ORIGINAL PAPER

Assessment of salt tolerance of Nasturtium officinale R. Br.using physiological and biochemical parameters

Rym Kaddour • Emna Draoui • Olfa Baatour •

Hela Mahmoudi • Imen Tarchoun • Nawel Nasri •

Margaret Gruber • Mokhtar Lachaal

Received: 27 March 2013 / Revised: 22 August 2013 / Accepted: 30 August 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract Nasturtium officinale R. Br. seedlings were

treated with a range of NaCl concentrations (0, 50, 100 and

150 mM) for 21 days after seedling emergence. Physio-

logical analysis based on growth and mineral nutrition,

showed a substantial decrease in leaf dry matter with

150 mM NaCl treatment. The growth decrease was corre-

lated with nutritional imbalance and a reduction in potas-

sium accumulation and transport to the leaves. At the same

time, we noted an increase in leaf sodium and chloride

accumulation and transport. Salt tolerance of N. officinale

under 100 mM NaCl was associated with osmotic adjust-

ment via Na? and Cl- and the maintenance of high K?/

Na? selectivity. Salt decreased carotenoid content more

than chlorophylls and also disturbed membrane integrity by

increasing malondialdehyde content and electrolyte leak-

age. At 150 mM NaCl, an increase in antioxidant enzyme-

specific activities for superoxide dismutase, catalase and

guaiacol peroxidase occurred in concert with a decrease in

ascorbic acid, polyphenol, tannin and flavonoid content.

These results indicate that N. officinale can maintain

growth and natural antioxidant defense compounds such as,

vitamin C, carotenoids, and polyphenols, when cultivated

in 100 mM NaCl, but not at higher salt levels.

Keywords N. Officinale R. Br. � Salinity response �Growth � Antioxidant enzymes � Carotenoids �Phenolics

Abbreviations

DW Dry weight

FW Fresh weight

D Day

Chl Chlorophylls

CAR Carotenoids

EL Electrolyte leakage

MDA Malondialdehyde

SOD Superoxide dismutase

CAT Catalase

POD Guaiacol peroxidase

Introduction

Salinity of soil and irrigation water are major factors that

limit global plant growth and productivity (Flowers 2004).

Salt tolerance involves the coordination of many functions,

such as ion sequestration, osmotic and metabolic adjust-

ment and antioxidative defense (Mahajan and Tuteja 2005).

In Tunisia, salinity currently affects about 10 % of the land

area. Moreover, climate change and water resource prob-

lem increased soil salinity of agricultural and horticulture

fields (Hachicha 2007).

Salt stress increases the generation of reactive oxygen

species (ROS) in plants (Abogadallah 2010), and scav-

enging of ROS depends on both enzymatic and non-

enzymatic components. The enzymatic antioxidant system

Communicated by J. Kovacik.

R. Kaddour and E. Draoui have equally participated in the elaboration

of the manuscript.

R. Kaddour (&) � E. Draoui � O. Baatour � H. Mahmoudi �I. Tarchoun � N. Nasri � M. Lachaal

Physiologie et Biochimie de la Tolerance des Plantes aux

Contraintes Abiotiques, Faculte des Sciences de Tunis, Campus

Universitaire, 2090 Tunis, Tunisia

e-mail: rym_kaddour@yahoo.fr

M. Gruber

Saskatoon Research Centre, Agriculture and Agri-Food Canada,

Saskatoon, SK S7N0X2, Canada

123

Acta Physiol Plant

DOI 10.1007/s11738-013-1377-8

is mainly represented by superoxide dismutases (SOD),

peroxidases (PRX), and catalases (CAT) (Harinasut et al.

2000).

Superoxide dismutases are metalloenzymes with three

known classes, each depending on active metal cofactor

(Cu–Zn, Fe or Mn) (Fridovich 1975). SOD is involved in

the detoxification of O2- leading to the formation of H2O2

which is subsequently removed by CAT and peroxidases

and reduced into water. Increased levels of antioxidant

enzymes have been correlated to the salt tolerance of plant

species, including wheat, rice, maize, cotton, tomato and

potato (Ashraf 2009). However, such a correlation is not

always evident in other plants such as Arabidopsis (Ka-

tsuhara et al. 2005) and strawberry (Turhan et al. 2008).

Plants are valuable sources of potent natural antioxidant

metabolites, including vitamins, carotenoids, phenolic

acids, tannins, flavonoids, and phenolic diterpenes (El-

Ghorab et al. 2007). Because of the potential carcinoge-

nicity of some synthetic antioxidants (Imaida et al. 1983),

their utilization is restricted in European countries (Mıkova

2002). Hence, there is increasing interest in the identifi-

cation and evaluation of natural antioxidants of plant ori-

gin. Phenolics play an important role in human health

owing to their antioxidant, and anti-cancer potential (Dai

and Mumper 2010). Ascorbate, which has been shown to

play multiple roles in plant growth, such as in cell sig-

naling, cell division, cell wall expansion, and other

developmental processes, is involved in the protection of

the photosystem by reacting with singlet oxygen and other

free radicals (Asada 2006) and in the suppression of per-

oxidation (Bielski et al. 1975). Carotenoids also protect the

photosystem, play an important role in fruit coloring, and

act as antioxidants to ‘‘defuse’’ free radicals, mainly per-

oxide radicals and singlet molecular oxygen (Namiki

1990). In the case of medicinal plants, abiotic stress may

modulate the level of these secondary metabolites (Cramer

et al. 2011), since plants defend themselves against

changing conditions by raising their antioxidant defense

systems.

The objective of this study was to evaluate the effect of

NaCl stress on growth, biomass, mineral composition,

chlorophyll, leaf membrane integrity, antioxidant enzymes,

and potentially valuable antioxidant phytochemicals pres-

ent of N. officinale R. Br. (watercress) cultivated in Tuni-

sia. This member of the Brassicaceae mustard family is one

of the oldest known vegetables. N. officinale is native to

north Africa (including Tunisia) and parts of temperate and

tropical Asia and Europe, and is naturalized elsewhere

(GRIN 2012). It has been declared a noxious weed in the

USA. In Tunisian villages where traditional used medicinal

plants are still familiar to ordinary people, N. officinale is

used to treat rheumatism and serious diseases, such as liver,

spleen and kidney vesicles, and is also largely claimed to

prevent diabetes (Lemordant 1977; Boulos 1983). The

development of a biochemical and physiological knowl-

edge base for N. officinale growing on saline soil will

enable agricultural specialists to develop a plan for the on-

going maintenance of this vital Tunisian crop under

changing environmental conditions.

Materials and methods

Plant growth and salinity treatment

Nasturtium officinale (commonly called ‘‘Habb Arrached’’

in Tunisia) usually grows in wet habitats or near water

sources. Seeds of watercress (N. officinale) V-409 were

provided by the National Agriculture Research Center

(Tunisia). Seeds were soaked in water for 24 h, then placed

in pots containing a 1:2 (v:v) mixture of sand and peat (a

mixture yielding 100 % germination). Experiments were

conducted in a culture chamber with 22/18 �C day/night,

and a 12-h photoperiod (150 mmol m-2 s-1 photosyn-

thetically active radiation), and irrigated with distilled

water. Emerged seedlings were thereafter transferred into

plots containing hydroponic nutrient solution (Hoagland

and Arnon 1950) diluted to 1/5th strength, such that the

final solution was composed of: (1.25 mM KNO3,

1.25 mM Ca(NO3)2�4H2O, 0.50 mM MgSO4�7H2O,

0.25 mM KH2PO4, 10 lM H3BO3, 1 lM MnSO4�4H2O,

0.5 lM CuSO4�5H2O, 0.5 lM ZnSO4�6H2O, and 0.05 lM

(NH4)6Mo7O24�4H2O) and 3 lM Fe2?–EDTA. Plants were

grown in pots with one plant per pot (Fig. 1). At time t1, 8

plants were harvested on day 24. Thereafter, the remaining

plants (usually eight per treatment) were treated with NaCl

(0, 50, 100 or 150 mM) for 21 days. Leaves were harvested

on day 45 (time t2) when the plants were in a mid-vege-

tative stage.

Tissue biomass and ion analyses

Fresh and dry matter of mature rosette leaves and roots

were determined. Leaves of each plant were cut, then laid

flat and photographed to measure individual leaf surface

Fig. 1 Phenotypes of N. officinale plants treated with different NaCl

concentrations (0, 50, 100 and 150 mM) for 21 days

Acta Physiol Plant

123

areas using Optimas� imaging software, version 6.1

(Optimas Corporation, USA). Four leaves from the same

position of each plant were air-dried, then separately

digested to clarity in 0.1 N HNO3. For Na? and K? content

determination, four rosette leaves of N. officinale were

rinsed three times with deionized water and dried at 70 �C

for 3 days. The dried material was ground with a mortar

and pestle and 20 mg dry powder samples were extracted

with 5 mL 0.1 N HNO3 for 48 h. Na? and K? content in

the clear extracts were determined by flame photometry

(Jenway PFP7, using butane air flame, UK) (Brody and

Chaney 1966), while Cl- was performed by coulometry

(Haake-Buchler Chloridometer, USA) (DeFord 1960). The

rates of ion net transport (Jtrp) were calculated according to

Pitman (1988): Jtrp ¼DIS Ln Wr2

=Wr1ð Þt2�t1ð ÞDWr

, where DIs is the

average content of K?, Na? or Cl- ions per leaf between

time t2 (45 days) and time t1 (24 days). IS was calculated

by the ratio of ion concentrations (mmol g-1 DW) and

shoot dry weights (g); Wr is the mean root dry weight (g).

Results are the mean of eight replicates (8 plants).

Pigment content

Pigments were extracted from three fresh rosette leaf

laminas per plant in acetone 80 %. The absorbance was

determined with a UV/visible spectrophotometer (Beck-

man DU 640, USA) at A470, A646 and A663 after incubation

of acetone extracts for 48 h in the dark at 4 �C. Chloro-

phyll and carotenoid concentrations were calculated

according to Lichtenthaler (1988). Pigment extraction and

determination were conducted on four replicates (8 plants).

Membrane permeability measurements

Leaf electrolyte leakage was determined on 0.2 g of fresh

Nasturtium detached rosette leaves per plant. Leaf samples

were rinsed three times with deionised water and incubated

in hermetic tubes containing 10 mL of deionised water for

1 h at 32 �C. Electrical conductivity of the leaf solution

(ECL1) was determined with a Consort C832 conductivity

meter (LABCOR, USA). Thereafter, the tubes containing

leaf samples were autoclaved at 120 �C for 20 min to

determine electrical conductivity after release of all elec-

trolytes (ECL2). Leaf electrolyte leakage was determined

according to Dionisio-Sese and Tobita (1998) and calcu-

lated as: ELL = (ECL1/ECL2) 9 100. Results are the mean

of four replicates (8 plants).

Malondialdehyde measurement

Malondialdehyde (MDA) was determined in 0.2 g of fresh

rosette leaves of N. officinale. Samples were homogenized

in 2 mL of 200 g L-1 2-thiobarbituric acid and 5 g L-1

trichloroacetic acid, and the extracts were incubated at

95 �C for 30 min. After a brief passage in ice, the samples

were centrifuged at 4,000g for 30 min at 4 �C, and the

absorbance of the supernatant was measured at 532 and

600 nm. The concentration of MDA (mol g-1 fresh

weight) in rosette leaves was calculated as reported by

Heath and Packer (1968), using a molar extinction coeffi-

cient of 155 mmol L-1 cm-1 at 532 nm. Analyses were

performed on four replicates (8 plants).

Extraction of leaf proteins

Salinity-treated Nasturtium rosette leaves (two leaves per

plant) were separately ground in liquid N2. The resulting

powder was resuspended according to M’rah et al. (2007)

in a 50 mM, pH 7.5 phosphate buffer containing 1 mM

EDTA, 1 mM DTT, 5 % glycerol and 5 % polyvinylpyr-

rolidone, and centrifuged for 20 min at 15,000g. Leaf

soluble protein content was determined in the supernatant

according to the method of Bradford (1976) using bovine

serum albumin (BSA) as the standard. Protein leaf

extraction and determination were conducted on four rep-

licates (8 plants).

Enzyme extraction and activity assays

Total SOD activity was assayed according to Beyer and

Fridovich (1987) by adding 20 lL of the 15,000g super-

natant above to a reaction mixture containing 1.5 lm

riboflavin, 50 lm nitroblue tetrazolium (NBT), 10 mM DL-

methionine and 0.025 % (v/v) Triton-X100 in 50 mM

phosphate buffer. The reaction was started by exposing the

mixture to white fluorescent light for 15 min, and reduced

NBT (blue color) was measured at 560 nm, such that one

unit of SOD activity caused 50 % inhibition of NBT

reduction per min. CAT activity was measured in extrac-

tion buffer containing 50 mM, phosphate, 1 mM EDTA,

1 mM DTT, 5 % glycerol and 5 % polyvinylpyrrolidone

pH 7.5 using a modified Chance and Maehly (1955)

method. The reaction mixture containing 25 mM K?

phosphate buffer pH 7.0, 30 mM H2O2 and enzyme extract

was monitored for the decomposition of H2O2 (decrease in

absorbance) at 240 nm. POD was extracted in a 100 mM

phosphate, 1 mM EDTA, 1 mM DTT, 5 % glycerol and

5 % polyvinylpyrrolidone, pH 7.8 buffer according to

Fielding and Hall (1978). POD reaction mixtures contained

50 mM K? pH 7.0 phosphate, 0.1 mM EDTA, 5 mM

H2O2, enzyme extract with 10 mM guaiacol as an electron

donor. The increase of absorbance (tetraguaiacol forma-

tion) was recorded at 470 nm, and all enzyme activities

were expressed per mg of total soluble protein. Each

parameter was studied in four replicates (8 plants).

Acta Physiol Plant

123

Total ascorbate determination

Total ascorbate content was assayed as described by

Kampfenkel et al. (1995). Samples of fresh rosette leaf

(0.25 g, *four leaves per plant) were homogenized in ice-

cold 6 % (w/v) TCA, using a cold mortar and pestle and

centrifuged at 15,000g for 10 min at 4 �C. Ascorbic acid

(AsA) was detected in the supernatant at 525 nm as a red-

colored complex of bipyridine and Fe2? ion produced by

AsA reduction of Fe3?. Dihydroascorbate (DHA) was

determined in the same supernatant by detecting ascorbate

after 10 mM DTT reduction [after excess DTT was

removed with 4 % (w/v) N-ethylmaleimide]. A standard

curve covering the range of 10–50 lmol ascorbate was

used. Results are the mean of four replicates (8 plants).

Total phenolic content determination

Total phenolic content of Nasturtium rosette leaves was

determined using the Folin–Ciocalteu method (Singleton

et al. 1999) as modified by Dewanto et al. (2002). Leaf

methanol extract (0.125 mL) was incubated at 23 �C with

0.5 mL of deionized water and 0.125 mL of Folin–Cio-

calteu reagent for 1 min, then 1.5 mL of 7 % sodium

carbonate (Na2CO3) solution, and samples were incubated

for 90 min at 23 �C. The absorbance was measured at

760 nm using a HACK UV–Vis spectrophotometer, and

expressed as mg gallic acid equivalents (GAE g-1 DW).

Total phenolic content determination was conducted in

four replicates (8 plants).

Total flavonoid content determination

Total flavonoid content was measured using a colorimetric

assay developed by Dewanto et al. (2002). Diluted etha-

nolic Nasturtium extracts of four rosette leaves or a stan-

dard solution of (?)-catechin were mixed with a 75 lL of

5 % NaNO2 (w/v) for 6 min and for 5 min with 0.15 mL

10 % AlCl3 (w/v), then with 0.5 mL of 1 M NaOH and

adjusted to 2.5 mL with distilled water. The absorbance of

the mixture was determined at 510 nm against a control

mixture without plant extract. Total flavonoid content was

expressed as mg catechin equivalents (CE) g-1 (DW) using

a calibration curve of (?)-catechin (50–400 lg mL-1).

Analyses were performed on four replicates (8 plants).

Total condensed tannin determination

Total condensed tannin content (proanthocyanidin) was

determined according to a modified vanillin assay descri-

bed by Sun et al. (2002). Diluted methanolic Nasturtium

leaf extract (50 lL from a 1 mL extract of four leaves) was

added to 3 mL of 4 % vanillin solution (in 100 % MeOH)

and 1.5 mL of H2SO4 and the absorbance measured at

500 nm against the extract solvent (100 % MeOH) as a

blank. Condensed tannin was expressed as mg (?)-catechin

g-1 (DW) using a calibration curve ranging from 50 to

400 lg mL-1. Results are the mean of four replicates (8

plants).

Statistical analysis

All data were initially analyzed for normal distribution by a

student Fisher test, then by analysis of variance (one-way

ANOVA) using Statistica� (StatSoft France). Means

(±standard error) were separated and ranked by a Turkey’s

post hoc test (P B 0.05).

Results

Biomass indicators and K/Na mineral analysis

Rosette leaf biomass of N. officinale plants grown in the

absence or presence of NaCl showed a 62 % reduction in

dry matter only after 3 weeks of exposure to 150 mM NaCl

but only slight changes during this exposure period at

lower NaCl levels (50, 100 mM) (Table 1). This biomass

reduction correlated strongly with a decrease in the total

leaf area per plant, which declined as a result of the pro-

gressive decrease in leaf number with salt increase and a

lower individual leaf area at the highest salt level

(Table 1). At maximum NaCl level (150 mM), we also

observed rosette leaf chlorosis (data not shown). Leaf

protein content also declined only slightly with the lower

salinity levels, but was strongly decreased with the highest

salt concentration in a fashion similar to leaf biomass

(Table 1).

Endogenous ion concentration and transport was affec-

ted with increasing salinity treatment, but most strongly

with the 150 mM NaCl treatment. N. officinale leaves

showed a progressive rise in Na? cation accumulation with

increased NaCl application levels so that this ion was

threefold higher (7.5 mmol g-1 DM) in leaves at 150 mM

NaCl compared with the lower and mid-range NaCl levels

(Fig. 2). Na? ion transport also reached its highest value (at

1.0 mmol day-1 g-1 DM root) with 150 mM NaCl treat-

ment, and Cl- ion displayed an identical pattern to Na? for

these two parameters (Fig. 2). Water content in rosette

leaves declined only slightly with increased Na? accumu-

lation until NaCl was applied at 150 mM (Fig. 2). At this

latter concentration, water content dropped substantially in

leaves and the drop was consistent with leaf dehydration

and leaf growth perturbation.

Potassium transport on the control medium was esti-

mated at 2.1 mmol day-1 g-1 DW roots (Fig. 2). After salt

Acta Physiol Plant

123

treatment, K? concentration showed only a slight decrease

when treated with low-to-moderate NaCl concentrations

(50 and 100 mM), followed by a large decrease (down to

12 %) compared with the no-salt control when 150 mM

NaCl was applied. Leaf K? transport was decreased in a

similar pattern to K? concentration as a function of applied

NaCl levels (Fig. 2). The contrasting patterns of Na? and

K? accumulation and distribution were a reflection of

differences in K?/Na? selectivity in N. officinale following

NaCl treatments. In the absence of salt, the plants showed a

selectivity value close to 1 and this declined slowly with

increasing NaCl treatment levels and then decreased

substantially with 150 mM NaCl treatment (*81 %

decrease) (Table 1).

Membrane integrity and chlorophyll changes

Overall membrane integrity was evaluated in rosette leaves of

N. officinale after 3 weeks of salt treatment by measuring

electrolyte leakage and malondialdehyde (MDA) levels

(Fig. 3). Both parameters remained stable after low-to-med-

ium (50–100 mM) NaCl treatment. Leaf electrolyte leakage

was only affected after high salt treatment (150 mM), with

values reaching 2.3-fold higher than the untreated control. In

parallel, MDA concentration was increased by twofold after

150 mM salt treatment. Photosystem pigments were also

changed in N. officinale rosette leaves. Leaves were normally

richer in chlorophyll a than chlorophyll b (Fig. 4). Although

both types of chlorophyll responded similarly to salt and were

not significantly affected at the lower NaCl treatment levels,

chlorophyll b was reduced at 150 mM by 48 % compared to

no-salt plants, whereas chlorophyll a showed a somewhat

greater reduction (64 %) (Fig. 4).

Antioxidant capacity

Regarding lipid-soluble photosystem antioxidants, N. offi-

cinale rosette leaves contained 75 % less total carotenoid

than total chlorophyll even in the absence of NaCl treatment

(Fig. 5). Carotenoid levels were insensitive up to 100 mM

salt, but declined by 83 % after treatment with 150 mM

NaCl (Fig. 5). Of the polar antioxidant metabolites, ascor-

bate was [2.0-fold higher (reaching *3.8 lmol g-1 FW)

than either flavonoids or condensed tannins in the absence

of NaCl treatment (Fig. 6). After NaCl treatment, there was

only a slight decrease in ascorbate concentration up to

100 mM NaCl. Beyond this dose (150 mM NaCl), the

levels of total ascorbate decreased substantially down to

1.2 lmol g-1 FW, which corresponded to a reduction of

67.3 % compared with the untreated control plants. Flavo-

noids decreased much less with increasing salinity than

ascorbate, while the dramatic non-linear decrease was also

seen with condensed tannins and total phenolics (Fig. 6).

Table 1 Biomass indicators and K/Na selectivity of N. officinale treated with increasing NaCl concentrations

NaCl (mM) 0 50 100 150

Leaf DM (mg DW plant-1) 254.4 ± 28.8a 208.5 ± 15.4b 200.0 ± 27.2b 95.3 ± 11.5c

Total leaf area (cm2 plant-1) 89.2 ± 13.8a 75.5 ± 7.9b 65.1 ± 2.7b 24.1 ± 5.4c

Individual leaf area (cm2 leaf-1) 10.1 ± 1.4a 10.2 ± 1.7a 9.6 ± 0.7a 6.0 ± 1.3b

Leaf number (plant-1) 8.8 ± 0.4a 7.5 ± 0.6b 6.5 ± 0.6b 4.0 ± 0.0c

Protein (mg g-1 DW-1) 3.2 ± 0.9a 2.6 ± 0.7a 2.6 ± 0.7a 1.1 ± 0.2b

K/Na selectivity 0.9 ± 0.0a 0.8 ± 0.0b 0.7 ± 0.0c 0.2 ± 0.0d

Protein is the mean of four replicates. All the other parameters are the means of eight replicates. Different letters across NaCl treatments indicate

significant differences of the means (±standard error) at P B 0.05 using one-way ANOVA and a Turkey’s test (Statistica�)

0.0

0.5

1.0

1.5

0

2

4

6

8 Na+

d

a

cb

c

bb

a

0

4

8

12

0

1

2

3

4

a abb

c

0

1

2

0

3

6

9

c

a

b b

c

bb

a

NaCl (mM) 0 50 100 1500 50 100 150

Na+

K+ K+

Cl- Cl-3

a

c

a

b

Ion transport (mmol d-1 g-1 DW)Ion content (mmol g-1 DW)

Fig. 2 Effect of NaCl treatment on Na?, K? and Cl- leaf accumu-

lation and their transport from roots into leaves of N. officinale. NaCl

treatments (0, 50, 100 and 150 mM) were applied to 24-day-old

individual plants and lasted for 21 days. At harvest time, the plants

were in mid-vegetative (rosette leaf) stage. Different letters (within

each panel) indicate significantly different means (±standard error) at

P B 0.05 (8 replicates/treatment)

Acta Physiol Plant

123

Three antioxidant enzymes, SOD (superoxide dismu-

tase), guaiacol peroxidase (POD) and catalase (CAT), were

measured in rosette leaves of N. officinale treated for

3 weeks with increasing NaCl concentrations (Table 2).

All three enzymes showed increased specific activity with

increasing salt treatment levels. However, POD and SOD

activities increased most dramatically, such that with

150 mM NaCl, POD was threefold higher and SOD 2.5-

fold higher than in the absence of salt treatment. Catalase

followed the same trend as SOD and POD, but did not rise

until the highest NaCl dose, during which CAT achieved

only 1.8-fold higher specific activity than the no-salt

control.

Discussion

The response of glycophytes to excess salt is often mani-

fested by a decrease in plant growth and yield (Horie et al.

2001). In N. officinale, leaf growth was largely decreased at

150 mM NaCl as measured by the decrease in total and

individual leaf area and leaf number. These effects could

be related to an inhibition of new leaf initiation and a

reduction of leaf expansion (Patel et al. 2009).

Salinity also induces a disturbance in mineral balance,

limiting absorption and transport of ions required for

growth (Niu et al. 1995). Our results showed that the

addition of salt into the culture medium resulted in an

inhibition in K? transport (down by an estimated 88 %) at

150 mM NaCl. This limitation to a supply of K? ions by

NaCl was observed in other Brassicaceae, such as Arabi-

dopsis thaliana (Kaddour et al. 2009). Any changes in the

status of this cation (particularly strong K? deficiency) will

affect growth by limiting cell expansion and inhibition of

photosynthetic processes (Lebaudy et al. 2007).

0

5

10

15

20

2 4 6 8Na+ accumulation (mmol g-1 DW)

% W

ater

co

nte

nt

(ml g

-1D

W)

NaCl (0 mM) NaCl (50 mM)

NaCl (100 mM)

NaCl (150 mM)

NaCl

Fig. 3 Water content of N. officinale leaves as a function of Na?

accumulation. The 24-day-old plants were exposed to NaCl for

21 days. The values are expressed as % of the mean value of the

control (0 mM NaCl). Each symbol corresponds to the mean of all the

rosette leaves of one individual plant (8 plants per treatment). In the

control condition (0 mM NaCl), the water content is

14.07 ± 1.27 mL g-1 DW

0

2

4

6

8Electrolyte leakage Malondialdehyde (µmol g-1 FW)

0

30

60

90

b

a

bb

b

a

b b

NaCl (mM) 0 50 100 1500 50 100 150

Fig. 4 Effect of NaCl treatment on electrolyte leakage and malondi-

aldehyde in the leaves of N. officinale. NaCl treatments (0, 50, 100

and 150 mM) were applied to 24-day-old individual plants and lasted

for 21 days. Different letters (within each panel) indicate significantly

different means (±standard error) at P B 0.05 (4 replicates/treatment)

0

1

2

3

aa

a

ba a

b

a

aa

ba ab b

c

a

Pig

men

t (m

g g

-1F

W)

Chl a Chl b Chl tot Car

NaCl (0 mM) NaCl (50 mM) NaCl (100 mM) NaCl (150 mM)

NaCl

Fig. 5 Effect of NaCl treatment on chlorophyll (chl) and carotenoid

(Car) content in the leaves of N. officinale. NaCl treatments (0, 50,

100 and 150 mM) were applied to 24-day-old individual plants and

lasted for 21 days. Different letters (within each parameter) indicate

significantly different means (±standard error) at P B 0.05 (4

replicates/treatment)

0

1

0

2

4 ab

c

d

0

2

4b

ac

d

ab

bc

0

1

2a

bb

c

NaCl (mM) 0 50 100 1500 50 100 150

Co

nte

nt

(µm

ol

g-1F

W)

Co

nte

nt

(mg

GA

E g

-1D

W)

Co

nte

nt

(mg

CE

g-1

DW

)

lonehpyloPlatoTetabrocsAlatoT

TanninFlavonoid

Co

nte

nt

(mg

CE

g-1

DW

)

2

Fig. 6 Effect of NaCl treatment on total ascorbate, polyphenol,

flavonoid and tannin content in the leaves of N. officinale. NaCl

treatments (0, 50, 100 and 150 mM) were applied to 24-day-old

individual plants and lasted for 21 days. Different letters (within each

panel) indicate significantly different means (±standard error) at

P B 0.05 (4 replicates/treatment)

Acta Physiol Plant

123

K? absorption from soil into living plant cells and K?

transport inside plants are mediated by high-affinity K?

transporters and low-affinity K? channels (Ashley et al.

2006). At least 35 genes present in the Arabidopsis genome

are thought to encode various K? channels or transporters

and at least some appear to be tolerant to increasing Na?

levels (Qi and Spalding 2004). However, salt treatment

reduces the expression of other potassium transporters,

such as AKT 1 (Kaddour et al. 2009). Maintenance of high

cytosolic K?/Na? ratios (especially in shoots) is an

important parameter in tolerance to salt in glycophytic

plants (Gorham et al. 1990; Ren et al. 2005; Hauser and

Horie 2010). Our results showed that N. officinale is able to

maintain a high K/Na selectivity until 100 mM NaCl. In A.

thaliana, the high-affinity K? AtHKT1 transporter is

known to be a Na?/K? transporter localized in the xylem

parenchyma cells of leaves. This protein mediates salt

tolerance by maintaining high Na?/K? loading from xylem

vessels into xylem parenchyma cells (Hattori et al. 2005).

Salt tolerance involves osmotic adjustments, and water

content and mineral ions play important roles in this pro-

cess. In N. officinale, 150 mM NaCl reduced rosette leaf

water content. Salinity also has deleterious effects on Vicia

faba plants growth due to reduced water availability

(osmotic effect) and accumulation of ions (particularly Na?

and Cl-) to toxic concentrations (Tavakkoli et al. 2010).

Na? ions at high NaCl concentration are likely to be

deposited in the leaf apoplast instead of being compart-

mentalized in vacuoles, which can protect the cell against

deleterious effects (Munns and Tester 2008). In addition,

accumulation of Na? in the cytoplasm may cause ion

imbalance and affect critical biochemical processes.

The molecular basis of the high-affinity plant Na?

transport system and its role in tolerance to salinity is still

not well understood (Horie et al. 2001). However, certain

transporters are proposed to play a role in Na? transport,

e.g. cyclic nucleotide-gated transport channels (CNGCs)

and AtAKT1 which regulates Na? distribution between

root and shoot (Berthomieu et al. 2003; Hauser and Horie

2010). Na? recirculation by the phloem is important for

salinity tolerance (Berthomieu et al. 2003). Moreover,

HKTs are known to transport Na? in a variety of species,

including A. thaliana, wheat, and rice (Gassmann et al.

1996; Maser et al. 2002; Jabnoune et al. 2009).

In the presence of NaCl, leaves of N. officinale were

found to accumulate large amounts of leaf Cl-. This

increase in chloride concentration was the result of a rise in

chloride transport from roots to shoots, especially at the

150 mM treatment level, and the rise in Cl- correlates with

a reduction in growth. Munns and Tester (2008) reported

that the competition between chloride and other anions

(such as nitrate and sulphate) induced an inhibitory effect

on the absorption and long-range transport of these

essential anions to organs. Excess of Cl– also decreases

growth and photosynthetic capacity (chlorophyll degrada-

tion) in two varieties of faba bean (Vicia faba), var. Nura

and line 1487/7 (Tavakkoli et al. 2010). Control of Cl-

transport and exclusion from shoots is correlated with salt

tolerance in many species, such as Citrus (Romero-Aranda

et al. 1998) and lotus (Teakle et al. 2007).

Salinity may induce the loss of the structural and

functional integrity of biological membranes, and this may

be the consequence of oxidative stress in addition to direct

ion toxicity (Zhu 2001). In our study, high concentrations

of NaCl (150 mM) induced leaf chlorosis (yellowing) in

salt-treated N. officinale, and this was caused by the

decrease in leaf chlorophyll and carotenoids. Zorb et al.

(2004) also showed that the presence of 150 mM NaCl in

the culture medium caused leaf chlorosis in maize. Toxic

levels of salt and oxidative stress can directly cause

increased plasma membrane and chloroplast membrane

injury, causing perturbation of the photosystem and its

light-harvesting activity (Dionisio-Sese and Tobita 1998).

The damage from this secondary stress can be evaluated by

determining the products of membrane lipid peroxidation,

such as malondialdehyde content and electrolyte leakage.

Our results showed that MDA content and electrolyte

leakage also increased in N. officinale leaves, especially at

150 mM NaCl. Yang et al. (2009) reported that an increase

in H2O2, MDA, and electrolyte leakage are also cellular

damages caused by salinity in Populus cathayana.

Phenolic compounds normally contribute directly to

antioxidation and protection from stress (Awika et al.

2003), but in the leaves of N. officinale, phenol content was

Table 2 Specific activity of antioxidant enzymes in N. officinale leaves treated with increasing NaCl concentrations

NaCl (mM) 0 50 100 150

SOD (U mg-1 protein) 4.5 ± 1.1c 4.6 ± 0.9c 6.8 ± 1.0b 11.4 ± 0.6a

Catalase (U mg-1 protein) 1.7 ± 0.4b 2.0 ± 0.3b 2.1 ± 0.5b 3.5 ± 0.5a

POD (U mg-1 protein) 3.3 ± 1.0c 3.9 ± 1.0c 5.7 ± 1.8b 9.3 ± 1.2a

Values are the means of four replicates. Different letters between salt levels indicate significant differences of the means (±standard error) at

P B 0.05 using one-way ANOVA and a Turkey’s test (Statistica�)

SOD superoxide dismutase, POD guaiacol peroxidase

Acta Physiol Plant

123

maintained only until 100 mM NaCl. Above this concen-

tration, a decrease in polyphenol, flavonoids, ascorbate,

and tannin content was observed. This decrease in leaf

phenolics at 150 mM NaCl could be related to accumula-

tion of toxic levels of Na? and Cl- ions from the enhanced

transport of these ions from roots to shoots, as explained by

Hussain et al. (2009). A negative effect of high NaCl

treatment was also reported on phenolic content in the

tomato cultivar Solanum esculentum M82 (Frary et al.

2010). Athar et al. (2008) also reported a reduction in

endogenous ascorbic acid in wheat leaves under salt stress.

These latter authors proposed direct destruction of wheat

ascorbate by salt, and showed an increase in tolerance to

salt by supplementation with exogenous ascorbate. ROS

destruction of ascorbate was observed earlier in peas under

water stress conditions (Iturbe-Ormaetxe et al. 1998).

Several studies have demonstrated that salt-tolerant wild

tomato (Lycopersicon pennellii) and barley (Hordeum

vulgare) increase their antioxidant enzyme activities in

response to salt stress (Mittova et al. 2004; Jin et al. 2009).

However, these defense mechanisms can become inade-

quate under high saline conditions and lead to growth

inhibition (Munns and Tester 2008). In rosette leaves of

N. officinale, the strongest increase in SOD, CAT and POD

activities at NaCl 150 mM occurs in concert with the

important decrease in rosette leaf growth. It is likely that

Nasturtium is up-regulating the antioxidant enzymes

because it is producing more ROS (Abogadallah 2010), as

shown by the increase in malondialdehyde content. These

large increases in the antioxidant enzyme fraction also

occur when the levels of chemical antioxidants (such as

total ascorbate content) are declining.

Conclusion

Nasturtium officinale has a physiological and biochemical

defense system to counteract the inhibitory impact of rising

salinity; however, this defense system appears to be lim-

ited. Up to 100 mM NaCl, N. officinale responses to salt

included growth maintenance, osmotic adjustment via Na?

and Cl-, and high K?/Na? selectivity. In contrast, all

physiological parameters were disrupted by 150 mM NaCl.

In the same way, natural antioxidant defense compounds

(vitamin C, carotenoids, and polyphenols) functioned also

up to 100 mM NaCl. However, by 150 mM NaCl, strong

inhibition of protective mechanisms occurred, with the

exception of three enzymes (SOD, CAT and POD) that

rose at the higher salt concentrations as protective chemi-

cals were being reduced. These antioxidant enzymes were

insufficient for N. officinale to physiologically manage the

higher salt concentration. Our findings provide a road map

for plant breeders to improve this crop by selecting for

germplasm with increased phenolics and carotenoids,

improved K?/Na? selectivity, and a higher and earlier titre

of antioxidant enzymes.

Author contribution The two first authors Rym Kad-

dour, Emna Draoui contributed equally in the culture,

experiments and writing of this manuscript. Olfa Baatour,

Hela Mahmoudi, ImenTarchoun, Nawel Nasri, helped us

with protocols and chemicals. Margaret Gruber and Mo-

khtar Lachaal contributed to the English language correc-

tions and critical reading of this article.

Acknowledgments Authors would like to thank all who partici-

pated in the elaboration of this work, with chemicals, instruments or

critical reading.

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