Biologia 69/2: 193—201, 2014

Section Botany
DOI: 10.2478/s11756-013-0304-1

Growth, sodium uptake and antioxidant responses of coastal plants

differing in their ecological status under increasing salinity

Karim Ben Hamed1*, Farhat Chibani1, Chedly Abdelly1 & Christian Magne2
1
Laboratoire des Plantes Extrêmophiles, Centre de Biotechnologie à la Technopôle de Borj Cédria, BP 901, Hammam Lif
2050, Tunisia; e-mail: kbenhamed@yahoo.fr
2
EA2219 Géoarchitecture, Université de Brest, 6 Avenue Victor Le Gorgeu, CS 93837, 29238 BREST Cédex 3, France

Abstract: In the present study, we compared the response to salinity of three plants from Brittany coast with contrasted
ecological status: Limonium latifolium (salt marshes), Matricaria maritima (beach tops and sand dunes) and Crambe
maritima (fixed dunes). Under controlled glasshouse conditions, the growth of the three plants decreased with increasing
external salinity. L. latifolium and C. maritima exhibited the highest and lowest resistance to severe salt stress (400 mM),
respectively. M. maritima could be considered as an intermediate species, since it tolerated salinity up to 200 mM. The same
observation could be made with sodium absorption and acuumulation in plant tissues, the most tolerant species (L. latifolium
being the least Na accumulator. Hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA), commonly produced in conditions
of stress, accumulated significantly in salt treated C. maritima and M. maritima while not in the tolerant L. latifolium. The
latter used glutathione reductase to maintain constant H2 O2 levels under salt stress while peroxidases were very low and
ascorbate peroxidase did not respond to salinity stimulation. The medium tolerant halophyte M. maritima used peroxidases
to protect from NaCl-induced H2 O2 , while the sensitive C. maritima failed to detoxify H2 O2 despite a sharp increase in
catalase activity. Results showed that the three coastal species differ in resistance to salinity. They also suggested that the
level of plant resistance to salinity could be attributed to differing mechanisms to manage the accumulation of sodium and
cope with the oxidative damages.
Key words: antioxidant enzymes; coastal plants; ecological status; growth; salinity

Introduction arid regions, particularly at Chott and Sebkhas, soil

Salty arid areas and salt marshes are considered as
wasteland. However, such habitats are home of many
salt tolerant species with a high economic potential for
increased food and fiber production, as well as medic-
inal sources (Flowers et al. 2010). Besides, they are a
crucial part of the complex natural ecosystems which
they help to sustain. Natural habitats are being pre-
served and disturbed communities are restored in or-
der to preserve the biodiversity. Such actions can bet-
ter be accomplished by understanding the distribution
and eco-physiology of species already adapted to diverse
saline environment (FAO 2008). Several halophytes are
reported along the coastal areas of France but little em-
pirical data is available about mechanisms underlying
their growth and development capacity under environ-
mental conditions such as salinity.
Plants in coastal areas are constantly exposed to
high salt concentrations. At the seaside, dune vegeta-
tion mainly receives salt spray, while brackish marsh
plants live on a salty soil and can be, if the sea has
tides, rhythmically submerged by salt water (Freipica
& Levinsh 2010). In salt deserts of the arid and semi-
is a source of salt particles easily carried by the wind
towards the land and surrounding cultures (Mtimet
2001). Thus, plants in these environments have cer-
tainly acquired characteristics to adapt to soils whose
chemical composition varies in time and space, depend-
ing on salinity and associated stress (Ben Hamed et
al. 2013). Therefore, the adaptations of these coastal
plants are complex and varied. Furthermore, coastal
and semi-arid areas could be economically productive
if we could understand the physiology of salt resistance
of both conventional and non-conventional crops.
Many abiotic stresses have been shown to secon-
darily cause oxidative stress in plants through the en-
hanced production of reactive oxygen species (ROS)
like OH. , O2−. and H2 O2 (Ozgur et al. 2013). These
ROS can damage essential membrane lipids as well as
proteins and nucleic acids of cells. Levels of ROS in
plant cells are normally mediated by protective antiox-
idant compounds or activities (Foyer & Noctor 2005;
Foyer & Noctor 2013; Ozgur et al. 2013). The primary
components of these systems include ascorbate and glu-
tathione, as well as antioxidant enzymes such as cata-
lase (CAT), peroxidases (POX), superoxide dismutase

* Corresponding author

c 2013 Institute of Botany, Slovak Academy of Sciences
194
(SOD) and the enzymes of the ascorbate glutathione cy-
cle including ascorbate peroxidase (APX), monodehy-
droascorbate reductase (MDHAR), dehydroascorbate
reductase (DHAR) and glutathione reductase (GR)
(Noctor & Foyer 1998).
Recently, many authors addressed investigations
on halophytes in which the mechanisms of salt resis-
tance are fully developed and functional to keep them
productive under stress. The regulation of ROS by an-
tioxidant enzymes under salt stress has been reviewed
in halophytes (Jithesh et al. 2006; Ozgur et al. 2013). It
was found that the activities of major antioxidant en-
zymes such as SOD, APX, CAT and POX can be sig-
nificantly increased in some halophytes. For example,
in Sesuvium portulacastrum, 1 M NaCl treatment in-
creased SOD, APX, CAT enzyme activities (Lokhande
et al. 2011). On the other hand, no significant change or
a decrease in activity of some antioxidant enzymes were
reported in Crithmum maritimum (Ben Hamed et al.
2007). Moreover, in Mesembryanthemum crystallinum,
400 mM NaCl treatment induced the H2 O2 content but
no significant difference was determined in CAT activ-
ity whereas a slight increase in peroxidase activity was
observed (Shevyakova et al. 2006). All halophytes have
unique antioxidant defence system and response differ-
ently to salinity which might be based on their physio-
logical differences and tolerance mechanisms.
Three coastal plant species of the Atlantic coast
in Brittany (France) were selected for the current ex-
periments. Limonium latifolium (sea lavender) grows
spontaneously in salt marshes, and is therefore period-
ically in contact with seawater. Matricaria maritima
(sea chamomile) grows in sandy littoral dunes, occa-
sionally exposed to NaCl through salt sprays. Finally,
Crambe maritima (seakale) is a characteristic species
of fixed dunes, only slightly exposed to the influence of
salt.
The purposes of the present paper were: i) to ana-
lyze growth response of coastal plants differing in their
ecological status to increasing salinity, ii) to evaluate
Na+ uptake, oxidative damage and antioxidative de-
fence stimulation; and iii) to define level of salt stress
tolerance of studied plant species as well as outline
mechanisms enabling it
Material and methods
Plant material and growth conditions
Seeds of Matricaria maritima (sea chamomile, Asteraceae),
Crambe maritima (wild seakale, Brassicaceae) and Limo-
nium latifolium (sea lavender, Plumbaginaceae) were col-
lected on the Atlantic shoreline of Brittany in late Septem-
ber, then kept at 4 ◦C until use. Seeds of each species were
sown in covered Petri dishes, on two filter paper layers im-
bibed with distilled water. To improve seed germination in
Crambe maritima, seeds were previously soaked in a solution
of 0.025% gibberellic acid for 18 h (Fusheng et al. 1998). Af-
ter three weeks, young seedlings were transferred in 250 mL
pots filled with a mixture of sand and sterile loam (1:1 v/v),
and watered daily with Hewitt nutrient solution (Hewitt
1966). Two-month old seedlings were partitioned into four
K. Ben Hamed et al.
sets of plants, treated with 0, 100, 200 or 400 mM NaCl. Ex-
periments were performed in a glasshouse under controlled
conditions: 21 ◦C ± 2 ◦C, 50–70% relative humidity, 700 µmol
m−2 s−1 PAR and 16 h/8 h day/night photoperiod. After
one month of NaCl treatment, plants were separated into
shoots and roots, and weighted. Plant parts were frozen at
–20 ◦C and freeze-dried before dry weight (DW) was deter-
mined. A part of the fresh shoot material was used for en-
zyme extraction and analysis.
Sodium assay
The dried plant material was finely ground to fine powders
with Dangoumau blender. Approximately 20 mg of pow-
der were digested with 50 mL of 0.5% HNO3, for one week
at room temperature. After filtration through ashless fil-
ter papers, particle free filtrates were diluted appropriately
and sodium was determined by flame emission photometer
(Corning, UK).
H2 O2 assay
H2 O2 quantification is based on the formation of a titanium
peroxide complex (Ellouzi et al. 2011). The dry leaf samples
were homogenized in cold acetone (1:6, w/v). After filtra-
tion through eight layers of gauze cloth, the extracts were
centrifuged twice at 3000 g for 15 min. To an aliquot of
the supernatant, 20% TiCl4 in concentrated HCl was added
to give a final titanium concentration of 4%, followed by
the addition of NH4 OH to precipitate the titanium perox-
ide complex. After 10 min of centrifugation at 3000 g, the
supernatant was discarded and pellet was dissolved in 3 mL
of 2 N H2 SO4 . The absorbance of the solution was read at
410 nm and H2 O2 content was calculated using a standard
curve with concentration ranging from 0.1 to 1 mM.
Lipid peroxidation
The extent of lipid peroxidation (LP) was estimated by
determining the concentration of malondialdehyde (MDA)
(Ben Hamed et al. 2007). Leaf material was homogenized in
0.1% (w/v) TCA solution. The homogenate was centrifuged
at 15,000 g for 10 min and 1 mL of the supernatant ob-
tained was added to 4 mL 0.5% (w/v) TBA in 20% (w/v)
TCA. The mixture was incubated at 90 ◦C for 30 min. The
reaction was stopped by placing the reaction tubes in ice.
Samples were centrifuged at 10,000 g for 5 min, and the
absorbance of supernatant was read at 532 nm. The value
for non-specific absorption at 600 nm was subtracted. The
concentration of MDA was calculated from the extinction
coefficient 155 mM−1 cm−1 .
Enzyme extractions
All of the following operations were performed at 4 ◦C. Fresh
shoot samples were rapidly extracted in a pre-chilled mortar
with 10% (w/w) PVP (polyvinylpyrrolidone) in 100 mM K-
phosphate buffer (pH 7 for CAT and POD; pH 7.8 for SOD,
APX and GR), containing 5 mM EDTA (ethylenediaminete-
traacetic acid), 2 mM DTT (dithiothreitol), glycerol 10%
(v/v) and protease inhibitor cocktail (leupeptin 10 µM, 4-
(2-aminoethyl) benzenesulfonyl fluoride 0.2 mM, pepstatin
A 1 mM, bestatin 1 mM, E-64 1 µM, 1,10-phenanthroline
1 mM). For APX activity, 10 mM ascorbate was added
to the extraction medium to maintain the enzyme active
during the extraction procedure. The homogenate was cen-
trifuged at 14,000 g for 30 min. Three replicates per treat-
ment were made. The supernatants were collected and pro-
tein concentration was determined according to Bradford
(1976), using bovine serum albumin as a standard.
Coastal plants under salinity 195
Table 1. Effect of NaCl salinity the fresh (FW) and dry (DW) weights, and water content of the shoots and roots of studied halophytes.
The results are the means of 5 replicates. For a plant tissue, means followed by different letters are significantly different at P ≤ 0.05
according to student’s LSD test.

Limonium latifolium Matricaria maritima Crambe maritima

Shoots Roots Shoots Roots Shoots Roots

FW (g/plant)
0 mM 33.25 ± 5.69 11.75 ± 5.03 25.70 ± 4.42 8.16 ± 0.46 37.94 ± 7.52 9.03 ± 2.51
100 mM 24.12 ± 5.86 10.20 ± 1.53 22.33 ± 3.83 6.65 ± 0.49 8.05 ± 3.25 1.41 ± 0.18
200 mM 11.90 ± 1.37 6.01 ± 3.17 13.34 ± 3.74 4.44 ± 0.99 1.85 ± 0.29 0.20 ± 0.09
400 mM 9.05 ± 0.43 6.12 ± 1.69 3.59 ± 1.16 0.81 ± 0.40

DW (g/plant)
0 mM 8.49 ± 2.30 2.28 ± 0.08 3.40 ± 0.59 0.642 ± 0.08 5.30 ± 0.59 2.42 ± 0.86
100 mM 4.80 ± 1.21 1.32 ± 0.05 3.15 ± 0.82 0.475 ± 0.04 1.23 ± 0.46 0.46 ± 0.32
200 mM 2.59 ± 0.29 0.94 ± 0.42 2.72 ± 0.76 0.38 ± 0.07 0.31 ± 0.07 0.04 ± 0.00
400 mM 2.08 ± 0.25 1.25 ± 0.46 0.68 ± 0.22 0.11 ± 0.06

Shoot/Root DW
0 mM 3.72 5.30 2.19
100 mM 3.64 9.97 2.68
200 mM 2.75 7.03 7.75
400 mM 1.66 6.34

H 2 O (mL/g DW)
0 mM 2.96 ± 0.40 4.13 ± 2.02 6.55 ± 0.60 11.76 ± 0.80 6.16 ± 0.82 2.72 ± 0.58
100 mM 3.70 ± 0.00 5.59 ± 0.82 6.09 ± 1.01 13.00 ± 0.11 5.52 ± 0.79 2.04 ± 0.71
200 mM 3.59 ± 0.00 5.31 ± 0.55 3.9 ± 0.22 10.52 ± 0.47 4.97 ± 1.25 3.73 ± 0.14
400 mM 3.37 ± 0.74 3.96 ± 0.47 4.3 ± 0.19 6.28 ± 1.00

Enzyme assays
All enzyme activities were measured at 25 ◦C using a
Shimadzu UV-160 spectrophotometer. Soluble peroxidase
(POD) activity was determined spectrophotometrically by
measuring the oxidation of o-dianisidine (3,3’-dimethoxy-
benzidine) at 460 nm (Ranieri et al. 2000). The reaction
mixture contained 20 mM Na-acetate buffer (pH 5.0), 1 mM
dianisidine, 3 mM H2 O2 and 50 µL of extract. POD activ-
ity was expressed as units (µmoles of oxidized dianisidine
per min ute) per g FW. CAT (EC 1.11.1.6) activity was
measured according to the method of Lück (1965), by mon-
itoring the decline in absorbance at 240 nm as H2 O2 was
consumed. The 3 ml reaction mixture contained 66 mM
sodium phosphate buffer (pH 7.0), to which 30% (w/v)
H2 O2 , was added (the optical density should be about 0.5
at 240 nm and with a 1 cm light path). The reaction was
initiated by adding an appropriate dilution of the shoot or
root crude extract to this solution. The time ∆t required
for a decrease in the absorbance from 0.45 to 0.4 is used for
CAT activity calculations. The activity was expressed as
units (µmol of H2 O2 decomposed per min) per g FW. APX
(EC 1.11.1.11) activity was measured spectrophotometri-
cally according to Nakano & Asada (1981) by following the
decline in absorbance at 290 nm as ascorbate was oxidized
(Σ = 2.8 mM−1 cm−1 ). The oxidation rate of ascorbate was
estimated between 1 and 60 s after starting the reaction
with the addition of H2 O2 . The 1 mL reaction mixture con-
tained 50 mM HEPES-NaOH (pH 7.6), 0.22 mM ascorbate,
1 mM H2 O2 , and an enzyme sample. The control reaction
mixture was prepared without the enzyme extract. Correc-
tions were made for the low, non-enzymatic oxidation of
ascorbate by H2 O2 and for the oxidation of ascorbate in
the absence of H2 O2 . The activity was expressed as units
(µmol of oxidized ascorbate per min) per g FW. GR (EC
1.6.4.2) activity was determined by following the rate of
NADPH oxidation as measured by the decrease in the ab-
sorbance at 340 nm. The assay mixture (1 mL final volume)
was composed of 0.4 M potassium phosphate buffer pH 7.5,
0.4 mM Na2 EDTA, 5 mM GSSG, 2 mM NADPH, and 100 µl
of crude extract. GR activity was determined by monitor-
ing GSSG-dependent oxidation of NADPH at 340 nm and
30 ◦C (Di Baccio et al. 2004). Corrections were made for the
background absorbance at 340 nm, without NADPH. Ac-
tivity was expressed as units (µmoles of oxidized NADPH
per minute) per g FW.
Statistical analysis
Analysis of variance (ANOVA) using AV1W MSUSTAT
program with orthogonal contrasts and mean comparison
procedures was performed to detect significant differences
between treatments. Mean separation procedures were car-
ried out using the multiple range tests with Student’s least
significant difference (LSD) (P ≤ 0.05).
Results
Effect of salt treatment on plant morphology and
biomass production
Salt treatment led to a different morphological re-
sponse in Limonium latifolium, Matricaria maritima
and Crambe maritima plants (Fig. 1). NaCl-treated
L. latifolium and M. maritima, although smaller than
control plants (on a dose-dependent manner), stayed
vigorous. In contrast, C. maritima size decreased dra-
matically in the presence of NaCl and the shoots of
these plants showed visible necrosis.
Tissue biomass analysis confirmed the previous
observation on growth slowing upon salinity (Ta-
ble 1). The fresh (FW) and dry weight (DW) of
the whole plant of L. latifolium decreased by 43,
196 K. Ben Hamed et al.

Fig. 1. Phenotypes of Limononium latifolium, Matricaria maritima and Crambe maritima exposed to salt stress treatment during the
period of study. From left to right, 0, 100 mM, 200 mM and 400 mM NaCl.

67, 69% (as compared to control plants), at 100,
200 and 400 mM NaCl, respectively. In M. mari-
tima, FW and DW decreased by 10 and 31% at
100 and 200 mM NaCl, respectively, and by 87% at
400 mM NaCl. C. maritima showed the highest growth
inhibition compared to the other two species, and
the measurements of biomass became difficult start-
ing from 200 mM NaCl due to the death of some
plants.
Shoot and root biomass decreased in salt-treated
plants when compared to controls (Table 1). As men-
tioned previously with whole plants, shoot biomass de-
Coastal plants under salinity
Fig. 2. Effect of NaCl treatments on the concentration of sodium in the roots and leaves of L. latifolium, M. maritima and C. maritima.
The results are the means of 5 replicates. Means followed by different letters are significantly different at P ≤ 0.05 according to student’s
LSD test.
creased rapidly under mild salt stress in L. latifolium
or C. maritima, and more slowly in M. maritima. How-
ever, the former tolerated more the higher salt level
since a significant root biomass was still observed at
400 mM NaCl. In Crambe, both root and shoot biomass
declined markedly at each salt level, and root biomass
could hardly been measured at 200 or 400 mM NaCl.
As a consequence of tissue biomass analysis upon
salinity, shoot/root ratio decreased in salt-treated L. la-
tifolium (by 50% at 400 mM) while it did not change
much in M. maritima. Noteworthy was the dramatic
increase of shoot/root ratio in C. maritima due to the
very low root biomass under mild or high salinity.
Effect of salt treatment on Na accumulation in plant
tissues
Sodium accumulated in the shoots of all the tested
halophytes in the presence of NaCl (Fig. 2). However,
Na+ concentrations were always lower in L. latifolium
shoots than in those of M. maritima and C. maritima.
Thus, Na+ levels in those plant shoots at 200 mM NaCl
reached 1.69, 4.47 and 4.75 mmol g−1 DW, respectively,
197
corresponding to respective Na+ mean concentrations
in shoot tissue water of 503 mM, 1014 mM and 954 mM.
Similar observation could be made in the roots, with
Na+ levels 2.3 and 3 times lower in L. latifolium than
in those of M. maritima and C. maritima, respectively.
Effect of salt treatment on H2 O2 and malodialdehyde
contents
Hydrogen peroxide (H2 O2 ) and MDA levels increased
significantly in the shoots of C. maritima when treated
with 100 and 200 mM NaCl (Table 2). The levels of
these molecules were higher in the shoots of M. mari-
tima treated with 200 mM and 400 mM NaCl than in
control and 100 mM treated plants. In L. latifolium, no
change in the level of H2 O2 and MDA could be observed
under salinity.
Effect of salt treatment on H2 O2 detoxifying enzyme ac-
tivities
The activity of dianisidine peroxidases (POD) upon
salt treatment decreased significantly in L. latifolium
shoots (–60%) while it increased significantly in M.
198 K. Ben Hamed et al.
Table 2. Effect of NaCl treatment on the concentration of H2 O2 and of malondialdéhyde in the shoots of L. latifolium, M. maritima
and C. maritima. The results are the means ± SD of 5 replicates. In a column, means followed by different letters are significantly
different at P ≤ 0.05 according to student’s LSD test.

NaCl (mM) Limonium latifolium M. maritima C. maritima

H2 O2 (µmol g−1 FW)

0 1.38 ± 0.24a 0.68 ± 0.08a 0.64 ± 0.03a
100 1.47 ± 0.10a 0.75 ± 0.14a 3.75 ± 0.26b
200 1.43 ± 0.13a 1.26 ± 0.06b 5.35 ± 0.44c
400 1.58 ± 0.10a 2.56 ± 0.27c —
MDA (nmol g−1 FW)
0 15.59 ± 2.41a 10.23 ± 1.58a 10.64 ± 2.35a
100 16.51 ± 4.24a 10.75 ± 1.14a 28.75 ± 2.61b
200 16.25 ± 3.63a 12.68 ± 2.56b 35.35 ± 4.45c
400 16.68 ± 2.17a 25.21 ± 2.12c —

Fig. 3. Absolute activities of POD (A), CAT (B), APX (C) and GR (D) in the leaves of L. latifolium, M. maritima and Crambe
maritima, expressed in percentage of the activity in control plants. The results are the means of 5 replicates. Means followed by
different letters are significantly different at P ≤ 0.05 according to student’s LSD test. POD activity in control plant (U g−1 FW):
3.69 ± 0.19 for L. latifolium, 2.17 ± 0.06 for M. maritima, and 38.23 ± 2.76 for C. maritima. CAT activity in control plant (U g−1
FW): 23.75 ± 0.94 for M. maritima, and 5.75 ± 1.00 for C. maritima. APX activity in control plant (U g−1 FW): 33 ± 4.20 for
L. latifolium, 45 ± 5.49 for M. maritima, and 72 ± 8.54 for C. maritima. GR activity in control plant (U g−1 FW): 2.43 ± 0.73 for
Limonium latifolium, 230.95 ± 4.43 for M. maritima, and 180.86 ± 33.80 for C. maritima.

maritima ones (+ 75 to 130%), compared to control
plants (Fig. 3A). In C. maritima plants, peroxidase ac-
tivity did not vary significantly under salinity. Catalase
(CAT) activity could be detected neither in control nor
in salt-stressed L. latifolium plants (Fig. 3B). In M. ma-
ritima, CAT activity decreased only in 400 mM NaCl-
treated plants, while it was significant stimulated in C.
maritima even under slight treatment (7-fold higher at
100 mM NaCl, compared to control plants). APX activ-
ity in L. latifolium did not change upon NaCl treatment
(Fig. 3C). In M. maritima and C. maritima, APX ac-
tivity increased only at 100 mM NaCl, but recovered
control levels under higher salinity.
GR activity increased significantly in salt treated
plants of L. latifolium and, to a lower extent, of Ma-
tricaria (Fig. 3D). The highest increase was found in
Coastal plants under salinity
L. latifolium at 400 mM NaCl (+ 123% of control).
In C. maritima, GR activity decreased with increasing
salinity.
Discussion
The growth of coastal plants investigated in this work
decreased with increasing external salinity, demonstrat-
ing that such halophytes grow optimally in the ab-
sence of salt (Vicente at al. 2004; Grigore et al. 2012).
Actually, most of the naturally tolerant plants are in
that case and are called facultative halophytes. Besides,
only a few dicotyledonous halophytes show some growth
stimulation at moderate NaCl concentrations from 50
to 250 mM (Flowers et al. 2008). The three studied
species were differently affected by salt treatment. L. la-
tifolium thus appeared as the most salt tolerant, with
sustained growth and vigorous tissues at 400 mM NaCl.
M. maritima tolerated well the light and medium salt
levels, but its growth was markedly inhibited (–90%)
by 400 mM NaCl. Plants of C. maritima were the most
sensitive, as shown by the drastic reduction of biomass
and the development of leaf necrosis in the presence of
salt.
Observations on whole plant biomass were con-
firmed by those of shoot biomass. Roots showed differ-
ent responses to NaCl in the three halophytes. In L. la-
tifolium, root biomass production decreased slightly in
treated plants when compared to control plants, even
under high salinity. As a consequence, shoot to root ra-
tio decreased with increasing NaCl concentration. Pre-
vious studies carried out with cotton (Meloni et al.
2001), as well as in Prosopis alba (Meloni et al. 2004)
and Aeluropus littorlais (Barhoumi et al. 2007) also
showed that shoot growth was more inhibited by NaCl
than root growth. Thus, increased root/shoot ratio ap-
pears to be an adaptation to salinity, resulting in a more
efficient water and nutrient uptake under saline stress
(Gorham et al. 1985). Little work has been done on root
physiology with regard to either salt or water stress
(Munns 2002). Roots would appear to be the most vul-
nerable part of the plant as they are directly exposed to
salt or to drying soil, but nevertheless they are surpris-
ingly robust. Our data show that root growth of L. la-
tifolium, under NaCl concentrations, was less affected
than shoot growth. This observation was confirmed by
the morphology of root system in L. latifolium. Thus,
root architecture was conserved even after a prolonged
high salinity treatment (data not shown). This original
result rather explains the resistance of L. latifolium to
high salinity. The same observations can be reported for
the roots of M. maritima that maintained their vigor at
100 mM and 200 mM NaCl. Heidari and Sarani (2012)
found that increasing salinity from 0 to 150 mM de-
creased FW of shoots and increased that of roots in
M. chamomilla.
Conversely, growth of C. maritima roots was more
affected by NaCl than that of the shoots, resulting in an
increased shoot to root ratio in salt-treated plants. This
root degradation, occurring from the lowest NaCl con-
199
centration (100 mM NaCl), could be the major cause
for the poor resistance of C. maritima to the inflicted
treatment. De Vos et al (2010) reported that the sensi-
tivity of C. maritima to salinity was mainly caused by
the reduction in the specific leaf area.
On the basis of these results, it is possible to range
the three species according to their degree of salt resis-
tance. Under moderate salt stress (NaCl ≤ 100 mM),
M. maritima appeared to be the most resistant species.
Additional experiments even showed a stimulation of
growth after the first two weeks of treatment (data not
shown). In another species of the genera Matricaria,
Kovačik et al. (2009) reported that M. chamomilla can
tolerate up to 100 mM NaCl. Conversely, under severe
stress (NaCl ≥ 200 mM), L. latifolium was the most
resistant species owing to its capacity to maintain root
development. Whatever the concentration, C. maritima
was sensitive to salt stress with particular susceptibility
of root apparatus.
Interestingly, our results obtained under controlled
conditions could be compared to the conditions experi-
enced by plants in their natural habitats. Thus, tolerant
L. latifolium plants grow spontaneously in salt marshes,
and are therefore periodically in contact with seawater.
M. maritima grows in sandy littoral dunes, occasion-
ally exposed to NaCl through salt sprays. Finally, the
sensitive C. maritima is a characteristic species of fixed
dunes, only slightly exposed to the influence of root
zone salinity. This species is however tolerant to sat
spray levels which it encounters in its natural habitats
(de Vos et al. 2010).
In order to see whether there is a relationship be-
tween salt resistance of plants and their capacity to
accumulate sodium, we have measured Na+ ions in the
shoots and roots of the tested plant species. On the
whole, all the plants accumulated sodium in their aerial
parts proportionally to the concentration of salt in the
medium, but with different intensities, and mainly with
different consequences on the survival of plants. M. ma-
ritima and C. maritima accumulated high and equiv-
alents levels of Na in their shoots. However, only the
latter developed necrosis signs suggesting that M. ma-
ritima is able to tolerate the presence of high levels of
sodium in shoot tissues, like the majority of dicotyle-
donous halophytes (Flowers & Colmer 2008). This hy-
pothesis needs to be ascertained by additional investi-
gations on the mechanisms of sodium resistance in M.
maritima. Conversely, NaCl-treated L. latifolium plants
accumulated less Na in their shoots at equivalent salin-
ity levels. This result suggested that this plant possesses
a better capacity to control the absorption of sodium
ions and/or the excretion of such ions through its leaves,
probably in relation to its high capacity to accumulate
osmoregulatory solutes (Gagneul et al. 2007). In addi-
tion, we observed the presence of salt crystals on both
sides of L. latifolium leaves, proportionally to the salin-
ity concentrations in the medium (data not shown).
These observations confirmed that L. latifolium had
morphological characteristics that allow it to exclude
salt. Salt excretion is generally mediated by specific
200
glands scattered on the leaf surfaces (Barhoumi et al.
2007) and it is a typical strategy of several plant gen-
era and families (e.g., Plumbaginaceae, Avicenniaceae,
Tamaricaceae, Frankeniaceae and Poaceae) to detoxify
NaCl (Waisel 1972; Barhoumi et al. 2007).
The activities of antioxidant enzymes were mea-
sured in the shoots of NaCl-treated plants and com-
pared to those of control plants. Differences were found
between the species in terms of antioxidant enzyme re-
sponses. In M. maritima, which previously appeared to
be resistant to moderate salinity, increase in dianisidine
peroxidases (POD) activities in salt-treated plants was
associated with high accumulation of H2 O2 . These re-
sults suggest that POD could play a crucial role in the
resistance of M. maritima to moderate saline condi-
tions. Similar results were reported in other halophytes
like Crithmum maritimum (Ben Hamed et al. 2007),
Cakile maritima (Ben Amor et al. 2006) and Mesem-
bryanthemum crystallinum (Shevyakova et al. 2006).
POD enzymes protect cells against harmful concentra-
tions of hydroperoxides and may also play a role in the
oxidation of phenolic metabolites in leaves under NaCl
stress (Ben Hamed et al. 2012).
In L. latifolium, which appeared more resistant to
high salinity, the activities of dianisidine peroxidases
were very low in the shoots where H2 O2 is not produced
in large amounts. In addition, the low levels of H2 O2
explain the undetectable catalase activity, that enzyme
being known for its high affinity to H2 O2 (Mittler 2002).
Li et al. (2008) reported an increase in POD and CAT
activities under salinity in the shoots of another species
of Limonium, L. bicolor. These results showed an exam-
ple of intra-specific variation in the antioxidant behav-
ior of both enzymes under salinity. The increased glu-
tathione reductase (GR) activities observed in 400 mM
NaCl-treated plants understudy indicated that this en-
zyme is involved in the resistance of L. latifolium to
high salinity level. The activity of GR is known to in-
crease the ratio of NADP+ /NADPH, thereby ensuring
availability of NADP+ to accept electrons from pho-
tosynthetic electron transport chain and min imizing
the formation of superoxides (Chaparzadeh et al. 2004).
Moreover, the higher GR activity in salt treated L. la-
tifolium plants would result in a higher pool of reduced
glutathione (GSH) which, in turn, could be used to
deplete dehydroascorbate and produce a more favor-
able ascorbate to dehydroascorbate ratio (Reddy et al.
2004).
In C. maritima, the high activity of CAT found
under salt treatment indicated that H2 O2 is highly pro-
duced in this species in response to NaCl. This activ-
ity seems, however, insufficient to allow plant survival.
The decrease of GR activities observed in salt treated
C. maritima indicated that GR is not crucial for the
detoxification of activated oxygen in that species.
In conclusion, the effect of NaCl on the growth of
the three studied plants was reported here for the first
time. Those halophytes exhibited different degrees of
salt resistance on the basis of biomass production (e.g.,
growth), L. latifolium and C. maritima appearing to
K. Ben Hamed et al.
be the most tolerant and sensitive species, respectively.
M. maritima could be considered as an intermediate
species, tolerating only moderate salinity (100 mM). All
the tested plants accumulated sodium in their shoots
but differed in its management. L. latifoilum excluded
sodium outside the leaves. M. maritima behaved like
a Na+ -includer tolerating the accumulated sodium, in
contrast to C. maritima. The study of plant antioxi-
dant defense under salt stress showed that the most
tolerant species (L. latifolium) uses GR to protect from
stress-induced ROS whereas peroxidases were very low
and ascorbate peroxidase did not respond to salinity
stimulation. The medium tolerant halophyte M. ma-
ritima uses POD for that purpose, while the sensitive
Crambe maritime, in spite of a strong stimulation of
CAT activity, failed to detoxify H2 O2 produced in its
cells. Results showed that L. latifolium, M. maritima
and C. maritima used different antioxidant enzymes
to cope with salinity. Additional experiments are in
progress to ascertain i) whether antioxidant molecules
such as ascorbate and glutathione are used by those
halophytes to protect plants from salinity-induced ROS
and ii) whether there is a common strategy of defense
against oxidative stress in the naturally tolerant species
(including facultative and obligate halophytes).
Acknowledgements
The authors are indebted to the Brittany Regional Council
(France) for financial support to K.B.H.
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Received July 3, 2013
Accepted October 11, 2013