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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.




15 *: Corresponding author:


17 Laboratory of Biochemistry and Environmental

18 Toxicology, ISA Chott-Meriem, Tunisia

19 Tel +216 99 923 652

20 Email :




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.


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

48 stress




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

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.


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:

149 𝐃𝐌(%) = ( ) ∗ 100

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).

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

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

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


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

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).

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

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).

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»

348 References
349 Adriano D C (2001) Trace elements in terrestrial environments: Biochemistry, bioavailability and risks of
350 metals. 2ed. Springer-Verlag, New York. pp 4-12, 64-65
352 Aebi H (1984) Catalase in vitro. Method. Enzymol. 105, 121-126.
353 6879(84)05016-3
355 Ahmad P, Jaleel A C, Salem A M, Nabi G, Sharma S (2010) Roles of enzymatic and Non enzymatic
356 antioxidants in plants during abiotic stress. Crit. Rev. Biotechnol. 30, 161-175.
359 Ali H, Khan E, Sajad MA (2013) Phytoremediation of heavy metals- Concepts and applications. Chemosphere
360 91, 869–881.

362 Ana P G V M, António O S S, Rangel & Paula M L Castro (2009) Remediation of heavy metal contaminated
363 soils: phytoremediation as a potentially promising clean-up technology. Crit Rev Environ Sci Technol 39,
364 622–654. https://
366 Anjum N A, Ahmad I, Mohmooda I, Pachecob M, Duartea AC, Pereiraa E, Umarc S, AAhmadc, Khand N A,
367 Iqbalc M, Prasade M NV (2012) Modulation of glutathione and its related enzymes in plants’ responses
368 to toxic metals and metalloids 75, 307-324.
370 Ansari MH, Verma BK, Ansari MA, Mishra D, Srivastava AK, Khan N, et al (2015) Impact of cropping
371 pattern on growth, yield attributes and system productivity of citronella (Citronella winterianus) pulses
372 intercropping system in Central India. The Indian Journal of Agricultural Sciences. 85(3)
374 Arnon DT (1949) Copper enzymes in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiol
375 24:1–15

376 Awasthi K, Sinha P (2013) Nickel stress induced antioxidant defence system in sponge gourd (Luffa
377 Cylindrical). J. Plant Physiol. Pathol. 1:1.

378 Baccouch S, Chaoui A, El Ferjani E (1998) Nickel toxicity: Effects on growth and metabolism of maize. Jour.
379 Plant Nutr. 21: 577-588.

380 Baccouch S, Chaoui A, El Ferjani E (2001) Nickel toxicity induces oxidative damage in Zea mays roots J Plant
381 Nutr 24:1085–1097. https:// doi: 101081/PLN-100103805

382 Bajguz A, Hayat S (2009) Effects of brassino steroids on the plant responses to environmental stresses Plant
383 PhysiolBiochem 47:1–8. https:// doi: 101016/jplaphy200810002

384 Boughattas I, Hattab S, Boussetta H, Sappin-Didier V, Viarengo A, Banni M, Sforzini S (2016) Biomarker
385 responses of Eisenia andrei to a polymetallic gradient near a lead mining site in North Tunisia. Environ
386 Pollut. 2016;218:530–41. 2016.07.033.

387 Bradford M M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein
388 utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254.
389 doi: 90527-3.

390 Bradl H (2002) Heavy metals in the environment: origin, interaction and remediation. London

391 Carrasco-Gil S, Estebaranz-Yubero M, Medel- Cuesta D, Millan R, Hernandez L E (2012) Influence of nitrate
392 fertilization on Hg uptake and oxidative stress parameters in alfalfa plants cultivated in a Hg-polluted soil.
393 Environ Exp Bot. 75, 16-24.

394 Carrasco-Gil S, Siebner H, LeDuc D L, Webb S M, Millán R, Andrews J C, Hernández L E (2013) Mercury
395 localization and speciation in plants grown hydroponically or in a natural environment. Environ. Sci.
396 Technol. 3082–3090. doi:

397 Cho UH, Park JO (2000) Mercury-induced oxidative stress in tomato seedlings Plant Sci 156:1–9. https:// doi:
398 101016/S0168-9452.00.00227-2

399 Choi C Y, An K W, An M I (2008) Molecular characterization and mRNA expression of glutathione peroxidase
400 and glutathione S transferase during osmotic stress in olive flounder (Paralichthys olivaceus). Comparative
401 Biochemistry and Physiology A 149, 330-337.

402 Choudhary S P, Bhardwaj R, Gupta B D, Dutt P, Gupta R K, Biondi S, Kanwar M (2010) Epibrassinolide
403 induces changes in indole-3-acetic acid, abscisic acid and polyamine concentrations and enhances

404 antioxidant potential of radish seedlings under copper stress. Plant Physiol. 140, 280-296.

406 Dixit V, Pandey V, Shyam R (2001) Differential antioxidative responses to cadmium in roots and leaves of pea
407 (Pisum sativum L. cv. Azad). Jour of Exper Botany, 52, Issue 358, 1: 1101–1109.

409 Dourado MN, Franco MR, Peters LP (2015) Antioxidant enzymes activities of Burkholderiaspp strains—
410 oxidative responses to Ni toxicity Environ SciPollut Res 22:19922–19932. https:// doi: 101007/s11356-
411 015-5204-1. https:// doi: 10.1007/s11356-015-5204-1

412 Elstner EF (1982) Oxygen activation and oxygen toxicity Annu Rev Plant Physiol 33:73–96. https:// doi:
413 101146/annurevpp33060182000445.

414 Erakhrumen A A (2007) State of forestry research and education in nigeria and subsaharan africa: implications
415 for sustained capacity building and renewable natural resources devel . Jour of Sust Dev in Africa 9: 1520-
416 5509.

417 Fabiano C, Tezotto T, Favarin JL, Polacco JC, Mazzafera P (2015) Essentiality of nickel in plants: a role in plant
418 stresses. Front Plant Sci 6:754.

419 Flores-Cáceres M L, Hattab S, Hattab S, Boussetta H, Banni M, Hernández L E (2015) Specific mechanisms of
420 tolerance to copper and cadmium are compromised by a limited concentration of glutathione in alfalfa
421 plants. Plant Sci. 233, 165–173.

422 Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in plants - reevaluation of the concept 1056–
423 1071. https:// doi: 101111/j1365-3040200501327x

424 Gajewska E, Sklodowska M, Slaba M, Mazur J (2006) Effect of nickel on antioxidative enzyme activities,
425 proline and chlorophyll contents in wheat shoots, Biol. Plant, 50, 653 – 659.

426 Gajewska E. Wielanek M. Bergier K. Sklodowska M (2009) Nickel-induced depression of nitrogen assimilation
427 in wheat roots. Acta Physiol. Plant. 31, 1291-1300. https:// doi: 10.1007/s11738-009-0370-8

428 Gill SS, Anjum NA, Hasanuzzaman M (2013) Glutathione and glutathione reductase: A boon in disguise for
429 plant abiotic stress defense operations Plant PhysiolBiochem 70:204–212. https:// doi:
430 101016/jplaphy201305032

431 Gill S S, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop
432 plants. Plant Physiol. Biochem. 48, 909-930.

433 González C I, Maine M A, Cazenave J, Hadad H R, Benavides M P (2015) Ni accumulation and its effects on
434 physiological and biochemical parameters of Eichhornia crassipes. Environ. Exp. Bot. 117, 20-27.

436 Gratão P L, Pompeu G B, Capaldi F R, Vitorello V A, Lea, PJ, Azevedo R A (2008) Antioxidant response of
437 Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress. Plant Cell Tiss. Organ Cult. 94,
438 73–83.

439 Greger M, Ögren E (1991) Direct and indirect effects of Cd2+on photosynthesis in sugar beet .Beta
440 vulgaris.Physiol Plant 83:129–135. https:// doi: 101111/j1399-30541991tb01291x

441 Guo XY, Zuo YB, Wang BR (2010) Toxicity and accumulation of copper and nickel in maize plants cropped on
442 calcareous and acidic field soils Plant Soil 333:365–373. https://doi: 101007/s11104-010-0351-0

443 Habig H W, Michael J, Pabst M J and William B, Jakoby W J (1974) Glutathione S-Transferases: The first
444 enzymatic step in mercapturic acid formation. The Journal of Biological Chemistry. 249, 7130-7139.

445 Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. Jor Exp Bot 53:1–1. https://

447 Harada E, Kim JA, Meyer AJ, Hell R, Clemens S, Choi YE (2010) Expression profiling of tobacco leaf
448 trichomes identifies genes for biotic and abiotic stresses. Plant Cell Physiol 51:1627–1637. https://

450 Hattab S, Hattab S, Boussetta H and Banni M (2014) Influence of nitrate fertilization on Cd uptake and oxidative
451 stress parameters in alfalfa plants cultivated in presence of Cd Jour of Soil Sci and Plant Nutr , 14(1), 89-
452 99. https://

453 Hattab S, Hattab S, Flores-Casseres M L, Boussetta H, Doumas P, Hernandez L E, Banni M (2016)

454 Characterisation of lead-induced stress molecular biomarkers in Medicago sativa plants. Envir and Exper
455 Botany 123, 1–12. https://

456 Heath RL PL (1968) Photoperoxidation in isolated Chloroplasts of Fatty Acid Peroxidation chlorophyll Arch
457 Biochembiophisics 126:189–198

458 Hussain M B, Ali S, Azam, A, Hina, S, Farooq A M, Ali B, Bharwana A S, Gill M B (2013) Morphological,
459 physiological and biochemical responses of plants to nickel stress: A review. Afr. J. Agric. Res. 8, 1596-
460 1602. https:// doi:10.5897/AJAR12.407

461 Järup L (2003) Hazards of heavy metal contamination Br Med Bull 68:167–182. https:// doi:
462 101093/bmb/ldg032

463 Jiang Q Y et al (2016) Can arbuscular mycorrhizal fungi reduce Cd uptake and alleviate Cd toxicity of
464 Lonicera japonica grown in Cdadded soils? Sci Rep-uk 6, 21805. https:// doi 10.1038/srep21805

465 Khaliq A, Ali S, Hameed A, Farooq MA, Farid M, Shakoor MB, Mahmood K, Ishaque W, Rizwan M (2016)
466 Silicon alleviates nickel toxicity in cotton seedlings through enhancing growth, photosynthesis, and
467 suppressing Ni uptake and oxidative stress. Arch Agron Soil Sci 62:633–647.

469 Kamran M A, Eqani Samas, Bibi S (2016) Bioaccumulation of nickel by E sativa and role of plant growth
470 promoting rhizobacteria.PGPRs. under nickel stress Ecotoxicol Environ Saf 126:256–263. https:// doi:
471 101016/jecoenv201601002

472 Karn B, Kuiken T and Otto M (2009) Nanotechnology and in Situ Remediation: A Review of the Benefits and
473 Potential Risks. https:// doi:10.1289/ehp.0900793

474 Kuo M C and Kao C H (2004) Antioxidant enzyme activities are upregulated in response to cadmium in
475 sensitive, but not in tolerant, rice (Oryza sativa L.) seedlings 45: 291-299

476 Küpper H, Kroneck PMH (2007) Nickel in the environment and its role in the metabolism of plants and
477 cyanobacteria. In: Sigel A, Sigel H, Sigel RKO (eds) Metal ions in life sciences, vol 2. Wiley, Chichester,
478 pp 31–62

479 Laemmli (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature,
480 volume 227, Issue 5259, pp. 680-685.

481 Madhava Rao K V, Sresty TVS (2000) Antioxidative parameters in the seedlings of pigeonpea. Cajanuscajan.L.
482 Millspaugh. in response to Zn and Ni stresses Plant Sci 157:113–128.https:// doi: 101016/S0168-
483 9452.00.00273-9

484 Mathieu S, Soussou S, Cleyet-Marel J C, Brunel B, Mauré L , Lefèbvre C, Escarré J (2012) Local adaptation of
485 metallicolous and non-metallicolous Anthyllis vulneraria populations: their utilization in soil restoration.
486 Restoration Ecology in press.DOI: 10.1111/j.1526-100X.2012.00927.x

487 Majid N M, Islam M M, Justin V, Abdu A, Ahmadpour P (2011) Evaluation of heavy metal uptake and
488 translocation by Acacia mangium as a phytoremediator of coppe contaminated soil. African Journal of
489 Biotechnology, 10(42): 8373-8379.

490 Murphy R O, Ackermann K A, and Handgraaf M J J (2011) Measuring Social Value Orien- tation," Judgment
491 and Decision Making, 2011, 6 (8), 771-781.

492 Nazir H, Asghar HN, Zahir ZA (2016) Judicious use of kinetin to improve growth and yield of rice in nickel
493 contaminated soil Int J Phytoremediation 18:651–655. https:// doi: 101080/1522651420151094444

494 Öquist G, Wass R (1988) A portable, microprocessor operated instrument for measuring chlorophyll
495 fluorescence kinetics in stress physiology Physiol Plant 73:211–217. https://doi:101111/j1399-
496 30541988tb00588x

497 Ortega-Villasante C, Rellan-Alvarez R, Del Campo F F, Carpena-Ruiz R O, Hernandez L E (2005) Cellular

498 damage induced by cadmium and mercury in Medicago sativa. J Exp Bot. 56, 2239-2251.

500 Page V, Feller U (2005) Selective transport of zinc, manganese, nickel, cobalt and cadmium in the root system
501 and transfer to the leaves in young wheat plants. Ann Bot 96:425–434. https://doi:10.1093/aob/mci189

502 Pandey N, Sharma CP (2002) Effect of heavy metals Co2+, Ni2+ and Cd2+ on growth and metabolism of
503 cabbage. Plant Sci 163, 753–758.

504 Pandolfinet, Gabbriellir, Comparinic C (1992) Nickel toxicity and peroxidase activity in seedlings of
505 Triticumaestivum L Plant Cell Environ 15:719–725. https:// doi: 101111/j1365-30401992tb01014x

506 Parida BK, Chhibba IM, Nayyar VK (2003) Influence of nickel contaminated soils on fenugreek (Trigonella
507 corniculata L.) growth and mineral composition. Sci Hort 98, 113–119.
508 4238(02)00208-X

509 Peuke AD, Rennenberg H (2005) Phytoremediation. EMBO Reports 6, 497. https://doi:
510 10.1038/sj.embor.7400445

511 Powell M J, Davies MS, Francis D (1986) The influence of zinc on the cell cycle in the root meristem of a zinc-
512 tolerant and a non-tolerant cultivar of festuca rubra l. New Phytologist 102:419–428. https://doi:
513 10.1111/j.1469-8137.1986.tb00819.x

514 Prabha K, Padmavathiamma & Loretta Y Li (2007) Phytoremediation Technology: Hyper-accumulation Metals
515 in Plants Water Air Soil Pollut 184:105–126. https://doi: 10.1007/s11270-007-9401-5

516 Rizwan M, Mostofa MG, Ahmad MZ, Imtiaz M, Mehmood S, Adeel M, Dai Z, Li Z, Aziz O, Zhang Y, Tu S
517 (2018) Nitric oxide induces rice tolerance to excessive nickel by regulating nickel uptake, reactive oxygen
518 species detoxification and defenserelated gene expression. Chemosphere 191:23–35.

520 Robertson A A I, Meakin MER (1980) The effect of nickel on cell division and growth of growth of
521 brachystegia spiciformis seedlings Published by National Herbarium & Botanic Garden Stable URL
522 12:115–125. https://wwwjstororg/stable/23502173

523 Rooney CP, Zhao FJ, McGrath SP (2007) Phytotoxicity of nickel in a range of European soils: Influence of soil
524 properties, Ni solubility and speciation Environ Pollut 145:596–605. https:// doi:
525 101016/jenvpol200604008

526 Samarakoon AB, Rauser WE (1979) Carbohydrate Levels and Photoassimilate Export from Leaves of Phaseolus
527 vulgaris Exposed to Excess Cobalt, Nickel, and Zinc Plant Physiol 63:1165–1169. https:// doi:
528 101104/pp6361165

529 Seregin I V, Ivanov VB (2001) Physiological aspects of cadmium and lead toxic effects on higher plants Russ J
530 Plant Physiol 48:523–544. https:// doi: 101023/A:1016719901147

531 Seregin IV, Kozhevnikova AD, Kazyumina EM, Ivanov VB (2003) Nickel toxicity and distribution in maize
532 roots. Russ J Plant Physiol 50:711–717

533 Seregin I V, Kozhevnikova AD (2006) Roles of root and shoot tissues in transport and accumulation of
534 cadmium, lead, nickel, and strontium Russ J Plant Physiol 55:1–22. https:// doi: 101007/s11183-008-10018

535 Sharma P, Bhardwaj R, Arora N, Arora H K, Kumar A (2008) Effects of 28-homobrassinolide on nickel uptake,
536 protein content and antioxidative defence system in Brassica juncea. Biol. Plant. 52, 767-770.

537 Shi G and Cai Q (2009) Cadmium tolerance and accumulation in eight potential energy crops. Biotechnol Adv.
538 27, 555–561.

539 Sobrino-Plata J, Ortega-Villasante C, Laura Flores-Cáceres M, Escobar C, Del Campo F F, Hernández L E

540 (2009) Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in
541 alfalfa. Chemosphere 77, 946–954. https:// chemosphere.2009.08.007.

542 Sobrino-Plata J, Herrero J, Carrasco-Gil, S, Pérez-Sanz A, Lobo C, Escoba C, Millán R, Hernández L E (2013)
543 Specific stress responses to cadmium, arsenic and mercury appear in the metallophyte Silene vulgaris when
544 grown hydroponically. RSC Adv. 3, 4736. doi:

545 Sobrino-Plata J, Meyssen D, Cuypers A, Escobar C, Hernández L E (2014) Glutathione is a key antioxidant
546 metabolite to cope with mercury and cadmium stress. Plant Soil 377, 369–381.
547 doi: 4.

548 Su D C, Wong J W C, Jagadeesan H (2004) Implications of rhizospheric heavy metals and nutrients for the
549 growth of alfalfa in sludge amended soil. Chemosphere. 56, 957-965.

551 Sun F H, Zhou Q X (2008) Oxidative stress biomarkers of the polychaete Nereis diversicolor exposed to
552 cadmium and petroleum hydrocarbons. Ecotoxicol. Environ. Saf. 70, 106-114.

554 Thioulouse J and Dray S (2007) Interactive Multivariate Data Analysis in R with the ade4 and ade4TkGUI
555 Packages. J of Stat Soft ware. 22 (5).

556 Ullah A, Heng S, Munis MFH, Fahad S, Yang X (2015) Phytoremediation of heavy metals assisted by plant
557 growth promoting (PGP) bacteria: A review. Environ Exp Bot 117:28–40

558 Vangronsveld J, Herzig R, Weyens N (2009) Phytoremediation of contaminated soils and groundwater: Lessons
559 from the field Environ SciPollut Res 16:765–794. https:// doi: 101007/s11356-009-0213-6

560 Wang S, Shi X (2001) Molecular mechanisms of metal toxicity and carcinogenesis Mol Cell Biochem 222:3–9.
561 https:// doi: 101023/A:1017918013293

562 Wang C R, Xiao J J, Tian Y, Bao X, Liu L, Yu Y, Wang X R, Chen T Y (2012) Antioxidant and prooxidant
563 effects of lanthanum ions on Vicia faba L. seedlings under cadmium stress, suggesting ecological risk.
564 Environ. Toxicol. Chem. 31, 1355–1362.

565 Yang Y, Yichen Ge, Hongyuan Zeng, Xihong Zhou, Liang Peng and Qingru Zeng (2017) Phytoextraction of
566 cadmium-contaminated soil and potential of regenerated tobacco biomass for recovery of cadmium.
567 Scientific Reports, 7: 7210. https:// doi:10.1038/s41598-017-05834-8

568 Yusuf M, Fariduddin Q, Hayat S, Ahmad A (2011) Nickel: An overview of uptake, essentiality and toxicity in
569 plants. Bull. Environ. Contam. Toxicol. 86, 1-17

570 Zhao C, Ye D, Wei D, Chen B L D (1994) Tertiary in Oil and Gas Province of China (III). Oil Industry Press,
571 Beijing, pp. 1–124

572 Zhi-xin N, Sun L Sun T, Li Y, Wang H (2007) Evaluation of phytoextracting cadmium and lead by sunflower,
573 ricinus, alfalfa and mustard in hydroponic culture. J. Environ. Sci. (China) 19, 961–967. https://