Physiological, biochemical and transcriptomic responses of Medicago sativa to nickel exposure

Journal Pre-proof Physiological, biochemical and transcriptomic responses of Medicago sativa to nickel exposure Sondes Helaoui, Iteb Boughattas, Sabrine Hattab, Marouane Mkhinini, Vanessa Alphonse, Alexandre Livet, Nourreddine Bousserhine, Mohamed Banni PII:

S0045-6535(20)30314-3

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126121

Reference:

CHEM 126121

To appear in:

ECSN

Received Date: 27 November 2019 Revised Date:

1 February 2020

Accepted Date: 3 February 2020

Please cite this article as: Helaoui, S., Boughattas, I., Hattab, S., Mkhinini, M., Alphonse, V., Livet, A., Bousserhine, N., Banni, M., Physiological, biochemical and transcriptomic responses of Medicago sativa to nickel exposure, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.126121. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

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Physiological, biochemical and transcriptomic responses of Medicago sativa to Nickel

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exposure

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Sondes HELAOUI 1, Iteb BOUGHATTAS 1*, Sabrine HATTAB 2, Marouane

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MKHININI 1, Vanessa ALPHONSE 3, Alexandre LIVET 3, Nourreddine

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BOUSSERHINE 3, Mohamed BANNI 1

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

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Tunisia

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

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Tunisia

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

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Cedex, 94010, France.

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*: Corresponding author:

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Iteb BOUGHATTAS

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Laboratory of Biochemistry and Environmental

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Toxicology, ISA Chott-Meriem, Tunisia

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Tel +216 99 923 652

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Email: [email protected]

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Abstract

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Metal accumulation in soil could lead to severe damage to plants, animals, and humans. The

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present work aims to evaluate the effects of nickel (Ni) exposure on Medicago sativa at

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physiological, biochemical, and transcriptomic levels. Plants were exposed to five increasing

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concentrations of Ni (0, 50, 150, 250, and 500 mg/kg) for 60 days. Agronomic parameters

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(fresh and dry matter) and chlorophyll content (Chl) were determined in an alfalfa plant.

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Chemical analyses were conducted, involving the determination of Ni loads in plants (roots

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and shoots). Moreover, malondialdehyde accumulation (MDA), glutathione-S-transferase

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(GST), and peroxidase activities, termed as oxidative stress biomarkers, were measured. The

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gene expression levels of Prx1C, GST, and phytochelatins (PCs) were determined at different

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nickel concentrations. Our results showed that Ni concentration in plants increased

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significantly along with Ni concentration in the soil. Regarding oxidative stress biomarkers,

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Ni contamination caused an increase in peroxidase and GST activities, with a remarkable

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accumulation of MDA, especially for the highest Ni concentration (500 mg/kg of Ni). Our

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data showed also a significant upregulation of Prx1C and GST genes in shoots and roots. The

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PCs’ gene expression was significantly enhanced in response to the different nickel

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concentrations, suggesting their important role in Ni detoxification in alfalfa plants. Our data

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provided evidence about the clear toxicity of Ni, an often-underestimated trace element

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Key words: Nickel, Medicago sativa, agronomic parameters, oxidative stress, gene

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expression

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1. Introduction

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Increasing trace element levels are being recorded in soils, and such pollution has become a

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critical problem for the environment (Shi and Cai, 2009; Jiang, 2016). Unlike organic

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pollutants, which can be metabolized by soil organisms and plant roots, most heavy metals are

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persistent in the environment (Järup, 2003). Two types of heavy metals are distinguished,

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according to their physiological and toxic effects: essential and toxic. Essential metals like

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Cu, Zn, and Fe are necessary for numerous cellular processes and are found in very minor

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quantities in biological tissues (Adriano, 2001). Conversely, non-essential metals such as Hg,

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Cd, and Pb are toxic to plants, animals, and humans at high levels (Baize, 2009).

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Interestingly, Ni has been classified among the essential micronutrients, since it is involved in

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many biological processes of plants and microorganisms in trace amounts (Bradl, 2002;

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Küpper and Kroneck, 2007; Fabiano et al., 2015). Notwithstanding this, and regardless of

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whether this metal is found in soil ecosystems naturally or following anthropogenic activities,

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it can cause several problems (Rooney et al., 2007; Wang et al., 2012). Ni reaches soils

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naturally through the alteration of the parent rock. Moreover, numerous human activities like

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industries, mining, and agricultural practices lead to Ni accumulation in the soil ecosystem

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(Yusuf et al., 2011; Hussain et al., 2013).

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Once in the soil, Ni can have undesirable effects on plants at high concentrations, which has

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been widely proven in several works (Parida et al., 2003; Gratão et al., 2008; Dourado et al.,

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2015; Rizwan et al., 2018). Among these effects are chlorosis, leaf necrosis, growth

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retardation, and induction of reactive oxygen species’ (ROS) production (Madhava Rao and

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Sresty, 2000; Pandey and Sharma, 2002; Foyer and Noctor, 2005; Rooney et al., 2007;

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Gajewska et al., 2009; Dourado et al., 2015). It is well known that excessive ROS production

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could lead to increased protein oxidation, cell membrane degradation, and DNA damage

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(Choudhury and Panda SK, 2004; Gill and Tuteja, 2010). Despite the imbalance between 3

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oxidants and antioxidants, cells could face ROS damage through the detoxification system

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involving a battery of enzymes, such as peroxidase and GST, which play a key role in the

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degradation and decomposition of reactive species (Sun and Zhou, 2008; Ahmad et al., 2010).

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Peroxidase is a ubiquitous antioxidant enzyme known for its ability to decompose hydrogen

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peroxide in cells (Nirala et al., 2015). Furthermore, MDA, one of the products of lipid

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peroxidation of the cell membrane (LPO), is usually used as a biomarker to assess ROS

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damage (Lucca et al., 2016; Tsao et al., 2017). GST is a phase II enzyme known to be

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involved in cellular protection against oxidative stress and ROS (Anjum et al., 2012).

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Conversely, several studies have indicated that PCs’ synthesis may be involved in the

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protective functions against Ni and other toxic elements in plants (Pinter and Stillman, 2014;

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Talebi et al., 2019). PCs are linear polymers of the 3,-Glu-Cys portion of glutathione

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synthesized by the enzyme phytochelatin synthase (Clemens et al., 1999; Pochodylo and

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Aristilde, 2017). In fact, there are various ways of metal remediation of soils. Nowadays,

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biological methods are a sustainable alternative to reduce the amount of pollutants in soils,

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such as through the use of animals like earthworms (Boughattas et al., 2016) or vegetables

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like Medicago sativa (Hattab et al., 2016). Compared to physicochemical methods, which

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cause a significant decrease in soil fertility and productivity, bioremediation is considered a

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friendly environmental technology. Furthermore, the cost of bioremediation is lower than that

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of traditional processes, both in situ and ex-situ (Peuke and Rennenberg, 2005; Vangronsveld

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et al., 2009; Ali et al., 2013).

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Phytoremediation is a green method based on natural processes to counteract metal

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contamination of soil ecosystems (Prabha and Loretta, 2007; Mathieu et al., 2012). This

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method allows the retention of metal (loid) from the soil in the plant (Ullah et al., 2015).

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Roots are the first container of heavy metals from the contaminated soil, although heavy

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metals will be found in different parts of the plant. In this context, these plants called

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hyperaccumulators are able to accumulate, degrade, and decrease the harmful effects of trace

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elements in soils (Karn et al., 2009). The alfalfa plant (Medicago sativa) is known to be a

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good model of phytoremediation plants, and this has been reported in many recent works

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(Gardea-Torresdey et al., 1998; Sobrino-Plata et al., 2009; Hattab et al., 2014; Flores Cáceres

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et al., 2015), which have indicated this plant’s tolerance of a wide variety of heavy metals like

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Cd, Cu, Zn, and Pb. Moreover, the alfalfa plant does not require any special cultural practices,

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and is characterized by its adaptation to a wide range of climates (Su et al., 2004). For these

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reasons, Medicago sativa represents a good candidate to palliate heavy metal contamination in

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terrestrial ecosystems.

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The first objective of the present study was to evaluate the effect of five progressively

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increased Ni concentrations on the agronomic and physiologic parameters of M. sativa plants.

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Second, we evaluated the oxidative stress status of shoot and root cells through anti-oxidative

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enzyme activities and the related gene expression levels. Finally, we examined the potential

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role of alfalfa in the phytoextraction of nickel from polluted soils.

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2. Material and methods

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2.1. Soil sampling

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Soil was sampled from an organic soil located in the higher institute of agronomy of Chott

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Meriem, in Tunisia. Then, soil samples were homogenized and dried over night at 25°C.

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2.2. Plant material

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Medicago sativa seeds were obtained from the Tunisian Seed Control Agency and they were

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germinated on wet paper in Petri dishes in darkness for48 hours at room temperature of

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24±1°C.

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2.3. Experimental design

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Soil samples were moistened with de-ionized water and brought to 70% of its holding

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capacity and this was maintained during the experimentation. Then, four increasing

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concentration of NiCl26H2O were prepared (C1: 50, C2: 150, C3: 250, and C4: 500 mg/kg)

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against a control condition (Control: Non-contaminated soil) (Kamran et al., 2016). Eight

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

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were used. Finally, the plastic pots were maintained under controlled temperature (20±1°C,

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with 12 h light-period). After 60 days of exposure, the plants were harvested, and biometric

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measurements were done. Subsequently, shoots and roots were separated than washed into

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water and Na2-EDTA (20 mM) in order to eliminate superficial Ni. Finally, plants were stored

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at -80°C for further analysis.

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2.4. Agronomic measurements

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

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the alfalfa plants after 60 days of culture. Then, from each sample, 100 g of fresh material was

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placed in oven at 60°C for 48 hours, and then the percentage of dry matter was calculated as

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following:
% = 

DM  ∗ 100 FM

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DM: Dry matter of plants; FM: Fresh matter of plants.

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2.5. Nickel content in soil and plants

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After 60 days, plants were carefully rinsed with distilled water. Then collected soil and plant

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samples were dried in the oven at 40°C for 24h. After that, samples were ground to a fine with

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a diameter (< 0.02µm). 1 g of each subsample has been subject to an acid digestion 37% HCl

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and 63% HNO3 (3/1: v/v) at 100°C (Zhao et al., 1994). After that, the suspensions were

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diluted in 0.6% HNO3 and the analysis was performed using inductively coupled plasma

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atomic emission spectrometry (ICP-AES; Fisons ARL Accuris) (Pekin Elmer 2380).

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2.6. The bioconcentration factor, translocation factor and phytoextraction efficiency of

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Ni

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The capacity of alfalfa plants to accumulate nickel from soils and transfer this metal from

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roots to shoots was evaluated by the bioconcentration factor (BCF) and translocation factor

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(TF), respectively (Majid et al., 2011).

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

BCFETM =  

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

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FTETM =  

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The phytoextraction efficiency (PEE) of Medicago sativa under different Ni-contamination in

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the pot experiment was estimated as suggested Yang et al. (2017):

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(PEE) =

 !""#$$%& '().+),- ./ 0 !"1234 "$ 65 ,78# ().+) / 078#%$&1#!"898"&:9&2#;&!"65

X 100

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ETM>?@ABBCDDEF = Ni-concentration in plant tissue (mg/kg)

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W>?@ABHIJKFCL

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ETMMNC? = Ni-concentration in soil (mg/kg)

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

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2.7. Chlorophyll content

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For this purpose, 1g of fresh leaves was extracted by grinding in a mortar using 20 ml of 80%

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acetone, with small amount of pure Silica Quartz, and 0.5 g calcium carbonate to equalize the

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cellular acidity. The extract was filtered and collected in Eppendorf’s tube. After that, the

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measurements of optical density (OD) were carried out using spectrophotometer VWR UV-

BD =

Total dry weight biomass of plant (kg)

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3100 PC at 663nm for the Chlorophyll a (ChlO) and 645 nm for the Chlorophyll b (Chlb)

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(Sobrino-Plata et al., 2013). Total chlorophyll as well as Chla and Chlb concentrations were

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calculated according to (Arnon, 1949) as following: ()

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ChlO O( ) = [(12, 7 x A663) − (2, 69 x A645)] )P0

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Chlb (

()

)P0

) = [(22, 9 x A645) − (4, 68 x A663)]

µ)

x

(

µ) (

x

QR( S ()

R,RU)P0SQRV: QR( S ()

R,RU)P0SQRV:

WXYZ + \ = Chla + Chlb 172

2.8. Oxidative stress biomarkers

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2.8.1. Total protein content

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According to Laemmli (1970), 0.5 g of frozen leaves and roots were ground in mortar with

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1ml of extraction buffer prepared instantly with: 30mM MOPS at pH 7.5 mM, 5mM Na2-

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EDTA, 10 mM DTT, 10 mM ascorbic acid, 0.6% PVP, 10 mL 100 mM PMSF and 1mL

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protease inhibitors cocktail (P2714, Sigma-Aldrich, St. Louis MO, USA) and 10 µl of the

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ascorbic acid solution. The suspension was centrifuged at 12.000 rpm for 15 min at 4°C.

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Protein was assayed according to the method of Bradford (1976) using bovine serum albumin

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as a standard.

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2.8.2. Peroxidase activity

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The specific activity of peroxidase was determined according to the Aebi (1984). In a quartz

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vat, 50 µl of protein extract for leaves and 100 µl for the roots extract were added to 750 µl of

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potassium phosphate buffer (0.05M, pH 7.8) and 200 µl of H2O2 (0.5M) added respectively.

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Peroxidase activity was evaluated by kinetic measurement at 20°C using a VWR UV-

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3100spectrophotometer (λ = 240nm). The results were expressed as µmole of H2O2

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

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2.8.3. Glutathion-S-Transferase activity

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

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of protein extract, 1 mM 1-chloro- 2,4 dinitrobenzene (CDNB) (Sigma-Aldrich, Saint Louis,

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MO, USA) as a substrate, and 4 mM glutathione (reduced form; GSH) in100 mMK phosphate

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buffer (pH 7.4). GST activity was determined by kinetic measurement at 20°C using a VWR

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UV-3100 spectrophotometer (λ =340 nm). The results were expressed as µmole GSH-CDNB

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produced per min and per mg of protein using 9.6 mM/cm as extinction coefficient.

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2.8.4. MDA content in alfalfa plants

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Lipid peroxidation in M. sativa apprehended by MDA amount was measured as described by

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Ortega-Villasante et al. (2005). For that, 0.3 g of fresh tissues (shoots/roots) was homogenized

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in 3 ml of 15% Trichloroacetic and 0.37% Thiobarbituric acids (TCA/TBA), 0.25 MHCl and

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0.01 % butylated hydroxytoluene. After that, the homogenate was incubated at 90°C for 30

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min and cooled for 5 min in an ice bath. Then, the obtained solution was centrifuged for 10

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min at 12.000 g and it was measured at 535 and 600 nm. Results were expressed as nmol of

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Thiobarbituric Acid Reactive Substances (TBAR’s) per g of fresh weight (FW).

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2.8.5. Gene expression analysis

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RNA was isolated from 0.04g of alfalfa samples using the RNeasy Plant mini kit (Qiagen).

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The RNA of three independent biological replicates was extracted and DNase treated using

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Turbo DNA-free (Ambion-Thermo Fisher Scientific Inc., Waltham, MA, USA). The quality

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and quantity of RNA samples were confirmed by UV spectroscopy as well as gel

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electrophoresis. First-strand synthesis was carried out using 1 mg of total RNA with the

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reverse transcription system (Promega, Madison, WI, USA) and oligo (dT) 15 primers

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(Promega). The first strand was amplified in 5 mL of LightCycler1 480 SYBR Green I

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(Roche Diagnostics) and 500 mM of each primer (Eurogenetec) of a total volume of 10 mL. 9

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The qPCR conditions consisted of initial denaturation at 95°C for 10 min, followed by 42

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cycles of 5 s at 95°C, at 60°C for 5 s, and at 72°C for 10 s. Immediately after amplification,

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the melt curve analysis was executed at 95°C for 5 s, followed by step wise annealing process

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at temperatures ranging from 60 to 95°C for 1min, and then constant temperature increasing

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to 97°C with continuous fluorescence reading. The efficiency of each primer pair was

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obtained by amplifying serial dilutions of cDNA. Specific primers were designed based on

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the sequences obtained from FernBase database NCBI database and then evaluated under the

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different samples and conditions (Table S1). The specificity of each amplicon was confirmed

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by the presence of a single peak in the melting curve and the visualization of a single band on

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the agarose gel electrophoresis. The relative gene expression was calculated for each target

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gene using the 2-DDCT method as described by Livak and Schmittgen (2001).

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2.9. Statistical analysis

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The results for nickel content, antioxidant enzymatic activities and chlorophyll content were

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presented as the mean ± standard deviations (SD) of 5 samples. SPSS Software, version

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20.0was used for statistical analysis. The normality of the distribution was tested using the

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Shapiro–Wilk test. For multiple comparisons, a parametric one-way analysis of variance

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(ANOVA) was performed on data along with Tukey's test. All calculations were made using

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

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To compare the evolution of plant response after Ni contamination, principal component

231

analyses (PCAs) were performed using the R software and the package ADE4TkGUI

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(Thioulouse and Dray, 2007). Moreover, a correlation matrix was performed using the R

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software and the package CORRPLOT.

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3. Results

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3.1. Nickel content in soils, roots and shoots of Medicago sativa plants 10

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Ni accumulation in soils is reported in Table 1. Ni-accumulation increased significantly along

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with

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(112.51±1.5mg/kg). Moreover, Ni loads in plants are reported in Table 1, showing a

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significant increase, with a maximum of 21.93±3.33 and 75.2±0.16mg/kg respectively in

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shoots and roots after 60 days of exposure to 500 mg/Kg of nickel.

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

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Ni

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The bioconcentration factor, translocation factor, and phytoextraction efficiency of Ni are

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presented in Table 1. Our study showed that the significant nickel concentration was

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accumulated in roots with TF < 1. BCF values increased in plants exposed to C1, C2, C3, and

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C4,with values of0.55±0.015; 0.67±0.035; 0.82±0.04, and 0.87±0.007 respectively against a

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control value 0.19±0.001. The PEE percentage of Medicago sativa increased significantly

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with the different nickel concentrations. Indeed, values reached 12.51±0.012%, 14.63±0.05%,

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9.19±0.014%, and 8.12±0.094% respectively for C1, C2, C3, and C4 against a control value

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5.42±0.014%.

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3.3. Growth parameters

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The effect of Ni exposure on the root and shoot lengths of Medicago sativa after 60 days of

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exposure are presented in Table 2. Our results revealed that the length of plants grown in Ni

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contaminated soils decreased significantly in comparison with control plants. The decrease

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was more pronounced with the highest concentration of Ni (500 mg/kg), with a 33% and

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32.25% reduction respectively in the length of roots and shoots.

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The effect of Ni treatment on root and shoot weight of Medicago sativa after 60 days’

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exposure is reported in Table 2. A significant decrease in root weight was observed with

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increasing Ni concentration. The most significant decrease was observed in the roots of plants

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exposed to the highest concentration (500 mg/kg), with a value of 5.11±1.0 g. Indeed, means

Ni

concentrations.

The

soil

Ni

reached

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its

maximum

concentration

at

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reached 9.33±0.5 g, 7.66±0.57 g, 5.33±0.54 g, and 3.66±0.56 g respectively for C1, C2, C3,

262

and C4 against a value of 11.25±0.95 g for the control plant.

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3.4. Effect of Ni on the dry matter of plants

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Variation of dry weight after Ni application is reported in Table 2. The results presented a

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significant decrease in the shoots’ dry matter for C2, C3, and C4 concentrations with

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percentages of 37.96±0.7%, 36.25±0.8%, and 34±1% respectively, against a control value of

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41.76±1%. Moreover, our results showed a significant decrease in the roots’ dry matter in

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plants treated with C2, C3, and C4, where the means reached 24.78±1.28%, 22.5±1.47%, and

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21.81±1.38% respectively, against a control value of 59.13±1.77%.

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3.5. Effect of Ni on chlorophyll content

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Chlorophyll content in plants exposed to increasing Ni concentrations for 60 days is reported

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in Table 3. Our data indicates a crucial decrease in Chla and Chlb content. Depending on the

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doses, Chla content decreased from 2.27±0.05 mg/g FM in the control plants to 2.25±0.06,

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2.08±0.03, 1.95±0.032, and 1.75±0.023mg/g FM respectively in plants exposed to C1, C2,

275

C3, and C4. The Chlb content also decreased from 1.62±0.14 mg/g FM in control to 1.1±0.13,

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0.95±0.04, 0.67±0.11, and 0.43±0.07 mg/g FM respectively in plants exposed to C1, C2, C3,

277

and C4. Chl a/b ratio increased, while total chlorophyll decreased, and the highest ratio was

278

recorded in plants exposed to C4 (4.01±0.31).

279

3.6. Effect of Ni on alfalfa oxidative stress biomarkers

280

Peroxidase activity increased in leaves after 60 days of exposure to increasing Ni

281

concentrations (Fig. 1A). The most important mean was observed in plants exposed to C4

282

(835.43±55.83 µmole/min mg of proteins against a control value of 24.19±1.96

283

µmole/min/mg of proteins). Similarly, for alfalfa roots, a significant difference was observed

284

for C1 (151.1±1.99 µmole/min/mg of proteins), C2 (255.27±5.65 µmole/min/mg of proteins),

285

C3 (191.16±2.15 µmol/min/mg of proteins), and C4 (176.64±8.21 µmol/min/mg of proteins)

12

286

compared to the control plants (65.37±2.98 µmol/min/mg of proteins) (Fig. 1A). Moreover,

287

our results showed a significant increase in the specific activity of GST (Fig. 1B).

288

Indeed, GST activity in plants exposed to C4 reached a value of 0.34±0.013 µmol/min/mg of

289

proteins. However, in roots, Ni treatment led to a strong activity at C3, with a value of

290

0.555±0.005 µmol/min/mg of proteins. Ni-induced oxidative damage on membranes was

291

evaluated by measuring changes in lipid peroxidation by quantifying the MDA formation in

292

the plants’ leaves and roots. Ni toxicity caused a significant increase in TBAR’s content in

293

leaves and roots (Fig. 1C). In leaves, the highest MDA content was observed for the

294

concentration C4 (500 mg/kg), with a value of 11.87±0.87 nmol TBAR’s/g FW. Similarly,

295

lipid peroxidation was enhanced in root parts along with increased Ni concentration, where

296

we recorded 5.49±0.26 for C1, 6.0±0.76 for C2, 11.31±0.47 for C3, and 13.74±0.58 nmol

297

TBAR’s/g FW for C4.

298

3.7. Effect of Ni on gene expression

299

Transcriptomic data (Fig. 2) for Prx1C, GST, and PCs revealed a significant increase in the

300

mRNA levels of all target genes. In the case of Prx1C, the most significant increase in

301

expression level was observed in the case of C4 and C2, in shoots and roots respectively.

302

Moreover, this expression was more significant in roots as compared to shoots, in the case of

303

C1 and C2. For GST expression level, the most significant upregulation was observed in

304

plants exposed to C4 and C3 in shoots where values reached 2.97±0.19 and 3.74±0.24 fold

305

respectively. For PCs, the gene expression was most significant in roots than in shoots and the

306

most significant value was observed in plants exposed to C4, being 4.82±0.28 fold.

307

3.8. Analysis of the evolution of alfalfa response after treatment with different Ni

308

concentrations

309

Evolution of alfalfa plant response after exposure to different Ni concentrations is shown as a

310

PCA biplot (Fig. 3). The first and second axes of the PCA explain, respectively, 85.61% and

13

311

7.47% of the variance. Results showed a strong separation between control plants and those

312

exposed to C3 and C4. Moreover, plants contaminated with C4 are mostly characterized by

313

high peroxidase and GST activity and pronounced Ni accumulation in shoots and roots.

314

Moreover, the correlation matrix (Fig. 4) shows that the concentration of Ni in soils is

315

positively and highly correlated with the concentration of Ni in shoots and roots of the alfalfa

316

plant as well as the augmentation of the activities of GST, peroxidase, and MDA in alfalfa

317

plant (root and shoot). In addition, the concentrations of Ni in the soil, shoot, and root are

318

negatively correlated with the content of chlorophyll a/b in plants.

319

4. Discussion

320

Phytoremediation is a sustainable technique involving plants, which helps to remove heavy

321

metals from contaminated soils (Peuke and Rennenberg, 2005; Ali et al., 2013; Hattab et al.,

322

2016). In this present investigation, we used the alfalfa plant (Medicago sativa) to assess its

323

potential for phytoremediation. For the purpose, biochemical, physiological, and

324

transcriptomic responses were assessed after60 days of exposure to four Ni concentrations

325

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

326

As hypothesized, alfalfa plants revealed an excellent capacity for massive metal

327

accumulation. Moreover, the results of Gardea-Torresdey et al. (1996) showed that more than

328

90% of Ni was removed from contaminated soil by alfalfa plants. Also, they preferentially

329

accumulated Ni in roots than in shoots with TF <1. These results are in agreement with

330

numerous works which have assessed the phytoremediation potential of other plants such as

331

Eruca sativa (Kamran et al., 2016), cotton (Khaliq et al., 2016), Medicago lupulina (Amer et

332

al., 2013), and Indian mustard (Ansari et al., 2015). Page and Feller (2005) and Seregin et al.

333

(2003) indicated that Ni is a highly mobile metal in plants and is mostly accumulated in roots.

334

This may be because roots could develop a barrier against toxic elements and could provide a

335

wide surface to absorb and accumulate heavy metals (Hall, 2002; Harada et al., 2010). 14

336

Conversely, the BCF values of all plants increased with Ni concentrations. According to Zhi-

337

xin et al. (2007), BCF can indicate the ability of alfalfa plants to accumulate heavy metals,

338

depending on numerous factors such as heavy metals concentration, the accumulative ability

339

of plants, and environmental properties. Additionally, the phytoextraction efficiency (PEE) of

340

Medicago Sativa increased significantly with different Ni concentrations. Similar results were

341

observed in alfalfa plants exposed to heavy metals such as Ni (Gardea-Torresdey et al., 1999),

342

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

343

et al., 2014).

344

Conversely, Amer et al. (2013) reported that Ni is an essential element for Medicago lupulina

345

at low concentrations, but becomes toxic at high concentrations. However, our results

346

revealed that there were no morphological or agronomical diseases in alfalfa plants after 60

347

days of exposure, except for a slight decrease in the growth of shoots and roots. Pandey and

348

Sharma (2002) have noted that the toxic effects of Ni are manifested by chlorosis, necrosis,

349

and wilting. Evidently, alfalfa plants tolerate high concentrations of Ni. The slight reduction

350

in the plants’ weight and length could be explained by the fact that Ni affected fundamental

351

processes such as photosynthesis and the transport of mineral nutrition, as proved by

352

numerous authors (Samarakoon and Rauser, 1979; Gajewska et al., 2009; Hussain et al.,

353

2013). Recently, Nazir et al. (2016) showed that Ni decreased the absorption of nitrogen (N),

354

phosphorus (P), and potassium (K) concentrations in rice plants. Moreover, Peralta et al.

355

(2001) reported that the fresh weight of Medicago sativa shoots and roots showed a

356

significant decline when exposed to 40mg/kg of Ni. In another previous work, Gajewska et al.

357

(2006) explained the decrease in fresh weight by the decline in water content under metallic

358

stress. Moreover, the growth inhibition in plants could be the result of a general metabolic

359

disorder and direct inhibition of cell division (Seregin and Kozhevnikova, 2006). Thus, in

360

shoots the dry matter was reduced for different Ni concentrations compared to control

15

361

condition, in concordance with Guo et al.’s (2010) results, which indicated that Ni had

362

reduced fresh and dry matter of soybean and maize. In fact, Ni can disturb numerous

363

mechanisms in plants by disrupting the mitotic division and cell elongation (Robertson and

364

Meakin, 1980; Powell et al., 1986; Serigin et al., 2001; Guo et al., 2010).

365

Chlorophyll content in Medicago sativa plants was significantly affected and decreased by

366

rising Ni concentrations. Furthermore, several studies have proved that heavy metals are a

367

potent inhibitor of Chl synthesis (Greger and Ogren, 1991; Bajguz and Hayat, 2009; Carrasco-

368

Gill et al., 2012; Kamaran et al., 2016). Besides, the reduction of Chl content could be a

369

result of a disruption in pigment synthesis (Somashekaraiah et al., 1992), a modification in

370

chloroplasts’ structure, and an inhibition of Calvin cycling enzymes. Furthermore, heavy

371

metals could lead to stomatal closure and eventually to a deficit of CO2 (Seregin et al., 2001).

372

Therefore, in order to assess the impact of Ni on the oxidative status of Medicago sativa,

373

peroxidase and GST activities were evaluated in addition to MDA content. Results showed

374

that lipid peroxidation increased significantly in plants exposed to different concentrations of

375

Ni, especially in roots. In this context, growth inhibition could be explained by cell membrane

376

destruction through lipid peroxidation (Baccouch et al., 2001). Our results are in concordance

377

with numerous studies which found a crucial increase in MDA in different contaminated

378

plants (Pandolfini et al., 1992; Madhava Rao and Sresty, 2000; Gonzálezet al., 2015).

379

Moreover, our results showed that peroxidase activity increased significantly in plants

380

exposed to Ni. A similar result was observed in Zea Mays exposed to Ni (Baccouch et al.,

381

1998). Other works have also found that Cd, Cu, Cr, and Hg could enhance peroxidase

382

activity in plants (Cho and Park, 2000; Kuo and Kao, 2004; Hattab et al., 2016). The

383

expression of Prx1C gene also increased, relative to the elevated Ni concentrations. This is in

384

agreement with the accumulation of Ni in alfalfa plants, indicating the key role of this gene in

385

the antioxidant response of plants. 16

386

The correlation matrix as well as the PCI analysis clearly indicated interconnections between

387

the study parameters in the plants’ tissues. Overall, the statistical analysis underlined that Ni

388

loads in the different plant tissues were positively correlated with the cellular alteration

389

termed as MDA accumulation. Indeed, this parameter, which reflects a higher cytotoxicity

390

(Mithofer et al., 2004), is an indication of cell alteration that may explain tissue and plant

391

growth decrease.

392

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

393

alignment with several works dealing with the impact of heavy metal pollution on different

394

species of plants (Dixit et al., 2001). Additionally, Choi et al. (2008) revealed that GST

395

played a fundamental role in plants against oxidative stress induced by metals. GST could

396

also modulate oxidative stress through numerous processes such as the translocation of lipid

397

peroxidation products, xenobiotics, and other secondary metabolites to the vacuole (Marrs and

398

Walbot, 1997; Marrs, 1996; Edwards and Dixon, 2004). In fact, GSTs are a superfamily of

399

enzymes, principally known for their role in detoxification reactions. Different classes of

400

GSTs have been used to develop plants with an improved detoxification mechanism (Kumar

401

et al., 2013). Furthermore, Wu et al. (2019) suggested that plants’ resistance to heavy metals

402

is clearly linked to the efficiency of GST in the detoxification process and the key role that it

403

may play in anti-oxidative stress. As shown in Fig. 4, our data clearly indicate a negative

404

correlation between GST/peroxydase activities and Chl content. This may act in favor of the

405

impairment of Chl production; consequently, an increase of the ROS production may be

406

counterbalanced by the antioxidant enzymes’ activities. Maiti et al. (2012) demonstrated that

407

overproduction of ROS in plants under metal contamination could degrade the permeability of

408

biological membrane structure facilitating the absorption of Ni. We also reported that GST

409

gene expression was induced in shoots and roots of Ni-treated plants. It is possible that a

17

410

specific activation of GST activity detected in alfalfa plants could be involved in preventing

411

cell damage induced by low concentrations of Ni (Nepovim et al., 2004).

412

Phytochelatins detoxify Ni and other heavy metals by their sequestration in plants such as

413

Ceratophyllum demersum L. (Shukla et al., 2012) and Azolla filiculoides (Talebi et al., 2019).

414

In this study, the highest expression of PCs genes was detected in shoots and roots of alfalfa

415

plants treated with C4 (500mg/kg) after 60 days. This is in consonance with numerous works

416

which have assessed the effect of heavy metals like Cd, Zn, Cu, Ag, Hg, and Pb on plants, and

417

this could explain the important role of PCs in the phytoremediation of trace elements in soils

418

(Talebi et al., 2019; Cobbett and Goldsbrough, 2002). Similar behavior was observed by

419

Hattab et al. (2016), who found that alfalfa plants exposed to Pb (100mg/kg) presented an

420

increase in PC levels. Seth (2012) showed more thoroughly that PCs have a significant

421

potential for heavy metal accumulation, indicating their protective role in plants. Sobrino-

422

Plata et al. (2009) showed that PCs are possibly involved in the protective effects in alfalfa

423

plants treated with low concentrations of Hg. Furthermore, it has been reported that Ni is able

424

to induce PCs’ gene expression in numerous species such as Thlaspi japonicum and Nicotiana

425

tabacum (Mizuno et al., 2003; Nakazawa et al., 2001). From a physiological point of view,

426

PCs could bind heavy metals present in cells in order to reduce their toxicity (Yousefi et al.,

427

2018; Luo et al., 2016). In addition, Nuzhat et al. (2019) indicated that Ni binding unfolds in

428

the cytosol, leading to the formation of a Ni-PCs complex which will be transferred to

429

vacuoles. For this reason, PCs represent a potent biomarker for the detection of Ni

430

accumulation in plants (Javed et al., 2019). These results reported that PCs are possibly

431

involved in cellular modification under metallic stress (Cobbett, 2000; Jozefczak, 2014),

432

which may explain the upregulation of PCs genes. Moreover, the obtained results show an

433

important synchronization between PCs’ synthesis and the antioxidant system in alfalfa plants

434

under Ni stress. 18

435

5. Conclusion

436

The present study evaluated the response of the alfalfa plant to increasing Ni concentrations,

437

in order to understand its capabilities in soil decontamination. In fact, this work demonstrated

438

that the alfalfa plant could be useful in the phytoremediation of heavy metal contaminated

439

soils. Furthermore, the results highlighted that PCs’ production associated with the

440

antioxidant enzymes plays an important role in the protection of plants against ROS

441

production under metallic stress.

442

Acknowledgements

443

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

444

de la Recherche Scientifique; UR04A6R05. Biochimie et Toxicologie Environnementale»

445

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831

832

833 834 835

29

Table 1 Nickel content in soil (ppm), uptake by alfalfa shootsand roots, bioconcentration factor (BCF), translocation factor (TF) and phytoextraction efficiency (PEE) of alfalfa’s plants after 60 days of exposure to: Control (0mg/kg), C1 (50mg/kg), C2 (150mg/kg), C3 (250mg/kg), C4 (500mg/kg).

Treatment (mg/kg) control C1 C2 C3 C4

Ni soil

Ni shoot

Ni root

BCF

TF

PEE(%)

11.53±0.26a 19.64±0.37b* 39.32±1.51c* 66.14±10.79d* 112.51±1.50e*

0.61±0.04a 1.96±0.26 b* 9.97±1.00 c* 11.68±0.50d* 23.65±2.14 e*

1.58±0.17a 8.92±0.21b* 22.64±0.93c* 32.84±0.15d* 75.20±0.17e*

0.19 ±0.001a 0.55±0.015b* 0.67±0.035c* 0.82±0.044d* 0.87±0.007e*

0.29±0.0014a 0.38±0.021b* 0.44±0.005c* 0.35±0.021d* 0.31±0.01e*

5.42 ±0.004a 12.51±0.012b* 14.63±0.05c* 9.19±0.14d* 8.12 ±0.094e*

* Statistically significant in comparison to control soil; the different letters denote significant differences between treatment at p<0.05.

Table 2 Effect of Ni on alfalfa’s shoots and roots lengths(cm), weight (g) and dry matter (%) after 60 days of exposure to: Control (0mg/kg), C1 (50mg/kg), C2 (150mg/kg), C3 (250mg/kg) and C4 (500mg/kg). Treatment (mg/kg)

Shoots length (cm)

Roots length (cm)

Shoot weight (g)

Roots weight (g)

DM (%)

Control

31 ±0.81a

31±1.154a

17±1a

11.25±0.95a

41.76±1.2a

28.44±1.77a

C1

25.75±0.5b*

25.5±0.57b*

13±1b*

9.33±0.50b*

39.23±1a

26.78±1.78a

C2

24.75±0.95b*

24.75±0.95b*

10.66±0.70c*

7.66±0.57c*

37.96±0.7 b*

24.76±1.28b*

C3

23.25±0.44b*

23 ±1b*

8±0.81d*

5.33±0.54d*

36.25±0.81c*

22.5±1.47c*

C4

20.75±0.57c*

21±0.95c*

5± 1e*

3.66± 0.56e*

34 ± 1.1d*

21 ±1.38d*

* Statistically significant in comparison to control soil; the different letters denote significant differences between treatment at p<0.05.

Shoots

DM (%)

Roots

Table 3 Effect of Ni on alfalfa’s chlorophyll content after 60 days of exposure to: Control (0mg/kg), C1 (50mg/kg), C2 (150mg/kg)., C3 (250mg/kg) and C4 (500mg/kg).

Treatment (mg/kg) Control

Chla (mg/g FM) 2.27±0.05a

Chlb (mg/g FM) 1.62±0.14a

Chl a+b (mg/g FM) 3.89±0.19a

1.39±0.34a

C1

2.25±0.06b*

1.1±0.13b*

3.36±0.18b*

2.03±0.51b*

C2 C3

2.08±0.03c* 1.95±0.032d*

0.95±0.04c* 0.67±0.11d*

3.04±0.07c* 2.63±0.15d*

2.17±0.64b* 2.88±0.26 b*

C4

1.75±0.023e*

0.43±0.07e*

2.18±0.09e*

4.01±0.31c*

*Statistically significant in comparison to control soil; the different letters denote significant differences between treatment at p<0.05.

Chl (a/b)

A

B

C

250

*

200

*

150

*

100 50

µmol/min/mg proteins

0

C1

C2

C3

*

0.25

*

*

0.2 0.15 0.1 0.05

C4

800 700 600 500 400 300 200

*

100

*

*

0

C1

C2

0.6

µmol/min/mg proteins

900

C3

C1

C2

C3

C4

*

12

*

10

*

*

8 6 4 2

*

0.5

*

C1

C2

C3

C4

16

*

0.4

Control

C4

*

0.3 0.2 0.1

*

14

*

12 10

*

8

*

6 4 2 0

0

Control

14

0

T

*

1000

µmol/min/mg proteins

0.3

0

Control

Roots

*

0.35

TBAR's (Content nmol /g FW)

µmol/min/mg proteins

Leaves

*

TBAR's (Content nmol /g FW)

0.4 300

Control

C1

C2

C3

C4

Control

C1

C2

C3

Fig. 1. The effect of metal exposure on peroxidase activity (A), GST activity (B) (µg/mn/mg protein) and MDA accumulation TBAR's (Content nmol /g FW) (C) in leaves and roots of alfalfa plants exposed to nickel: C1, C2, C3 and C4 concentrations and grown under control conditions for 60 days. *: Statistically significant differences at p < 0.005 in comparison with control.

C4

A

B Control

C1

C2

C3

C4

6

Control

C1

C2

C3

C4

5

*

4

* **

*

3.5 3

2.5

5

*

*

**

2

*

* *

1.5 1

Gene expression level

Gene expression level

4.5

4

**

* *

* *

3 2

*

* *

*

**

1

0.5 0 Prx1C

GST

PCs

0 Prx1C

GST

PCs

Fig. 2. Quantitative real time PCR expression-analysis of Prx1C, GST and PCs related genes of M. sativa shoots (A) and roots (B) treated with Ni (0, 50, 150, 250 and 500 mg/kg) during 60 days. Values were normalized against EF1-a and tubulin (used as a housekeeping genes) and represent the mean mRNA expression value ±SD (n = 3) relative to those of the control. *: significant up-regulation

A

B

Axis 2 : 7.47 %

PeroxidaseR

GSTR MDAS GSTS Nishoot MDAR Nisoil Niroot

Chlora

Axis 1 : 85.61 % Chlorb

PeroxidaseS

Fig. 3. PCA Biplot of plant response to the different Nickel concentration used in this study. The plot of the parameter tested (a) and the plot of the four concentrations tested (b) are represented. Sample name: control soil: red ellipses, Concentration 1: orange ellipses, Concentration 2: purple ellipses, Concentration 3: cyan ellipses, Concentration 4: blue ellipses.

GSTR

PeroxidaseR

Niroot

Nisoil

Nishoot

MDAR

GSTS

MDAS

PeroxidaseS

Chlorb

Chlora

1

Chlora 0.8

Chlorb

0.6

PeroxidaseS

0.4

MDAS

0.2

GSTS MDAR

0

Nishoot

-0.2

Nisoil

-0.4

Niroot

-0.6

PeroxidaseR -0.8

GSTR -1

Fig. 4. Correlation Matrix between the different parameters studied after Ni contamination. The parameters are: Chlora: Chlorophyll a, Chlora: Chlorophyll b, carote: Carotenoides, CATS: Activity of Catalase in shoot, MDAS: malondialdehydes in shoot, GSTS; Activity of Glutathion-S-Transferase in shoot, MDAR: malondialdehydes in roots, CATR: Activity of Catalase in root, GSTR: Activity of Glutathion-STransferase in root

- Medicago sativa accumulated a high rate of Nickel in roots than in shoots

- Peroxidase and GST had been induced to protect cells from oxidative damages - The PCs gene expression was enhanced in response to the different nickel concentrations suggesting their important roles in the detoxification of Ni in alfalfa plants

Sondes HELAOUI

Iteb BOUGHATTAS Sabrine HATTAB Marouane MKHININI Vanessa ALPHONSE Alexandre LIVET Nourreddine BOUSSERHINE Mohamed BANNI

Contribution Conceptualization, Methodology, Investigation, Preparation, creation and/or presentation of the published work, specifically writing the initial draft (including substantive translation) Formal analysis, Writing - Review & Editing Resources Software, Writing - Review & Editing Investigation, Validation Investigation, Validation Resources, Supervision, Writing - Review & Editing Conceptualization, Resources, Supervision, Writing - Review & Editing

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: