CO2 mol)1 photon) Rd (lmol m)2 s)1) Lcp (lmol m)2 s)1)

control 13.27 1.49a 0.044 0.006a 0.36 0.15a 7.74 2.37a

control + 100 mM NaCl 7.52 0.20c 0.033 0.000b 0.07 0.02b 2.29 0.78b
10)7 M SA 12.60 2.19ab 0.038 0.001a 0.25 0.04a 6.62 0.06a
10)7 M SA + 100 mM NaCl 9.59 0.07bc 0.036 0.001a 0.33 0.12a 8.98 3.18a
10)4 M SA 11.97 1.25ab 0.041 0.001a 0.48 0.04a 12.04 1.45a
10)4 M SA + 100 mM NaCl 8.97 0.75bc 0.037 0.001a 0.28 0.02a 7.61 0.88a

Amax = maximal assimilation (lmol CO2 m)2 s)1); Q = apparent quantum yield (mol CO2 mol)1 photon); Rd = dark respiration (lmol CO2 m)2 s)1);
Lcp = light compensation point; (lmol m)2 s)1).
Mean SE, n = 5. Means denoted by different letters are significantly different at P 0.05 as determined by Duncans multiple range test.

Table 3. Chl a fluorescence parameters. Steady state Chl a fluorescence parameters (Fv Fm, qP, FPS2 and NPQ) of the youngest mature leaf of SA pre-
treated tomato plants exposed to 100 mM NaCl for 7 days. After 20 min of dark adaptation, the plants were illuminated with 700 lmol m)2 s)1 actinic
light for 20 min.

treatments Fv F m qP FPS2 NPQ

n.s. a bc
control 0.765 0.01 0.582 0.05 0.252 0.01 0.599 0.01a
control + 100 mM NaCl 0.773 0.01n.s. 0.447 0.03b 0.232 0.02c 0.607 0.01a
10)7 M SA 0.784 0.01n.s. 0.625 0.06a 0.349 0.03a 0.538 0.01ab
10)7 M SA + 100 mM NaCl 0.764 0.00n.s. 0.561 0.01a 0.311 0.01ab 0.534 0.01b
10)4 M SA 0.775 0.00n.s. 0.519 0.02ab 0.298 0.02ab 0.508 0.02b
10)4 M SA + 100 mM NaCl 0.793 0.01n.s. 0.637 0.02a 0.374 0.02a 0.549 0.03b

Mean SE, n = 5. Means denoted by different letters are significantly different at P 0.05 as determined by Duncans multiple range test.
n.s. = not significant.

or the plant organ (Guan & Scandalios 1995). Lower concen-
trations (e.g. 10)7 m SA) may enhance photosynthesis even at
low gs, as was found in the 24-h samples. However, this con-
centration was not effective in alleviation of salt stress elicited
by 100 mm NaCl (Szepesi et al. 2009). In plants grown in
SA-containing culture solution, control values of gs and rate
of CO2 fixation were restored after 3 weeks.
In the long-term experiments, the beneficial effect of SA on
photosynthetic performance in tomato could be detected only
under the influence of the stressor. These plants exhibited
higher Amax and CE or Amax and Q values, calculated from the
A Ci and A PPFD curves, respectively. This means that the
plants could maintain higher maximum photosynthetic capac-
ity at saturating Ci and light intensity, and the significantly
enhanced CE values indicate that carboxylase activity of Rubi-
sco is more efficient under CO2-limited conditions.
The activity of carbonic anhydrase (CA), an enzyme
involved in CO2 transfer and photosynthetic CO2 fixation, was
significantly enhanced at low concentrations of SA in Brassica
juncea leaves (Fariduddin et al. 2003). The close association of
CA with Rubisco increases the availability of CO2 at the site of
carboxylation and may lead to elevation of net CO2 fixation
(Hatch & Burnell 1990) and CE. Any change in CA activity
directly affects the rate of photosynthetic CO2 fixation under
CO2-limiting conditions (Imamura et al. 1981). The significant
increment of CE detected in SA pre-treated plants during salt
stress in our experiments may support this hypothesis.
Different sugars can accumulate due to infection by patho-
gens, and sugar accumulation is a salt-tolerance trait and a
prerequisite for osmotic adaptation of tomato during salt
stress (Juan et al. 2005). There have been reports of the pres-
ence of HXK and glucokinase activity in L. esculentum
(Martinez-Bajaras & Randall 1998). We also detected HXK
activity when glucose or fructose was used as substrates in
the second leaf of tomato plants. Both short- (1 day) and
long-term (4 weeks) application of SA enhanced the total sol-
uble sugar content of leaf and or root tissues. This coincided
with tissue-specific decreases in HXK activity in the short-
term experiments and under the influence of high salinity.
Since such treatments may affect carbohydrate metabolism at
several points, we cannot expect a very close correlation with
accumulation of one or more hexose species, but it can be
concluded that in these tissues, the decreases in activities of
glucokinase and fructokinase may promote accumulation of
soluble sugars in leaves under salt stress.
Sugars not only participate in osmotic adaptation but are
also important signals of plant metabolism and development.
Hexose concentrations rise drastically in senescing leaves of
tomato, and exogenous glucose may induce a decrease in
photosynthetic activity (decrease in Amax, FPSII, qP and
increase in qN) (Dorais et al. 2001) or may control gene
expression in HXK-dependent or -independent pathways
(Wingler & Roitsch 2008). Thus, manipulation of HXK activ-
ity affects the trigger of leaf senescence. Over-expression of
Arabidopsis HXK1 in tomato accelerated senescence (Dai
et al. 1999), while a mutant in hexokinase 1 (HXK1) showed
delayed senescence (Moore et al. 2003). While the accumula-
tion of sugars as compatible osmolytes exerts a beneficial
effect on acclimation to high salt concentrations, it may trig-
ger senescence of leaves.

Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 111
Effects of salicylic acid on photosynthesis
A A
B B
Fig. 6. Changes in sucrose (A), glucose (B) and fructose (C) content of
samples harvested from the second leaf and from roots of tomato plants
pre-treated with 10)710)4 M salicylic acid. Pre-treated plants were then
exposed to 100 mM NaCl for 1 week. Mean SE, n = 3. Bars with differ-
ent letters indicate significant differences at the 0.05 level (Duncans multi-
ple range test). The tests were performed separately for leaf and root
samples and are indicated by normal and italic letters, respectively.
It can be concluded that apart from enhancement of pho-
tosynthetic performance under salt stress, the reduction of
hexokinase activity concomitant with accumulation of soluble
sugars is an important aspect of improved salt tolerance of
plants pre-treated with SA. Besides its putative role in delay-
ing senescence, decreased hexokinase activity may divert car-
bon skeletons of hexoses from catabolic reactions, e.g. from
glycolysis, preserving them for osmotic adaptation. By
increasing soluble sugar, and especially sucrose accumulation
in the root tissues, treatment with 10)4 m SA enhanced the
sink status of roots when it was applied in the culture
solution. The osmotic adjustment elicited by SA through
accumulation of compatible osmolytes appears to be an
112 Plant Biology 13 (2011) 105114 2010 German Botanical Society and The Royal Botanical Society of the Netherlands
Poor, Gemes, Horvath, Szepesi, Simon & Tari
Fig. 7. Changes in fructose (A) and glucose (B) phosphorylation activity
due to 10)710)4 M salicylic acid and 100 mM NaCl treatments, in leaves
and roots of tomato plants. Mean SE, n = 3. Bars with different letters
indicate significant differences at the 0.05 level (Duncans multiple range
test). The tests were performed separately for leaf and root samples and
are indicated by normal and italic letters, respectively.
essential, but not sufficient, prerequisite for the successful
acclimation of tomato to high salinity.
ACKNOWLEDGEMENTS
We thank Kispalne Szabo Ibolya for excellent technical assis-
tance. This work was supported by a grant from the Hungar-
ian National Scientific Research Foundation (OTKA K76854).
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