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INFLUENCE OF DIFFERENT SEED PRIMING

METHODS FOR IMPROVING SALT STRESS
TOLERANCE IN LETTUCE PLANTS
a a a
Hela Mahmoudi , Raouia Ben Massoud , Olfa Baatour , Imen
a b a a
Tarchoune , Imen Ben Salah , Nawel Nasri , Wissal Abidi ,
a c a
Rym Kaddour , AbdelAli Hannoufa , Mokhtar Lachaâl & Zeineb
a
Ouerghi
a
Unité de Physiologie et Biochimie de la Tolérance au Sel des
Plantes, Département des Sciences Biologiques, FST, Campus
Universitaire, Tunis, Tunisia
b
Laboratoire des Plantes Extrêmophiles, CBBC, Hammam-Lif,
Tunisia
c
Southern Crop Protection and Food Research Centre, Agriculture
and Agri-Food Canada, London, Ontario, Canada
Accepted author version posted online: 17 Jul 2012.Published
online: 27 Aug 2012.

To cite this article: Hela Mahmoudi , Raouia Ben Massoud , Olfa Baatour , Imen Tarchoune , Imen
Ben Salah , Nawel Nasri , Wissal Abidi , Rym Kaddour , AbdelAli Hannoufa , Mokhtar Lachaâl
& Zeineb Ouerghi (2012) INFLUENCE OF DIFFERENT SEED PRIMING METHODS FOR IMPROVING
SALT STRESS TOLERANCE IN LETTUCE PLANTS, Journal of Plant Nutrition, 35:12, 1910-1922, DOI:
10.1080/01904167.2012.711410

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 Journal of Plant Nutrition, 35:1910–1922, 2012
Copyright
C Taylor & Francis Group, LLC

ISSN: 0190-4167 print / 1532-4087 online

DOI: 10.1080/01904167.2012.711410

INFLUENCE OF DIFFERENT SEED PRIMING METHODS FOR

IMPROVING SALT STRESS TOLERANCE IN LETTUCE PLANTS

Hela Mahmoudi,1 Raouia Ben Massoud,1 Olfa Baatour,1 Imen Tarchoune,1

Imen Ben Salah,2 Nawel Nasri,1 Wissal Abidi,1 Rym Kaddour,1 AbdelAli
Hannoufa,3 Mokhtar Lachaâl,1 and Zeineb Ouerghi1
Downloaded by [University of York] at 07:16 07 October 2013

1
Unité de Physiologie et Biochimie de la Tolérance au Sel des Plantes, Département des
Sciences Biologiques, FST, Campus Universitaire, Tunis, Tunisia
2
Laboratoire des Plantes Extrêmophiles, CBBC, Hammam-Lif, Tunisia
3
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada,
London, Ontario, Canada

2 Seeds of lettuce, variety Romaine were subjected to different priming treatments such as water,
potassium nitrate (KNO3 ) and gibberellic acid (GA3 ). Seedlings obtained from primed (Pr) and
nonprimed (NP) seeds were grown in a hydroponic culture system supplemented with 0, 100 or
200 mM sodium chloride (NaCl). The different physiological and biochemical responses were studied
15 days after treatment. Under NaCl, the dry weight was higher in plants derived from hydro-
primed (HP) seeds when compared to NP, osmoprimed (KNO3 P), and hormonal primed (GA3 P)
ones. Under control and 100 mM NaCl treatment, malondialdehyde (MDA) content and EL did
not show any correlation with activities of gaiacol peroxidase (GPX) and catalase (CAT), but did
with the increase in reduced ascorbate (AsA) and total ascorbate contents. The results indicated that
plants derived from HP seeds exhibited higher adaptive potential under salinity stress. Our findings
suggest that a hydropriming technique can be used as a simple commercial approach to alleviate the
effects of NaCl induced stress in lettuce plants.

Keywords: lettuce, priming technique, growth, antioxidant activities, ascorbate, salinity

INTRODUCTION
Vegetables are important for human nutrition and their dietary signif-
icance is growing around the world. Good quality and high volume crop
yield is one of the major challenges to crop production in the world, and its

Received 28 September 2011; accepted 17 January 2012.

Address correspondence to Hela Mahmoudi, Unité de Physiologie et Biochimie de la Tolérance au
Sel des Plantes, Département des Sciences Biologiques, FST, Campus Universitaire, 2092 Tunis, Tunisia.
E-mail: mahmoudihela@yahoo.fr

1910
 Seed Priming Methods in Lettuce Plants 1911

importance is recognized by farmers as well as researchers (Murungu et al.,

2004). Seed quality can have a profound influence on the yield and the
quality of crops. In particular, seeds of high viability and vigor are essential
for achieving high yield and quality.
Salinity can affect plant physiological processes resulting in reduced
growth and yield (Yamaguchi and Blumwald, 2005). Increased tolerance to
salinity stress in crop plants is necessary in order to increase productivity
with limited water supplies and high salinity. Seed priming is one of a variety
of strategies used to cope with the issue of salinity. Seed priming is an easy,
low cost, and low risk technique, and this approach has recently been used
to overcome the salinity problems in agricultural lands (Iqbal and Ashraf,
2006).
The beneficial effects of priming have been demonstrated in many crops
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like wheat (Iqbal and Ashraf, 2007), chickpea (Kaur et al., 2002), sunflower
(Kaya et al., 2006) and cotton (Casenave and Toselli, 2007). To enhance
seed germination, several treatments were adopted, and performance en-
hancements with beneficial value added techniques were used on seeds
after harvest, but prior to sowing (Taylor and Harman, 1990). The purpose
of these treatments was to shorten the emergence period and to protect the
seeds from biotic and abiotic factors during the critical phase of seedling
establishment, so as to synchronize emergence, which led to uniform stand
and improved yield. These priming treatments, which enhanced seed germi-
nation include hydropriming, osmopriming, and hormonal priming (Afzal
et al., 2006). The aim of the present study was to compare different prim-
ing technique (hydro-, hormonal-, osmo-priming) in order to overcome the
negative effects of sodium chloride (NaCl) stress in lettuce plants.

MATERIALS AND METHODS

Plant Material and Seed Priming Treatment
Seeds used in this study were obtained from the Ministry of Agriculture
of Tunisia. Three different techniques were used for the purpose of seed
priming: distilled water (hydro-primed), potassium nitrate (KNO3 ) (0,5%;
osmo-primed), and gibberellic acid (GA3 ) (3 mM; hormonal-primed). Seeds
were first surface-sterilized with 95% ethanol for 5 min, washed thoroughly
to remove any traces of ethanol, and were then distributed in four different
sets. Three sets were separately immersed in different priming solutions for
2 h, 2 h and 12 h at 25◦ C under dark conditions, respectively, for KNO3 ,
dH2 O and GA3 . The remaining seeds were unsoaked (untreated) and used
as non-primed control. After the priming period, seeds were rinsed three
times with distilled water and then dried (at room temperature; 25 ± 2 ◦ C)
for two days.
 1912 H. Mahmoudi et al.

Plant Growth and Treatment Conditions

Seeds for each treatment were germinated in Petri dishes at room tem-
perature in the dark. Seven-day old seedlings were irrigated with distilled
water during the first week. Uniform seedlings were subsequently cultured
individually in a hydroponic Hoagland’s medium (Hoagland and Arnon,
1950) diluted eight-fold in a culture chamber with a 16 h photoperiod
(150 µmol m−2 s−1). The temperature and average relative moisture were
22◦ C and 40% during the day and 18◦ C and 86% at night, respectively. Indi-
vidual plants were grown in control (without salt), 100 mM NaCl, or 200 mM
NaCl for 15 days prior to harvest. Fresh weight (FW) and dry weight (DW)
of roots and rosette leaves were separately recorded for six plants randomly
selected from each variety and treatment combination and used to calculate
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the percent of tissue water [(FW - DW)/FW] ∗ 100. The remaining plants
were frozen at −80◦ C for biochemical analysis.

Chlorophyll and Carotenoid Extraction

Concentrations of Chlorophylls (a and b) and carotenoids were mea-
sured as described by Lichtenthaler and Wellburn (1983), after extraction
with 80% acetone.

Membrane Permeability (Electrolyte Leakage)

Electrolyte leakage (EL) was determined as described by Quartacci et al.
(2002). Leaf discs of fresh seedlings were cut into 2–3 mm pieces and placed
in test tubes containing 25 ml double-distilled water at room temperature.
After one hour the initial electrical conductivity of the medium (EC1) was
measured using a digital conductimeter type Metrohm (Metrohm, Herisau,
Switzerland). The samples were placed in liquid nitrogen and then replaced
on the same tube for an additional hour of shaking and the final electrical
conductivity (EC2) was measured. The electrolyte leakage (EL) was calcu-
lated from EL (%) = (EC1/EC2)∗ 100.

Malondialdehyde Content
Lipid peroxidation was measured as the amount of malondialdehyde
(MDA) determined as described by Mahmoudi et al. (2011). Frozen samples
(200 mg) were homogenized with a mortar and pestle in 2 mL of a mixture
containing 20% 2-thiobarbituric acid (TBA) and 0.5% trichloroacetic acid
(TCA). Assay mixture containing 2 mL of the supernatant and 2 mL of 0.5%
(w/v) TBA in 20% (w/v) TCA was heated at 95◦ C for 30 min and then rapidly
cooled in an ice bath. After centrifugation (4000 g for 30 min at 4◦ C), the
supernatant absorbance was read at 532 nm, and the values corresponding
to nonspecific absorption (600 nm) were subtracted. Lipid peroxidation
 Seed Priming Methods in Lettuce Plants 1913

products were measured as the content of TBA-reactive substances. The MDA

content (µmol g−1 FW) was calculated according to the molar extinction
coefficient of 155/(mM cm−1).

Enzyme Extraction and Assay

All steps in the preparation of enzyme extracts were performed at 4◦ C.
Frozen tissue (0.5 g) was ground in a mortar with liquid nitrogen with
5 mL extraction buffer containing 50 mM potassium phosphate buffer, pH
7.6 and 0.1 mM sodium (Na)-ethylenediaminetetraacetic acid (EDTA). The
homogenate was centrifuged at 15,000 g for 15 min and the supernatant
fraction was used to assay for the various enzymes.
Catalase (CAT) activity was measured spectrophotometrically according
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to Cakmak and Marschner (1992) by monitoring the disappearance of hy-

drogen peroxide (H2 O2 ) at 240 nm at 25◦ C for 1 min. The reaction mixture
(1 mL) contained 50 mM potassium phosphate buffer (pH 7.0) and 10.6 mM
H2 O2 . The reaction was initiated by adding 10 µL of the enzyme extract.
Gaiacol peroxidase (GPX) activity was determined according to Srini-
vas et al. (1999) by monitoring the increase in absorbance at 470 nm for
1 min. The reaction consisted of 20 mM potassium phosphate buffer (pH
7) and 30 µM H2 O2 . The increase of absorbance, due to tetraguaiacol for-
mation, was recorded at 470 nm. One unit of peroxidase activity catalyzes
the oxidation of 1 µmol of guaiacol, ascorbate (AsA), and dehydroascorbate
(DHA).
AsA and total AsA (AsA + DHA) were determined in the supernatants
according to the methods of Kampfenkel et al. (1995). Fresh leaf tissue was
immediately homogenized at 4◦ C in 6% (w/v) trichloroacetic acid. After
centrifugation at 12 100 g for 15 min, based on the reduction of Fe3+ by AsA,
followed by complex formation between Fe2+ and 2,2’-dipyridyl, that absorbs
at 525 nm. Total ascorbate was determined through a reduction of DHA
to AsA by dithiothreitol. DHA content was estimated from the difference
between total ascorbate and AsA. A standard curve covering the range of
550 nmol AsA was used.

RESULTS
Effect of Priming on Plant Growth
Under control conditions, primed and nonprimed seedlings looked sim-
ilar, and no differences in dry weight, chlorophyll and carotenoid contents
were observed (Table 1; Figure 1). Sodium chloride treatment reduced leaf
DW in all plants derived from primed (Pr) and nonprimed (NP) seeds. Fur-
thermore, leaf DW reduced by 46%, 24%, 26% and 42% in NP, hydroprimed
(HP), hormonal primed (GA3 P), and osmoprimed (KNO3 P), respectively,
 1914 H. Mahmoudi et al.

TABLE 1 Growth (per plant), water content, total chlorophyll (mg −1 chl g−1 FW tissue), and
carotenoids contents (µg g−1 FW tissue) of NP, HP, GA3 P and KNO3 P lettuce seedlings grown in the
presence of 0, 100 and 200 mM NaCl for 15 d (mean ± SE).

Physiological response

Parameter Priming 0 mM NaCl 100 mM NaCl 200 mM NaCl

Leaf DW (g) (n = 6) NP 0.36a ± 0.05 0.20b ± 0.01 0.12 c ± 0.02

HP 0.38 a ± 0.03 0.29 b ± 0.03 0.22 c ± 0.02
GA3 P 0.31 a ± 0.03 0.23 b ± 0.02 0.12 c ± 0.02
KNO3 P 0.31 a ± 0.06 0.18 b ± 0.01 0.12 c ± 0.01
Leaf number (n = 6) NP 17.17a ± 2.34 12.33b ± 0.01 8.33c ± 1.43
HP 16.00a ± 1.99 13.33b ± 1.71 10.33c ± 1.27
GA3 P 14.33a ± 1.58 14.50b ± 0.57 11.00c ± 1.15
KNO3 P 17.67a ± 1.43 12.17b ± 1.23 8.67c ± 1.43
Leaf area (cm2, n = 6) NP 89.40a ± 6.86 58.98b ± 1.95 24.07c ± 2, 76
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HP 83.17a ± 16.22 72.98a ± 15.41 53.59b ± 3.15

GA3 P 80.76a ± 7.12 59.78b ± 16.17 42.74c ± 1.41
KNO3 P 70.38a ± 15.52 43.79b ± 2.82 21.69c ± 3.04
Leaf water content (%, n = 6) NP 11.96b ± 0.97 12.90ab ± 0.68 7.83d ± 1.02
HP 9.85b ± 0.46 11.80ab ± 2.65 11.88c ± 1.71
GA3 P 13.27b ± 1.66 13.90a ± 1.61 7.85d ± 1.53
KNO3 P 11.25c ± 0.76 11.41b ± 0.38 8.69d ± 0.50
Total chlorophyll (n = 4) NP 1.15a ± 0.55 0.96a ± 0.43 0.62a ± 0.37
HP 0.67a ± 0.19 0.69a ± 0.15 0.90a ± 0.21
GA3 P 0.84a ± 0.29 0.93a ± 0.20 0.52a ± 0.26
KNO3 P 0.91a ± 0.19 1.22a ± 0.58 1.20a ± 0.41
Total carotenoids (n = 4) NP 3.28 a ± 1.03 4.12a ± 0.34 2.68a ± 1.01
HP 3.14a ± 1.83 3.67a ± 1.39 2.30a ± 1.12
GA3 P 2.81a ± 0.57 3.30a ± 0.82 3.09a ± 1.48
KNO3 P 3.54a ± 1.67 3.70a ± 0.91 2.52a ± 1.10

Means followed by different letters are significantly different (P ≤ 0.05) as determined by analysis of
variance (ANOVA).

under 100 mM NaCl. At the highest NaCl concentration (200 mM), leaf
DW was reduced by 67%, 42%, 61%, and 61%, respectively, under the same
condition. This reduction may be explained by the leaf number and leaf
area reductions (Table 1). This result suggests that HP alleviates the effect
of salt on lettuce growth under both NaCl concentrations.
Leaf water content was significantly reduced under 200 mM NaCl treat-
ment (Table 1), suggesting tissue dehydration occurred following exposure
to severe high NaCl, except for leaf water content of plants derived from HP
seeds.

Parameters of Oxidative Stress

Two markers of oxidative damage: the extent of lipid peroxidation deter-
mined as MDA content and electrolyte leakage index (EL), were measured.
Under control conditions, leaf MDA content was similar for all treatments
(NP and Pr plants) (Figure 2A). Under NaCl, 100 mM the MDA content was
 Seed Priming Methods in Lettuce Plants 1915
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FIGURE 1 Phenotypes of Romaine plants derived from unprimed (NP), osmoprimed (KNO3 P), hy-
droprimed (HP) and hormonalprimed (GA3 P) seeds grown under 0, 100 and 200 mM NaCl for 15 days.
(Color figure available online).

FIGURE 2 Effect of NaCl on MDA content (µmol g−1FW, n = 12) in A) leaves and B) roots and
electrolyte leakage (%, n = 5) in C) leaves and D) roots of plants derived from NP, KNO3 P, HP and
GA3 P seeds grown under 0, 100 and 200 mM NaCl for 15 days. Means followed by different letters are
significantly different at P ≤ 0.05 level as determined by analysis of variance (ANOVA).
 1916 H. Mahmoudi et al.

not affected in Pr plants but increased by 1.47-fold in NP ones. The maxi-

mum lipid peroxidation was observed in leaves of plants subjected to 200 mM
NaCl treatment, except for the HP plants. However, the MDA increased by
2.1-, 2.6- and 2.0-fold, respectively, in NP, KNO3 P, and GA3 P plants.
In roots (Figure 2B), MDA level remained unchanged for all Pr and
NP plants treated with 100 mM NaCl. Furthermore, MDA accumulation
increased in response to 200 mM NaCl with only one exception (GA3 P), by
1.85-, 1.62- and 2.39-fold compared to nonNaCl treated, respectively, in NP,
KNO3 P, and HP plants.
The EL index of leaves of NP and Pr plants (with only one exception
for GA3 P plants) was not affected by NaCl (100 mM). At 200 mM NaCl, EL
index progressively increased with increasing NaCl concentration (Figure
2C). In roots (Figure 2D), salt treatment increased EL by 1.7- and 1.9-fold
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in NP roots, by 1.7- and 1.5-fold in KNO3 P roots, and by 3.1- and 3.7-fold in
GA3 P ones under 100 and 200 mM NaCl, respectively.

Antioxdative Enzymes
Under control conditions, leaf GPX activity was similar for all plants
derived from NP, KNO3 P, HP, and GA3 P seeds (Figure 3A). NaCl, 100 mM
had no effect on GPX activity in all plants. In contrast, GPX activity was

FIGURE 3 A,B) Leaf and C, D) root anti-oxidative enzyme activity (U mg−1 protein) of plants derived
from NP, KNO3 P, HP and GA3 P seedlings (14 d) grown in Hoagland nutrient solution supplemented
with 0 (control), 100 or 200 mM NaCl for 15 days. Means followed by different letters are significantly
different at P ≤ 0.05 level as determined by analysis of variance (ANOVA).
 Seed Priming Methods in Lettuce Plants 1917

enhanced by about 2.7-, 1.1-, 5.7-and 14.3-fold compared to their controls in

NP, KNO3 P, HP, and GA3 P seeds, respectively, under NaCl, 200 mM. The
same trend was observed for leaf CAT activity (Figure 3B). However, CAT
was enhanced by 3.1, 3.1-, 3.3- and 5.7-fold in NP, KNO3 P, HP, and GA3 P
seeds, respectively, under NaCl, 200 mM.
In 100 mM treated roots, GPX activity was significantly enhanced by
2.4-, 3.8- and 1.5-fold as compared to their control in NP, HP, and GA3 P
seedlings, respectively (Figure 3C). Whereas, a decrease by 6-fold was de-
tected in KNO3 P roots. High GPX activity was observed at 200 mM NaCl.
However, a significant increase by 5.8-, 2.1-, 4.9- and 4.1-fold was determined
in NP, KNO3 P, HP, and GA3 P plants.
Under control conditions, KNO3 P roots showed the highest CAT activity
as compared to the other three priming types (Figure 3D). At 100 mM
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NaCl, CAT activity was enhanced by 2.35-, 1.84- and 2.54-fold and remained
unchanged at high NaCl concentration, in NP, HP, and GA3 , respectively.
In contrast, CAT activity was decreased by about 16-fold compared to the
control in plants derived from KNO3 P seeds.

Effect of Priming and Salt on Ascorbate Content

Under control conditions, seedlings derived from KNO3 P seeds have
the highest AsA (Figure 4A) and total ascorbate (AsA+DHA) (Figure 4B)
compared to the other seedlings derived from NP, HP and GA3 P seeds.
Reduced ascorbate (AsA) was increased by 7.91-, 1.75-, 6.17-and 4.31- fold,
respectively, in seedlings of NP, KNO3 P, HP, and GA3 P subjected to 100 mM
NaCl and by 4.0-, 1.75-, 6.70- and 1.75-fold under 200 mM NaCl (Figure 4A).
By contrast, NaCl had no effect on the content of total ascorbate (Figure
4B) for all seedlings derived from NP, KNO3 P and GA3 P seeds. However,
total ascorbate increased by 2.51- and 2.62-fold in HP ones. Sodium chloride
treatment increased AsA/DHA (Figure 4C) levels in all plants.

DISCUSSION
Salinity caused growth inhibition in Romaine lettuce due to both ionic
and osmotic impacts of NaCl (Mahmoudi et al., 2011). Although prim-
ing is one of the physiological methods which improves seed performance,
and provides faster and synchronized germination (Sivritepe and Dourado,
1995), it has been shown that different priming methods could be used
as an adaptation method to improve salt tolerance of seeds. Nevertheless,
the beneficial effects of seed priming on the later growth and development
stages of plants remain unclear. In our experiment, both GA3 P with gib-
berellic acid and HP on lettuce seeds alleviated the effect of NaCl stress on
plant growth. However, it was determined that HP diminished the inhibiting
effect of salinity on plant growth in lettuce.
 1918 H. Mahmoudi et al.
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FIGURE 4 Effect of NaCl on A) ascorbate (AsA, µmol.g−1FW), B) total ascorbate (AsA+DHA,

µmol.g−1FW), and C) AsA/DHA ratio in Romaine plants derived from NP, KNO3 P, HP and GA3 P
seedlings (14 d) grown in Hoagland nutrient solution supplemented with 0 (control), 100 or 200 mM
NaCl for 15 d. Means of 6 replicates ± SE. Means followed by different letters are significantly different
(P ≤0.05) as determined by analysis of variance (ANOVA).

These results suggest that in Romaine lettuce, seedlings derived from

HP seeds have higher adaptation capacity to salinity than of those derived
from KNO3 P seeds and NP ones.
Resistance to salinity occurs when a plant withstands the imposed stress
and this may arise from either tolerance, or a mechanism that permits avoid-
ance of the stress. Even under optimal conditions many metabolic processes
produce reactive oxygen species (ROS). The production of toxic derivatives
is increased as a result of all types of abiotic or biotic stresses. The lipid perox-
idation of membranes, which results from a degradation of polyunsaturated
fatty acids and severely affects membrane functionality causing irreversible
 Seed Priming Methods in Lettuce Plants 1919

damage, has been considered a good marker for oxidative damage (Halli-
well and Gutteridge, 1989). Our data showed a marked increase of leaf MDA
content under 200 mM NaCl except seedlings derived from HP seeds, which
is indicative of severe oxidative injury to membrane lipids in these plants
due to the inadequate response of the antioxidative systems. Similar results
were observed in tomato, cucumber, pepper (Kaya et al., 2002), and wheat
plants (Zheng et al., 2008).
Fortunately, plants possess several anti-oxidant enzyme systems that pro-
tect their cells from the negative effects of ROS. These include non-enzymatic
anti-oxidants such as ascorbic acid, glutathione and carotenoids, as well as
antioxidative enzymes such as superoxide dismutase (SOD), ascorbate per-
oxidase (APX), and glutathione reductase (GR; Lee et al., 2002).
Catalase is essential for the removal of H2 O2 produced in the perox-
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isomes by photorespiration (Noctor et al., 2000). However, CAT activity

decreases under conditions that suppress photorespiration, such as elevated
carbon dioxide (CO2 ) (Azevedo et al., 1998). The importance of CAT in
photosynthetic cells is demonstrated by observations in CAT-deficient mu-
tants (Kendall et al., 1983) and in transformed tobacco in which the major
leaf CAT isoform is decreased by antisense technology (Willekens et al.,
1994).
To understand the cellular events during different methods of priming,
and in order to explore their relationship with the stress tolerance during let-
tuce growth seedling establishment, we investigated the antioxidant system
activity. Our results showed that the activities of two antioxidant enzymes
GPX and CAT showed a progressive increase in all Pr and NP plants, es-
pecially at high NaCl concentration (200 mM). Many changes have been
observed in the activities of antioxidant enzymes in plants under salt stress.
However, the activity of antioxidant enzymes has been reported to increase
under saline conditions in the case of salt tolerant cotton (Meloni et al.,
2003), shoot cultures of rice (Fadzilla et al., 1997), cucumber (Lechno et al.,
1997) and salt tolerant lettuce (Mahmoudi et al., 2011). It should be noted
that the MDA accumulation (Figure 2), which represents the level of lipid
peroxidation and thus the accumulation of ROS, remained unchanged in
leaves and roots treated with moderate NaCl concentration (100 mM) dur-
ing hydropriming, despite the lower activities of CAT and GPX (Figure 3).
Therefore, it is possible that hydropriming represses or maintains the activ-
ities of CAT and GPX, but it enhances alternative antioxidant mechanisms
such as SOD and APX, which might be responsible for lower lipid peroxida-
tion as compared to the other priming technique.
Beside the roles established by antioxidative enzymes, non enzymatic
antioxidants were also implicated in the detoxification of ROS. Our results
demonstrated that the AsA and total AsA contents of control plants were sim-
ilar, and no significant differences were found between NP, HP and GA3 P
plants except KNO3 P ones. Generally, NaCl treatment increased the AsA
 1920 H. Mahmoudi et al.

content of all plants used in this experiment with the exception of osmo-
primed plants. Our results demonstrated that the total ascorbate content
was increased in plants derived from hydroprimed seeds. This could be ex-
plained by a stimulation of its synthesis, or a diminution of its catabolism.
Higher AsA contents under salt-stress were also reported in salt-tolerant
pepper (Aktas, 2002) and citrus species (Arbona et al., 2003).
Regeneration of the oxidized ascorbate is a critical component of the
antioxidant scavenging system in plant. An important indicator of the redox
status of the cell is a sign of oxidative stress (Meneguzzo et al., 1999). The
increase in AsA to DHA ratio observed in plants subjected to NaCl treatment
could be due to an enhancement of reduced ascorbate regeneration.
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CONCLUSIONS
In conclusion, our study showed that NaCl treatment decreased plant
growth, but seed priming with GA3 or H2 O may have alleviated this effect.
NaCl treatment also led to oxidative stress as indicated by increases in CAT
and GPX activities and MDA content with the exception of HP plants. These
findings suggest that hydropriming could be used as a simple and cost-
effective strategy to alleviate the NaCl induced stress in lettuce.

ACKNOWLEDGMENT
Mahmoudi Hela and Ben Masaoud Raouia have equally participated on
the elaboration of this work. We are grateful to Bibi HAKIM for English
assistance

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Biology 8: 23–28.
Aktas, H. 2002. Selection and physiological characterization of pepper for resistance to salinity stress.
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