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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


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

* Corresponding author


c 2013 Institute of Botany, Slovak Academy of Sciences
194 K. Ben Hamed et al.

(SOD) and the enzymes of the ascorbate glutathione cy- sets of plants, treated with 0, 100, 200 or 400 mM NaCl. Ex-
cle including ascorbate peroxidase (APX), monodehy- periments were performed in a glasshouse under controlled
droascorbate reductase (MDHAR), dehydroascorbate conditions: 21 ◦C ± 2 ◦C, 50–70% relative humidity, 700 µmol
reductase (DHAR) and glutathione reductase (GR) m−2 s−1 PAR and 16 h/8 h day/night photoperiod. After
one month of NaCl treatment, plants were separated into
(Noctor & Foyer 1998).
shoots and roots, and weighted. Plant parts were frozen at
Recently, many authors addressed investigations –20 ◦C and freeze-dried before dry weight (DW) was deter-
on halophytes in which the mechanisms of salt resis- mined. A part of the fresh shoot material was used for en-
tance are fully developed and functional to keep them zyme extraction and analysis.
productive under stress. The regulation of ROS by an-
tioxidant enzymes under salt stress has been reviewed Sodium assay
in halophytes (Jithesh et al. 2006; Ozgur et al. 2013). It The dried plant material was finely ground to fine powders
was found that the activities of major antioxidant en- with Dangoumau blender. Approximately 20 mg of pow-
zymes such as SOD, APX, CAT and POX can be sig- der were digested with 50 mL of 0.5% HNO3, for one week
nificantly increased in some halophytes. For example, at room temperature. After filtration through ashless fil-
in Sesuvium portulacastrum, 1 M NaCl treatment in- ter papers, particle free filtrates were diluted appropriately
and sodium was determined by flame emission photometer
creased SOD, APX, CAT enzyme activities (Lokhande
(Corning, UK).
et al. 2011). On the other hand, no significant change or
a decrease in activity of some antioxidant enzymes were H2 O2 assay
reported in Crithmum maritimum (Ben Hamed et al. H2 O2 quantification is based on the formation of a titanium
2007). Moreover, in Mesembryanthemum crystallinum, peroxide complex (Ellouzi et al. 2011). The dry leaf samples
400 mM NaCl treatment induced the H2 O2 content but were homogenized in cold acetone (1:6, w/v). After filtra-
no significant difference was determined in CAT activ- tion through eight layers of gauze cloth, the extracts were
ity whereas a slight increase in peroxidase activity was centrifuged twice at 3000 g for 15 min. To an aliquot of
observed (Shevyakova et al. 2006). All halophytes have the supernatant, 20% TiCl4 in concentrated HCl was added
unique antioxidant defence system and response differ- to give a final titanium concentration of 4%, followed by
the addition of NH4 OH to precipitate the titanium perox-
ently to salinity which might be based on their physio-
ide complex. After 10 min of centrifugation at 3000 g, the
logical differences and tolerance mechanisms. supernatant was discarded and pellet was dissolved in 3 mL
Three coastal plant species of the Atlantic coast of 2 N H2 SO4 . The absorbance of the solution was read at
in Brittany (France) were selected for the current ex- 410 nm and H2 O2 content was calculated using a standard
periments. Limonium latifolium (sea lavender) grows curve with concentration ranging from 0.1 to 1 mM.
spontaneously in salt marshes, and is therefore period-
ically in contact with seawater. Matricaria maritima Lipid peroxidation
(sea chamomile) grows in sandy littoral dunes, occa- The extent of lipid peroxidation (LP) was estimated by
sionally exposed to NaCl through salt sprays. Finally, determining the concentration of malondialdehyde (MDA)
Crambe maritima (seakale) is a characteristic species (Ben Hamed et al. 2007). Leaf material was homogenized in
0.1% (w/v) TCA solution. The homogenate was centrifuged
of fixed dunes, only slightly exposed to the influence of
at 15,000 g for 10 min and 1 mL of the supernatant ob-
salt. tained was added to 4 mL 0.5% (w/v) TBA in 20% (w/v)
The purposes of the present paper were: i) to ana- TCA. The mixture was incubated at 90 ◦C for 30 min. The
lyze growth response of coastal plants differing in their reaction was stopped by placing the reaction tubes in ice.
ecological status to increasing salinity, ii) to evaluate Samples were centrifuged at 10,000 g for 5 min, and the
Na+ uptake, oxidative damage and antioxidative de- absorbance of supernatant was read at 532 nm. The value
fence stimulation; and iii) to define level of salt stress for non-specific absorption at 600 nm was subtracted. The
tolerance of studied plant species as well as outline concentration of MDA was calculated from the extinction
mechanisms enabling it coefficient 155 mM−1 cm−1 .

Enzyme extractions
Material and methods All of the following operations were performed at 4 ◦C. Fresh
shoot samples were rapidly extracted in a pre-chilled mortar
Plant material and growth conditions with 10% (w/w) PVP (polyvinylpyrrolidone) in 100 mM K-
Seeds of Matricaria maritima (sea chamomile, Asteraceae), phosphate buffer (pH 7 for CAT and POD; pH 7.8 for SOD,
Crambe maritima (wild seakale, Brassicaceae) and Limo- APX and GR), containing 5 mM EDTA (ethylenediaminete-
nium latifolium (sea lavender, Plumbaginaceae) were col- traacetic acid), 2 mM DTT (dithiothreitol), glycerol 10%
lected on the Atlantic shoreline of Brittany in late Septem- (v/v) and protease inhibitor cocktail (leupeptin 10 µM, 4-
ber, then kept at 4 ◦C until use. Seeds of each species were (2-aminoethyl) benzenesulfonyl fluoride 0.2 mM, pepstatin
sown in covered Petri dishes, on two filter paper layers im- A 1 mM, bestatin 1 mM, E-64 1 µM, 1,10-phenanthroline
bibed with distilled water. To improve seed germination in 1 mM). For APX activity, 10 mM ascorbate was added
Crambe maritima, seeds were previously soaked in a solution to the extraction medium to maintain the enzyme active
of 0.025% gibberellic acid for 18 h (Fusheng et al. 1998). Af- during the extraction procedure. The homogenate was cen-
ter three weeks, young seedlings were transferred in 250 mL trifuged at 14,000 g for 30 min. Three replicates per treat-
pots filled with a mixture of sand and sterile loam (1:1 v/v), ment were made. The supernatants were collected and pro-
and watered daily with Hewitt nutrient solution (Hewitt tein concentration was determined according to Bradford
1966). Two-month old seedlings were partitioned into four (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 sorbance at 340 nm. The assay mixture (1 mL final volume)
All enzyme activities were measured at 25 ◦C using a was composed of 0.4 M potassium phosphate buffer pH 7.5,
Shimadzu UV-160 spectrophotometer. Soluble peroxidase 0.4 mM Na2 EDTA, 5 mM GSSG, 2 mM NADPH, and 100 µl
(POD) activity was determined spectrophotometrically by of crude extract. GR activity was determined by monitor-
measuring the oxidation of o-dianisidine (3,3’-dimethoxy- ing GSSG-dependent oxidation of NADPH at 340 nm and
benzidine) at 460 nm (Ranieri et al. 2000). The reaction 30 ◦C (Di Baccio et al. 2004). Corrections were made for the
mixture contained 20 mM Na-acetate buffer (pH 5.0), 1 mM background absorbance at 340 nm, without NADPH. Ac-
dianisidine, 3 mM H2 O2 and 50 µL of extract. POD activ- tivity was expressed as units (µmoles of oxidized NADPH
ity was expressed as units (µmoles of oxidized dianisidine per minute) per g FW.
per min ute) per g FW. CAT (EC 1.11.1.6) activity was
measured according to the method of Lück (1965), by mon- Statistical analysis
itoring the decline in absorbance at 240 nm as H2 O2 was Analysis of variance (ANOVA) using AV1W MSUSTAT
consumed. The 3 ml reaction mixture contained 66 mM program with orthogonal contrasts and mean comparison
sodium phosphate buffer (pH 7.0), to which 30% (w/v) procedures was performed to detect significant differences
H2 O2 , was added (the optical density should be about 0.5 between treatments. Mean separation procedures were car-
at 240 nm and with a 1 cm light path). The reaction was ried out using the multiple range tests with Student’s least
initiated by adding an appropriate dilution of the shoot or significant difference (LSD) (P ≤ 0.05).
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
Results
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- Effect of salt treatment on plant morphology and
cally according to Nakano & Asada (1981) by following the biomass production
decline in absorbance at 290 nm as ascorbate was oxidized Salt treatment led to a different morphological re-
(Σ = 2.8 mM−1 cm−1 ). The oxidation rate of ascorbate was sponse in Limonium latifolium, Matricaria maritima
estimated between 1 and 60 s after starting the reaction and Crambe maritima plants (Fig. 1). NaCl-treated
with the addition of H2 O2 . The 1 mL reaction mixture con- L. latifolium and M. maritima, although smaller than
tained 50 mM HEPES-NaOH (pH 7.6), 0.22 mM ascorbate, control plants (on a dose-dependent manner), stayed
1 mM H2 O2 , and an enzyme sample. The control reaction vigorous. In contrast, C. maritima size decreased dra-
mixture was prepared without the enzyme extract. Correc-
matically in the presence of NaCl and the shoots of
tions were made for the low, non-enzymatic oxidation of
ascorbate by H2 O2 and for the oxidation of ascorbate in these plants showed visible necrosis.
the absence of H2 O2 . The activity was expressed as units Tissue biomass analysis confirmed the previous
(µmol of oxidized ascorbate per min) per g FW. GR (EC observation on growth slowing upon salinity (Ta-
1.6.4.2) activity was determined by following the rate of ble 1). The fresh (FW) and dry weight (DW) of
NADPH oxidation as measured by the decrease in the ab- 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, the measurements of biomass became difficult start-
200 and 400 mM NaCl, respectively. In M. mari- ing from 200 mM NaCl due to the death of some
tima, FW and DW decreased by 10 and 31% at plants.
100 and 200 mM NaCl, respectively, and by 87% at Shoot and root biomass decreased in salt-treated
400 mM NaCl. C. maritima showed the highest growth plants when compared to controls (Table 1). As men-
inhibition compared to the other two species, and tioned previously with whole plants, shoot biomass de-
Coastal plants under salinity 197

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 corresponding to respective Na+ mean concentrations
or C. maritima, and more slowly in M. maritima. How- in shoot tissue water of 503 mM, 1014 mM and 954 mM.
ever, the former tolerated more the higher salt level Similar observation could be made in the roots, with
since a significant root biomass was still observed at Na+ levels 2.3 and 3 times lower in L. latifolium than
400 mM NaCl. In Crambe, both root and shoot biomass in those of M. maritima and C. maritima, respectively.
declined markedly at each salt level, and root biomass
could hardly been measured at 200 or 400 mM NaCl. Effect of salt treatment on H2 O2 and malodialdehyde
As a consequence of tissue biomass analysis upon contents
salinity, shoot/root ratio decreased in salt-treated L. la- Hydrogen peroxide (H2 O2 ) and MDA levels increased
tifolium (by 50% at 400 mM) while it did not change significantly in the shoots of C. maritima when treated
much in M. maritima. Noteworthy was the dramatic with 100 and 200 mM NaCl (Table 2). The levels of
increase of shoot/root ratio in C. maritima due to the these molecules were higher in the shoots of M. mari-
very low root biomass under mild or high salinity. tima treated with 200 mM and 400 mM NaCl than in
control and 100 mM treated plants. In L. latifolium, no
Effect of salt treatment on Na accumulation in plant change in the level of H2 O2 and MDA could be observed
tissues under salinity.
Sodium accumulated in the shoots of all the tested
halophytes in the presence of NaCl (Fig. 2). However, Effect of salt treatment on H2 O2 detoxifying enzyme ac-
Na+ concentrations were always lower in L. latifolium tivities
shoots than in those of M. maritima and C. maritima. The activity of dianisidine peroxidases (POD) upon
Thus, Na+ levels in those plant shoots at 200 mM NaCl salt treatment decreased significantly in L. latifolium
reached 1.69, 4.47 and 4.75 mmol g−1 DW, respectively, 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 100 mM NaCl, compared to control plants). APX activ-
plants (Fig. 3A). In C. maritima plants, peroxidase ac- ity in L. latifolium did not change upon NaCl treatment
tivity did not vary significantly under salinity. Catalase (Fig. 3C). In M. maritima and C. maritima, APX ac-
(CAT) activity could be detected neither in control nor tivity increased only at 100 mM NaCl, but recovered
in salt-stressed L. latifolium plants (Fig. 3B). In M. ma- control levels under higher salinity.
ritima, CAT activity decreased only in 400 mM NaCl- GR activity increased significantly in salt treated
treated plants, while it was significant stimulated in C. plants of L. latifolium and, to a lower extent, of Ma-
maritima even under slight treatment (7-fold higher at tricaria (Fig. 3D). The highest increase was found in
Coastal plants under salinity 199

L. latifolium at 400 mM NaCl (+ 123% of control). centration (100 mM NaCl), could be the major cause
In C. maritima, GR activity decreased with increasing for the poor resistance of C. maritima to the inflicted
salinity. treatment. De Vos et al (2010) reported that the sensi-
tivity of C. maritima to salinity was mainly caused by
Discussion the reduction in the specific leaf area.
On the basis of these results, it is possible to range
The growth of coastal plants investigated in this work the three species according to their degree of salt resis-
decreased with increasing external salinity, demonstrat- tance. Under moderate salt stress (NaCl ≤ 100 mM),
ing that such halophytes grow optimally in the ab- M. maritima appeared to be the most resistant species.
sence of salt (Vicente at al. 2004; Grigore et al. 2012). Additional experiments even showed a stimulation of
Actually, most of the naturally tolerant plants are in growth after the first two weeks of treatment (data not
that case and are called facultative halophytes. Besides, shown). In another species of the genera Matricaria,
only a few dicotyledonous halophytes show some growth Kovačik et al. (2009) reported that M. chamomilla can
stimulation at moderate NaCl concentrations from 50 tolerate up to 100 mM NaCl. Conversely, under severe
to 250 mM (Flowers et al. 2008). The three studied stress (NaCl ≥ 200 mM), L. latifolium was the most
species were differently affected by salt treatment. L. la- resistant species owing to its capacity to maintain root
tifolium thus appeared as the most salt tolerant, with development. Whatever the concentration, C. maritima
sustained growth and vigorous tissues at 400 mM NaCl. was sensitive to salt stress with particular susceptibility
M. maritima tolerated well the light and medium salt of root apparatus.
levels, but its growth was markedly inhibited (–90%) Interestingly, our results obtained under controlled
by 400 mM NaCl. Plants of C. maritima were the most conditions could be compared to the conditions experi-
sensitive, as shown by the drastic reduction of biomass enced by plants in their natural habitats. Thus, tolerant
and the development of leaf necrosis in the presence of L. latifolium plants grow spontaneously in salt marshes,
salt. and are therefore periodically in contact with seawater.
Observations on whole plant biomass were con- M. maritima grows in sandy littoral dunes, occasion-
firmed by those of shoot biomass. Roots showed differ- ally exposed to NaCl through salt sprays. Finally, the
ent responses to NaCl in the three halophytes. In L. la- sensitive C. maritima is a characteristic species of fixed
tifolium, root biomass production decreased slightly in dunes, only slightly exposed to the influence of root
treated plants when compared to control plants, even zone salinity. This species is however tolerant to sat
under high salinity. As a consequence, shoot to root ra- spray levels which it encounters in its natural habitats
tio decreased with increasing NaCl concentration. Pre- (de Vos et al. 2010).
vious studies carried out with cotton (Meloni et al. In order to see whether there is a relationship be-
2001), as well as in Prosopis alba (Meloni et al. 2004) tween salt resistance of plants and their capacity to
and Aeluropus littorlais (Barhoumi et al. 2007) also accumulate sodium, we have measured Na+ ions in the
showed that shoot growth was more inhibited by NaCl shoots and roots of the tested plant species. On the
than root growth. Thus, increased root/shoot ratio ap- whole, all the plants accumulated sodium in their aerial
pears to be an adaptation to salinity, resulting in a more parts proportionally to the concentration of salt in the
efficient water and nutrient uptake under saline stress medium, but with different intensities, and mainly with
(Gorham et al. 1985). Little work has been done on root different consequences on the survival of plants. M. ma-
physiology with regard to either salt or water stress ritima and C. maritima accumulated high and equiv-
(Munns 2002). Roots would appear to be the most vul- alents levels of Na in their shoots. However, only the
nerable part of the plant as they are directly exposed to latter developed necrosis signs suggesting that M. ma-
salt or to drying soil, but nevertheless they are surpris- ritima is able to tolerate the presence of high levels of
ingly robust. Our data show that root growth of L. la- sodium in shoot tissues, like the majority of dicotyle-
tifolium, under NaCl concentrations, was less affected donous halophytes (Flowers & Colmer 2008). This hy-
than shoot growth. This observation was confirmed by pothesis needs to be ascertained by additional investi-
the morphology of root system in L. latifolium. Thus, gations on the mechanisms of sodium resistance in M.
root architecture was conserved even after a prolonged maritima. Conversely, NaCl-treated L. latifolium plants
high salinity treatment (data not shown). This original accumulated less Na in their shoots at equivalent salin-
result rather explains the resistance of L. latifolium to ity levels. This result suggested that this plant possesses
high salinity. The same observations can be reported for a better capacity to control the absorption of sodium
the roots of M. maritima that maintained their vigor at ions and/or the excretion of such ions through its leaves,
100 mM and 200 mM NaCl. Heidari and Sarani (2012) probably in relation to its high capacity to accumulate
found that increasing salinity from 0 to 150 mM de- osmoregulatory solutes (Gagneul et al. 2007). In addi-
creased FW of shoots and increased that of roots in tion, we observed the presence of salt crystals on both
M. chamomilla. sides of L. latifolium leaves, proportionally to the salin-
Conversely, growth of C. maritima roots was more ity concentrations in the medium (data not shown).
affected by NaCl than that of the shoots, resulting in an These observations confirmed that L. latifolium had
increased shoot to root ratio in salt-treated plants. This morphological characteristics that allow it to exclude
root degradation, occurring from the lowest NaCl con- salt. Salt excretion is generally mediated by specific
200 K. Ben Hamed et al.

glands scattered on the leaf surfaces (Barhoumi et al. be the most tolerant and sensitive species, respectively.
2007) and it is a typical strategy of several plant gen- M. maritima could be considered as an intermediate
era and families (e.g., Plumbaginaceae, Avicenniaceae, species, tolerating only moderate salinity (100 mM). All
Tamaricaceae, Frankeniaceae and Poaceae) to detoxify the tested plants accumulated sodium in their shoots
NaCl (Waisel 1972; Barhoumi et al. 2007). but differed in its management. L. latifoilum excluded
The activities of antioxidant enzymes were mea- sodium outside the leaves. M. maritima behaved like
sured in the shoots of NaCl-treated plants and com- a Na+ -includer tolerating the accumulated sodium, in
pared to those of control plants. Differences were found contrast to C. maritima. The study of plant antioxi-
between the species in terms of antioxidant enzyme re- dant defense under salt stress showed that the most
sponses. In M. maritima, which previously appeared to tolerant species (L. latifolium) uses GR to protect from
be resistant to moderate salinity, increase in dianisidine stress-induced ROS whereas peroxidases were very low
peroxidases (POD) activities in salt-treated plants was and ascorbate peroxidase did not respond to salinity
associated with high accumulation of H2 O2 . These re- stimulation. The medium tolerant halophyte M. ma-
sults suggest that POD could play a crucial role in the ritima uses POD for that purpose, while the sensitive
resistance of M. maritima to moderate saline condi- Crambe maritime, in spite of a strong stimulation of
tions. Similar results were reported in other halophytes CAT activity, failed to detoxify H2 O2 produced in its
like Crithmum maritimum (Ben Hamed et al. 2007), cells. Results showed that L. latifolium, M. maritima
Cakile maritima (Ben Amor et al. 2006) and Mesem- and C. maritima used different antioxidant enzymes
bryanthemum crystallinum (Shevyakova et al. 2006). to cope with salinity. Additional experiments are in
POD enzymes protect cells against harmful concentra- progress to ascertain i) whether antioxidant molecules
tions of hydroperoxides and may also play a role in the such as ascorbate and glutathione are used by those
oxidation of phenolic metabolites in leaves under NaCl halophytes to protect plants from salinity-induced ROS
stress (Ben Hamed et al. 2012). and ii) whether there is a common strategy of defense
In L. latifolium, which appeared more resistant to against oxidative stress in the naturally tolerant species
high salinity, the activities of dianisidine peroxidases (including facultative and obligate halophytes).
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 Acknowledgements
being known for its high affinity to H2 O2 (Mittler 2002).
The authors are indebted to the Brittany Regional Council
Li et al. (2008) reported an increase in POD and CAT (France) for financial support to K.B.H.
activities under salinity in the shoots of another species
of Limonium, L. bicolor. These results showed an exam-
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