Assessment of salt tolerance of Nasturtium officinale R. Br. using physiological and biochemical...
Transcript of Assessment of salt tolerance of Nasturtium officinale R. Br. using physiological and biochemical...
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: [email protected]
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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