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1 Title: Phytoremediation capabilities and oxidative status of Medicago Sativa exposed to increased Nickel

2 concentrations.

4 Authors: Sondes HELAOUI 1, Iteb BOUGHATTAS 1*, Sabrine HATTAB 2, Marouane MKHININI 1,

5 Vanessa ALPHONSE 3, Alexandre LIVET 3, Nourreddine BOUSSERHINE 3, Mohamed BANNI 1

8 1 Laboratory of Biochemistry and Environmental Toxicology, ISA, Chott-Meriem, Sousse, Tunisia

9 2 Regional Research Centre in Horticulture and Organic Agriculture, Chott-Mariem, Sousse, Tunisia

10 3 Laboratory of Water Environment and Urban systems, University Paris-Est Créteil, Créteil Cedex, 94010,

11 France.

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15 *: Corresponding author:

16 Iteb BOUGHATTAS

17 Laboratory of Biochemistry and Environmental

18 Toxicology, ISA Chott-Meriem, Tunisia

19 Tel +216 99 923 652

20 Email : iteb.boughattas@yahoo.fr

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32 Abstract:

33 Metal accumulation in soil ecosystem could lead to severe damages on plants, animals and humans.

34 Phytoremediation is a useful technology serving to remove contaminants from the soils. In this context, the

35 present work aimed to evaluate the potential use of Medicago sativa for phytoremediation purposes in soils

36 contaminated with Nickel. Plants were exposed to five increasing concentrations of NiCl2 (0, 50, 150, 250 and

37 500 mg/kg) for 60 days. Agronomic parameters were determined by measuring fresh and dry matter in root and

38 shoot plant. Chemical analyses were assessed through the determination of Ni accumulation in plants (roots and

39 shoots) and soil. Moreover, oxidative stress biomarkers such as malondialdehyde accumulation (MDA),

40 glutathione-S-transferase (GST) and catalase (CAT) in roots and shoots were measured. Our results showed that

41 Ni concentration increased significantly in roots along with the Ni concentration in the soil. Regarding oxidative

42 stress biomarkers, Ni contamination caused an increase on CAT and GST activity in M.sativa plants

43 accompanied with an important accumulation of MDA especially under the exposure to 500 mg/kg of Ni.

44 Our findings showed that Medicago sativa can be used in the phytoremediation process of metal contaminated

45 soils.

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47 Key words: Metal accumulation, Nickel, Medicago sativa, phytoremediation, Agronomic parameters, oxidative

48 stress

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64 1. Introduction

65 Increasing trace elements levels in soils are recorded and such pollution has a critical problem on the

66 environment (Shi and Cai 2009; Jiang 2016). Unlike organic pollutants which can be metabolized by soil

67 organisms and plant roots, most heavy metals are persistent in the environment (Järup 2003). Two types of heavy

68 metals were distinguished according to their physiological and toxic effects: essential and toxic heavy metals.

69 Essential metals like i.e. Cu, Zn and Fe are necessary for many cellular processes and they are founded in very

70 low proportion in biological tissues (Adriano2001). Then, nonessential metals such as Hg, Cd and Pb at high

71 levels are toxic to plants, animal and human.

72 Interestingly, Ni has been classified among essential micronutrients since it was involved in many biological

73 processes of plants and microorganisms at a low amount (Bradl 2002; Küpper and Kroneck 2007; Fabiano et al.

74 2015). Despite that, this metal could be founded in soil ecosystem naturally or through anthropogenic causing

75 several problems (Rooney et al. 2007; Wang et al. 2012). Ni could reach soils naturally through the alteration of

76 the parent rock. Also, numerous human activities like industries, mining sites and agricultural practices could

77 lead to the Ni accumulation in soil ecosystem (Yusuf et al. 2011; Hussain et al. 2013).

78 Once in soils, at high concentrations, Ni cause undesirable effects on plants and this was widely proved in

79 several works (Parida et al. 2003; Gratão et al. 2008; Dourado et al. 2015; Rizwan et al. 2018). Among these

80 effects, chlorosis, leaves’ necrosis, growth retardation and induction of Reactive Oxygen Species (ROS)

81 production (Madhava Rao and Sresty 2000; Pandey and Sharma 2002; Foyer and Noctor 2005; Rooney et al.

82 2007; Gajewska et al. 2009; Dourado et al. 2015). It’s well known the excessive of ROS production could lead to

83 the increase of protein’s oxidation, cell membranes’ degradation, and DNA damages (Choudhury and Panda SK

84 2004; Gill and Tuteja 2010).Although the imbalance between oxidants and antioxidants, cells could face ROS

85 damages through the detoxification system involving a battery of enzymes such as CAT and GST which plays a

86 key role in the degradation and the decomposition of reactive species (Sun and Zhou 2008; Ahmad et al. 2010).

87 Catalase (CAT) is aubiquitous antioxidant enzyme known for its ability to decompose hydrogen peroxide in

88 cells. Furthermore, Malondialdehyde (MDA) one of the products of lipid peroxidation of cell’s membrane (LPO)

89 is usually used as a biomarker to assess ROS damages. Glutathione-S-transferase (GST) is a phase II enzyme

90 known to be involved in cellular protection against oxidative stress and ROS (Anjum et al. 2012). In fact, there

91 are various ways of metal remediation from soils. Nowadays, the biological methods are a sustainable

92 alternatives to reduce the amount of pollutants in soils and this through the use of animals like earthworms

93 (Boughattas et al. 2016) or vegetables like M. sativa (Hattab et al. 2016). Comparing to physicochemical

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94 methods which could cause a significant decrease in soil fertility and productivity, bioremediation is considered

95 as a friendly environmental technology. Also, the cost of bioremediation is lower than that of traditional

96 processes both in situ and ex-situ (Peuke and Rennenberg 2005; Vangronsveld et al. 2009; Ali et al. 2013).

97 Phytoremediation is a green method based on natural processes to face metals contamination of soil ecosystems

98 (Prabha and Loretta 2007; Mathieu et al. 2012). This method allows the retention of metal (loid) from the soil to

99 the plant (Ullah et al. 2015). So, roots are the first container of heavy metals in contaminated soil, further, heavy

100 metals will be in different parts of the plant. In this context, theses plants called hyperaccumulators are able to

101 accumulate, degrade and decrease the harmful effects of trace elements in soils (Karn et al. 2009). Alfalfa plants

102 (Medicago sativa) are known as a good model of phytoremediation plants and this was reported in numerous

103 recent works (Sobrino-Plata et al. 2009; Hattab et al. 2014; Flores Cáceres et al. 2015) which approved the

104 tolerance of this plant to a wide variety of heavy metals like Cd, Cu, Zn and Pb. Moreover, alfalfa plant doesn’t

105 require any special cultural practices, and it is characterized by its adaptation to a wide range of climates (Su et

106 al. 2004). For these purposes, Medicago sativa could represent a good candidate to palliate heavy metal

107 contamination in terrestrial ecosystems.

108 The present study aims first to evaluate the effect of five increasing Ni concentrations on the agronomic and

109 physiologic parameters of M.sativa plants. Second, we assessed the impact of Ni on the oxidative status of

110 Medicago sativa. Finally, we examined its potential role in the phytoextraction of nickel from polluted soils.

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128 2. Material and methods

129 2.1. Soil sampling

130 Soilwas sampled from an organic soil located in the higher institute of agronomy of Chott Meriem, in Tunisia.

131 Then, soil samples were homogenized and dried at 25°C.

132 2.2. Plant material

133 Medicago sativa seeds were obtained from the Tunisian Seed Control Agency and they were germinated on wet

134 paper in Petri dishes in darkness for48 hours at room temperature of 24±1°C.

135 2.3. Experimental design

136 Soil samples were moistened with deionized water and brought to 70 % of its holding capacity and this was

137 maintained during the experimentation. Then, four increasing concentration of NiCl26H2O were prepared (C1:

138 50, C2: 150, C3: 250, and C4: 500 mg/kg) against a control condition (Control: Non-contaminated soil). Eight

139 alfalfa seeds were kept in 1 Kg of soil placed in plastic pots and five replicates per soil sample were used.

140 Finally, the plastic pots were maintained under controlled temperature (20 ± 1 ° C, with 12 h light-period). After

141 60 days of exposure, the plants were harvested, and biometric measurements were done. Subsequently, shoots

142 and roots were separated than washed into water andNa 2-EDTA (20 mM) in order to eliminate superficial Ni.

143 Finally, plants were stored at -80°C for further analysis.

144 2.4. Agronomic measurements

145 Length and fresh weight (FW) of plants were measured immediately after harvesting of the alfalfa plants after

146 60 days of culture.

147 Then, from each sample, 100 g of fresh material was placed in oven at 60 °C for 48 hours, and then the

148 percentage of dry matter was calculated as following:

DM
149 𝐃𝐌(%) = ( ) ∗ 100
FM

150 DM—Dry matter of plants; FM— Frech matter of plants.

151 2.5. Nickel content in soil, shoot and roots

152 After 60 days, plants were carefully rinsed with distilled water. Then collected soil and plant samples were dried
153 in the oven at 40°C for 24h. After that, samples were ground to a fine with a diameter (< 0.02μm). 1 g of each
154 subsample has been subject to an acid digestion 37% HCl and 63% HNO3 (3/1: v/v) at 100 °C (Zhao et al. 1994).

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155 After that, the suspensions were diluted in 0.6% HNO3 and the analysis was performed using inductively coupled
156 plasma atomic emission spectrometry (ICP-AES; Fisons ARL Accuris) (Pekin Elmer 2380).
157 2.6. The bioconcentration factor, translocation factor and phytoextraction efficiency of Ni

158 The capacity of alfalfa plants to accumulate nickel from soils and transfer this metal from roots to shoots was

159 evaluated by the bioconcentration factor (BCF) and translocation factor (TF), respectively (Majid et al.2011).

160 BCF is the ratio of the metal concentration in the plants to those in the soil.

ETM (Plant)
161 BCFETM =
ETM (soil)

162 TF is the ratio of the metal concentration in the shoots to those in the roots of plants.

ETM (Shoots)
163 FTETM =
ETM (Roots)

164 The phytoextraction efficiency (PEE) of Medicago sativa under different Ni-contamination in the pot experiment

165 was estimated as suggested Yang et al., (2017):

ETM𝑃𝑙𝑎𝑛𝑡 𝑡𝑖𝑠𝑠𝑢𝑒 (mg.Kg−1 )X W𝑃𝑙𝑎𝑛𝑡 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡𝑠 (𝐾𝑔)


166 (PEE) =
ETM𝑆𝑜𝑖𝑙 (mg.Kg−1 )X W𝑆𝑜𝑖𝑙 𝑢𝑠𝑒𝑑 𝑖𝑛 𝑡𝑜 𝑝𝑜𝑡 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡(𝐾𝑔)
X 100

167 ETM𝑃𝑙𝑎𝑛𝑡 𝑡𝑖𝑠𝑠𝑢𝑒 = Ni-concentration in plant tissue (mgkg-1)

168 W𝑃𝑙𝑎𝑛𝑡 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡𝑠 = Total dry weight biomass of plant (kg)

169 ETM𝑆𝑜𝑖𝑙 = Ni-concentration in soil (mgkg-1)

170 Weight = Weight of soil used to fill into pot experiment (kg)

171 2.7. Chlorophyll content

172 For this purpose, 1g of fresh leaves was extracted bygrinding in a mortar using 20 ml of 80% acetone, with

173 smallamount of pure (Silica Quartz), and 0.5 g calcium carbonate to equalize the cellular acidity. The extractwas

174 filtered and collected in Eppendorf’s tube. After that, the measurements of optical density (OD) were carried out

175 using spectrophotometer VWR UV-3100 PC at 663nm for the Chlorophyll a (Chl𝑎) and 645 nm for the

176 Chlorophyll b (Chlb) (Sobrino-Plata et al. 2013). Total chlorophyll as well as chlorophyll a and b concentrations

177 were calculated according to (Arnon 1949) as following

mg µg 10ml x mg
178 Chl𝑎 ( ) = [(12,7 x A663) − (2,69 x A645)] x
gFW ml 0,05gFWx103𝑥

mg µg 10ml x mg
179 Chlb ( ) = [(22,9 x A645) − (4,68 x A663)] x
gFW ml 0,05gFWx103𝑥

180 𝐂𝐡𝐥(𝐚 + 𝐛) = Chla + Chlb

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181 2.8. Oxidative stress biomarkers

182 2.8.1. Total protein content

183 According to Laemmli (1970), 0.5 g of frozen leaves and roots were ground in mortar with1 ml of extraction

184 buffer prepared instantly with: 30 mM MOPS at pH 7.5 mM, 5mM Na2-EDTA, 10 mM DTT, 10 mM ascorbic

185 acid, 0.6% PVP, 10 mL 100 mM PMSF and 1 mL protease inhibitors cocktail (P2714, Sigma–Aldrich, St. Louis

186 MO, USA) and 10 μl of the ascorbic acid solution. The suspension was centrifuged at 12.000 rpm for 15 min at

187 4°C. Protein was assayed according to the method of Bradford M.M (1976) using bovine serum albumin as a

188 standard.

189 2.8.2. Catalase activity

190 The specific activity of CAT was determined according to the Aebi (1984). In a quartz vat, 50 µl of protein

191 extract for leaves and100 µl for the roots extract were added to 750 µl of potassium phosphate buffer (0.05M, pH

192 7.8) and 200 µl of H2O2 (0.5M) added respectively. CAT activity as evaluated by kinetic measurement at 20 °C

193 using a VWR UV-3100spectrophotometer (λ = 240 nm).The results were expressed as µmole of H2O2

194 transformed per min and per mg of protein using 0.036 mM-1 cm-1as extinction coefficient

195 2.8.3. Glutathion-S-Transferase activity

196 The GST activity was determined according to the protocol of Habig et al. (1974) using 50 μl of protein extract,

197 1 mM 1-chloro- 2,4dinitrobenzene (CDNB) (Sigma-Aldrich, Saint Louis, MO, USA) as as ubstrate, and 4 mM

198 glutathione (reduced form; GSH) in100 mMK phosphate buffer (pH 7.4).GST activity was determined by kinetic

199 measurementat 20 °C using a VWR UV-3100 spectrophotometer (λ =340 nm). The results were expressed as

200 µmole GSH-CDNB produced per min and per mg of protein using 9.6 mM/cm as extinction coefficient.

201 2.8.4. Malondialdehydes accumulation

202 Lipid peroxidation in M.sativa apprehended by MDA amount was measured as described by Ortega-Villasante et

203 al. (2005). For that, 0,3 g of fresh tissues (shoots/roots) was homogenized in 3 ml of 15% Trichloroacetic and

204 0.37% Thiobarbituric acids (TCA/TBA), 0.25 M HCl and 0.01 % butylated hydroxytoluene. After that, the

205 homogenate was incubated at 90 ° C for 30 min and cooled for 5 min in an ice bath. Then, the obtained solution

206 was centrifuged for 10 min at 12,000 g and it was measured in absorbance at 535 and 600 nm.

207 2.9. Statistical analysis

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208 The results for nickel content, antioxidant enzymatic activities and chlorophyll content are presented as the mean

209 ± standard deviations (SD) of 5 samples. SPSS Software, version 20.0was used for statistical analysis. The

210 normality of the distribution was tested using the Shapiro–Wilk test. For multiple comparisons, a parametric one-

211 way analysis of variance (ANOVA) was performed on data along with Tukey's test. All calculations were made

212 using Statistica V6.1 software (StatSoft). Differences were considered significant for p <0.05.

213 To compare the evolution of plant response after Ni contamination, principal component analyses (PCAs) were

214 performed using the R software and the package ADE4TkGUI (Thioulouse and Dray, 2007). Moreover, a

215 correlation matrix was done used the R software and the package CORRPLOT.

216 3. Results

217 3.1. Nickel content in soils, roots and shoots of Medicago sativa plants

218 Ni accumulation in soils was presented in Figure 1A. Ni-concentrations increased gradually along with Ni doses

219 increase. However, the concentration reached its maximum in soils treated with the high Ni concentration (C4)

220 with an amount of 112, 51 ±1,5 mg/kg.

221 Moreover, Ni contents in root and shoot were given in Figure 1B and C and showed a progressive increase of Ni

222 accumulation and reach a maximum of 21, 93±3, 33 and 75, 2 ± 0, 16mg/kg respectively in shoots and roots

223 after 60 days of exposure to 500 mg/Kg of NiCl2.

224 3.2. The bioconcentration factor, translocation factor and phytoextraction efficiency of Ni

225

226 The bioconcentration factor, translocation factor and phytoextraction efficiency of Ni were presented in Table.1.

227 Our study showed that the important nickel concentration was accumulated in roots with TF < 1. BCF values

228 increased in plants exposed to C1, C2, C3 and C4 with values respectively 0,55±0,015; 0,67± 0,035; 0,82± 0,04

229 and 0,87±0,007against a control value 0,19 ±0,001. The PEE percentage of Medicago sativa increased

230 significantly with the different nickel concentrations. Indeed, values reached12,51±0,012%, 14,63±0,05%,

231 9,19±0,014% et 8,12±0,094% respectively for C1, C2, C3 and C4 against a control 5,42±0,014%.

232 3.3. Growth parameters

233 The effect of Ni treatments on the root and the shoot length of Medicago sativa after 60 days of exposure were

234 presented in Table2. Results revealed that plants length grown in Ni contaminated soils had significant

235 differences in comparison to control plants. Indeed, it decreased progressively with the increase of Ni levels

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236 (P<0.005). The decrease was more pronounced with the highest dose of Ni (500 mg/kg) where the length was

237 reduced by 33% in shoots and 32, 25% in roots.

238 The effect of Ni treatments on root and shoot weight of Medicago sativa after 60 days exposure was presented in

239 Table3. The most significant decrease was observed in roots contaminated with the highest concentration C4

240 (500 mg/kg) with the value of 5± 1 g. Moreover, a significant decrease in root weight was observed with the

241 increasing of Ni concentration. Indeed, means reached 9,33 ± 0,5 g; 7,66 ± 0,57 g; 5,33 ± 0,54 g and 3,66±

242 0,56 g respectively for C1, C2, C3 and C4 against a value of 11,25 ± 0,95 g for the control plant.

243 3.4. Effect of Ni on the dry matter of plants

244 Variation of dry weight after Ni application was expressed in Table4. Results presented a significant decrease in

245 shoots dry matter for C2, C3 and C4 concentrations with percentages respectively 37.96 ± 0.7%, 36, 25 ± 0.8%

246 and 34 ± 1%, against a control value of 41.76± 1%. Moreover, our results showed a significant decrease in root

247 dry matter in plants treated with C2, C3 and C4 where means reached respectively 24,78 ± 1.28%, 22,5 ± 1.47%

248 and 21,81± 1.38%, against a control value of 59.13 ± 1.77%.

249 3.5. Effect of Ni on chlorophyll content

250 The variation of chlorophyll content of plants after Ni contamination is shown in Table5. Results showed that

251 chlorophyll a, chlorophyll b and total chlorophyll contents decreased in plants exposed to C4 with a percentage

252 respectively of40 %, 73 % and 44 % (P < 0.05). Whatever the Ni concentration appreciated, Chlorophyll b

253 decreased more than chlorophyll a. Also, an increase in chlorophyll a/b ratio indicated that the toxic effect of

254 nickel is more pronounced in chlorophyll b.

255 3.8. Effect of Ni on alfalfa oxidative stress biomarkers

256 CAT activity increased in leaves after 60 days of exposure to the rise of Ni concentration (Figure 2A). The most

257 important mean was observed in plants exposed to C4 (835, 43± 55, 83μmole / min / mg of protein) against a

258 control value of 24, 19± 1, 96 μmole / min / mg of protein. Similarly, for alfalfa roots, a significant difference

259 was observed for C1 (151,1 ± 1,99 μmole / min / mg of proteins), C2 (255,27 ± 5,65 μmole / min / mg of

260 protein), C3 (191,16 ± 2,15 μmol / min / mg protein) and C4 (176,64 ± 8,21 μmol / min / mg protein) compared

261 to the control plants (65,37 ± 2,98 μmol / min / mg) (Figure 2B).

262 Moreover, our results showed a significant increase in the specific activity of GST. Indeed, GST activity reached

263 in plants exposed to C4 a value of 0, 34 ±0,013 (mmol/ min / mg of protein) (Figure 3A). However, in roots, Ni

264 treatments led to a strong activity at C3 with a value of 0.555 ± 0.005 nmol / min/mg of protein (Figure 3B).

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265 Ni-induced oxidative damage on membranes was evaluated by measuring changes in lipid peroxidation by

266 quantifying the malondialdehydes (MDA) formation in the plant leaves and roots. Ni toxicity caused a

267 significant increase in TBARS content in leaves (Figure 4A) and roots (Figure 4B). In leaves, the highest MDA

268 content was observed for the concentration C4 (500 mg/kg) with the value of 11, 87 ± 0,87 nmol TBAR's/g

269 PF(Figure 4a).Similarly for foot lipid peroxidation, a significant difference was observed for C1 (5,49 ± 0,26,

270 C2 6,0 ± 0,76, C3 11,31 ± 0,47and C4 13,74 ± 0,58 nmol TBAR's/g PF) compared to the control plants (4,92±

271 0,25 nmol TBAR's/g PF).

272 3.9. Analyses of the evolution of alfalfa response after treatment with different Ni concentrations

273 Evolution of alfalfa plant response after exposition to different Ni concentration was shown in a PCA biplot

274 (Figure 5). The first and second axis of the PCA explained, respectively 85.61 % and 7.47% of the variance.

275 Results showed a strong separation between control plants and those exposed to C3 and C4. Moreover, plants

276 contaminated with C4 are mostly characterized by a high CAT and GST activity and an important Ni

277 accumulation in shoots and roots.

278 Moreover, the correlation matrix Figure 6 showed that the concentration of Ni in soils is positively and highly

279 correlated with the concentration of Ni in shoot and root of the alfalfa plant and also the augmentation of the

280 activities of GST, CAT and MDA in root and shoot of alfalfa plant. Also, the concentrations of Ni in the soil,

281 shoot and root are negatively correlated with the content of chlorophyll a/ b and carotenoids in plants.

282 4. Discussion

283 Phytoremédiation is sustainable technique involving plants in order to remove heavy metals from contaminated

284 soils (Peuke and Rennenberg 2005; Ali et al. 2013; Hattab et al. 2016). In this present study, we used alfalfa

285 plants (Medicago sativa) to assess its phytoremediation potential of Ni from soils. For that, we analyzed

286 chemical changes, agronomic parameters and oxidative stress biomarkers after60 days of exposure to four Nicl 2

287 (50, 150, 250 and 500 mg/kg) against a control condition.

288 In line with our findings, alfalfa plants showed an excellent capacity for massive metal accumulation. Also, our

289 calculationshowed that alfalfa plants preferentially accumulated Ni in roots than in shoots with TF <1 . This

290 results are in agreement with numerous works that assessed the phytoremediation potentiality of other plants like

291 Eruca sativa (Kamran et al. 2016), cotton (Khaliq et al. 2016), and Indian mustard (Ansari and al. 2015). Page

292 and Feller (2005) indicated that Ni is a highly mobile metal in plants but it is mostly accumulated in roots. This

293 is in accordance with several authors (Seregin et al. 2003). This may be explained by the fact that roots could

294 develop a barrier against toxic elements and could provide a wide surface to absorb and accumulate heavy metals

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295 (Hall 2002; Harada et al. 2010).The BCF values of all plants increased with the increasing Ni-concentrations.

296 Zhi-xin et al. 2007 showed that BCF can estimate the ability of alfalfa plants to accumulate heavy metals

297 according to the bioconcentration. Also, BCF values of plants depend on many factors such as concentrations of

298 heavy metals, the accumulation ability of plants and environmental properties. The phytoextraction efficiency

299 (PEE) of M.Sativa increased significantly with the different Ni concentration. In fact, the use of plants to remove

300 heavy metals from contaminated soil has been described in several studies (Ana et al. 2009; Murphy et al. 2011).

301 Similar results was observed in alfalfa plants contaminated with other heavy metals such as Pb (Zhi-xin et al.,

302 2007; Hattab et al. 2016), Hg (Sobrino-Plata et al., 2014) and Cd (Hattab et al., 2014).

303 Although, Our results showed that no morphologic or agronomic diseases were recorded in alfalfa plants after 60

304 days of exposure, except a slight decrease in shoots and roots growing, Mean while, Pandey and Sharma (2002)

305 had shown that the toxic effects of Ni was manifested by chlorosis, necrosis and wilting. So, alfalfa plants could

306 tolerate high concentrations of Ni and continue its growing. On the other hand, the slight reduce of plant’s

307 weight and length could be explained by the fact that Ni had affected fundamental processes such as the

308 transport of photosynthetic process and mineral nutrition as proved by numerous authors (Samarakoon and

309 Rauser 1979; Gajewska et al. 2009; Hussain et al. 2013). Recently, Nazir et al (2016) showed that nickel

310 decreased the absorption of nitrogen (N), phosphorus (P) and potassium (K) concentrations in rice plants. In

311 another previous work Gajewska et al. (2006) explained the decrease in fresh weight by the decline in the water

312 content under metallic stress. Moreover, the growth inhibition in plants could be the result of a general

313 metabolic disorder rand direct inhibition of cell-division (Seregin and Kozhevnikova 2006).Thus, in shoots, the

314 dry matter was reduced for the different nickel concentrations comparing to control condition and this was in

315 concordance with Guo et al. (2010) results which founded that Ni had reduced fresh and dry matter of soybean

316 and maize. In fact, Ni can disturb numerous mechanisms in plants through disrupting the mitotic division and

317 cell elongation (Robertson and Meakin 1980; Powell et al. 1986; Serigin et al. 2001; Guo et al. 2010).

318 Then, chlorophyll (Chl) content in Medicago sativa plants was significantly affected and shown an important

319 decrease along with Ni concentration. Also, several studies have proved that heavy metals are a potent inhibitor

320 of Chl synthesis (Greger and Ogren 1991; Bajguz and Hayat 2009; Carrasco-Gill et al. 2012; Kamaran et al.

321 2016). Besides, the reduction of chl content could be a result of a disruption in pigment synthesis

322 (Somashekaraiah et al. 1992), a modification in chloroplasts structure and an inhibition of Calvin cycling

323 enzymes. Furthermore, heavy metal could lead to a stomatal closure and eventually to a deficit in CO 2 (Seregin

324 et al. 2001).

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325 Therefore, in order to assess of the impact of Ni on the oxidative status of Medicago sativa, CAT and GST

326 activities were evaluated, in addition of MDA content. Results showed that lipid peroxidation in plants exposed

327 to different concentrations of Ni were significantly increased, especially in roots. In this context, growth

328 inhibition could be explained by cell membranes destruction through the lipid peroxidation (Baccouch et al.

329 2001). Our results are in concordance with numerous studies who founded a crucial increase in MDA content

330 and a high lipid peroxidation in different plants (Pandolfini et al. 1992; Madhava Rao and Sresty 2000; González

331 et al. 2015). Moreover, our results showed that CAT activity had significantly increased in both parts of plants

332 treated with Ni. Similarly, an increase in CAT enzymatic activity was also observed after the application of Ni in

333 Zea Mays (Baccouch et al. 1998). Besides, other works founded that Cd, Cu, Cr and Hg could increase CAT

334 activity in vegetables (Cho and Park 2000; Kuo and Kao 2004; Hattab et al. 2016).

335 Alfalfa plants exposed to Ni showed a remarkable increase in GST activity. Our results are in concordance with

336 several works dealing with the impact of heavy metal pollution on different species of plants (Dixit et al. 2001).

337 Additionally, Choi et al. (2008) revealed that GST could play a fundamental role in plants against oxidative

338 stress induced by metals. Nonetheless, GST could modulate oxidative stress through numerous processes such as

339 the translocation of lipid peroxidation products, xenobiotics and other secondary metabolites to the vacuole.

340 5. Conclusions

341 The present study evaluated the response of alfalfa plant to increasing Ni concentrations in order to apprehend its

342 capabilities in soil’s decontamination. Results highlighted the important role of antioxidant enzymes in the

343 protection of plants against ROS production under metallic stress. Also, this work demonstrated that alfalfa plant

344 could be useful in the phytoremediation of heavy metal contaminated soils.

345 Acknowledgements

346 This work was also supported by funds from the «Ministère de l’Enseignement Supérieur et de la Recherche

347 Scientifique; UR04A6R05. Biochimie et Toxicologie Environnementale»

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