South African Journal of Botany 142 (2021) 124
130

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Morphological, physiological, and biochemical responses of Tunisian

Urtica pilulifera L. under salt constraint

dia, Ben Nasri- Ayachi Mouhiba
Ghazouani Soumaya*, Hannachi He
Laboratoire de Productivite
Ve
ge
tale et Contraintes environnementales LR18ES04, De
partement de Biologie, Faculte
des Sciences de Tunis Universite
de Tunis El
Manar Campus Universitaire, 2092 Tunis, Tunisie

A R T I C L E I N F O A B S T R A C T

Article History: Urtica pilulifera from Urticaceae family was known by its benefit effects on human health and used in tradi-
Received 10 January 2021 tional medicine and recently largely used in pharmacological and food fields. In other hand, the salinization
Revised 4 April 2021 of soils is caused by several environmental factors leading to crop losses around the World. The ability of
Accepted 4 June 2021
plants to tolerate salt stress is determined by multiple mechanisms. In the present study, morphological,
Available online xxx
physiological, and biochemical modulations in Urtica pilulifera under salt stress was evaluated to assess its
Edited by L Sebastiani tolerance or sensitization potential to salt stress using different concentrations of NaCl (0, 50, 100 and 150
mM). The studied parameters of Urtica pilulifera were evaluated by cultivating the plant on a hydroponic
Keywords:
medium. Results showed that after 15 days of treatment, a reduction in plant growth was noted correlating
Urtica pilulifera
with leaf chlorosis under 100 mM NaCl and an increased level of proline and soluble sugar. Regression analy-
salinity
sis showed that the applied salt concentration was proportional with the activity of antioxidant enzymes
growth
photosynthetic pigments including catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX), both on the arial and
osmolytes the root parts.
antioxidant enzymes Cellular damage induced by salinity is strongly correlated with the generation of reactive oxygen species,
osmotic damage, and reduction in relative water content. Accelerated antioxidant activity and osmotic
adjustment through training osmolytes, are effective salinity tolerance mechanisms developed by Urtica
pilulifera.
© 2021 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Gilliham, 2015), low rainfall, high evapotranspiration, irrigation with

Salinity is considered among the main abiotic constraint limiting
growth, survival, and plant production (Acosta-Motos et al. 2017). This
environmental constraint leads to loss of organic matter from the soil
and other forms of soil degradation (Egamberdieva et al. 2017), subse-
quently affecting seed germination, plant growth and vigor
(Mathur et al. 2007; Plouznikoof et al. 2016), and agricultural produc-
tion worldwide (Torbaghan et al. 2017). 800 million hectares of the
earth's surface suffer from soil salinization (Yun et al. 2018) including
20% of cultivated land and 33% of irrigated agricultural land
(Machado and Serralheiro, 2017). Thus, soil salinity is a serious problem
worldwide which are becoming really acute especially in arid and
semi-arid areas (Li et al. 2014; Bencherif et al. 2015). Among the main
factors that intensified salt constraint problem are the use of ground-
water and an inappropriate agricultural practice, including "inadequate
drainage on irrigated land" (Jamil et al. 2011; Munns and
Abbreviations: CAT, Catalase; GPX, Guaiacol peroxidase; APX, Ascorbate peroxidase
* Corresponding author. Soumaya Ghazouani, Phone (+216) 51 629 061
E-mail addresses: soumayaghazouani92@gmail.com (G.
hannachi_hedia@yahoo.fr (H. He
dia), benasri@gmail.com (B.N.-A. Mouhiba).
salt-laden water, as well as the exaggeration use of fertilizers to
increase crop yields. It has been estimated that more than 50% of arable
land will be salinized by 2050 (Shrivastava and Kumar, 2015;
Machado and Serralheiro, 2017).
Depending on their tolerance level, plants showed different physi-
ological and biochemical mechanisms of salt stress response which
are quite complex including osmotic adjustment, compartmentation
of toxic ions (Munns and Tester, 2008) and compatible solutes accu-
mulation (Porcel et al. 2012; Plouznikoof et al. 2016). All these pro-
cesses involved in salt stress responses are highly complex and
related to many factors such as salt stress duration, plant develop-
ment stage, genotype tolerance and also the genotype x environment
interaction (Manaa et al. 2011; Negra ~o et al. 2017). Discrimination
between resistant and sensitive genotypes within species is clearly a
challenging but tricky issue and should allow for a better understand-
ing of the physiological and biochemical mechanisms of salt toler-
ance. Several studies on the effect of salinity in plants and the
different mechanisms adopted to cope with this constraint have been
published, but to our knowledge, little was known on the effects of
salinity in Urtica pilulifera from Urticaceae family. The Urtica species
Soumaya),
constitute a potential sources of important nutriments

https://doi.org/10.1016/j.sajb.2021.06.017
0254-6299/© 2021 SAAB. Published by Elsevier B.V. All rights reserved.
G. Soumaya, H. He
dia and B.N.-A. Mouhiba
(Wetherilt, 1992) because their richness on minerals, vitamines and
phenolic compounds (Wetberilt, 2003; Ozen € et al., 2010) and have
beneficts effect on human health such as the hypoglycemic, anti-
inflammatory, therapeutic properties, antitumor, antifungal, and
antimicrobial activities (Kavalalı et al. 2003).
The main objective of this study was to investigate the effect of
different NaCl concentrations on growth, physiological and some bio-
chemical traits of Urtica pililifera to determine its ability to avoid salt
constraint.
2. Material and methods
2.1. Plant material and culture conditions
Seeds of Urtica pulilifera were collected from Beja in North West of
Tunisia (36°43’32” N, 9°10’54’’E). The collected seeds were disin-
fected with commercial bleach diluted to 20% for 2 minutes, then
washed thoroughly and rinsed twice with distilled water. Then, they
were sown in Petri dishes 10 cm in diameter, lined with two layers of
filter paper soaked in distilled water (at the rate of 25 seeds per dish).
Petri dishes were placed at 28°C in the dark. The seeds were watered
daily (every 24 hours rehydrated with distilled water) until the 18th
day when the seedling growth was finished by the presence of coty-
ledons, hypocotyls and radicles.
The 18-day-old seedlings were transferred to a culture chamber
(16 h of light / 8 h of darkness at 22/18°C). They were transplanted
on hydroponic medium, containing a nutritive solution of Hoagland
(Hoagland and Arnon, 1950) diluted eight times. After 21 days of
acclimatization, homogeneous plants with four leaf stages (same
height) were subjected to treatments with NaCl (0 mM, 50 mM, 100
mM and 150 mM NaCl) for 15 days. The aerial and root parts of Urtica
pilulifera were harvested after 15 days of saline treatment.
2.2. Growth parameters
Water content (WC); lengths of aerial part (LAP) and root part
(LRP), number of leaves were determined. After 15 days of treatment,
plants were harvested, separated into aerial and root parts which
were rinsed with cold distilled water 3 successive times then
sponged between 2 strips of filter paper. The aerial and root parts for
each treatment were quickly put in pre-tared aluminum foil bags.
The fresh matter weight (FW) was determined using a precision bal-
ance type Mettler type, to 1 / 10th of mg. The dry matter weight
(DW) was determined after drying the sample at 40°C for 3 days.

Water content (WC), expressed as mL.g
1 DW, was determined by
the difference between the fresh weight and the dry weight com-
pared to the fresh weight:

WC (mL.g-1) = (FW-DW)/FW

Lengths of aerial part (LAP) and root part (LRP) were determined
using a rule graduated in centimeters.

Number of leaves was determined on plant having four leaves
stages.
2.3. Quantification of photosynthetic pigments
The photosynthetic pigments were determined using fresh leaf
samples cleaned with distilled water to remove any surface contami-
nation. Briefly, 100 mg of fresh leaf discs (0.2 cm2) were immersed in
5 mL of 80% (v/v) acetone and kept in dark for 72 h at room tempera-
ture. The absorbance was measured with a Beckman spectrophotom-
eter. Absorbance of acetone extracts were measured at 470 nm, 646
nm and 663 nm to calculate chlorophyll a (Chla), chlorophyll b
(Chlb), total chlorophyll (Chl-tot), and total carotenoids (Car) con-
tents, according to the equation proposed by (Lichtenthaler 1987).
125
South African Journal of Botany 142 (2021) 124
130
The chlorophylls and carotenoid contents are expressed as mg. g
1
FW.
Chla ¼ 12:25 A663
2:79 A646
Chlb ¼ 21:50 A646
5:10 A663
Chl
tot ¼ 7:15 A663 þ 18:71 A646
Car ¼ ð1000 A470
1:82 Chla
85:02 ChlbÞ=198
A: absorbance
2.4. Determination of proline content
Proline in root and leaf tissues was extracted and analyzed
(Bates et al. 1973). Dry matter powder (10 mg) was mixed with 1.5
mL of 3% aqueous sulfosalicylic acid. The homogenate was centri-
fuged at 14.000 x g for 10 min and the supernatant was transferred
to a new 1.5 mL tube. The extracted solution was reacted with an
equal volume of glacial acetic acid and ninhydrin reagent (1.25 g of
ninhydrin in 30 mL of glacial acetic and 20 mL of 6 M H3PO4) and
incubated at 100°C for 1 h. The reaction was stopped by placing the
tube in an ice bath. The reaction mixture was mixed vigorously with
2 mL of toluene. After heating to 25°C, the chromophore was mea-
sured at 520 nm. Proline was used as a standard. The proline concen-
tration was determined using the calibration curve in mmol of
proline g
1 DW.
2.5. Determination of sugar content
The content of soluble sugars was estimated (Staub 1963). The
extract was prepared as follows: 10 mg of dry plant matter were
mixed with 5 mL of 80% ethanol. The mixture was placed in water
bath at 70°C for 30 minutes and stirred regularly. After cooling, the
extract is divided and placed in glass tubes, they were centrifuged at
3059 g for 15 min at 4°C. Then, 2 mL of anthronone (2 g of anthro-
none in 100 mL of 36 N H2SO4) were added to 1 mL of the superna-
tant. The resulting solution was stirred and placed in a water bath
boiling at 100°C for 10 minutes. At the end of this reaction, the solu-
tion was placed directly in ice. The absorbance was recorded at 640
nm. Sugar concentrations were determined based on standard curve
prepared using a glucose solution. The sugar content was expressed
as mg of sugars per gram of dry weight (mg.g
1 DW). The control
consists of a solution containing 5 mL of anthrone and 2.5 mL of 80%
ethanol.
2.6. Enzyme assays
2.6.1. Extraction of soluble proteins
The preparation of enzyme extracts was performed at 4°C. After
grinding the samples (1g) in a mortar with liquid nitrogen, the pow-
der was taken up in 1.5 mL extraction buffer. The volume was propor-
tional to the fresh material weight and its composition is as follows:
potassium phosphate (50 mM, pH 7.5), polyvinylpolypyrrolidone 5%
(PVPP), glycerol 5%, DL-Dithiothreitol 1mM (DTT), ethylene diamine
tetra acetic acid 100 mM (EDTA). The mixture was centrifugated at 4°
C for 20 min at 13.000 x g in cooled centrifuge. The supernatant con-
taining the soluble proteins was used for protein determination and
to measure the for enzymes activities.
2.6.2. Catalase activity (CAT)
Catalase (CAT) activity was determined by monitoring the disap-
pearance of H2O2 (Cakmak and Marschner, 1992). The final reaction
mixture contained 50 mM sodium phosphate buffer (pH 7.0) and 2%
H2O2 was used to determine the catalase activity. The catalase activ-
ity was expressed as U mg
1 protein min-1.
G. Soumaya, H. He
dia and B.N.-A. Mouhiba South African Journal of Botany 142 (2021) 124
130

2.6.3. Guaiacol Peroxidase activity (GPX)
The GPX activity was measured using guaiacol as an electron
donor with a reaction mixture containing 20 mM phosphate buffer
(pH 7.0), and 30 mM H2O2 (Srinivas et al. 1999). The increase of
absorbance due to tetraguaiacol formation was recorded at 470 nm.
One unit (U) of GPX activity catalyzed the oxidation of 1 mmol of
guaiacol. The GPX activity was expressed as U mg
1 protein min
1.
2.6.4. Ascorbate Peroxidase activity (APX)
The activity of ascorbate peroxidase (APX) was determined using
a reaction mixture of 1.5 mL containing 50 mM phosphate buffer (pH
7.0), 0.1 mM EDTA, 0.5 mM ascorbate, 2 mM H2O2 and 50 ml of
enzyme extract (Nakano and Asada, 1981) Nakano and Asada 1981).
The reaction was started by the addition of H2O2, and the oxidation
of ascorbate measured at 290 nm. The molar extinction coefficient
was 2.8 mM
1cm
1. This experiment was arranged as a factorial in
the framework of a completely randomized design with two factors:
salinity (0, 50, 100, and 150 mM) and cultivar Urtica pilulifera with
three replicates. APX was expressed as U mg
1 protein min
1.
2.7. Statistical analyses
All measurements were carried out in triplicate and results were
presented as means § Standard Deviation (SD). Analysis of variance
was used to compare salt effect on measured parameters. Significant
differences of the mean values (P < 0.05) were determined by Dun-
can’s multiple range test. Regression analyses were performed on
NaCl concentrations and antioxidant enzymes activities. The equation
and regression coefficient R2 were determined. Data analyses were
performed using Xlstat 2016. (www.xlstat.com).
3. Results
3.1. Effects of NaCl on growth and morphology parameters
Results showed that after 15 days of growth under different NaCl
concentrations (control (T0 = 0 mM), low (T1 = 50 mM), moderate
(T2 = 100 mM) and high (T3 = 150 mM)), Urtica pilulifera presented
some significant differences in terms of growth measures. The toxic
effects observed on leaves were mainly necrosis and loss of chloro-
phylls in the presence of a moderate and high concentration of NaCl
(T2 = 100 mM) (T3 = 150 mM). Death of some seedlings preceded by
necrosis and falling eaves, was mainly observed at 100 mM and espe-
cially at 150 mM of NaCl
Analysis of variance (ANOVA) to a single factor (salinity factor) fol-
lowed by the multiple comparison test of Duncan means showed that
salinity has a significant influence (p <0.05) on growth parameters of
the aerial and root part of Urtica pilulifera
Moderate (T2 = 100 mM) and high (T3 = 150 mM) salinities
affected negatively the production of fresh and dry biomass as well
as water content in leaves and roots of Urtica pilulifera (Tables 1, 2).
Low salt concentration (T1 = 50 mM) in the culture medium did not
affect significantly the LAP of Urtica pilulifera, while moderate
(T2 = 100 mM) and high salinity (T3 = 150 mM) significantly
decreased the LAP of the plant. Length of the plant roots reduced sig-
nificantly under studied saline concentrations (low, moderate or
high). The number of leaves decreased significantly at 100 mM and
150 mM of NaCl treatments in Urtica pilulifera (Table 1).
3.2. Effects of salinity on photosynthetic pigments
The chlorophyll a and total chlorophylls (Fig. 1A, 1C) contents
were affected significantly under salt concentrations compared to the
control medium. The lowest values for chlorophyll a, chlorophyll b
and total chlorophylls contents were 0.41, 0.20 and 0.61 mg.g
1 FW
respectively, detected in plants cultivated under 150 mM NaCl. How-
ever, the chlorophyll b and the carotenoids contents in Urtica piluli-
fera leaves were no affected significantly according the increase of
the salt concentration in the culture medium (Fig. 1B, 1D).
The carotenoid content has the highest value in plants growing on
a salt-free medium (control) with a value of 3.76 mg.g
1FW. The pres-
ence of salt in the culture medium doesn’t cause any significant
decrease in the total carotenoid contents, whatever the concentra-
tions of 3.56 at T1 (50 mM), 3.44 mg.g
1FW at T2 (100 mM) and 3.40
mg.g
1FW at T 3 (150 mM).
3.3. Effects of NaCl on the proline synthesis
The proline levels increased from 0.411 in medium T0 (0 mM) to
1.643 mmol.g
1FW in T3 medium (150 mM) for the aerial part and
from 0.27 in T0 medium (0 mM) to 1.259 mmol.g
1FW in T3 medium
(150 mM) for the root part (Fig. 2A). In the aerial and the root parts,
the presence of salt in the culture medium, stimulated the production
of proline.

Table 1
Effect of salinity on growth parameters of aerial part of Urtica pilulifera

[NaCl] FW (mg) DW (mg) WC (mL.g-1) LAP (cm) NF

A A A A
T0 (0mM) 9.924 § 0.151 5.310 § 0.274 46.507 § 2.280 39.800 § 0.819 8.000 § 0.000A
T1(50 mM) 7.031 § 0.342B 4.236 § 0.264B 39,767 § 0.606AB 37.233 § 0.493B 5.667 § 0.577B
T2 (100 mM) 5.811 § 0.061C 4.148 § 0.016BC 28,115 § 0,441B 34.767 § 0.252C 4.333 § 0.577C
T3 (150 mM) 4.286 § 0.329D 3.542 § 0.300C 17.389 § 0.707C 31.333 § 0.6110D 3.333 § 0.577D
The values are mean (n = 3) § SD; T: treatment; FW: fresh weight; DW: dry weight; WC: water content; LAP: length of
aerial part; NF: Number of leaves. Means with different letters within the same column differs significantly at P = 0.05.

Table 2
Effect of salinity on growth parameters of root part of Urtica pilulifera

[NaCl] FW (mg) DW (mg) WC (mL.g
1) LRP (cm)

T0 (0mM) 1.541 § 0.048A 0.686 § 0.065A 55.423 § 4.777A 54.067 § 1.007A

T1(50 mM) 1.035 § 0.082B 0.491 § 0.010B 52.330 § 3.528B 40.133 § 0.907B
T2 (100 mM) 0.522 § 0.020C 0.324 § 0.028C 42.601 § 6.828C 15.467 § 0.751C
T3 (150 mM) 0.288 § 0.010D 0.166 § 0.025D 37.735 § 7.501D 11.330 § 0.416D
The values are mean (n = 3) § SD;; T: treatment; FW: fresh weight; DW: dry weight; WC: water
content; LRP: length of root part. Means with different letters within the same column differs sig-
nificantly at P = 0.05.

126
G. Soumaya, H. He
dia and B.N.-A. Mouhiba South African Journal of Botany 142 (2021) 124
130

Fig. 1. Pigments variation: chlorophyll a (A), Chlorophyll b (B), total chlorophylls (C) and Carotenoids (D) in Urtica pilulifera cultivated under different NaCl concentrations during 15
days of treatment. Values are mean values § Standard deviation of three replicates. Bars with different letters showed significant differences at P  .05 (FW: fresh weight).

Fig. 2. Proline (A) and total soluble sugar (B) contents in aerial and root parts of Urtica pilulifera under salt constraint. Values are mean values § Standard Deviation of three repli-
cates. Bars with different letters show significant differences at P  .05 (FW: fresh weight; DW: dry weight).

3.4. Effect on NaCl on the soluble sugar synthesis illustrated in Figure 2B. The soluble sugars contents were signifi-
cantly increased. In all cases, the aerial part accumulated more solu-
The accumulations of soluble sugars in different organs of the con- ble sugars than the root part. The contents of this osmolyte increased
trol plants and plants subjected to different saline treatments were from 58.15 in medium T0 (0 mM) to 131.21 mg. g
1DW in T3 medium

127
G. Soumaya, H. He
dia and B.N.-A. Mouhiba South African Journal of Botany 142 (2021) 124
130

Table 3
Effect of salinity on catalase, glutathione peroxidases and ascorbate peroxydase activities in the arial and root
parts of Urtica pilulifera

CAT (U mg
1protein min
1) GPX (U mg
1protein min
1) APX (U mg
1protein min
1)

Aerial part
T0 (0 mM) 1.093 § 0.240D 2.542 § 0.677B 142.59 § 2.338A
T1 (50 mM) 3.103 § 0.697B 4.880 § 1.095A 143.28 § 2.22A
T2 (100 mM) 1.849 § 1.583C 2.823 § 2.403B 151.1 § 4.08A
T3 (150 mM) 3.737 § 0.607A 5.860 § 1.002A 156.51 § 5.06A
Root part
T0 (0 mM) 1.044 § 0.098C 1.06 § 0.095C 121.83 § 1.26A
T1 (50 mM) 1.528 § 0.086B 2.443 § 0.531B 126.53 § 2.04A
T2 (100 mM) 2.972 § 0.175AB 4.743 § 0.476AB 136.52 § 4.54A
T3 (150 mM) 3.499 § 1.144A 5.797 § 2.190A 139.31§ 5.24A
T : treatment ; CAT : catalase ; GPX : guaiacol peroxydase ; APX : ascorbate peroxydase

Table 4 showed that intense symptoms of sensitivity to salt constraint such

Regression analysis of salinity and GPX, CAT, APX activities in the aerial and root
parts of Urtica pilulifera
Equation

1
1
Aerial part GPX (U mg protein min ) y = 0.0241x + 2.2206
CAT (U mg
1 protein min
1) y = 0.0184x + 1.0671
APX (U mg-1 protein min
1) y = 0.0992x + 140.93
Root part GPX (U mg
1 protein min
1) y = 0.0330x + 1.0340
CAT (U mg
1 protein min
1) y = 0.0170x + 0.9370
APX (U mg
1 protein min
1) y = 0.1249x +121.680
CAT: catalase ; GPX : guaiacol peroxydase ; APX : ascorbate peroxydase
(150 mM) for the aerial part and from 43 in medium T0 (0 mM) to
100.99 mg. g
1 DW in T3 medium (150 mM) for the root part.
3.5. Effects of NaCl on antioxidants enzymatic activities
In order to better understand the effect of different saline treat-
ments on antioxidant activity, a quantitative analysis of the activities
catalase (CAT), guaiacol peroxidases (GPX) and ascorbate peroxydase
(APX) were carried out from extraction of soluble proteins of aerial
and root parts of plants. The stimulation of the studied enzymes
activities was related to the applied salt treatment during the culture.
The salt stress induced the CAT synthesis in arial part and was signifi-
cantly affected by salt stress especially at 150 mM NaCl. The CAT syn-
thesis was very close under T1 (50 mM) and T2 (100 mM) in arial
part showing that at high salt treatment the synthesis of CAT was
more improved. The GPX and The APX don’t showed any significant
differences under different salt concentrations reflecting that the salt
stress doesn’t induce the GPX and APX syntheses in aerial part
(Table 3). For the root part, the salinity induced the CAT and GPX syn-
theses especially at 150 mM NaCl. The APX synthesis was no affected
by salt stress. It should be noted that APX activity was much higher
in the absence of salt and at all salinity levels for the whole plant
compared to other antioxidant enzymes (CAT, GPX).
Regression analysis showed that the applied salt concentration
was proportional with antioxidant enzymes activities including CAT,
GPX and APX, both on the aerial and the root parts reflection by
higher values of R2 (Table 4).
4. Discussion
At 150 mM NaCl, there is an inhibition of growth observed in the
both aerial and roots parts of Urtica pilulifera. This inhibition is com-
monly reported for glycophytes such as wheat (Alaoui et al. 2013),
rice (Hussain et al. 2017), sugar cane (Gandonou et al. 2012;
Gandonou and Skali Senhaji, 2015), tomato (Albacete et al. 2008),
amaranth (Omami and Hammes 2006; Wouyou et al. 2017). Results
as necrosis and early leaf fall were observed at 100 and 150 mM in
Urtica pilulifera. These recorded morphological symptoms suggested
R2 certain vulnerability of this plant to salt constraint at these two con-
centrations. The appearance of leaf necrosis is generally explained by
0.9317
0.9850 a specific toxicity to Cl- and / or Na + ions (Flowers and Yeo, 1988).
0.9239 However, early leaf senescence could be attributed as an adaptive
0.9810 response to salinity in order to reduce the total content of toxic ions
0.9573
at plant level (Acosta-Motos et al. 2017) as noted previously pistachio
0.9572
rootstocks (Karimi et al. 2014). Therefore, Urtica pilulifera seemed to
develop an adaptative response by early leaf senescence to reduce
the total toxic ions under 100 and 150 mM NaCl.
In our work, the chlorophylls a and total chlorophylls were
reduced significantly by increasing the salt concentrations. However,
no significant differences were observed on chlorophyll b and carote-
noids contents. This suggests that, as reported by Ashraf and Har-
ris, 2013), Urtica pilulifera seemd to conserve its photosynthetic
ability proving by its chlorophyll b content stability. In general, a
decrease in photosynthetic pigments under saline stress is consid-
ered to be the result of a slow synthesis or rapid breakdown of the
pigments in the cells (Ashraf 2003). The carotenoids content as pho-
tosynthetic pigments don’t decrease significantly under the salinity
treatments of Urtica pilulifera. Several studies have shown that under
saline conditions, carotenoids play an effective antioxidant role by
protection and stabilization of photosynthetic and photochemical
processes and play a vital role in the prevention of photosensitization
(Demmig-Adams and Adams, 1996). In the present study, moderate
(T2 = 100 mM) and high (T3 = 150 mM) salinity induced a significant
increase in the proline content in the aerial and root parts of Urtica
pilulifera. To survive and cope in saline environments, plants have
evolved salt tolerance mechanisms such as the synthesis of compati-
ble solutes or organic compounds (Abouelsaad and Renault, 2018)
including amino acids and soluble sugars which play an essential role
in reducing of the toxic effects of salt on plants (Chang et al. 2014).
Proline, a multifunctional amino acid, is one of the most important
compatible solutes, accumulated in response to various environmen-
tal constraints. It plays a key role in the tolerance of plants to salt
stress and in primary metabolism as a constituent of proteins
(Aroca et al. 2013). Under environmental constraint, the proline
seems to have various roles as the stabilization of proteins, mem-
branes and subcellular structures, and protect cellular functions by
cleaning reactive oxygen species (ERO) (Kaur et al. 2015). Therefore,
Urtica pilulifera with greater accumulation of proline under high
salinity could be a sign of resistance as noted previously that proline
accumulation is used as one of the most important physiological indi-
cators for salt resistance in plants (Mansour and Ali, 2017) as Pistacia
species (Chelli- Chaabouni et al. 2010; Karimi et al. 2014).
Results showed that the salt constraints applied on Urtica piluli-
fera induced the soluble sugars content. It is known, that during
osmotic stress caused by salinity, plants accumulate sugars in their
128
G. Soumaya, H. He
dia and B.N.-A. Mouhiba
leaves, for osmotic adjustment to maintain cell survival
(Thalmann et al. 2016). The accumulation of these organic compounds
has been demonstrated in several plants under saline stress (El Midaoui
et al. 2007; Islam et al. 2019). The sugars accumulation varied according
to the species, the stage of development and the salt concentration and
there is a strong correlation between the accumulation of sugars and the
level of tolerance to salinity (El Midaoui et al. 2007). The accumulation of
sugars, mainly following the hydrolysis of starch (Phillips et al. 2002), is
stimulated by salt (Bartels and Sunkar, 2005). Therefore, Urtica pilulifera
improved sugar synthesis for osmotic adjustment to avoid salt constraint
and to maintain cell survival.
Active oxygen species are very damaging at high doses for the cell,
to cope with this damage, plants have developed antioxidant defense
systems. This antioxidant defense system can be enzymatic or non-
enzymatic (Kim et al. 2017; Alam et al., 2020). In our study, we tested
three antioxidant detoxification enzymes including CAT, GPX APX in
aerial and root parts of Urtica pilulifera under salt stress. Results
showed that the CAT activity increased according the salt concentra-
tions in the both arial and root parts (Tables 3 and 4). The presence of
high levels of intracellular H2O2 leads to preferential activation of cat-
alase (Pamplona and Costantini, 2011) which is an enzyme found
mainly in peroxisomes, cytosol and mitochondria and plays a key
role in the catalytic trapping of H2O2 by breaking it down into water
and oxygen (Arora et al. 2002). It can break down millions of hydro-
gen peroxide molecules in a second (Ighodaro et al. 2019). Catalase
prediction plays an important role in the adaptive response of
cells (Gebicka et al. 2019) and its activity increased in plants, under
various unfavorable conditions, as an antioxidant defense
(Gupta et al. 2018). Results showed that under salt constraint, Urtica
pilulifera improved the GPX and APX activities to prevent, trap, elimi-
nate and sequester the toxicity of EROs imposed by salinity. The CAT
and GPX were the most among the H2O2 scavenging enzymes in the
both arial and root parts; however, the APX seemed to be more
important in arial parts. Results were in agreement with others
(Azevedo Neto et al., 2006; Liang et al., 2003) who suggested that
CAT and GPX were the most important H2O2 scavenging enzymes
leading to salt tolerance. In other hand, it was noted that the CAT and
GPX had much higher H2O2 scavenging activity than APX in leaves
and roots of salt-stressed maize plants (Azevedo Neto et al., 2006).
The GPX and APX peroxidases are distributed throughout the cell and
catalyzed the reduction of H2O2 to H2O. The GPX is less specific to
electron donor substrate and decomposes H2O2 by oxidation of co-
substrates such as phenolic compounds and/or ascorbate (Bray et al.,
2000) and APX utilises ascorbate as an electron donor (Noctor and
Foyer, 1998).
We noted that Urtica pilulifera cultivated under saline constraint,
showed a higher activity of antioxidant enzymes in the aerial than in
the root parts for the same saline concentrations, suggesting that the
aerial part is more damaged than the roots by the salinity. Salt stress
may induce more severe oxidative stress in the leaves than in the
roots (Dang Hee et al., 2001) as noted for other species as wheat
(Meneguzzo et al. 1999), cotton cultivars (Meloni et al. 2002) rice and
lettuce plants (Lee et al. 2001; Mahmoudi et al. 2011). Therefore, to
defend salt constraint, plants have evolved specific protective mecha-
nisms, including antioxidant and enzyme system which is recognized
as the main mechanism of plant tolerance to environmental stress
(Bor et al., 2003). Results showed that CAT, GPX and APX activities
coordinated with salt induced oxidative stress tolerance of Urtica
pilulifera as noted previousely for other species (Liang et al., 2003).
The accumulation of H2O2 has been reported to function as an inter-
cellular signal, and it stimulates several genes and proteins involved
in stress responses, such as catalase, peroxidase, and alternative oxi-
dase.
Results showed that the overexpression of CAT, GPX and APX
reflected that the Urtica pilulifera reacted to saline stress by its enzy-
matic antioxidant system reflecting that it was able to avoid salt
129
South African Journal of Botany 142 (2021) 124
130
stress by improvement its antioxidant enzymes system. A high APX
activity in Urtica pilulifera in the absence or presence of salinity, sug-
gesting less cellular damage caused by oxygen radicals, as previously
demonstrated (Waqas et al. 2019).
5. Conclusion
Urtica pilulifera developed morphological and physiological mech-
anisms to avoid the effect of saline constraint. Therefore, the growth
parameters decreased significantly accompanied by a decrease on
chlorophyll a and total chlorophylls. However, insignifiant differen-
ces were noted on chlorophyll b and carotenoids content. Urtica pilu-
lifera accumulated proline and soluble sugar to regulate and adjust
the osmotic pressure. To improve its capacity to prevent cell damage,
this specie improved antioxidant enzymes under saline constraint. In
order to better understand the behavior of Urtica pilulifera at salinity,
it is desirable to broaden our study on other methods of analysis
(quantitative and qualitative analyzes of proteins based on different
proteomic approaches).
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgements
The authors thank Madame Cheibi Wided Professor at the Faculty
of Sciences Tunis for providing seeds of Urtica pilulifera
References
Abouelsaad, I., Renault, S., 2018. Enhanced oxidative stress in the jasmonic acid-defi-
cient tomato mutant def-1 exposed to NaCl stress. Journal of Plant Physiology 226,
136–144.
Acosta-Motos, J.R., Ortun ~ o, M.F., Bernal-Vicente, A., Diaz-Vivancos, P.,
Sanchez Blanco, M.J., Hernandez, J.A., 2017. Plant responses to salt stress: adaptive
mechanisms. Agronomy 7, 1–38.
Alam, P., Kohli, Kaur, S., Al Balawi, T., Altalayan, F., H., Alam, P., Ashraf, M., Ahmad, P.,
2020. Foliar Application of 24-Epibrassinolide Improves Growth, Ascorbate-Gluta-
thione Cycle, and Glyoxalase System in Brown Mustard (Brassica juncea (L.) Czern.)
under Cadmium Toxicity. Plants 9, 1487 24 pages.
Alaoui, M.M., El Jourmi, L., Ouarzane, A., Laza, rS., El Antri, S., Zahouily, M., Hmyene, A.,

te

s maro-
2013. Effet du stress salin sur la germination et la croissance de six varie
caines de ble
. Journal of Materials and Environmental Science 4, 997–1004.
Albacete, A., Ghanem, M.E., Martínez-Andu
jar, C., Acosta, M., Sa
nchez-Bravo, J.,
Martínez, V., Lutts, S., Dodd, I.C., Pe
rez-Alfocea, F., 2008. Hormonal changes in rela-
tion to biomass partitioning and shoot growth impairment in salinised tomato
(Solanum lycopersicum L.) plants. Journal of Experimental Botany 59, 4119–4131.
Aroca, R., Ruiz-Lozano, J.M., Zamarren ~ o, A.M., Paz, J.A., García-Mina, J.M., Pozo, M.J.,
2013. Arbuscular mycorrhizal symbiosis influences strigolactone production under
salinity and alleviates salt stress in lettuce plants. Journal of Plant Physiology 170,
47–55.
Arora, A., Sairam, R., Srivastava, G., 2002. Oxidative stress and antioxidative system in
plants. Current Science 82, 1227–1238.
Ashraf, M., 2003. Relationships between leaf gas exchange characteristics and growth
of differently adapted populations of blue panic grass (Panicum antidotale Retz.)
under salinity or waterlogging. Plant Science 165, 69–75.
Ashraf, M., Harris, P., 2013. Photosynthesis under stressful environments: an overview.
Photosynthetica 51, 163–190.

as-Filho, J., Braga de Abreu, C.E.,
Azevedo Neto, A.D., Tarquinio Prisco, J., Ene
Gomes-Filho, E., 2006. Effect of salt stress on antioxidative enzymes and lipid per-
oxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes.
Environmental and Experimental Botany 56, 87–94.
Bartels, D., Sunkar, R., 2005. Drought and salt tolerance in plants. CRC. Critical Reviews
in Plant Sciences 24, 23–58.
Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water
stress studies. Plant Soil 39, 205–207.
Bencherif, K., Boutekrabta, A., Fontaine, J., Laruelle, F., Dalpe
, Y.,
Loune s-Hadj Sahraoui, A., 2015. Impact of soil salinity on arbuscular mycorrhizal
fungi biodiversity and microflora biomass associated with Tamarix articulata Vahll
G. Soumaya, H. He
dia and B.N.-A. Mouhiba
rhizosphere in arid and semi- arid Algerian areas. Science of Total Environment
533, 488–494.
€
Bor, M., Ozdemir, € rkan, I., 2003. The effect of salt stress on lipid peroxidation and
F., Tu
antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L.
Plant Science 164, 77–84.
Bray, E.A., Bailey-Serres, J., Weretilnyk, E., 2000. Responses to abiotic stresses.
In: Buchanan, B.B., Gruissem, W., Jones, R.L. (Eds.), Biochemistry and Molecular
Biology of Plants. American Society of Plant Physiologists, Rockville Md,
pp. 1158–1203.
Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity
enhance activities of superoxide dismutase, ascorbate peroxidase and gluthatione
reductase in bean plants. Plant Physiology 98, 1222–1227.
Chang, B.W., Cong, W.W., Chen, Q., Zu., YG., Tang, Z.H., 2014. The influence of different
forms and concentrations of potassium nutrition on growth and alkaloid metabo-
lism in Catharanthus roseus seedlings. Journal of Plant Interactions 9, 370–377.
Chelli-Chaabouni, A., Ben Mosbah, A., Maalej, M., Gargouri, K., Gargouri-Bouzid, R.,
Drira, N., 2010. In vitro salinity tolerance of two pistachio rootstocks: Pistacia vera
L. and P. atlantica Desf. Environmental and Experimental Botany 69, 302–312.
Demmig-Adams, B., Adams, W.W., 1996. Xanthophyll cycle and light stress in nature:
uniform response to excess direct sunlight among higher plant species. Planta 198,
460–470.
Dong Hee, L., Young Sang, K., Chin Bum, L., 2001. The inductive responses of the antiox-
idant enzymes by salt stress in the rice (Oryza sativa L.). Journal of Plant Physiology
158, 737–745.
Egamberdieva, D., Davranov, K., Wirth, S., Hashem, A., Abd-Allah, E., 2017. Impact of
soil salinity on the plant-growth
promoting and biological control abilities of
root associated bacteria. Saudi Journal of Biological Sciences 24, 1601–1608.
El midaoui, M., benbella, M., Aït houssa, A., ibriz, M., Talouizte, A., 2007. Contribution a

tude de quelques me
l''e
canismes d''adaptation a la salinite
chez le tournesol
cultive
(Helianthus annuus L.). Revue HTE 136, 29–34.
Flowers, T.J., Yeo, A.R.,1988. Ion relations of salt tolerance, in: Baker D., and Halls J.
(Eds.), Solute transport in plant cells and tissues. Harlow, UK, pp. 392
413.
Gandonou, C.B., Gnancadja, S.L., Abrini, J., Skali Senhaji, N., 2012. Salinity tolerance of
some sugarcane (Saccharum sp.) cultivars in hydroponic medium. International
Sugar Journal 114, 190–196.
Gandonou, C.B., Skali Senhaji, N., 2015. Sugarcane (Saccharum sp.) salt tolerance at var-
ious developmental levels. In: Chakraborty, U., Chakraborty, B (Eds.), Abiotic
Stresses in Crop Plants. CABI Publishing, United Kingdom, pp. 102–111.
Gebicka, L., Krych-Madej, J., 2019. The role of catalases in the prevention/promotion of
oxidative stress. Journal of Inorganic Biochemistry 197, 110699.
Gupta, D.K., Palma, J.M., Corpas, F.J., 2018. Antioxidants and antioxidant enzymes in
Higher Plants. Springer-Verlag, Germany.
Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants with-
out soil. California Agricultural Experiment Station Circular 347, 1–32.
Hussain, S., Zhang, J.H., Zhong, C., Zhu, L.F., Cao, X.C.Yu., Hu, S.M., JABJ, J., Jin, Q.J., 2017.
Effects of salt stress on rice growth, development characteristics, and the regulat-
ing ways: A review. Journal of Integrative Agriculture 16, 2357–2374.
Ighodaro, O.M., Akinloye, O.A., 2019. First line defence antioxidants-superoxide dismu-
tase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental
role in the entire antioxidant defence grid. Alexandria Journal of Medecine 54,
287e293.
Islam, F., Wang, J., Farooq, M.A., Yang, C., Jan, M., Mwamba, T.M., 2019. Rice responses
and tolerance to salt stress, in: Advances in Rice Research for Abiotic Stress Toler-
ance. Elsevier, Amsterdam, The Netherlands, pp. 791–819.
Jamil, A., Riaz, S., Ashraf, M., Foolad, M.R., 2011. Gene expression profiling of plants
under salt stress. Critical Reviews in Plant Sciences 30, 435–458.
Karimi, H.R., Maleki Kuhbanani, R., 2014. Evaluation of inter-specific hybrid of P.
atlantica£P.vera cv. ‘Badami-Rize-Zarand’ as pistachio rootstock to salinity stress
according to some growth indices, echophysiological and biochemical parameter.
Journal of Stress Physiology & Biochemistry 10, 5–17.
Kaur, U., Banerjee, P., Bir, A., Sinha, M., Biswas, A., Chakrabarti, S., 2015. Reactive oxy-
gen species, redox signalling and neuroinflammation in Alzheimer's disease: The
NF-kB connection. Current Topics in Medicinal Chemistry 15, 446–457.
Kavalali, G.M., 2003. Urtica: therapeutic and nutritional aspects of stinging nettles, in:
The Chemical and Pharmacological Aspects of Urtica. Taylor & Francis, New York,
pp. 25–39.
Kim, M.J., Kim, H.J., Pak, J.H., Cho, H.S., Choi, H.K., Jung, H.W., Lee, D.H., Chung, Y.S.,
2017. Overexpression of AtSZF2 from Arabidopsis showed enhanced tolerance to
salt stress in soybean. Plant Breed Biotech 5, 1–15.
Lee, D.H., Young, S.K., Lee, C.B., 2001. The inductive responses of the antioxidant
enzymes by salt stress in the rice (Oryza sativa L.). Journal of Plant Physiology 158,
737–745.
Liang, Y., Chen, Q., Liu, Q., Zhang, W., Ding, R., 2003. Exogenous silicon (Si) increases
antioxidant enzyme activity and reduces lipid peroxidation in root of salt-stressed
barley (Hordeum vulgare L.). Journal of Plant Physiology 160, 1157–1164.
Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: Pigments of photosynthetic
biomembranes. Methods in Enzymology 148, 350–382.
Li, X.J., Han, B.Xu.XY., Han, L.P., Zhao, Y.Y., Liu, Z.L., Dong, H.S., Zhang, C.L., 2014.
Plant growth enhancement and associated physiological responses are co-reg-
ulated by ethylene and gibberellin in response to harpin protein. Hpa1. Planta
239, 831–846.
South African Journal of Botany 142 (2021) 124
130
Machado, R.M.A., Serralheiro, RP., 2017. Soil Salinity: Effect on Vegetable Crop Growth
Management Practices to Prevent and Mitigate Soil Salinization. Horticulturae 3,
1–13.
Mahmoudi, H., Kaddour, R., Huang, J., Nasri, N., Bouattour, O., M’Rah, S., Hannoufa, A.,
Lachaal, M., Ouerghi, Z., 2011. Varied tolerance to NaCl salinity is related to bio-
chemical changes in two contrasting lettuce genotypes. Acta Physiologiea Planta-
rum 33, 1613–1622.
Manaa, A., Ben Ahmed, H., Valot, B., Bouchet, J.P., Aschi-Smiti, S., Causse, M.,
Faurobert, M., 2011. Salt and genotype impact on plant physiology and root prote-
ome variations in tomato. Journal of Experimental Botany 62, 2797–2813.
Mansour, M.M.F., Ali, E.F., 2017. Evaluation of proline functions in saline conditions.
Phytochemistry 140, 52–68.
Mathur, N., Singh, J., Bohra, S., 2007. Arbuscular mycorrhizal status of medicinal halo-
phytes in saline areas of Indian Thar desert. International Journal of Soil Science 2,
119–127.
Meloni, D.A., Oliva, M.A., Martinez, C.A., Cambria, J., 2002. Photosynthesis and activity
of superoxide dismutase, peroxidase and glutathione reductase in cotton under
salt stress. Environmental and Experimental Botany 49, 69–76.
Meneguzzo, S., Navari-Izzo, F., Izzo, R., 1999. Antioxidative responses of shoots and
roots of wheat to increasing NaCl concentrations. Journal of Plant Physiology 155,
274–280.
Munns, R., Gilliham, M., 2015. Salinity tolerance of crops—what is the cost? New Phy-
tologist 208, 668–673.
Munns, R., Tester, M., 2008. Mechanisms of Salinity Tolerance. Annual Review of Plant
Biology 59, 651–681.
Nakano, Asada, 1981. Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxi-
dase in Spinach Chloroplasts. Plant and Cell Physiology 22, 867–880. https://doi.
 org/10.1093/oxfordjournals.pcp.a076232.
Negra ~o, S., Schmo € ckel, S.M., Tester, M., 2017. Evaluating physiological responses of
plants to salinity stress. Annals of Botany 119, 1–11.
Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under
control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–
279.
Omami, E.N., Hammes, P.S., 2006. Differences in salinity tolerance for growth and
water use efficiency in some amaranth (Amaranthus spp.) genotypes. New Zealand
Journal of Crop Horticultural Science 34, 11–22.
€
Ozen, €llu
T., Ç o € , Z., Korkmaz, H., 2010. Antioxidant Properties of Urtica pilulifera Root,
Seed, Flower, and Leaf Extract. Journal of Medicinal Food 13, 1224–1231.
Pamplona, R., Costantini, D., 2011. Molecular and structural antioxidant defenses
against oxidative stress in animals. American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301, 843–863.
Phillips, J.R., Oliver, M.J., Bartels, D., 2002. Molecular genetics of desiccation and toler-
ant Systems. In: Black, M., Pritchard, H.W. (Eds.), Desiccation and survival in plants:
drying without dying. CAB International, Wallingford, pp. 319–341.
Plouznikoof, K., Declerck, S., Calonne-Salmon, M., 2016. Mitigating abiotic stresses in
crop plants by arbuscular mycorrhizal fungi. In: Vos, C.M.F., Kazan, K. (Eds.), Below-
ground defence strategies in plants, signaling and communication in plants.
Springer, Cham, pp. 341–400.
Porcel, R., Aroca, R., Ruíz-Lozano, J.M., 2012. Salinity stress alleviation using arbuscular
mycorrhizal fungi
a review. Agronomy for Sustainable Development 32, 181–200.
Shrivastava, P., Kumar, R., 2015. Soil salinity: a serious environmental issue and plant
growth promoting bacteria as one of the tools for its alleviation. Saudi Journal of
Biologicla Sciences 22, 123–131.
Srinivas, N.D., Rshami, K.R., Raghavarao, K.S.M.S., 1999. Extraction and purification of a
plant peroxidise by aqueous two-phase extraction coupled with gel filtration. Pro-
cess Biochemistry 35, 43–48.
Staub, A., 1963. Extraction, identification et dosages des glucides dans les extraits d’or-
ganes et les corps bacte
riens, in: Masson et Compagnie (Eds.), Techniques de labo-
ratoire (Tome 1 et 2), Paris, pp. 1307
1366.
Thalmann, M., Pazmino, D., Seung, D., Horrer, D., Nigro, A., Meier, T., 2016. Regulation
of leaf starch degradation by abscisic acid is important for osmotic stress tolerance
in plants. Plant Cell 28, 1860–1878.
Torbaghan, M.E., Lakzian, A., Astaraei, A.R., Fotovat, A., Besharati, H., 2017. Salt and
alkali stresses reduction in wheat by plant growth promoting haloalkaliphilic bac-
teria. J Soil Sci Plant Nutrition 17, 1058–1087.
Waqas, M., Yaning, C., Iqbal, H., Shareef, M., urRehman, H., Iqbal, S., Mahmood, S., 2019.
Soil drenching of paclobutrazol: An efficient way to improve quinoa performance
under salinity. Physiology Plantarum 165, 219–231.
Wouyou, D.A., 2017. Re
ponse de l’amarante (Amaranthus cruentus, l.) au stress salin:
caracte
risation de cultivars, me
canisme physiologique de re
sistance et qualite

nutritionnelle des feuilles, the se de doctorat. Universite

d’Abomey-Calavi, p. 155.
Wetberilt, H., 2003. Nutritional evaluation of Urtica species. In: Kavalali, G.M. (Ed.),
Urtica : therapeutic and nutritional aspects of stingin nettles. Taylor & Francis Ltd,
pp. 56–66.
Wetherilt, H., 1992. Evaluation of URTICA Species as Potential Sources of Important
Nutrients. Developments in Food Science 92, 15–25.
Yun, P., Xu, L., Wang, S.S., Shabala, L., Shabala, S., Zhang, W.Y., 2018. Piriformospora indica
improves salinity stress tolerance in Zea mays L. plants by regulating Na+ and K+ loading
in root and allocating K+ in shoot. Plant Growth Regulation 86, 323–331.
130