ORIGINAL PAPER

Salt tolerance of the annual halophyte Cakile maritima as affectedby the provenance and the developmental stage

Wided Megdiche Æ Nader Ben Amor Æ Ahmed Debez ÆKamel Hessini Æ Riadh Ksouri Æ Yasmine Zuily-Fodil ÆChedly Abdelly

Received: 19 September 2006 / Revised: 9 January 2007 / Accepted: 11 January 2007 / Published online: 21 March 2007

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2007

Abstract Though halophytes are naturally adapted to

salinity, their salt-tolerance limits are greatly influenced by

their provenance and developmental stage. In the present

study, physio-biochemical responses of two Tunisian eco-

types of the oilseed coastal halophyte Cakile maritima

(Brassicaceae) to salinity (0–400 mM NaCl) were moni-

tored during germination and vegetative growth stages.

Tabarka and Jerba seeds were collected from humid or arid

climatic areas, respectively. Plant response to salinity ap-

peared to depend on the ecotype and salinity levels.

Increasing salinity inhibited germination process. Jerba

seeds were found to be more salt tolerant than the Tabarka

ones. At the autotrophic stage of growth and under salt-free

conditions, Jerba was less productive than Tabarka (in

terms of dry matter accumulation), but plant biomass

production and leaf expansion (area and number) of the

former ecotype were progressively improved by 100 mM

NaCl, as compared to the control. In contrast, at the same

salt concentration, these parameters decreased under

increasing salinity in Tabarka (salt sensitive). Leaf

chlorophyll content was reduced at severe salinity, but this

effect was more conspicuous in the sensitive Tabarka

plants. Na+ contents in the Jerba and Tabarka leaves col-

lected from the 400 mM NaCl-treated plants were 17- and

12-fold higher than in the respective controls. This effect

was accompanied by a significant reduction in the leaf K+,

Mg2+ and Ca2+ contents, especially in the salt-treated

Tabarka. A significant accumulation of proline and soluble

carbohydrates in leaves was found during the period of

intensive leaf growth. These organic compounds likely

play a role in leaf osmotic adjustment and in protection of

membrane stability at severe salinity.

Keywords Cakile maritima � Compatible solute �Germination � Ion accumulation � Provenance �Salt tolerance � Vegetative growth stage

Introduction

Salinity is an increasing environmental problem throughout

the world. According the estimation of the United Nations

Environment Program, 20% of cultivated land worldwide

is adversely affected by a high salt concentration, which

inhibits plant growth and yield (Flowers and Yeo 1995).

The deleterious effects of salinity on plant growth are

associated with low osmotic potential of soil solution

(water stress), nutritional imbalance, specific ion effects

(salt stress), or a combination of these factors (Ashraf

1994; Ashraf and Harris 2004; Marshner 1995).

Saline soils occur naturally in both coastal region, where

groundwater is contaminated by seawater, and in areas

subjected to inadequate irrigation and/or draining (De

Souza Filho et al. 2003). One of the most common

approaches suggested for dealing with salinity is the

Communicated by A. Kacperska-Lewak.

W. Megdiche � N. B. Amor � A. Debez � K. Hessini �R. Ksouri (&) � C. Abdelly

Laboratoire d’Adaptation des Plantes aux Stress Abiotiques,

Centre de Biotechnologie a la Technopole de Borj-Cedria

(CBBC), BP 901, 2050 Hammam-lif, Tunisia

e-mail: physiologiste@yahoo.com; riadh.ksouri@cbbc.rnrt.tn

Present address:

A. Debez

Institut fur Botanik, Universitat Hannover,

Herrenhauser Str. 2, 30419 Hannover, Germany

Y. Zuily-Fodil

Laboratoire d’Ecophysiologie Moleculaire de l’Universite Paris

XII - Val de Marne, Paris, France

123

Acta Physiol Plant (2007) 29:375–384

DOI 10.1007/s11738-007-0047-0

utilization of genetic engineering to increase salt tolerance

of common crop species (Maggio et al. 2000). The out-

come of such an approach, although promising, remains

still non significant, because salt tolerance is a complex

trait. An alternative approach may consist in the utilization

of salt-tolerant species (halophytes), being native of the

region (Lieth et al. 1999). Indeed, halophytic species are

known to represent a potentially important crop (O’Leary

1984; Glenn et al. 1999), in addition to their ecological role

in rehabilitation of damaged ecosystems and/or in land-

scaping (Debez et al. 2004). Salt-tolerant plants, growing

normally in saline coastal ecosystems, evolved a number of

adaptive traits expressed at various levels of organization,

which allow them to grow and achieve their complete cycle

of development under such hostile conditions (Flowers

et al. 1986; Tipirdamaz et al. 2005). Such behaviour im-

plies a complexity of salt tolerance mechanisms, among

which osmotic adjustment (usually accomplished by taking

up inorganic ions), as well as an accumulation of com-

patible solutes plays the most important role (Meloni et al.

2004). Nevertheless, at the earliest stages of the life cycle

(germination, seedling establishment), halophytes are

generally as salt-sensitive as glycophytes (Debez et al.

2001; Khan et al. 2002).

Cakile maritima (sea rocket) is a succulent, annual

halophyte, confined to maritime strandlines on sand or

shingle and associated fore dunes. This species shows

considerable variability, within and between subspecies

(Davy et al. 2006). Genotypic differences play a major role

in adaptation of plants to their specific environments

(Gunasekera et al. 2006). As a part of a national project

aiming at the evaluation of local halophytes with eco-

nomical and/or ecological potentials, the physiology of salt

tolerance of C. maritima was investigated using a single

Tunisian ecotype (Debez et al. 2004). In continuation of

this work, the present study was aimed at monitoring the

salt-induced responses in plants of two Tunisian ecotypes

of C. maritima at different developmental stages, with a

special emphasis put on seed-germination capacity, accu-

mulation of ions and compatible solutes (soluble carbo-

hydrates and proline) in leaves and the role of these

substances in osmotic adjustment. So far, differences in C.

maritima behaviour with respect to its developmental stage

and geographic origin are poorly documented.

Materials and methods

Plant material and culture conditions

Seeds of C. maritima were collected at two Tunisian

littoral sites: at Tabarka (extreme north, humid climatic

area) and at Jerba (south, arid climatic area). After their

disinfection, seeds were germinated in Petri dishes (20

seeds each) containing two layers of filter paper moistened

with salt solutions of different concentrations (in the

0–400 mM NaCl range). Petri dishes (three replicates per

treatment) were incubated in the dark at 20 ± 2�C for

2 weeks. Germination was considered as occurred when

emerging radicule was visible. In addition to the final

germination percentage, a theoretical model (Debez et al.

2004) was used for a more accurate assessment of salt

impact on seed germination. Such a mathematical simula-

tion assumes that germination is composed of two phases: a

latency phase of duration t0, during which the seeds acquire

the aptitude to germinate, and the germination itself. Suc-

ceeding the latency phase, the probability k of germination

per unit of time is equal and constant with time for all

seeds. The model was formulated as:

yðtÞ ¼ ymaxð1þ e�kðt�t0ÞÞ: ð1Þ

With y(t)- the number of germinated seeds at time t and

ymax-the plateau reached by y(t), the number of viable seeds

is determined. The time interval between the end of latency

and reaching the plateau is determined by t0 and k together.

k depends on time needed for germination of 50% of viable

seeds and is calculated as:

t50% ¼ t0 þ lnð2Þ=k: ð2Þ

The values of ymax, k and t0 were determined by fitting

the observed values to the above equation using the non-

linear regression module of the StatisticaTM program.

In the second experiment, seeds were germinated in pots

filled with inert and humid sand. Ten-day-old seedlings,

selected for their uniformity in size and form, were culti-

vated in pots under greenhouse conditions (25 ± 5�C

temperature, 60 ± 10% relative humidity). Three-week-old

plants were then irrigated at alternate days with a nutrient

solution (Hewitt 1966), pH 7.3, containing 0, 100, 200, or

400 mM NaCl. Leaf samples (originating from four plants

per treatment) harvested at 4, 6, 10, 15, and 20 days after

salt addition to the culture medium, were used for moni-

toring the main growth parameters, ion status and organic

solute contents in the studied tissues.

Determination of chlorophyll content

Chlorophyll content (mg g–1 FW) in mature leaves was

determined according to Torrecillas et al. (1984), with a

slight modification (we used a pure acetone rather than

acetone 80%). Five ml of pure acetone was added to fresh

leaf samples (ca. 100 mg each), cut into discs. The

extraction took place in darkness at 4�C for 72 h. Extract

absorbance was measured at 649 and 665 nm and total

376 Acta Physiol Plant (2007) 29:375–384

123

chlorophyll content was calculated according to the fol-

lowing equation:

Total chlorophyll (lg ml�1Þ¼ 6:45 ðA665 nmÞ þ 17:72ðA649 nmÞ: ð3Þ

Leaf growth, water relations, and ion assay

Leaf growth was assessed by determining their total dry

weight (DW). Total leaf area was measured by a leaf area

meter (Portable Areameter LI/3000A LI-COR). Leaf water

content was calculated as ml H2O per g DW. Dry weight of

the samples was estimated after their drying at 60�C for

72 h.

After ion extraction from leaf samples (30 mg DW

each) in 0.5% HNO3, chloride was assayed by coulometry

(Buchler chloridometer), Na+ and K+ by flame emission

photometry (Corning, UK), and Ca2+ and Mg2+ by atomic

absorption spectrophotometry (Perkin Elmer 4000).

Proline and total soluble carbohydrate contents

Proline was determined following the ninhydrin method

described by Bates et al. (1973), using L-proline as a

standard. Leaf samples (100 mg FW) were homogenized in

1.5 ml of 3% (w/v) aqueous sulfosalicylic acid and cen-

trifuged for 30 min at 14,000g. To the supernatant

(500 ll), 2 ml of acid ninhydrin and 2 ml of glacial acetic

acid were added and the mixture was boiled for 1 h. After

extraction with toluene, the free proline was quantified

(k = 520 nm) from the organic phase using an Anthelie

Advanced 2, SECOMAN spectrophotometer. Proline in the

test samples was calculated from a standard curve prepared

against L-Proline (5–30 lg, from MERCK KGaA):

y = 0.059x – 0.014, R2 = 0.99.

The content of total soluble carbohydrates in the studied

samples was determined according to Mc Cready et al.

(1950) and Staub (1963), using glucose as a standard.

Twenty-five milligram (DW) leaf samples was homoge-

nized with 5 ml methanol 80% and boiled while shaking at

70�C for 30 min. The homogenate was centrifuged for

15 min at 6,000g. After decanting, the residue was re-

suspended in 5 ml of the extraction solution and centri-

fuged at 6,000 g for 10 min. The supernatant was decanted

and combined with the original extract. For measurement

of total soluble carbohydrates, anthrone–sulfuric acid assay

was used. An aliquot of 250 ll was added to 5 ml of an-

throne–sulfuric acid solution. The mixture was shaken,

heated in a boiling water-bath for 10 min and cooled at

4�C. The absorption was determined by spectrophotometry

(Anthelie Advanced 2, SECOMAN) at 640 nm. A standard

curve was prepared using different concentration of glu-

cose (0–100 lg, from MERCK KGaA). From the standard

curve, the concentrations of soluble carbohydrates in the

test samples were calculated (y = 0.0095x – 0.0299,

R2 = 0.979).

Experimental design and statistical analysis

The germination data were subjected to a two-way analysis

of variance (ANOVA), using salinity (S) and ecotype (A)

as the factors. The means were compared by Newman–

Keuls post-hoc test at 5% probability. Concerning the plant

culture in a greenhouse, the experimental design was a

completely randomized 2 · 2 factorial with four replicates

per treatment.

Results

Seed germination

Germination capacity was dependent on the plant ecotype

and applied salinity. In a salt-free medium, germination

percentages were significantly higher in Jerba as compared

to Tabarka (100% and ca. 40%, respectively) (Fig. 1a).

Salinity in the 50–300 mM NaCl range decreased signifi-

cantly germination percentages in both ecotypes, although

Jerba was more tolerant. In both ecotypes, germination was

virtually suppressed at 400 mM NaCl (Fig. 1b). On the

other hand, salinity delayed germination process, as indi-

cated by the increase of t0 and t50% (Fig. 1c), with a more

marked effect on Tabarka seedlings (sensitive).

Leaf development, water relations and total chlorophyll

content

In the control treatment (plants grown in the absence of

salt), Tabarka seedlings were more productive than Jerba,

as indicated by dry matter increases (Fig. 2). Yet, the latter

showed a maximal growth potential at a moderate salinity

level, i.e. at 100 mM NaCl, which indicates a typical hal-

ophytic behaviour. In contrast, growth of Tabarka seedlings

markedly decreased at 100 mM NaCl (Fig. 2). Salinity

levels higher than 100 mM NaCl restricted significantly the

plant growth, although to a greater extent in the sensitive

Tabarka plants. This ecotype-dependent salt response was

more pronounced along with the duration to salt exposure.

At 400 mM NaCl, plant biomass production represented 77

and 28% of the control values in Jerba and Tabarka eco-

types, respectively.

Leaf growth was also salt- and ecotype dependent. For

instance, the moderate salinity (100 mM NaCl) increased a

Acta Physiol Plant (2007) 29:375–384 377

123

leaf number and area in Jerba seedlings, comparing to the

control (Fig. 3a, b), but it reduced leaf growth in Tabarka.

However, the 400 mM NaCl-treated plants of both eco-

types showed significantly smaller leaf area, as compared

to other treatments (Fig. 3b).

Irrespective of the ecotype, total chlorophyll content in

the leaves increased during the culture period (Fig. 4). In

Jerba plants treated with 100 mM NaCl, the chlorophyll

content was maintained at the level similar to that in the

control. This parameter was significantly reduced by severe

salinity, and to a higher extent in Tabarka seedlings, as

seen after 20 days of the salt-treatment (by ca. 20 and 30%

in Jerba and Tabarka ecotypes, respectively). Nevertheless,

neither necrosis nor chlorosis symptoms were observed in

leaves of plants submitted to severe salinity.

Changes in leaf water content paralleled those of bio-

mass production. In both ecotypes, water content in seed-

lings subjected to moderate salinity was maintained near to

the control level (Fig. 5). Increasing salinity resulted in

a progressive decrease in leaf hydration, especially in

Tabarka plants (Fig. 5). At 400 mM NaCl, the reduction of

water content in Jerba and Tabarka leaves reached 30 and

51%, as compared to the respective controls.

Ion content and solute accumulation

In Jerba leaves, Na+ content increased with salinity along

with the exposure time (Fig. 6a). In Tabarka, Na+ content

reached a plateau 15 days after the start of the exposure. In

plants challenged with 400 mM, NaCl content in Jerba and

Fig. 1 Effects of NaCl on

germination parameters in two

ecotypes of C. maritima.

Observed germination

percentage (a), ymax (the

number of viable seeds, as % of

the sown seeds) (b), the time for

50% germination (t50%), given

by t0 + ln(2)/k (c), the

probability of germination per

time unit, k (d). The values of

ymax, t0, and k were determined

by fitting Eq. 1 to the observed

germination kinetic data. Means

of three replicates ± standard

error. Values followed by at

least one the same letter were

not significantly different at

P < 0.05, according to the

Newman–Keuls post-hoc test

Fig. 2 Effects of NaCl on the

leaf dry weight of Jerba and

Tabarka ecotypes, as a function

of the exposure period. Means

of four replicates ± standard

error

378 Acta Physiol Plant (2007) 29:375–384

123

Tabarka leaves was ca. 17- and 12-fold greater than in the

control values, respectively (Fig. 6a). Sodium accumula-

tion was associated with an increase in chloride uptake

(Fig. 6b), and with a marked decrease in K+, Ca2+ and

Mg2+ contents (Fig. 7). A more pronounced impact of

salinity was observed in the salt-sensitive Tabarka plants,

especially at high salinity levels (>100 mM NaCl). In fact,

both Na+ and Cl– were found to be the principal ions

Fig. 3 Effects of NaCl on leaf

area (a) and leaf number (b) in

Jerba and Tabarka ecotypes, as

a function of the exposure

period. Means of four

replicates ± standard error

Fig. 4 Effects of NaCl on total

chlorophyll content in Jerba and

Tabarka leaves, as a function of

the exposure period. Means of

four replicates ± standard error

Fig. 5 Effects of NaCl on water

content in Jerba and Tabarka

leaves, as a function of the

exposure period. Means of four

replicates ± standard error

Acta Physiol Plant (2007) 29:375–384 379

123

accumulated in the leaves of the salt-treated plants of both

ecotypes. Interestingly, in both ecotypes the Na+/K+ and

Na+/Ca2+ ratios increased with increasing salt concentra-

tion in the culture medium (Fig. 8).

No changes in proline content in the leaves were ob-

served up to 200 mM NaCl level, over the whole period of

the salt treatment (Fig. 9a). However, a sharp increase in

proline content was observed after 2 weeks of plant

exposure to the highest salt level (400 mM of NaCl). At the

end of the experiment, the proline content in Jerba leaves

reached ca. threefold higher level than that in the Tabarka

ones (12.83 and 4.11 lmol g–1 FW, respectively).

The total soluble carbohydrate concentrations in leaves

of Jerba plants were unaffected by a moderate salinity

(Fig. 9b). Plants of the salt sensitive Tabarka showed a

slight increase in soluble carbohydrate contents during leaf

development, but the contribution of this compatible solutes

to the ‘‘osmotic pool’’ was higher in the salt-tolerant Jerba

than in the salt-sensitive Tabarka seedlings, especially

during the period of intensive leaf growth at 400 mM (i.e.

after 15 days of the experiment, see Fig. 9b), when soluble

carbohydrate contents in the Jerba and Tabarka leaves

increased to 516 and 380.4 lmol NaCl g/DW, respectively.

Discussion

In the present study, plants of two ecotypes, normally

growing along Tunisian coasts in different climatic areas,

were exposed to the NaCl stress. Our findings indicate that

the plant responses to salt stress differed among the chosen

ecotypes, depending on the growth stage and severity of

salt stress.

As far as germination is concerned, salinity impaired

and delayed this process in both ecotypes, although Jerba

seedlings were more tolerant than Tabarka ones. The

finding confirms that of Debez et al. (2004) for another

Tunisian ecotype of the same species. Actually, several

studies have shown that halophytes are salt-sensitive at

the earliest establishment stage; however, they acquire

halophytic features during autotrophic stage of develop-

ment (Katembe et al. 1998; Noe and Zedler 2000; Ungar

1996).

At the vegetative growth stage, 100 mM NaCl treatment

increased both growth and leaf expansion in Jerba plants

(typical halophyte response) while these parameters were

reduced in Tabarka. In leaves of both C. maritima eco-

types, no toxicity symptoms appeared at severe salinity,

despite Jerba could be considered as more salt tolerant than

Tabarka. Similar observations have been reported for crop

plants such as sunflower and soybean (Ashraf et al. 1995;

Essa 2002).

High salinity reduced the leaf chlorophyll content in

both studied ecotypes, which might be due to the increased

activity of chlorophyllase (Iyengar and Reddy 1996).

However, this depressive effect was rather moderate, as the

maximal reduction in chlorophyll content did not exceed

30% in the sensitive ecotype (Tabarka), and might explain

Fig. 6 Effects of NaCl on Na+

(a) and Cl– (b) contents in Jerba

and Tabarka leaves, as a

function of the exposure period.

Means of four

replicates ± standard error

380 Acta Physiol Plant (2007) 29:375–384

123

the absence of chlorosis symptoms in the leaves, reported

for glycophytes (Gadallah 1999; Agastian et al. 2000).

Sodium accumulation in leaves, associated with the in-

creased chloride content, was prominent in plants of C.

maritima subjected to 400 mM NaCl, especially in the

Jerba ecotype. This finding points to easy transport of Na+

and Cl– from roots to leaves. On the other hand, the salt

dependent increases in the Na+/K+ and Na+/Ca2+ ratios in

the leaves of both ecotypes suggest that selective ion

transport mechanisms may have taken place in the studied

plants and favoured the Na+ transport against K+ and Ca2+

(Moghaieb et al. 2004). Presented results are in line with

our previous findings (Debez et al. 2004), as well with data

reported for some dicotyledonous halophytes, such as

Salicornia prostrata, Suaeda confusa, and Plantago mari-

tima (Tipirdamaz et al. 2005).

C. maritima plants, subjected to a high salt stress

(particularly at 400 mM NaCl) accumulated high con-

centrations of proline and total soluble carbohydrates.

This observation indicates that osmotic adjustment took

place in C. maritima plants grown under saline condition.

According to Cram (1976), soluble sugars may contribute

to up to 50% of the total osmotic potential in glycophytes

subjected to saline conditions. The accumulation of sol-

uble carbohydrates in plants has been commonly reported

as a response of plants to salinity or drought (Popp and

Smirnoff 1995; Murakeozy et al. 2003). Studies carried

out by Ashraf and Tufail (1995) on five sunflower culti-

vars, differing in salt tolerance, revealed that although

sugar content in seedlings of five lines was increased

significantly by increasing salt concentrations, the salt-

tolerant lines showed a higher accumulation of soluble

sugars than the salt sensitive ones. Similar results were

also reported for leaves of wild (salt tolerant) and culti-

vated population of Melilotus indica and Eruca sativa,

exposed to varying salt levels in the growth medium

(Ashraf 1994). The content of proline in leaves of C.

maritima increased in plants treated with 400 mM NaCl

only. Proline content doubled rapidly in Jerba plants to

reach the threefold higher level than that in Tabarka

Fig. 7 K+ (a), Ca2+ (b) and

Mg2+ (c) contents in leaves of

Jerba and Tabarka plants

exposed to NaCl for different

periods. Means of four

replicates ± standard error

Acta Physiol Plant (2007) 29:375–384 381

123

seedlings. Proline content has been found to be higher in

many salt-tolerant plants than in salt sensitive ones. This

amino acid, which occurs widely in the salt stressed

plants, accumulates in larger amounts than other amino

acids and could be considered as an indicator of salt

tolerance (Ali et al. 1999; Ashraf 1993, 1994). In line

with this suggestion, accumulation of proline was found to

be higher in salt-tolerant alfalfa plants than in the sensi-

tive ones (Fougere et al. 1991; Petrusa and Winicov

1997). Higher proline accumulation was observed also in

salt-tolerant ecotypes of Agrostis stolonifera than in salt

sensitive ones (Ahmad et al. 1981).

Fig. 8 Leaf Na+/K+ (a) and

Na+/Ca2+ (b) ratios in two

ecotypes of C. maritima grown

in nutrient solution containing

0, 100, 200 or 400 mM NaCl, as

a function of the exposure

period. Means of four

replicates ± standard error

Fig. 9 Proline (a), and soluble

carbohydrates (b) in leaves of

Jerba and Tabarka plants

exposed to NaCl for different

periods. Means of four

replicates ± standard error

382 Acta Physiol Plant (2007) 29:375–384

123

In our study, the increase in proline content occurred

when water content in the leaves decreased to 50% in

comparison with the control. Leaf water potential of plants

exposed to 400 mM NaCl was found to be fivefold lower in

Jerba than in Tabarka at the end of the experiment (data not

shown). Therefore, proline accumulation may be associ-

ated with a decrease in water potential of tissues, as re-

ported by Slama et al. (2007) for the salt-treated halophyte

Sesuvium portulacastrum. The difference in ww seemed to

be related to a high accumulation of sodium ions, soluble

carbohydrates and proline in Jerba and thus, may partially

explain its high salt tolerance. According to Ali et al.

(1999), the accumulation of these compatible solutes and

certain ions in response to osmotic stress represents an

important adaptive response to salt and drought stress.

In summary, presented experiments showed marked

differences in salt tolerance of C. maritima plants of two

Tunisian ecotypes. Jerba was more salt tolerant than Ta-

barka, at both germination and vegetative growth stages.

This was related to a greater ability of the former to

maintain osmotic adjustment resulting from its higher

ability to accumulate sodium as well as proline and car-

bohydrates within leaf cells in response to salinity. This

difference between plants of two ecotypes of C. maritima

may reflect the difference in climatic conditions of their

native habitats.

Acknowledgments This work was supported by the Tunisian-

French ‘‘Comite Mixte de Cooperation Universitaire’’ (CMCU)

network # 02FO924.

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