1 Ability of Atriplex halimus to tolerate high

2 concentrations of Zinc or Lead combined with

3 NaCl.

6 Insaf BANKAJI1, Rosa M. PREZ-CLEMENTE2, Isabel CAADOR3 and

7 Noomene SLEIMI1,*

91 UR: Matriaux, Nanomatriaux et Ecosystmes, Facult des sciences de

10Bizerte, Universit de Carthage, Tunisie.

112 Universitat Jaume I, Departamento de Ciencias Agrarias y del Medio

12Natural. Castelln, Spain.

133MARE Marine and Environmental Sciences Centre, Faculdade de

14Cincias, Universidade de Lisboa, Portugal.

15

16insafbanc@yahoo.fr, noomene.sleimi@gmail.com

17

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18

19 ABSTRACT

20An experiment in this study was designed to investigate the effect of different concentrations of Zn 2+ or Pb2+ (0,
21200, 400 and 600 M) with NaCl (0, 200 mM), on the growth, total chlorophyll, water status, nutrient uptake
22and antioxidant enzyme activities [ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT)]
23of the halophytic species Atriplex halimus. Results showed that Pb had no signicant impact on biomass
24production while Zn significantly affected plant development mainly at high concentation, 600 M. Zn and Pb
25contents in tissues were higher in the roots of this halophyte.Overall results pointed out that application of
26different concentrations of Zn2+ or Pb2+ disturbed status of nutrients in A. halimus. The activity of antioxidant
27enzymes, CAT, APX and GPX was diminished by increasing Zn2+ concentrations in the medium. Whereas, the
28addition of Pb2+ in the medium increased CAT activity and decreased APX activity.

29
30Keywords: Atriplex halimus, Halophyte, Pb and Zn accumulation potential, Antioxidant enzymes.
31
32

33 1. Introduction

34 Industrialization and urbanization have increased the anthropogenic contribution of trace metal elements
35(TME) in environment (Gill et al., 2014). These pollutants are characterized by their persistence in the
36environment and their highly toxic eects to all living organisms (Rajkumar et al., 2009). There are two kinds of
37metals found in soils, which are referred to as essential micronutrients for normal plant growth (Fe, Mn, Zn, Cu,
38Mg, Mo, and Ni) and nonessential elements with unknown biological and physiological function (Cd, Sb, Cr, Pb,
39As, Co, Ag, Se, and Hg) (Rascio and Navari-Izzo, 2011, Zhou et al., 2014). TME toxicity in plants varies with
40plant species, specific metal, mediumconcentration, speciation and soil pH (Gill et al., 2014).

41 Some of these heavy metals like Cu and Zn either serve as cofactor and activators of enzyme reactions
42(Mildvan, 1970). These TME are required in minute quantities by organisms, excessive amounts of these
43elements can become harmful to organisms. TME such as Pb, Cd, As, and Hgdo not have any beneficial effect on
44organisms and are thus regarded as the main threats since they are very harmful to both plants and animals,
45pollutant in the environment air, water and soil, may be poisonous or toxic and will cause harm to living things
46(Asati et al., 2016).

47 The TME exposure could lead to multiple toxic effects in plants by inducing reactive oxygen species
48(ROS), such as superoxide free radicals (O2), hydroxyl free radicals (OH), or non-free radical species
49(molecular forms) such as singlet oxygen (O 2) as well as hydrogen peroxide (H 2O2), which inhibit most cellular
50processes at various levels of metabolism and can cause oxidative stress via disturbing the equilibrium between
51prooxidant and antioxidant homeostasis within the plant cells (Hossain et al., 2012; Sytar et al., 2013). Metal
52ions play an important role in the antioxidant network, as these are essential cofactors of most antioxidant
53enzymes (Nagajyoti et al., 2010). TME are involved in the direct or indirect generation of free radicals and ROS
54in the following ways: 1. direct transfer of electron in single electron reduction, 2. disturbance of metabolic

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55pathways resulting in an increase in the rate of free radicals and ROS formation, 3. Inactivation and down
56regulation of the enzymes of the antioxidative defense system and 4. depletion of low molecular weight
57antioxidants (Aust et al., 1985).

58 Atriplex halimus (Chenopodiaceae) is a xerohalophyte which is perennial and native in arid and semi arid
59Mediterranean regions (Lotmani and Mesnoua, 2011). This species shows a high resistance to various abiotic
60stresses such as salinity (Bajji et al., 2002), drought (Martinez et al., 2004), cold (Walker et al., 2008) and TME
61(Mateos-Naranjo et al. 2013, Bankaji et al., 2014). The perennial plant species which are exposed to extreme
62conditions possess morphological, anatomical, biochemical and physiological adaptations (Walker and Lutts,
632014).

64 The present experiment was undertaken to investigate a change in physiological parameters and in
65antioxidant enzymes in A. halimus treated with zinc or lead combined with NaCl in order to contribute to an
66understanding of A. halimus adaptation to environmental stress and to assessing its potential of accumulation to
67TME.

68

69 2. Material and Methods

70 2.1. Plant sampling and experimental setup
71 Atriplex halimus, was propagated by cutting from mother plants cultivated in greenhouse under semi-
72controlled conditions with a natural photoperiod, mean temperature (night-day) of 15-25C and relative humidity
73between 60-90% (Bankaji et al., 2014). During a one month period of rooting, young rooted cuttings were
74irrigated every 48 h with nutritive solution (Hewitt, 1966) enriched with iron as complex EDTA-K-Fe and
75micronutrients as mixture of salts (Bankaji et al., 2016). The electrical conductivity of the nutritive solution was
761.7 dS.m-1. After the pretreatment period, plants were divided into 14 groups of twenty plants that were stressed
77for three months. Control plants were irrigated three times a week with the same nutritive solution and the rest of
78groups used the same solution supplemented TME and NaCl (Tab. 1).
79 90 days after the start of experiments, plants were randomly separated into groups of five plants for
80further analysis. In the first group, leaves and shoots were separated from roots, washed, frozen in liquid nitrogen
81and kept at -80C respectively for chlorophyll and enzymatic activities assays. The remaining plants (groups of
8210 plants), for the cation assays, were divided into shoots and roots, three times successively rinsed in cold water
83and blotted with filter paper. Treated roots were dipped in a cold solution of CaCl 2 during 5 min (Stolt et al.,
842003) to eliminate trace metal elements adsorbed to the root surface and then rinsed three times with cold
85distilled water. Fresh weight (FW) was immediately determined and dry weight (DW) was measured
86after plant material desiccation in an oven at 60 C until constant weight.
87 The relative growth rate (RGR) based on fresh biomass production, was calculated as formula defined by
88Hunt (1990): RGR = (ln (W2) - ln (W1))/(t2 - t1), where W is the fresh matter at the beginning of treatment (W 1)
89and at the end (W2), and (t2 - t1) is the duration of this period.
90 The tolerance index (TIN) which is a measure of the tolerance of the plant to TME, was determined by
91comparing the dry biomass of plants subjected to metal treatment with the control using the relationship outlined
92by Wilkins (1978):

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 93 Tolerance index = Biomass of treated plants/Biomass of control plant x100.

94 2.2. Chlorophyll analysis and estimation

95 To extract chlorophyll, leaves were washed with distilled water and homogenized
96with 10 ml of 80% ethanol solution. The mixture was placed in a water bath at 80C for
9720 min. The absorbance of the chlorophyll solution was measured at 654 nm using a
98spectrophotometer (Thermo Spectronic Genesis UV10, USA). The chlorophyll content was
99estimated using the formula of Arnon (1949).

100 2.3. Leaf water status

101 The tissue water content was calculated using this formula: WC = (FWDW)/DW and it expressed in ml
102of H2O g-1 DW).

103 2.4. Elemental analysis

104 Dry plant material was acid digested using a mixture of HNO 3:HClO4 (4:1, v/v) (Mediouni et al., 2006).
105 Mineralization was conducted gradually from 100 to 350C during 2 hours. The digestion products were diluted
106 with 50 ml of HNO3 0.5% and filtered through a filter paper (Whatman N 1).
107 Total contents of Pb2+, Zn2+, K+, Na+, Ca2+and Mg2+ in plants were determined by atomic absorption
108 spectrometry (Perkin Elmer PinAAcle 900T, USA). The blanks were processed as described above. It is used to
109 adjust the zero of appareil and to detect possible cross-contamination.

110 2.5. Enzyme Analysis

111 2.5.1. Enzyme extraction
112 Leaf tissues were homogenized in a mortar and pestle with a small amount of inert sand, in a solution
113(2.0 ml/0.4g FW) containing 50 mM KH2PO4/K2HPO4 (pH 7.0), 5 mM Na-ascorbate and 0.2 mM EDTA.
114After filtration through four layers of Miracloth, the homogenate was centrifuged at 6000 rpm for 15 minutes. All
115operations were performed at 4C and the samples were prepared for CAT, APX and GPX analysis.
116 2.5.2. Enzyme assay
117 The supernatant was used for the measurement of antioxidative enzyme activities by a UV/Visible
118spectrophotometer (TOMOS, UV-1200 Spectrophotometer).
119 Catalase activity (CAT, EC1.11.1.6) was measured in terms of rate of disappearance of H2O2 at 240 nm
120( molar extinction coefficient: = 0.036 mM-1.cm-1) according to Aebi (1984). The reaction mixture contained 25
121mM K- phosphate buffer (pH 7.0), 10 mM H2O2 and 2 ml of enzyme extract.
122 The activity of guaiacol peroxidase (GPX, EC1.11.1.7) was assayed by monitoring the increase in
123absorbance at 470 nm ( molar extinction coefficient: = 26.6 mM -1.cm-1) due to polymerization of guaiacol.
124(Fielding and Hall, 1978).The reaction mixture consisted of 25 mM K-phosphate buffer (pH 7.0), 10 mM
125H2O2, 9 mM guaiacol and 2 ml enzyme extract.
126 Ascorbate peroxidase (APX, EC1.11.1.11) was determined by measuring the decrease in absorbance at
127290 nm (absorbance coefficient of 2.8 mM-1.cm-1) due to oxidation of ascorbic acid to dehydroascorbate (Nakano
128and Asada 1981).The reaction mixture contained 25 mM K- phosphate buffer (pH 7.0), 0.5 mM ascorbate, 2 mM
129H2O2, 0.1 mM EDTA and 2 ml of enzyme extract.
130 All enzyme activities were expressed per gram fresh weight. One unit of enzyme was dened as the
131amount necessary to decompose 1 mol.min-1 of each substrate at 25C in the case of CAT and APX. For GPX

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132activity, one unit was dened as amount of enzyme producing 1 mol.min-1 of tetraguaiacol at 25C.

133 2.6. Statistical analysis

134 All data presented are the mean values of ve replicates, which are presented along with the standard
135error () in bars in the figures. The effects of TME and salinity treatments on the variability of the response
136parameters were assessed using regression analyses and One-way ANOVA. Normality was verified by using the
137Shapiro-Wilk test, and homogeneity of variance was tested using the Bartlett test. Statistical analyses were
138conducted using Statistica 8 (Statsoft). Tukey's HSD test was performed to define which specific mean pairs
139were significantly different (p < 0.05). The correlation factor r2 and the p value of correlation were determined
140by using the "origin 8.0 software".
141

142 3. Results
143 3.1. Plant morphology and growth

144 The effect of Zn2+ and Pb2+ applied separately on A. halimus development was evaluated based on dry
145biomass production. Our results indicated that biomass production significantly decreased in A. halimus
146cultivated in a medium supplemented with different concentrations of Zn 2+, mainly at 600 M. (Fig. 1). It varied
147from 3783.3 to 2273.3 mg DW and from 646.8 to 515.6 mg DW, respectively, in shoots and roots. The addition
148of Pb2+ to the irrigation solution alone or in combination with NaCl did not have a significant effect on A.
149halimus (Fig. 1).
150 Similarly, the mean relative growth rate of A. halimus was significantly affected by Zn2+ as compared to
151Pb2+. In fact, the RGR of shoots and roots significantly decreased in A. halimus cultivated in a medium
152supplemented with Zn2+, either alone or in combination with NaCl (Fig. 2).Interestingly, treatment with Pb 2+
153alone or in combination with NaCl did not have a significant effect on A. halimus ((Fig. 2).
154 Total chlorophyll concentration did not change significantly under dierent
155treatment concentrations of Zn2+ (200, 400 or 600 M) when compared to control (Fig.1).
156 However, there was a modest decrease in total chlorophyll concentrationin the leaves of A.
157halimus cultivated in a medium supplemented with 600 M Pb2+and with combined stress Pb/NaCl(Fig. 1).
158 The results showed that the plant morphology and the growth parameters were not affected by the
159addition of NaCl alone in the culture medium (Fig. 1 and Fig. 2).
160 Tolerance indexes values decreased in A. halimus treated with high concentration of Zn, 600 M,
161showing a net decrease in biomass, and growth inhibition. The value was decreased from 100% to 79.6 and to
16260%, respectively in the roots and shoots. Whereas, A. halimus treated with Pb2+ showed a significant increase in
163the TIN, from 100% to 127% in the roots (Tab. 2).

164 3.2. Water status

165 Generally, our results showed that the water content in the shoots decreases with increasing concentration
166of Pb in the culture medium (Fig. 2). Regarding plants grown in the presence of Zn 2+, the results showed no
2+

167significant variation in water content in the shoots and roots of A. halimus grown in different concentrations of
168Zn2+. We notice a clear improvement in hydration root tissue in the presence of NaCl, 200 mM. In the case of
169combined stress Zn/NaCl, a higher water content value was observed compared to the control (Fig. 2).
170

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171 3.3. Mineral uptake
172 In our experiments, we observed a decrease of shoots K + content in A. halimus treated with Zn2+ or Pb2+
173either alone or in combination with NaCl (Fig. 3). The shoots K + content varied from 55 to 39. 2 mg.g -1 DW and
174from 55 to 39. 9 mg.g-1 DW in A. halimus treated respectively by Zn2+ or Pb2+. Results show that the shoots and
175roots Mg2+ concentrations were significantly decreased in A. halimus under Zn2+ stress, supplemented or not with
176NaCl (Fig. 3).The same result was observed under Pb2+ treatment. In fact, the addition of different concentration
177of Pb2+ significantly decreased the shoots and roots Mg2+ concentrations.
178 Data given in Fig.3 indicate that Ca2+ levels were significantly decreased in the shoots and roots of plants
179with elevation of Zn2+ concentrations in the medium. On the other hand, the presence of Pb2+ induced a
180significant increase of Ca2+ contents in the shoots of A. halimus at different concentrations and in the roots,
181mainly at 400, 600 M of Pb2+.
182 In general, the presence of NaCl reduced K +, Ca2+ and Mg2+ concentrations in the vegetative organs of A.
183halimus, either alone or in combination with TME (Fig. 3).
184

185 3.4. Sodium and TME uptake

186 The addition of 200 mM NaCl in the irrigation solution increased the Na + concentration in roots and
187shoots of A. halimus (Fig. 4). Zn2+ or Pb2+ exposure did not modify the accumulation of Na + in its organs.
188However, the combined stress Zn2+/NaCl increased the Na+ content in this halophytic species.
189 The results on TME uptake showed an increase in roots Zn2+ and Pb2+ of A. halimus with TME medium
190concentrations (200, 400 or 600 M). A. halimus accumulated higher concentrations of Zn 2+and Pb2+ in the roots
191than in the shoots tissues. The levels of Zn2+ were 2510219g.g-1 DW in roots stressed with 600 M Zn2+.
192In the shoots, Zn2+ content represented 86988g.g-1 DW (Fig. 4). A. halimus also accumulated higher
193concentrations of Pb2+ in the roots. Levels of Pb2+ were 2005137g.g-1 DW in roots treated with 600 M
194Pb2+. In the shoots, Pb2+ content represented 34127 g.g-1 DW (Fig. 4).
195 In saline conditions, the endogenous Zn2+ levels in root tissues were significantly
196reduced. While, the Pb2+ levels in the roots were increased. In fact, in the presence of
197NaCl, Zn2+ concentration decreased from 2510219 g.g-1 DW to 1550222 g.g-1 DW in
198roots of A. halimus treated with Zn2+, 600 M (Fig. 4). On the contrary, the endogenous
199Pb2+content in root tissues increased from 2005137 g.g -1 DW to 2343353 g.g-1 DW in
200the presence of NaCl (Fig. 4).
201
202 3.5. Enzyme activities
203 The presented results show that the addition of Zn2+ to the irrigation solution induced a significant
204decrease in CAT activity in leaves of A. halimus. CAT activity decreased 33.2% under Zn2+ stress. On the other
205hand, addition of Pb2+ in the culture medium increased the activity of CAT, mainly at 400 and 600 M. APX
206activity in the leaves decreased 26.6% after either 400 M Zn2+ or Pb2+ treatments (Fig. 5). GPX activity in
207leaves of A. halimus decreased 75.6% under Zn2+ treatment (Fig. 5). Whereas, the addition of different dose of
208Pb2+in the culture medium did not appear to have any effect in the GPX activity.
209 The addition of NaCl to the irrigation solution, either alone or combined with Zn2+, decreased the

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210activities of the CAT and GPX in A. halimus. However, the activity of APX was increased under NaCl treatment
211(Fig. 5).
212 A negative correlation was observed between the activity of CAT and GPX and the concentration of Zn 2+
213in the medium, respectively with and without NaCl (Fig. 6). On the other hand, a positive correlation was
214signaled between the activity of GPX and the concentration of Pb2+ in the medium with NaCl (Fig. 7).
215

216 4. Discussion
217 A decrease in plant dry weight was noted in A. halimus due to Zn2+ treatments, mainly at 600 M.
218Compared to the control, the reduction in total dry mass was 40% and 20%, respectively in the shoots and roots.
219A similar trend was reported for RGR. Similar response to Zn 2+ treatment was previously noticed in halophyte
220plants, Spartina densiflora (Redondo-Gomez et al., 2011) and Juncus acutus (Mateos-Naranjo et al., 2014).
221Decreased plant growth might be associated with the inhibition of mitotic index noticed with TME (Vecchia et al.
2222005). Interestingly, the addition of Pb2+ to the medium either alone or in combination with NaCl did not have a
223significant effect on the growth of A. halimus. Similarly, the mean relative growth rate of A. halimus was not
224significantly affected by Pb2+ (200, 400 or 600 M) as compared to control.

225 Tolerance indexes have been useful to characterize plant tolerance. TIN values lower than 100% indicate
226a net decrease in biomass and suggest that plants are TME-stressed. At the same time, TIN values equal to 100%
227indicate no differences relative to non-TME control treatments. Also, TIN values greater than 100% indicate a
228net increase in biomass, and suggest that plants express a growth dilution effect (Audet and Charest, 2007). Our
229TIN values decreased in A. halimus treated with high concentration of Zn2+, 600 M. Whereas, A. halimus
230treated with Pb2+ showed a significant increase in the TIN in the roots.

231 A different behavior was observed in the total chlorophyll concentration. In fact, total
232chlorophyll concentration did not change significantly under dierent treatment
233concentrations of Zn2+ (200, 400 or 600 M) when compared to control. However, there was a modest
234decrease in total chlorophyll concentration in the leaves of A. halimus cultivated in a medium
235supplemented with 600 M Pb2+. It was suggested that Pb2+ could interfere with chlorophyll biosynthesis either
236through the direct inhibition of enzymatic steps or through the substitution of the central Mg ion (Cenkci et al.,
2372010; Pourraut et al., 2011). Previous authors showed that Pb 2+ stress results in a heavy reduction of chlorophyll,
238owing to both chloroplast disorganization and diminution in the amount of thylakoids and grana, and direct
239inhibition of chlorophyll synthesis, as well as changes of chlorophyll structure owing to replacement of key
240nutrients (Mg, Fe and Cu) by Pb2+ (Haider et al., 2006; Akinci et al., 2010; Lamhamdi et al. 2013).

241 The results show that the application of different Zn2+ concentrations had no significant effect on the water
242content in A. halimus. Similarly, Mateos-Naranjo et al. (2014) observed that Zn2+ increment did not affect
243intrinsic water use efficiency and tiller water content of Juncus acutus. Belhaj Sghaier et al. (2015) signaled that
244no significant variation of water content was observed in Tamarix gallica treated by different concentrations of
245arsenic alone or in combination with NaCl. On the other hand, our results show a decrease in water content in the
246shoots of A. halimus grown in the presence of different doses of Pb 2+ was signaled. In the roots, the water content
247was significantly decreased only at 600 M Pb2+. The same behavior was reported in Suaeda fruticosa grown in

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248the presence of 200, 400 and 600 M of Pb2+ (Bankaji et al., 2016). This suggests that the Pb2+-induced
249perturbation in the water status was linked to ion toxicity rather than to osmotic environment.

250 The addition of NaCl, alone or combined with Zn 2+, in the culture medium enhanced the hydration of
251plants, essentially in the roots and improved tissue hydration of A. halimus. This result can be associated with
252improved growth of this species in the presence of NaCl, 200 mM.
253 Phytotoxicity of TME may result in plant reduced nutrient uptake and disorder in plant metabolism (Dan
254et al, 2008). Overall results pointed out that application of different concentrations of Zn 2+ or Pb2+ disturbed
255status of nutrients in A. halimus. According to Lamhamdi et al. (2013), the reductions of ionic content can hardly
256result from just an inhibition of ion uptake, and they probably also result from additional ion leakage from the
257plants.

258 Results showed that both metals accumulated to a higher extent in the roots than in the shoots, reaching
259values 2510 g.g-1 of Zn2+ and 2005 g.g-1 of Pb2+ in the roots. Shahid et al. (2014) signaled that among defense
260mechanism plants adopt is to reduce the translocation of TME to aerial plant parts. Most of the TME absorbed by
261plants are sequestered in plant root cells. In root cells, toxic TME are detoxified by complexation with organic
262acids, amino acids or sequestered into vacuoles (Rascio and Navari-Izzo, 2011; Pourrut et al., 2011). Such
263complexation restricts the transfer of TME towards aerial plant parts, thus protecting leaf tissues, and particularly
264the metabolically active photosynthetic cells from TME damage (Rascio and Navari-Izzo, 2011).

265 The tolerance to heavy metals in plants could be controlled by two essential strategies: exclusion and
266accumulation (Mnasri et al., 2015). The exclusion one signies that a plant avoids or restricts the absorption of
267metals while accumulation is directly related to the ability of the plant to sequester metals inside the tissues. In
268the present study, we showed that A. halimus is able to accumulate Zn and Pb which conrm the previous results
269published by Lutts et al. (2004) and Manousaki and Kalogerakis (2009) demonstrating that this species adopts
270the second strategy.

271 It is noteworthy that the combination of Zn and NaCl in the nutrient solution significantly reduced the
272accumulation of Zn inside the root and the shoot tissues.

273 Results described in this work are in agreement with other previously described in Suaeda fruticosa
274(Bankaji et al., 2016). The same behavior was observed in S. fruticosa and A. halimus treated with Cd2+ and Cu2+
275combined with NaCl (Bankaji et al., 2014). Similarly, Lefvre et al. (2009) demonstrated that NaCl decreased Cd
276accumulation in the halophyte A. halimus. According to Lutts and Lefvre (2015), results suggest that these
277species are able to maintain root integrity under osmotic stress and that transporter remain able to discriminate
278against the TME-Cl complex. On the other hand, salinity did not influence in a clear way the uptake of Pb 2+ by
279the plant probably because of leads limited mobility in soils and plant tissues. The same result was observed by
280Manousaki and Kalogerakis (2009) in Atriplex halimus.

281 Plants generally face the oxidative damage when exposed to TME (Gupta et al., 2009).They synthesize
282numerous antioxidant molecules, such as glutathione and enzymes, including catalase, superoxide dismutase,
283ascorbate peroxidase and glutathione reductase, as a defense against oxidative stress (Sai Kachout et al., 2010).
284In the present study antioxidant enzymes such as CAT, GPX and APX decreased under Zn treatments. The

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285decrease in antioxidant enzymes at higher concentrations of TME which might be due to the severe stress of
286oxidative damage to antioxidant enzymes, hence, the activity of enzymes was diminished (Erdei et al., 2002).On
287the other hand, the addition of Pb in the medium increased the CAT activity and diminished the activity of APX
288in A. halimus.

289 Excess TME generates inhibition or stimulation in the activity of various antioxidant enzymes (Pl et al.,
2902006; Zhang et al., 2009; Zheng et al., 2010). In fact, Rastgoo et al. (2014) showed an increase in the CAT
291activity following treatment with 50 and 100 M Zn 2+ in the halophytic species Aeluropus littoralis. Kaur et al.
292(2013) showed an increase in CAT activity accompanied by a decrease in the activity of GPX and APX in
293Triticum aestivum treated with 500 M of Pb2+. A decrease in APX activity followed by an increase in CAT
294activity was reported under TME treatments (Cu 2+, Ni2+, Pb2+ and Zn2+) in Atriplex hortensis (Sai Kachout et al.,
2952010). Bankaji et al. (2015) signaled that the activities of the antioxidant enzymes CAT, APX, and GPX were
296diminished under Cd or Cu stress in Suaeda fruticosa.

297 Several literature data confirm the increase in the antioxidant enzyme activities under TME stress
298(Lotmani and Mesnoua, 2011; Chai al., 2013). However, above a certain TME concentrations, the antioxidant
299enzymes were found to be inhibited (Hegeds et al., 2001; Srivastava et al., 2004). Rastgoo and Alemzadeh
300(2011) showed a dose-dependent inhibition of CAT activity in Aeluropus littoralis treated with various
301concentrations of Pb2+. They reported that high concentration of TME, CAT is not able to protect cells against
302ROS. They also showed that CAT and GPX activities were increased in Aeluropus littoralis treated with 50 M
303Ag+. However, the increase of the TME concentration (100 M) reduced the activity of these two enzymes.

304 The diversity of the observed activity of antioxidant enzymes in plants subjected to environmental stress
305such as TME, depends on the species (physiological and genetic potential of the plant), duration of the treatment
306and the TME concentration (Schtzendbe and Polle, 2002; Tams et al, 2008). The decrease in enzyme
307activities at high doses of TME may be due to an imbalance in metabolism and the generation of reactive oxygen
308species (Luna et al., 1994; Shah et al., 2001; Rai et al. 2004).

309 5. Conclusion

310 The results obtained in this work indicated that zinc application significantly decreased shoots and roots
311dry weight of A. halimus at high concentration600 M. The reductions of shoots and roots dry weight of A.
312halimus were 40 and 20% respectively, when compared to control. The addition of Pb 2+ to the irrigation solution
313alone or in combination with NaCl did not have a significant effect on A. halimus. Similarly, the mean relative
314growth rate of A. halimus was significantly affected by Zn2+ as compared to Pb2+.

315 Zn2+ and Pb2+ concentrations in tissues were higher in the roots of this halophyte. Overall results pointed
316out that application of different concentrations of Zn 2+ or Pb2+ disturbed status of nutrients in A. halimus. The
317activity of antioxidant enzymes, CAT, APX and GPX was diminished by increasing Zn2+ concentrations.
318Whereas, the addition of Pb2+ in the medium increased CAT activity and decreased APX activity.

319

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466

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467 Table1. Treatments of plants with the Hewitt nutritive solution supplemented TME and NaCl
468
Treatments Hewitt nutritive Pb (M) Zn (M) NaCl
solution 200 mM
20 400 600 20 400 600
0 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
469
470
471
472 Table2. Tolerance index of shoots and roots of A. halimus measured in response to different
473concentrations of TME in the culture medium. (*p<0.05, values are significantly different).
474
Treatments Tolerance Indexes (%)
Shoots Roots
Pb (M)
0 100.00 100.00
200 91.54 107.13
400 102.77 127.13
600 107.57 126.638
Zn (M)
0 100.00 100.00
200 78.42* 96.75*
400 79.81* 102.79
600 60.08* 79.56*
475

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