Belkadhi Et Al.,R2

OMICS: A Journal of Integrative Biology
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OMICS: A Journal of Integrative Biology: http://mc.manuscriptcentral.com/omics
Salicylic acid improves root antioxidant defense system and

total antioxidant capacities of flax subjected to cadmium
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Manuscript ID:
OMI-2013-0030.R2
Abiotic
Manuscript Type:
Date Submitted by the Author:
belkadhi, aicha; Facult des Sciences de Tunis, Dpartement de Biologie,

Unit de Recherche de Physiologie et Biochimie de la tolrance des plantes
aux contraintes abiotiques,; Institute of Sustainable Agriculture, Spanish
Council for Scientific Research (CSIC), Department of Agronomy and Plant
Breeding
De Haro-Bailon, Antonio; Institute of Sustainable Agriculture, Spanish
Council for Scientific Research (CSIC), Department of Agronomy and Plant
Breeding
Soengas, Pilar; Misin Biolgica de Galicia, Spanish Council for Scientific
Research, Department of Plant Genetics
Obregn, Sara; Institute of Sustainable Agriculture, Spanish Council for
Scientific Research (CSIC), Department of Agronomy and Plant Breeding
Cartea, Mara Elena; Misin Biolgica de Galicia, Spanish Council for
Scientific Research, Department of Plant Genetics
Djebali, Wahbi; Facult des Sciences de Tunis, Dpartement de Biologie,
aux contraintes abiotiques
Chabi, Wided; Facult des Sciences de Tunis, Dpartement de Biologie,
aux contraintes abiotiques
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Complete List of Authors:
06-Apr-2013
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Integrative Biology
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Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
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Title: Salicylic acid improves root antioxidant defense system and total
antioxidant capacities of flax subjected to cadmium
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Authors: A. Belkadhi, A. De Haro, P. Soengas, S. Obregon, M. E. Cartea, W. Djebali and W.
Chabi
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Affiliations:
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Acha Belkadhi,
a. Dpartement de Biologie, Unit de Recherche de Physiologie et Biochimie de la tolrance
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des plantes aux contraintes abiotiques, Facult des Sciences de Tunis, Campus Universitaire,
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1060 Tunis, Tunisia
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b. Department of Agronomy and Plant Breeding, Institute of Sustainable Agriculture, Spanish
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Council for Scientific Research (CSIC), Alameda del Obispo s/n, 14080 Crdoba, Spain
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c. Department of Plant Genetics, Misin Biolgica de Galicia, Spanish Council for Scientific
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Research (CSIC), Apartado 28, E-36080 Pontevedra, Spain
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e-mail: aicha585@yahoo.ca
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Antonio De Haro,
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e-mail: adeharobailon@ias.csic.es
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Pilar Soengas,
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e-mail: psoengas@mbg.csic.es
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Sara Obregon,
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e-mail: obcas1979@yahoo.es
Mara Elena Cartea,

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e-mail: ecartea@mbg.cesga.es
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Wahbi Djebali,
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1060 Tunis, Tunisia
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e-mail: wahbi.djebali@fst.rnu.tn
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Wided Chabi,
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a .Dpartement de Biologie, Unit de Recherche de Physiologie et Biochimie de la tolrance
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1060 Tunis, Tunisia
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e-mail: wided.chaibi@fst.rnu.tn
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Corresponding author;
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Acha Belkadhi,
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1060 Tunis, Tunisia
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e-mail: aicha585@yahoo.ca
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Fax: +216 71 885 480

Phone: +216 20 964 826
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Abstract
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Cadmium (Cd) disrupts the normal growth and development of plants depending on their
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tolerance to this toxic element. The present study was focused on the impacts of exogenous
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salicylic acid (SA) on the response and regulation of the antioxidant defense system and
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membrane lipids to 16-d-old flax plantlets under Cd stress. Exposure of flax to high Cd
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concentrations led to strong inhibition of root growth and enhanced lipid peroxides,
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membrane permeability, protein oxidation and hydrogen peroxide (H2O2) production to
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varying degrees. Concomitantly, activities of the antioxidant enzymes catalase (CAT, EC
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1.11.1.6), guacol peroxydase (GPX, EC 1.11.1.7), ascorbate peroxydase (APX, EC 1.11.1.11)
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and superoxide dismutase (SOD, EC 1.15.1.1) and the total antioxidant capacities (2,2-
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diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and ferric reducing antioxidant power
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(FRAP)) were significantly altered by Cd. In contrast, exogenous SA greatly reduced the toxic
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effects of Cd on the root growth, antioxidant system and membrane lipid content. The Cd-
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treated plantlets pre-soaked with SA exhibited less lipid and protein oxidation and membrane
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alteration, as well as a high level of total antioxidant capacities and increased activities of
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antioxidant enzymes except of CAT. These results may suggest that SA plays an important
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role in triggering the root antioxidant system, thereby preventing membrane damage as well
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as the denaturation of its components.
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Key words: salicylic acid, cadmium, hydrogen peroxide, roots, membrane fatty acids,
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antioxidant activities.
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Abbreviations: Cd cadmium; SA salicylic acid; H2O2 hydrogen peroxide; CAT
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catalase; GPX guacol peroxydase; APX ascorbate peroxydase; SOD superoxide
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dismutase (SOD); MDA malondialdehyde; DPPH 2,2-diphenyl-1-picrylhydrazyl; FRAP
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ferric reducing antioxidant power; DW dry weight; TL total lipid.
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Introduction
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Roots are the first part of the plant to respond to heavy metal stress. Cadmium (Cd) stress may
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disturb plant nutrient balance (Belkhadi et al. 2010, Douchiche et al. 2010), inhibit growth
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(Hdiji et al. 2010, Gallego et al. 2012) and generate oxidative stress (Tams et al. 2007). It is
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well known that Cd is a non-redoxactive metal which does not trigger the direct formation of
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hydroxyl radicals (OH.) via Haber-Weiss and Fenton reactions but indirectly, by disturbing
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the chloroplast and mitochondria electron transport rates (Hendry et al. 1992) and/or by
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inducing loss in enzymatic and non enzymatic antioxidative system capacities (Grato et al.
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2005, Rodriguez-Serrano et al. 2009, Gallego et al. 2012).
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The oxidative stress induced by Cd can also be correlated with damage to membrane
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lipids (Djebali et al. 2005; Belkhadi et al. 2010), nucleic acids (Apel and Hirt 2004, Gichner
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et al. 2004) and proteins (Djebali et al. 2008, Douchiche et al. 2010). In response to reactive
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oxygen species (ROS), plants can induce a succession of detoxification reactions catalyzed by
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antioxidative enzymes (Grato et al. 2012). However, in comparison of some species, Cd
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treatment can be seen to have opposite effects on certain antioxidative enzymes. In Bacopa
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monnieri L., Cd induced elevated superoxide dismutase (SOD, EC 1.15.1.1), guacol
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peroxydase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione
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reductase (GR, EC 1.6.4.2) but decreased catalase (CAT, EC 1.11.1.6) activities (Mishra et al.
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2006). Severe diminution of peroxidase (POX, EC 1.11.1.7), and CAT activities were found
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in Oryza sativa roots, while in contrast to B. monnieri, SOD activity was also diminished
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(Guo et al. 2007). An increase in CAT activity under Cd stress has been found in other species
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(Balestrasse et al. 2001, Smeets et al. 2008).
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Salicylic acid (SA) is a biomolecule that may be involved in several cell processes
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under normal and stress conditions. Under biotic and abiotic stress conditions, increasing
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evidence indicates that SA, a phytohormone which acts at low concentrations, is produced and
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accumulates inside cells to function as a signaling molecule in plants (Choi et al. 2012, Hao et
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al. 2012). SA is involved in gene expression in response to multiple stresses (Durrant and
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Dong 2004, Fobert and Despres 2005, Foyer and Noctor 2005). When applied exogenously at
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appropriate concentrations, SA was found to enhance the efficiency of antioxidant system in
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plants (Radwan 2012, Saruhan et al. 2012). Furthermore, SA application has been reported to
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alleviate the symptoms of Cd toxicity observed in plants (Zawoznik et al. 2007; Zhang and
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Chen 2011). Previous studies have shown that SA treatment mitigates the oxidative stress
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generated by Cd in different plant species like barley (Metwally et al. 2003), soybean (Drazic
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and Mihailovic 2005), rice (Guo et al. 2007), maize (Krantev et al. 2008), pea (Popova et al.
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2009) and hemp (Shi et al. 2009); however, little paper has been reported about the role of SA
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in the increase of total antioxidant capacities under Cd stress. In the present study, we aim to
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characterize the mechanism by which SA protects plants against lipid and protein oxidative
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damage caused by Cd exposure.characterized that SA-induced protection against lipid and
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protein oxidative damage due to Cd exposure is mediated through hydrogen peroxide (H2O2).
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This is supported by the observations that 8-h SA pretreatment resulted in an increase in GPX,
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APX and SOD activities, but not CAT activity, and hence an enhancement of total antioxidant
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capacities of Cd-treated flax roots. In roots, Zhang et al. (2011) have demonstrated that SA-
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induced Cd tolerances in Phaseolus aureus and Vicia sativa were related to increases in
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symplastic and apoplastic antioxidant enzyme activities. Recent study also reported that the
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priming of seeds with lower concentrations of SA, before sowing, lowered the elevated levels
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of ROS due to Cd exposure and enhanced the activities of various antioxidant enzymes
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(CAT, GPX, GR and SOD) in Oryza sativa, thereby protecting the plants from oxidative burst
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(Panda and Patra 2007). However, contrary to this observation, Choudhury and Panda (2004)
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reported a decline in the activities of the antioxidant enzymes CAT, POX, SOD and GR in
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rice following the pre-treatment with SA.
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Taken together, this work provides novel avenues toward understanding the
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mechanisms of plant enhancement of tolerance to Cd-mediated oxidative stress by using SA
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priming event and its possible function in the restoration and maintenance of the cell
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membrane integrity in roots. To test this possibility, SA-related changes in hydrogen peroxide
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(H2O2) production, membrane integrity, antioxidant capacities and membrane lipid content
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and fatty acid profiles have been studied in roots of Cd-treated flax plantlets.
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Materials and methods
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Flax seeds (cv. Viking), were soaked for 8 h in SA solutions as previously shown in Belkhadi
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et al. (2010). After that they were germinated for four days at 25 C in the dark, uniform
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plantlets were transferred to a continuously aerated nutrient solution (pH 5.5) containing 1mM
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MgSO4, 2.5Mm Ca (NO3)2, 1mM KH2PO4, 2 mM KNO3, 2 mM NH4Cl, 50 mM EDTA Fe
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K, 30 mM H3BO3, 10 mM MnSO4, 1mM ZnSO4, 1mM CuSO4 and 30 mM (NH4)6Mo7O24.
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The nutrient solution was buffered with HCl/KOH and changed twice per week. After
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growing for 2 days, plantlets were subjected during 10 days to CdCl2 appropriately from
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moderate to high concentrations (50 to 100 M). Five replicates were in individual 6 l plastic
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beakers made for control and Cd treatments. Plantlets were grown in a growth chamber at a
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day/night cycle of 16 h/8 h, at 23 C/18 C, respectively, a relative humidity close to 75% and
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a light intensity of 200 mol photons m-2 s-1. Roots were then detached, washed with
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deionized water and immediately used or frozen at -80 C. Three independent culture
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experiments were performed.
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Roots of the harvested plantlets were washed carefully using distilled water to
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eliminate any contamination. Root diameter, surface area and volume were recorded by
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THSCSA Image Tool (IT) Version 3.0. Three plantlets from each replication of all treatments
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were selected for data collection.
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The level of lipid peroxidation in roots was determined as 2-thiobarbituric acid
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(TBA) reactive metabolites, chiefly malondialdehyde (MDA), as described previously by
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(Buege and Aust 1972). Membrane permeability of roots was determined as follows. Root
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tissue (0.1 g) was vibrated for 30 min in deionised water followed by measurement of
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conductivity of bathing medium (EC1). Then, the samples were boiled for 15 min and the
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final conductivity (EC2) of the medium was measured using a conductivity meter (Consort
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C832).
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Root samples were homogenized in 0.2 N perchloric acid (pH 7.5). The flax H2O2
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content in roots was determined as described by OKane et al. (1996).. Protein-bound
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carbonyl content was determined by using the dinitrophenylhydrazine (DNPH) method,
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according to Reznick and Packer (1994). The level of carbonyl groups was estimated using an
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extinction coefficient of 22 000 M-1 cm-1.
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Total SOD activity was assayed by monitoring the inhibition of the photochemical
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reduction of nitroblue tetrazolium (NBT) according to the method of Beauchamp and
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Fridovich (1971). One unit of SOD activity was defined as the amount of enzyme required to
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cause 50 % inhibition in the reduction of NBT as monitored at 560 nm. For the assay of APX
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activity, 2 mM ascorbate was added in the extraction medium. The reaction mixture
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containing 50 mM potassium phosphate (pH 7.0), 0.1% H2O2, 0.5 mM ascorbate (extinction
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coefficient 2.8 mM-1 cm-1)and root extract induced a linear decrease in absorbance at 290 nm
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for 25 s (Nakano and Asada 1981). GPX activity was measured according to Landberg and
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Greger (2002) following the change in absorption at 470 nm. CAT activity was determined by
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following the consumption of H2O2 (extinction coefficient 39.4 mM-1 cm-1) at 240 nm for 30 s
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(Aeby 1984). Soluble protein contents in enzyme extracts were determined by the method of
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Bradford (1976).
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For antioxidant potential analysis, extracts were prepared following Ferreres et al.
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(2007). 20 mg of powder material was suspended in 2 mL of Milli-Q water then boiled for 1 h
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at 100 C. Suspension was filtered and supernatant lyophilized (BETA 2-8 LD plus, Christ).
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The dry material was weighed and dissolved in Milli-Q water to reach a concentration of 10
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mg mL-1. Antioxidant potential of the samples was determined spectrophotometrically by
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monitoring the disappearance of radical DPPH accoding to the method of Brand-Williams et
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al. (1995). Readings were taken at 517 nm after 1 h of incubation at room temperature in a
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microplate reader (Spectra MR, DYNEX Technologies). After extracting subtracting the
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value of the blank from each sample, absorbance was plotted against concentration of
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samples, EC50 values (concentrations which produced 50% inhibition) were computed for
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each extract and were used to compare among samples. Antioxidant potential of the samples
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was also measured by the FRAP assay developed by Benzie et al. (1996). Readings were
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taken at 593 nm after 1 h at room temperature. Sample absorbance was plotted against
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concentration of samples and the concentration which produced an absorbance of 1.00 was
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computed and was used to compare among samples. For both assays, three replications were
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analyzed for each sample and standard prepared with different concentrations of Trolox
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were also measured. The results were expressed in mol Trolox g-1dry weight (DW).
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Lipids in roots were extracted according to Garcs and Mancha (1993) using 30 mg
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plant tissue. Fatty acids were identified by comparing the retention times of flax root methyl
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esters with those of known mixtures of standard fatty acids (Sigma) run on the same column
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under the same conditions.
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Statistical calculations were performed with SPSS-17 statistical software. Mean
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difference comparison among different treatments was done by ANOVA and Tukeys (HSD)
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test at a 0.05 probability level.
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Results
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When Cd was present in the nutrient solution, flax plantlets exhibited reduced root growth.
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Root dry weight, length, diameter, surface and volume decreased proportionally with
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increasing concentrations of Cd (Table 1). In contrast, pre-soaking with SA for 8 h led to an
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increase in the root growth parameters in Cd-dependent manner. As a result, already after 10
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days exposure to Cd, the whole root system appeared less short and thick (Fig.1).
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In roots, Cd treatments showed a significant increase of H2O2 content compared
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with control (Fig. 2A). It increased by about 37% over control at 50 M Cd and was further
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enhanced to nearly 62% more over control at 100 M Cd. However, upon the 8-h SA
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pretreatment, there was no significant decrease in the H2O2 content in flax roots compared to
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those with Cd only (Fig. 2A).
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The damage by Cd to membranes was investigated by monitoring MDA content and
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electrolyte leakage (Fig. 2B and C). Relative to the control, Cd-treated flax plantlets exhibited
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a higher rate of lipid peroxidation. However, it was observed that 8-h SA pretreatment
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exhibited a significant reduction of Cd-increased MDA content (Fig. 2B). Electrolyte leakage
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was also altered by the 8-h SA pretreatment, but the extent of change was not so great as for
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change in the MDA level (Fig. 2C). Only at 100 M Cd, the electrolyte leakage level in roots
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increased significantly by 1.6-fold as compared with the control. 8-h SA pretreatment

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counteracted the Cd-induced loss of membrane permeability. At the highest metal
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concentration, the most prominent effect was at 1000 M SA; a nearly 46% decrease was
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noticed (Fig. 2C). Significant increases in total protein content were observed between treated
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and control flax plantlets irrespective of the Cd concentration, (Fig. 2D). Within both Cd
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concentrations (50 and 100 M), 8-h SA pretreatment led to restoration of the protein content
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and that in a dose dependent way. Thus, at 100 M Cd-treated plantlets, SA (250 or 1000
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M) inhibited the amount of protein oxidation in roots by about 2.4- and 1.4-fold,
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respectively, compared to control (Fig. 2E).
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The effect of Cd on the activities of antioxidative enzymes (Fig. 3A-D) was
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significant in most Cd-exposed roots. CAT activity was significantly increased in Cd-treated
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roots while, 8-h SA pretreatment alone (1000 M) decreased it, and the effect was similar
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with the addition of Cd (50 or 100 M) (Fig. 3A). After 10 days of Cd stress, root GPX and
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APX showed significantly higher activities levels than control, especially at 100 M Cd. A
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significantly enhanced response in both enzymes activities occurred by 8-h SA pretreatment
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followed by Cd exposure (Fig. 3B and C). In contrast, SOD activity was suppressed by Cd
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addition in the medium. Cd treatment caused an increase of 28% and 50% upon exposure to
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50 and 100 M Cd, respectively (Fig. 3D). However, SA (250 or 1000 M) inhibited Cd-
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decreased activity of SOD (Fig. 3D).
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At 50 and 100 M Cd, the total antioxidant capacities increased with respect to the
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control plantlets (Fig. 4A and B). These Cd-induced increases were further improved by the
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8-h SA pretreatment but in a dose-dependent manner. In terms of 100 M Cd, the maximum
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values have been registered at 250 M SA for both analyzed tests (Fig. 4A and B).
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changed in Cd-treated roots and highly dependent on the metal concentration in the medium
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(Fig. 5). At 50 and 100 M Cd, TL content of roots was 40.5% and 79% reduced,
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respectively, compared to control. Only at the highest Cd dose did 8-h SA pretreatment
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significantly enhance TL content in roots of flax plantlets (Fig. 5). In controls, the fatty acids
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composition of TL in flax roots contained mostly linolenic (C18:3), linoleic (C18:2) and
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palmitic (C16:0) acids (about 87% of the total fatty acids). CdCl2 (50 and 100 M) showed a
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slight statistically significant increase in the degree of fatty acid unsaturation in the roots
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(Table 2). The impact of Cd on the fatty acid composition of root cell membranes became
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more evident at higher concentration. The SA-induced change in the lipid unsaturation was
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similar to that caused by Cd. Interestingly, when Cd was added in the medium (50 or 100
255
M), 8-h pretreatment with SA (250 M) significantly increased the total extent of fatty acid
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unsaturation (Table 2).
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Discussion
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In this work, we investigate the potential defense mechanisms enabling primed plantlets to
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overcome Cd-induced oxidative damage to proteins and membranes (oxidation, loss of
260
membrane fluidity and integrity and the degradation of its components). Flax roots exposed
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for 10 days to 50 and 100 M CdCl2 had reduced dry weight and the morphological
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characters such as surface, volume and diameter (Table 1). These alterations observed at the
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root level could be a consequence of Cd interference in plant metabolism (Liu et al. 2007), as
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well as modification in antioxidative enzyme activities (Rodriguez-Serrano et al. 2009).
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The results showed that Cd decreased the biomass and induced the generation of
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H2O2 (Fig. 2A) which agrees with previous reports by Schtzendbel and Polle (2002) for
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Scots pine, Ortega-Villasante et al. (2007) for alfalfa, Liu et al. (2008) for tomato, and
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Gonalves et al. (2009) for potato. Cd-increased H2O2 concentration led to lipid peroxidation,
269
causing membrane damage and leakage of electrolytes (Schtzendbel and Polle 2002).
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However, in this experiment, the reduction in biomass, induction of electrolyte leakage, lipid
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peroxidation and protein carbonylation were partially alleviated by applying SA to Cd-treated

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flax plantlets (Table 1 and Fig. 2B-E). This is linked to the SA- increased levels of GPX, APX
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and SOD activities under Cd stress (Fig. 3B, C and D).
274
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Superoxide radicals generated by oxidative metabolism were dismutated into H2O2
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and O2 by SOD which acts as a first line of defense response in planta (Grato et al. 2012,
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Radwan 2012). The increased SOD activity due to SA might be to protect biomolecules from
277
being attacked by superoxide radicals. It has been shown that exogenous SA application
278
resulted in the alleviation of Cd-induced ROS overproduction in Arabidopis thaliana (Zhang
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and Chen 2011) and Maize seedlings (Radwan 2012). Consequently, the lower level of lipid
280
peroxidation, and thus the lower degree of membrane damage, might result from the SA-
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increased activity of SOD enzyme in flax roots (Fig. 3D).
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Protein carbonylation may occur due to direct oxidation of amino acid side chains
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(e.g. proline, arginine, lysine and threonine) by ROS and/or by protein reactions with lipid
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peroxidation products, such as 4-hydroxy-2-nonenal (HNE). Numerous studies have shown
285
that Cd bioaccumulation lead to the oxidation of proteins in plant tissues (Romero-Puertas et
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al. 2002, Djebali et al. 2008, Douchiche et al. 2010) which is in agreement with our present
287
findings (Fig. 2E). On the other hand, SA-decrease of lipid peroxidation may partially
288
contribute to the Cd-alteration effect on protein content in roots (Fig. 2B). In fact, several
289
studies reported the increase in total protein content and formation of new proteins in pea due
290
to SA treatment (Katoch 2007, ag et al. 2009).
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metals; it is considered to play a critical role in metal tolerance of plants. The presence of
293
polyunsaturated fatty acids in the plasma membrane results in increased membrane
294
permeability to ions (Hanzel and Williams 1990). The depletion of unsaturated fatty acids
295
may also indicate lipid peroxidation elicited by Cd (Ben youssef et al. 2005; Djebali et al.
296
2005). In a study of pea roots, Hernndez and Cooke (1997) reported a similar decrease in the
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Plant membranes are the first functional structure to come into contact with toxic
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fatty acid saturation in response to Cd stress. SA acts in response to the Cd-induced changes
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in fatty acid saturation, i.e. the increased unsaturation in the roots (Table 2). Moreover, it can
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be assumed that the defensive role of SA against Cd toxicity might be credited to its function
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in regulating root membrane permeability.
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Other mechanisms involved in the prevention of Cd-induced membrane damage
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require the synthesis of antioxidant enzymes. In fact, an increase in antioxidant enzyme
303
activities after Cd treatment has been detected in Hordeum vulgare (Guo et al. 2004); O.
304
sativa (Hsu and Kao, 2004); Triticum aestivum (Khan et al. 2007); Brassica juncea (Mobin
305
and Khan 2007); Vigna mungo (Singh et al. 2008); C. arietinum (Hassan et al. 2008). In this
306
study, Cd affected SOD, CAT, APX and GPX activities in flax plantlets, with their impact
307
being higher at 100 M Cd (Fig. 3). In the roots, Cd, especially at this concentration, tended
308
to increase their activities (with the exception of SOD). The severe Cd-decrease in SOD
309
activity might be caused by the continuous competition between Zn and Cd for the same sites
310
in Cu/Zn SOD. Similarly, Rodriguez-Serrano et al. (2009) showed that Cd-decrease in Ca
311
lead to Cu/Zn SOD down-regulation which resulted in the overproduction of superoxide
312
radicals in Pisum sativum.
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However, our results showed that the 8-h SA pretreatment caused a significant
314
increase in GPX, APX and SOD, and a decrease in CAT activity in Cd-treated plantlets (Fig.
315
3A-D). In barley seedlings treated with Cd, Metwally et al. (2003) reported that total root
316
APX activity reflected the cytosolic isoforms and the Cd response was fully suppressed by
317
SA. Conversely, the same authors reported that CAT activity and expressional level decreased
318
upon SA pretreatment. According to Janda et al. (1999) a new peroxidase isoform was
319
detected in maize treated with SA even the total activity did not increase significantly.
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structures of root cells, because SOD is involved in the processes of lipid peroxidation
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The effect of SA on the activation of SOD may facilitate the integrity of membrane
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deactivation (Zenkov et al. 2001). But, the significance of catalase inhibition could be partly
323
due to the possible binding of SA to CAT. In fact, SA has proved capable of binding directly
324
to CAT enzyme (Chen et al. 1993), isolated from tobacco, inhibiting its activity (Conrath et
325
al. 1995). The in vitro SA catalase-inhibiting effect has also been demonstrated in many other
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plant species such as Arabidopsis, tomato, cucumber (Snchez-Casas and Klessig 1994) and
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tobacco (Horvth et al. 1998).
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Numerous studies showed that exogenous application of SA can influence the
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antioxidant capacity of plant cells (Janda et al. 2003, Ananieva et al. 2004, Radwan 2012).
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Besides, since adaptation to oxidative stress involves not only the regulation of the synthesis
331
and repair of proteins but also enhanced antioxidant capacity (Ananieva et al. 2004); two
332
different tests were used in this work to confirm the SA-induced changes in antioxidant
333
activities of Cd-treated plantlets (Fig.4A and B). In roots, Cd stress significantly increased
334
FRAP and DPPH-radical scavenging activity. This effect was improved by SA in Cd-stressed
335
plantlets.
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Conclusion
337
To summarize, it was demonstrated that the exogenous 8-h SA pretreatment may improve the
338
tolerance of flax roots to Cd-induced oxidative stress. Moreover, SA-enhanced antioxidant
339
enzymes SOD, APX and GPX maintained ROS such as superoxide radicals at minimum
340
levels with respect to 100 M Cd treatment. Therefore, SA-increase of the total antioxidant
341
capacities in Cd-treated flax roots avoids its deleterious effects in cell membranes. For these
342
reasons, we can conclude that due to Cd high toxicity and its potent free-radical scavenging
343
ability, SA-induced priming could be used as a potential preventive action taken to limit
344
oxidative damages.
345
Acknowledgments
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This research was supported by a grant from the Tunisian Ministry of Higher Education,
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Scientific Research and Technology. We gratefully acknowledge Dr. Michael Deyholos
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(University of Alberta, Canada) for his careful reading of the manuscript and Ms. Graldine
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Dambry (Service Recherche / Laboratoire Pathologie et Biologie du lin, France) for her
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gracious help to provide us the flax seeds.
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ie
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Landberg T, and Greger M (2002). Differences in oxidative stress in heavy metal resistant and
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514
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Liu XL, Zhang SZ, Shan XQ, and Christie P (2007). Combined toxicity of cadmium and
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Liu KL, Shen L, Wang JQ, and Sheng JP (2008). Rapid inactivation of chloroplastic ascorbate
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521
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/N
ly
522
ot
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Metwally A, Finkemeier I, Georgi M, and Dietz KJ (2003). Salicylic acid alleviates the
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cadmium toxicity in barley seedlings. Plant Physiol. 132, 272281.
525
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Mishra S, Srivastava S, Tripathi RD, Kumar R, Seth CS, and Gupta DK (2006). Lead
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529
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531
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532
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Nakano Y, and Asada K (1981). Hydrogen peroxide is scavenged by ascorbate-specific
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536
ie
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OKane D, Gill V, Boyd P, and Burdon R (1996). Chilling, oxidative stress and antioxidant
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Ortega-Villasante C, Hernandez LE, Rellan-Alvarez R, Del Campo FF, and Carpena-Ruiz RO
541
(2007). Rapid alteration of cellular redox homeostasis upon exposure to cadmium and
542
mercury in alfalfa seedlings. New Phytol. 176, 96107.
ly
543
/N
544
Panda SK, and Patra HK (2007). Effect of salicylic acid potentiates cadmium-induced
545
oxidative damage in Oryza sativa L. Leaves. Acta Physiol. Plant. 29, 567575.
ot
546
547
Popova LP, Maslenkova LT, Yordanova RY, Ivanova AP, Krantev AP, Szalai G, and Janda T
548
(2009). Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings.
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Plant Physiol. Biochem. 47, 224231.
fo
550
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Radwan DEM (2012). Salicylic acid induced alleviation of oxidative stress caused by
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553
23
bu
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ly
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/N
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569
tolerance and delays leaf rolling by inducing antioxidant systems in maize genotypes. Acta
570
Physiol. Plant. 34, 97-106.
ot
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577
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578
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584
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589
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/N
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ot
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Stress). Moscow., Nauka, pp. 280.
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598
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599
salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in
600
root apoplasts of Phaseolus aureus and Vicia sativa. Plant Cell Rep. 30, 14751483.
601
25
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Fo
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Zhang WN, and Chen WL (2011). Role of salicylic acid in alleviating photochemical damage
603
and autophagic cell death induction of cadmium stress in Arabidopsis thaliana. Photochem.
604
Photobiol. sci. 10, 947-955.
605
606
608
609
611
613
ly
On
612
610
ie
ev
607
rR
ee
614
/N
615
616
ot
617
618
fo
619
rD
620
621
is
622
623
624
26
bu
tri
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Page 27 of 35
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rR
ee
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Tables
627
628
629
Table 1
Effect of SA on the root growth of 16-d-old Cd-treated flax plantlets.
630
SA
(M)
Cd
(M)
ie
ev
Root
Length (cm)
Diameter
(mm)
Surface
2
-1
(dm plant )
Volume
3
-1
(cm plant )
2.44 0.15 c
14.16 1.19 d
19.34 1.17d
Dry Weight
-1
(mg plant )
5.51 0.27 e
31.78 1.81 bc
50
4.34 0.15 bc
16.09 0.8 e
1.33 0.03 ab
3.83 0.21 b
2.70 0.3 b
100
3.21 0.13 a
12.05 1.13 e
1.04 0.02 a
1.63 0.06 a
1.19 0.05 a
250
5.21 0.1 cd
45.69 2.3 cd
3.16 0.2 cd
13.39 0.7 d
51.78 2.3 g
250
50
5.55 0.2 ef
31.82 1.5 bc
2.69 0.11 b
7.08 0.26 c
21.38 1.5 e
250
100
4.26 0.11 ab
18.23 0.13 a
1.16 0.1 a
2.94 0.02 ab
3.03 0.3 bc
1000
5.59 0.21 ef
40.90 1.7 d
3.57 0.25 d
16.01 1.22 e
46.88 2.1f
1000
50
4.75 0.25 cd
25.80 2.31 b
1.41 0.03 ab
4.13 0.13 bc
7.37 0.24 c
1000
100
3.75 0.07 a
14.20 0.7 e
1.28 0.13 ab
1.81 0.02 a
2.03 0.19 ab
ot
Data are means of three independent experiments (SE).
/N
632
ly
On
631
Means with different letters indicate statistically different results at p 0.05, according to Tukeys (HSD) test.
633
634
fo
635
rD
636
637
638
is
639
640
641
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Page 28 of 35
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Table 2
644
Effect of SA on fatty acid of total lipids composition in root of 16-d-old flax plantlets
645
subjected during 10 days to Cd toxicity.
Fatty acids
(%TL)
SA (M) Cd (M)
Roots
C16:1
C18:0
ab
Tr
5.96 0.60
Tr
6.88 0.34
ab
Tr
bc
Tr
6.04 0.90
Tr
7.63 0.13
ab
Tr
8.11 0.06
Tr
6.02 0.67
Tr
7.22 0.12
Tr
8.02 0.35
C16:0
50
29.75 0.15
100
27.13 0.83
250
30.02 0.73
250
50
27.45 1.00
250
100
28.43 0.73
1000
29.72 0.15
1000
50
30.07 1.16
1000
100
28.41 0.45
ab
ab
6.62 0.82
ab
8.19 0.29
6.74 0.24
bc
8.15 0.32
43.78 0.58
6.21 0.77
7.73 0.04
bc
bc
8.14 0.09
bc
42.08 0.18
42.51 0.54
43.55 2.29
5.85 0.45
bc
7.52 0.19
bc
bc
8.09 0.28
bc
41.97 1.30
39.87 0.47
44.66 3.20
ab
13.40 0.33
14.55 0.52
ab
13.94 0.62
14.18 0.12
15.21 2.48
15.43 0.12
ab
13.74 2.23
/N
ly
30.22 1.68
C18:3 Uns Unsaturation
C18:2
On
C18:1
42.52 0.4
41.51 1.00
12.70 0.67
ab
69.77 1.66
70.25
13.98 0.04
0.13
72.78 0.54
69.99 0.74
72.55 1.00
71.55 0.73
70.28 0.15
ab
69.94 1.15
ot
ie
ev
71.59 0.41
ab
646
Data are means of three independent experiments (SE).
647
Means with different letters indicate statistically different results at p 0.05, according to Tukeys (HSD) test,
648
(Tr, trace).
fo
rD
649
650
is
651
652
653
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Page 29 of 35
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rR
ee
655
Figures
656
Figure 1
0 M Cd
100 M Cd
50 M Cd
657
659
250 M SA
1000 M SA
0 M SA
250 M SA
1000 M SA
ly
660
0 M SA
On
658
0 M SA
ie
ev
661
/N
662
ot
663
664
fo
665
666
rD
667
668
is
669
670
671
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tri
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1
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Page 30 of 35
rP
Fo
672
673
rR
ee
Figure 2
0 M SA
250 M SA
1000 M SA
H2O 2 content (mol g-1 DW)
0,6
cd cd cd
0,5
bc
b b
0,4
ie
ev
a
0,3
0,2
0,1
0 M Cd
0 M SA
250 M SA
100 M Cd
1000 M SA
25
0 M SA
70
c
b
10
ab ab
ab
ab
60
50
40
30
c c
ab
20
10
0 M Cd
0 M SA
50 M Cd
250 M SA
0 M SA
1000 M SA
50 M Cd
100 M Cd
250 M SA
1000 M SA
c c
ab
1,4
1,2
1
0,8
0,6
cd c
b
0,4
cd
cd
a
0,2
0
0 M Cd
50 M Cd
0 M Cd
100 M Cd
675
50 M Cd
100 M Cd
30
bu
tri
is
rD
Protein carbonyl content (nmol g-1 FW)
e
10
1,6
fo
674
1,8
14
Total protein content (mg g -1FW)
0 M Cd
100 M Cd
ab
ot
/N
1000 M SA
ly
15
Electrolyte leakage (%)
12
250 M SA
80
20
MDA (nmol g-1 FW)
50 M Cd
On
tio
1
2
3
4
5
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8
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Page 31 of 35
rP
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676
677
678
rR
ee
Figure 3
0 M SA
250 M SA
0 M SA
1000 M SA
cd cd
ab a
60
40
ab
CAT (mol H 2O2 mg protein -1 m in -1)
b
80
c cd
20
GPX (mol guaacol mg protein-1 min-1)
100
0 M SA
50 M Cd
250 M SA
0,2
d
d
bc
1,5
bc
0,5
100 M Cd
1000 M SA
ef
0 M Cd
0 M SA
12
e ef
10
0,16
c c
0,12
a a
6
4
e
d
c
50 M Cd
0 M Cd
100 M Cd
680
b b
50 M Cd
fo
0
0 M Cd
1000 M SA
100 M Cd
ot
0,04
250 M SA
/N
SOD (U mg protein-1)
50 M Cd
ly
APX (m ol ascorbate mg protein -1 min -1)
On
0 M Cd
679
1000 M SA
0,08
250 M SA
2,5
120
ie
ev
100 M Cd
rD
681
682
is
683
684
685
686
31
bu
tri
tio
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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Page 32 of 35
rP
Fo
687
688
689
rR
ee
Figure 4
0 M SA
250 M SA
1000 M SA
0 M SA
15
e e
10
bc
50 M Cd
100 M Cd
1,5
0,5
On
691
690
2,5
DPPH . scavenging activity
(mol Trolox g-1 DW)
20
1000 M SA
ef
FRAP (mol Trolox g-1 DW)
250 M SA
25
ie
ev
0 M Cd
50 M Cd
100 M Cd
0 M Cd
ly
692
693
/N
694
695
ot
696
697
fo
698
rD
699
700
701
is
702
703
704
705
32
bu
tri
tio
1
2
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50
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Page 33 of 35
rP
Fo
706
707
708
rR
ee
Figure 5
0 M SA
250 M SA
25
cd c
cd
15
10
0 M Cd
On
710
Lipid content (mg g -1DW)
20
709
1000 M SA
30
ie
ev
50 MCd
100 MCd
ly
711
712
/N
713
ot
714
715
fo
716
717
rD
718
719
is
720
721
722
33
bu
tri
tio
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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17
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Page 34 of 35
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723
724
725
rR
ee
Figure captions
726
Fig. 1. Root morphological aspects of SA-pretreated flax plantlets grown in hydroponics
727
culture and subjected for 10 days to increased CdCl2 concentrations.
ie
ev
728
729
Fig. 2. The effects of salicylic acid (SA) on hydrogen peroxide content (A), lipid peroxidation
730
(B), electrolyte leakage (C), total protein (D) and protein carbonyl (E) contents in roots of flax
731
plantlets under cadmium (Cd) stress. Values are the means of 5 replicate experiments SE.
732
Bars with different letters are statistically different at (P 0.05).
On
733
734
Fig. 3. The effects of salicylic acid (SA) on the activities of catalase (CAT, A), guacol
735
peroxidase (GPX, B), ascorbate peroxidase (APX, C), and superoxide dismutase (SOD, D) in
736
roots of flax plantlets under cadmium (Cd) stress. Values are the means of 5 replicate
737
experiments SE. Bars with different letters are statistically different at (P0.05).
ot
/N
ly
738
739
Fig. 4. The effects of salicylic acid (SA) on ferric reducing antioxidant power (FRAP) (A)
740
and DPPH scavenging activity (B) in roots of flax plantlets under cadmium (Cd) stress.
741
Values are the means of 5 replicate experiments SE. Bars with different letters are
742
statistically different at (P 0.05).
fo
743
Fig. 5. The effects of salicylic acid (SA) on total lipid content in roots of flax plantlets under
745
cadmium (Cd) stress. Values are the means of 5 replicate experiments SE. Bars with
746
different letters are statistically different at (P 0.05).

34
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744
is
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Belkadhi Et Al.,R2

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OMICS: A Journal of Integrative Biology

OMICS: A Journal of Integrative Biology: http://mc.manuscriptcentral.com/omics

Salicylic acid improves root antioxidant defense system and

OMICS: A Journal of Integrative Biology

Date Submitted by the Author:

belkadhi, aicha; Facult des Sciences de Tunis, Dpartement de Biologie,

Complete List of Authors:

OMICS: A Journal of Integrative Biology

antioxidant capacities of flax subjected to cadmium

Authors: A. Belkadhi, A. De Haro, P. Soengas, S. Obregon, M. E. Cartea, W. Djebali and W.

a. Dpartement de Biologie, Unit de Recherche de Physiologie et Biochimie de la tolrance

1060 Tunis, Tunisia

b. Department of Agronomy and Plant Breeding, Institute of Sustainable Agriculture, Spanish

Research (CSIC), Apartado 28, E-36080 Pontevedra, Spain

b. Department of Agronomy and Plant Breeding, Institute of Sustainable Agriculture, Spanish

Research (CSIC), Apartado 28, E-36080 Pontevedra, Spain

OMICS: A Journal of Integrative Biology

b. Department of Agronomy and Plant Breeding, Institute of Sustainable Agriculture, Spanish

Mara Elena Cartea,

Research (CSIC), Apartado 28, E-36080 Pontevedra, Spain

a. Dpartement de Biologie, Unit de Recherche de Physiologie et Biochimie de la tolrance

1060 Tunis, Tunisia

a .Dpartement de Biologie, Unit de Recherche de Physiologie et Biochimie de la tolrance

1060 Tunis, Tunisia

a. Dpartement de Biologie, Unit de Recherche de Physiologie et Biochimie de la tolrance

1060 Tunis, Tunisia

b. Department of Agronomy and Plant Breeding, Institute of Sustainable Agriculture, Spanish

Research (CSIC), Apartado 28, E-36080 Pontevedra, Spain

OMICS: A Journal of Integrative Biology

Fax: +216 71 885 480

membrane permeability, protein oxidation and hydrogen peroxide (H2O2) production to

varying degrees. Concomitantly, activities of the antioxidant enzymes catalase (CAT, EC

1.11.1.6), guacol peroxydase (GPX, EC 1.11.1.7), ascorbate peroxydase (APX, EC 1.11.1.11)

diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and ferric reducing antioxidant power

as the denaturation of its components.

OMICS: A Journal of Integrative Biology

Abbreviations: Cd cadmium; SA salicylic acid; H2O2 hydrogen peroxide; CAT

catalase; GPX guacol peroxydase; APX ascorbate peroxydase; SOD superoxide

dismutase (SOD); MDA malondialdehyde; DPPH 2,2-diphenyl-1-picrylhydrazyl; FRAP

ferric reducing antioxidant power; DW dry weight; TL total lipid.

2005, Rodriguez-Serrano et al. 2009, Gallego et al. 2012).

antioxidative enzymes (Grato et al. 2012). However, in comparison of some species, Cd

monnieri L., Cd induced elevated superoxide dismutase (SOD, EC 1.15.1.1), guacol

peroxydase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione

OMICS: A Journal of Integrative Biology

(Balestrasse et al. 2001, Smeets et al. 2008).

appropriate concentrations, SA was found to enhance the efficiency of antioxidant system in

damage caused by Cd exposure.characterized that SA-induced protection against lipid and

OMICS: A Journal of Integrative Biology

rice following the pre-treatment with SA.

mechanisms of plant enhancement of tolerance to Cd-mediated oxidative stress by using SA

Materials and methods

MgSO4, 2.5Mm Ca (NO3)2, 1mM KH2PO4, 2 mM KNO3, 2 mM NH4Cl, 50 mM EDTA Fe

K, 30 mM H3BO3, 10 mM MnSO4, 1mM ZnSO4, 1mM CuSO4 and 30 mM (NH4)6Mo7O24.

Formatted: Not Superscript/ Subscript

OMICS: A Journal of Integrative Biology

experiments were performed.

were selected for data collection.

The level of lipid peroxidation in roots was determined as 2-thiobarbituric acid

(TBA) reactive metabolites, chiefly malondialdehyde (MDA), as described previously by

content in roots was determined as described by OKane et al. (1996).. Protein-bound

carbonyl content was determined by using the dinitrophenylhydrazine (DNPH) method,

extinction coefficient of 22 000 M-1 cm-1.

reduction of nitroblue tetrazolium (NBT) according to the method of Beauchamp and