Accepted Manuscript Title: Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress Author: Jamel Manai Houda Gouia Francisco J corpas PII: DOI: Reference:

S0176-1617(14)00084-4 http://dx.doi.org/doi:10.1016/j.jplph.2014.03.012 JPLPH 51920

To appear in: Received date: Revised date: Accepted date:

14-2-2014 11-3-2014 11-3-2014

Please cite this article as: Manai J, Gouia H, corpas FJ, Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress, Journal of Plant Physiology (2014), http://dx.doi.org/10.1016/j.jplph.2014.03.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Revised version March 11 2014

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Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots

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under salinity-induced oxidative stress

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Jamel Manai1,2, Houda Gouia2, Francisco J corpas1*

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Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín,

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CSIC, Apartado 419, E-18080 Granada, Spain

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Faculty of Sciences of Tunisia, University Tunis El Manar, Tunis

*Corresponding author:

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Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture,

Dr. Francisco Javier Corpas

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Departamento de Bioquímica, Biología Celular y Molecular de Plantas

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Estación Experimental del Zaidín (CSIC)

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Apartado 419

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E-18080 Granada SPAIN

(Fax: 34 958 129600; e-mail: [email protected])

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Running title: Redox and NO homeostasis salinity-induced oxidative stress

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Summary

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The nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH)

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molecules play important roles in the redox homeostasis of plant cells. Using tomato

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(Solanum lycopersicum) plants grown with 120 mM NaCl, we studied the redox state of

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NADPH and GSH as well as ascorbate, nitric oxide (NO) and S-nitrosoglutathione (GSNO)

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content and the activity of the principal enzymes involved in the metabolism of these

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molecules in roots. Salinity caused a significant reduction in growth parameters and an

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increase in oxidative parameters such as lipid peroxidation and protein oxidation. Salinity also

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led to an overall decrease in the content of these redox molecules and in the enzymatic

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activities of the main NADPH-generating dehydrogenases, S-nitrosoglutathione reductase and

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catalase. However, NO content as well as gluthahione reductase and glutathione peroxidase

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activity increased under salinity stress. These findings indicate that salinity drastically affects

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redox and NO homeostasis in tomato roots. In our view, these molecules,  which show the

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interaction between ROS and RNS metabolisms, could be excellent parameters for evaluating

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the physiological conditions of plants under adverse stress conditions.

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Key-words: NADP-dehydrogenase; NADPH; GSH; nitric oxide; lipid oxidation; S-

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nitrosoglutathione; salinity.

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Abbreviations: G6PDH, glucose-6-phosphate dehydrogenase; GR, glutathione reductase;

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GSH, glutathione; ICDH, NADP-isocitrate dehydrogenase; NO, nitric oxide; ME, malic

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enzyme;

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nitrosoglutathione reductase; ONOO-, peroxynitrite; ROS, reactive oxygen species; RNS,

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reactive nitrogen species.

GPX,

glutathione

peroxidase;

GSNO,

S-nitrosoglutathione;

GSNOR,

S-

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Introduction Reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced

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glutathione (GSH, γ-L-Glutamyl-L-cysteinyl-glycine),  which are electron donors required in

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many enzymatic reactions, biosynthetic pathways and detoxification processes, are key

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molecules in the cellular redox homeostasis of plant cells under physiological and stress

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conditions (Barroso et al., 1998; Noctor, 2006; Noctor et al., 2006; Pollak et al.2007; Foyer

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and Noctor, 2011; Dubreuil-Maurizi and Poinssot, 2012). The regeneration of these molecules

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therefore needs to be adapted to cell requirements during plant development and under

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adverse conditions. In general, although the reduced form of glutathione and nicotinamide

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adenine dinucleotide phosphate predominates under physiological conditions, its equilibrium

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could be displaced to oxidized forms under adverse circumstances (Noctor et al., 2006, 2012;

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Pollak et al., 2007).

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Plant cells also generate the nitric oxide (NO) free radical, which belongs to a family

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of related molecules including S-nitrosoglutatione (GSNO) and peroxynitrite (ONOO-),

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collectively referred to as reactive nitrogen species (RNS). These molecules are involved in

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regulating

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environmental stresses (Corpas et al., 2011, 2013; Chaki et al., 2011; Airaki et al., 2012) such

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as salinity (Valderrama et al., 2007; Corpas et al., 2009; Bai et al., 2010; Tanou et al., 2009,

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2012; Leterrier et al., 2012). In addition, the involvement of enzymatic components regulating

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the production and use of essential antioxidant molecules such as GSH and NADPH indicates

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that the redox state of the cell is the cornerstone of the regulatory mechanism involved

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(Noctor, 2006). Research on the metabolism of these families of molecules suggests that there

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is a close relationship between redox homeostasis and the metabolism of reactive oxygen and

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a wide range of metabolic pathways and,

specifically, in

responding to

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nitrogen species (ROS and RNS) that affects signal and transcriptional processes in cells

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under physiological and stress conditions (Baudouin, 2011). The impact of environmental stress around the world caused by salinity is constantly

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expanding. Plant research has focused on the physiological, biochemical and molecular

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biological aspects of responses to salinity (Hasegawa et al., 2000: Sairam and Tyagi, 2004;

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Munns and Tester, 2008; Teakle and Tyerman, et al. 2010; Dinneny, 2010; Belver et al.,

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2012; Zhang et al., 2012). Salinity stress has been reported to contain an oxidative component

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due to the uncontrolled generation of reactive oxygen species (ROS) and damage to the

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antioxidative system (Hernández et al., 1995, 2000; Gapińska and Skłodowska, 2000; Miller

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et al. 2010; Abogadallah, 2010; Leterrier et al., 2011).

Our main aim was to study the relationship between different metabolites (GSH,

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NADPH and NO) in roots, which come into direct contact with NaCl, the compound

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responsible for stress. Our results show that cellular redox homeostasis in tomato roots is

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drastically affected by salinity.

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Material and methods

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Plant Material and Growth Conditions

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Tomato (Solanum lycopersicum L. cv. Micro-Tom) seeds were surface sterilized with 3%

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(v/v) commercial bleaching solution for 3 min,

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germinated in Petri dishes on moist filter paper at 24°C in the dark for 3 d. Healthy and

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vigorous seedlings were selected and grown in aerated Hoagland nutrient solution (Hoagland

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and Arnon, 1950) for 7 d. Hydroponic cultivation was carried out in a glasshouse, where

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natural light irradiance was supplemented with 122 µmol m-2 s-1 of artificial light (16/8 h

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photoperiod) at 24°C/18°C, day/night temperatures and 40-50% relative humidity. After 20 d

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washed with distilled water and then

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of cultivation under hydroponic conditions, 120 mM NaCl was applied to the nutrient solution

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for 6 d.

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Crude extracts of plant roots

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Plant roots were frozen in liquid N2 and ground in a mortar with a pestle. The powder was

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suspended in a homogenizing medium containing 50 mM Tris-HCl, pH 7.8, 0.1 mM EDTA,

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0.2 % (v/v) Triton X-100 and 10% (v/v) glycerol. Homogenates were centrifuged at 27,000 g

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for 20 min and the supernatants were used for assays.

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Estimation of photosynthetic pigment content

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Photosynthetic pigments were extracted in 80% acetone for 24 hr in darkness at 4°C. The

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resulting suspension was centrifuged for 15 min at 10,000 ×g, and supernatant absorbance

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was measured at 460, 645 and 663 nm using an UV/Vis spectrometer. The pigment

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concentrations were calculated using equations to enable simultaneous determination of

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chlorophyll a (Chl-a) and b (Chl-b) (Arnon, 1949).

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Determination of lipid peroxidation, protein oxidation and hydrogen peroxide content

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Lipid peroxidation was estimated in enriched membrane fractions, corresponding to the pellet

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obtained after homogenates were centrifuged at 27,000 g for 20 min, by determining the

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concentration of malondialhehide (MDA) with the aid of thiobarbituric acid, according to the

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method described by Buege and Aust (1978).

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spectrophotometric dinitrophenyl hydrazine (DNPH) method described by Levine et al.

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(1991) was followed for each sample using their respective blanks. Samples containing at

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least 0.5 mg protein were mixed with 500 μL of 10 mM DNPH in 2 M HCl and the blank was

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incubated in 2 M HCl. After 1 h incubation at room temperature in the dark, proteins were

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precipitated with 10% (w/v) trichloroacetic acid, and the pellets were washed three times with

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500 μL ethanol:ethylacetate (1:1). The pellets were finally dissolved in 1 mL of 6 M

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To determine carbonyl groups, the

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guanidine hydrochloride in 20 mM potassium phosphate at pH 2.3, and absorption at 370 nm

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was measured. Protein recovery was estimated for each sample by measuring the A280.

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Carbonyl content was calculated using a molar absorption coefficient for aliphatic hydrazones

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of 22,000 M−1 cm−1 (Levine et al., 1994).

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Hydrogen peroxide content was determined spectrophotometrically by a peroxidase

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coupled assay using 4-amininoantipyrine and phenol as donor substrates (Frew, et al., 1983).

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Soluble fractions (200 µL) were added to a reaction mixture containing 25 mM phenol, 5 mM

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4-aminoantipyrine, 0.1 mM potassium phosphate buffer (pH 6.9), 0.02 µM peroxidase and 2.5

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µM H2O2. Quinone-imine formation was measured at 505 nm.

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Enzymatic activity assays

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Catalase activity (EC 1.11.1.6) was determined by measuring the disappearance of H2O2, as

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described by Aebi (1984). Glutathione reductase (GR; EC 1.6.4.2) activity was assayed by

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recording NADPH oxidation, as described by Valderrama et al. (2006). Glutathione

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peroxidase (GPX; 1.11.1.9) activity was measured following the method developed by Flohé

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and Günzler (1984). The 2.0 ml reaction mixture consisted of 50 mM phosphate buffer (pH

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7.0), 0.5 mM EDTA, 0.24 unit GR, 1 mM sodium azide, 0.15 mM NADPH and 0.15 mM

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H2O2. After the addition of 10 mM reduced glutathione (GSH), the decrease in absorption at

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340 nm was measured at intervals of 10 s for 2 min.

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Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity was determined

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spectrophotometrically by recording the reduction of NADPH at 340 nm. The assays were

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performed at 25ºC in a reaction medium (1 ml) containing 50 mM HEPES, pH 7.6, 2 mM

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MgCl2 and 0.8 mM NADP+, and the reaction was triggered by the addition of 5 mM glucose-

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6-phosphate. To determine 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44)

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activity, the reaction mixture used was similar to that described for G6PDH, although the

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substrate used was 5 mM 6-phosphogluconate (Valderrama et al., 2006). NADP-isocitrate

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dehydrogenase (NADP-ICDH, EC 1.1.1.42) activity was also measured by monitoring

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NADP+ reduction according to the technique described by Leterrier et al. (2007). The assay

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was performed at 25ºC in a 1 ml reaction medium containing 50 mM HEPES, pH 7.6, 2 mM

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MgCl2 and 0.8 mM NADP+, and the reaction was triggered by the addition of 10 mM 2R 3S-

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isocitrate. NADP-malic enzyme (NADP-ME; EC 1.1.1.40) activity was also determined

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spectrophotometrically by recording the reduction in NADPH at 340 nm using the same

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reaction mixture (1 ml) as described above for other dehydrogenases. However, in this case,

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the reaction was triggered by the addition of 1 mM L-malate (Valderrama et al., 2006).

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S-nitrosoglutathione reductase (GSNOR; EC 1.2.1.1) activity

was assayed

spectrophotometrically at 25 ºC by monitoring the oxidation of NADH at 340 nm (Barroso et

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al., 2006). Plant samples were incubated in an assay mixture containing 20 mM Tris-HCl (pH

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8.0), 0.2 mM NADH and 0.5 mM EDTA, and the reaction was triggered by adding GSNO

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(Calbiochem) to the mixture at a final concentration of 400 mM. Activity was expressed as

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nmol NADH consumed per min per mg protein (ε340 6.22 mM-1 · cm-1).

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Determination of nicotinamide adenine dinucleotide phosphate (NADPH and NADP+)

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Tomato root samples were frozen in liquid N2 and ground in a mortar with a pestle. The

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powder was suspended in preheated 0.1 N NaOH and 0.1 N HCl solutions (1/4, w/v).

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NADPH remained stable in the alkaline solution, while NADP+ was stable in the acid

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solution. The extracts were incubated at 100ºC for 2 min, then cooled on ice and centrifuged

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at 12,000 g for 6 min. The supernatants obtained were used to quantify the nucleotides

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according to the enzyme cycling method described by Matsumura and Miyachi (1980).

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Detection and quantification of ascorbate, GSH, GSSG and GSNO by liquid

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chromatography-electrospray/mass spectrometry (LC-ES/MS)

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Tomato roots (300 mg) were ground alongside 1 ml of 0.1 M HCl using a mortar and pestle.

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Homogenates were centrifuged at 15,000g for 20 min at 4°C. The supernatants were collected

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and filtered through 0.22-μm polyvinylidene fluoride filters and immediately analyzed. All

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procedures were carried out at 4°C and protected from light to avoid potential degradation of

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the analytes (ascorbate, GSH, GSSG and GSNO). The LC-ES/MS system consisted of a

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Waters Alliance 2695 HPLC system connected to a Micromass Quattro Micro API triple

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quadrupole mass spectrometer, both obtained from the Waters Corporation. HPLC was

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carried out using an Atlantis® T3 3μm 2.1x100 mm column supplied by the same company.

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The Micromass Quattro Micro API mass spectrometer was used in positive electrospray

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ionization mode for simultaneous detection and quantification of ascorbate, GSH, GSSG and

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GSNO (Airaki et al., 2011).

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Spectrofluorimetric detection of nitric oxide

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NO was assayed using the spectrofluorimetric method (Airaki et al., 2011). A 2µM

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concentration of 4,5-diaminofluorescein diacetate (DAF-2) was added to crude extracts of

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tomato roots. The reaction mixtures were then incubated at 37ºC in the dark for 2h, and

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fluorescence was measured in a QuantaMaster

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obtained from Photon Technology International (PTI®), at excitation and emission

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wavelengths of 485 and 515 nm, respectively

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Other assays

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Protein concentrations were determined using the Bio-Rad Protein Assay (Hercules, CA),

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with bovine serum albumin as standard. To estimate the statistical significance between

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means, the data were analyzed using the Student’s t-test.

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QM-4 fluorescent spectrophotometer,

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Results

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Effect of salinity on tomato physiological parameters

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A previous study (Huertas et al., 2012) has shown that tomato plants exposed to 120 mM

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NaCl affected plant growth and presented clear symptoms of damage such as leaf chlorosis.

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To determine whether this concentration of NaCl had a similar effect in our experiments,

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several physiological parameters were evaluated. The phenotype of 26-day-old tomato plant

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growth in the presence of 120 mM NaCl for 6 d showed that salinity stress led to a reduction

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in leaf size and chlorophyll a content (Fig. 1, panels a and b, respectively). It also caused

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reductions in leaf fresh weight (Fig. 1c), root fresh weight (Fig. 1d) and root length (Fig. 1e).

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Parameters of oxidative stress

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Peroxidation of unsaturated lipids in biological membranes and protein carbonylation are both

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recognized markers of oxidative damage caused by ROS. Figure 2a and Fig. 2b show that the

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content of the malondialdehyde (MDA) and carbonil groups increased 54% and 38% in roots

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under salinity conditions, respectively. However, the hydrogen peroxide content decreased by

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22% under salinity stress (Fig. 2c).

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Content of nicotinamide adenine dinucleotide phosphate (reduced and oxidized),

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glutathione (reduced, oxidized and S-nitrosylated) and ascorbate in tomato roots under

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salinity stress conditions

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To determine how salinity affects the homeostasis of nicotinamide adenine dinucleotide

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phosphate, the content of NADPH and NADP+ was measured in roots (Table 1). The content

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of the reduced form was greater than that of the oxidized form, and salinity reduced NADPH

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and NADP+ content by 29% and 50%, respectively.

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To determine how salinity affects the status of non-enzymatic antioxidants, the content

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of ascorbate and glutathione (reduced, oxidized and S-nitrosylated) was studied using LC-

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ES/MS (Table 2). Under salinity stress conditions, ascorbate content decreased by 76%.

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However, GSH content decreased by 52%, with GSSG content showing a reduction of 39%.

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S-nitroglutathione (GSNO) content, which fell by 72%, behaved in a similar way.

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Activity of NADPH-generating dehydrogenases in tomato roots under salinity stress

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The NADPH enzymes with the greatest reductive capacity in plants are a group of four

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NADP-dehydrogenases: NADP-isocitrate dehydrogenase (NADP-ICDH), the NADP-malic

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enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate

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dehydrogenase (6PGDH) (the latter two belonging to the pentose phosphate pathway). Figure

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3 shows the activity of these NADP-dehydrogenases in the roots of tomato plants exposed to

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salinity stress. In all cases, a general diminution in activity was observed. In particular,

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NADP-ICDH activity in roots under salinity conditions showed a drastic reduction of 94%,

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whereas no changes were observed in NADP-ME activity. G6PDH and 6PGDH activity

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diminished by 33% and 37%, respectively.

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Analysis of glutathione reductase (GR), glutathione peroxidase (GPX), GSNO reductase

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(GSNOR) and catalase activity in tomato roots under salinity stress

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Given that glutathione in its transformed state is involved in multiple metabolic pathways, key

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enzymes active in oxidative and nitrosative stress mechanisms were analyzed. Figure 4 shows

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the enzymatic activity of GR, GPX and GSNOR in tomato plant roots exposed to salinity

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stress. While GR activity in roots increased by 30% (Fig. 4a), GSNOR activity fell by 25%

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(Fig. 4b), and GPX activity increased (Fig. 4c). Catalase is an important antioxidant enzyme

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in peroxisomes, and its activity decreased by 62% under salinity stress (Fig. 4d).

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Nitric oxide content in tomato roots under salinity stress

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As nitric oxide (NO) has been shown to be involved in the mechanism of response to different

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types of environmental stress, NO content was analyzed under our experimental conditions.

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Figure 5 shows an increase of 24% in NO content under salinity stress.

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Discussion

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Given that NADPH is a necessary reducing equivalent for the regeneration of reduced

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glutathione (GSH) by glutathione reductase involved in the ascorbate-glutathione cycle, both

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molecules are required in antioxidant systems in order to counter oxidative damage (Berger et

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al., 2004; Pollak et al., 2007; Foyer and Noctor, 2009). Nitric oxide (NO) is a free radical

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shown to be a multifunctional molecule involved in different metabolic processes under

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physiological and stress conditions (Baudouin, 2011; Corpas et al., 2011). Tomato is

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agronomically important, with many studies focusing on different aspects of its response to

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salinity stress (Cuartero et al., 2006; Huertas et al., 2012). It has been clearly established that

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salinity is accompanied by oxidative stress in different organs of tomato plants (Gapińska and

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Skłodowska, 2000; Shalata et al., 2001; Mittova et al., 2002, 2003, 2004).

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In a previous study, Gapińska et al (2008) showed that tomato roots were subject to

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oxidative stress caused by concentrations of 50 mM and 150 mM NaCl, and the antioxidative

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system was modulated according to short- or long-term salinity. Given these findings and the

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fact that roots are in direct contact with the agent (NaCl) causing the stress, the main aim of

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this study was to analyze the potential relationship between NADPH, GSH and NO on the one

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hand and the different enzymes involved in their metabolism such as NADP-dehydrogenases,

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GSNO reductase, GR and GPX on the other This could provide new information on the

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complex response mechanism in tomato plant roots during salinity stress.

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NADPH content and NADPH-regenerating system is down-regulated in roots under

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salinity-induced oxidative stress

Page 11 of 32

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Under our experimental salinity conditions (120 mM NaCl), the various growth parameters of

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tomato plants analyzed were observed to be drastically affected. Moreover, salinity stress was

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accompanied by oxidative stress, as demonstrated by the increase in the oxidative stress

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biomarkers, including lipid peroxidation and protein oxidation products such as

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malondialdehyde (MDA) and protein carbonyl group, respectively (Farmer and Mueller,

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2013; Jacques et al., 2013). These oxidative markers indicate that salinity leads to alterations

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in membranes and protein functionality. These findings are in line with salinity-induced

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oxidative stress described in previous studies of tomato (Mittova et al., 2002, 2003) and other

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plant species (Hernández et al., 1995; Valderrama et al., 2006; Begara-Morales et al., 2014)

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Thus, the total content of NADPH and NADP+ clearly decreased in roots under

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salinity stress. Given that the NADPH pool is a major source of electrons for reductive

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biosynthesis (Schafer and Buettner, 2001), the data indicate that salinity greatly affected the

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redox state of tomato roots, which closely correlates with the different growth parameters

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assayed. NADPH is also the primary source of reducing equivalents for the ascorbate-

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glutathione system, which plays a particularly important role under oxidative stress

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conditions. Despite the fact that NADPH content decreased, it is likely that this level was

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sufficient to keep up the activity of GR that was induced under our experimental conditions of

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salinity stress. Although GR takes part of the ascorbate-glutathione system, an important

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antioxidant mechanism for counteracting potential damage, the induction of GR activity was

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insufficient to offset the damage caused by salinity.

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The group of NADPH-generating dehydrogenases (G6PDH, 6PGDH, NAPD-ICDH

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and NADP-ME) is now regarded as a second barrier for antioxidative systems and, as noted

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previously, they provide the ascorbate-glutathione system with NADPH (Valderrama et al.,

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2006; Leterrier et al., 2012). Although the information available to carry out a comparative

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analysis on this group of NADP-dehydrogenases under stress conditions is very limited, in

Page 12 of 32

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recent years, research has begun to focus on how these NADP-dehydrogenases contribute to

2 

the mechanism of response to environmental stress. Under our experimental conditions, all

3 

NADP-dehydrogenases in roots were observed to be clearly affected by salinity, as shown by

4 

the reduced content of NADPH and especially NADP-ICDH whose activity decreased by

5 

94%. Nevertheless, under salinity stress conditions, available information shows that the

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activity of these enzymes behaves in quite different ways in herbaceous and woody plant

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species. Thus, in the leaves of olive plants, salinity caused an opposite reaction, with the four

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NADP-dehydrogenases (G6PDH, 6PGDH, ME and ICDH) recording a general increase in

9 

activity (Valderrama et al., 2006). Under in vitro conditions, the treatment of Arabidopsis

10 

seedlings with 100 mM NaCl caused an increase in NADP-ICDH activity in roots but not in

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leaves (Leterrier et al., 2012). However, in rice suspension cells under salt stress conditions,

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G6PDH activity was down-regulated (Zhang et al., 2013), which is in line with our findings.

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NADP-ICDH merits particular attention as, under biotic stress caused by

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Pseudomonas syringae, for example, this enzyme made a significant contribution to

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maintaining redox homeostasis in Arabidopsis leaves (Mhamdi et al., 2010b). Consequently,

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the drastic reduction (94%) in NADP-ICDH activity in tomato roots could partly explain the

17 

decrease in NADPH content under salinity conditions. In addition, apart from its ability to re-

18 

generate NADPH, this enzyme also serves an important metabolic function given its

19 

involvement in both carbon and nitrogen metabolisms. NADP-ICDH activity has recently

20 

been reported to decrease during natural root senescence, a process that is accompanied by the

21 

establishment of an oxidative stress and an increase in NO content. This inhibition occurs as

22 

root NADP-ICDH activity is subject to tyrosine nitration (Begara-Morales et al., 2013) which

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is a post-translational modification mediated by NO-derived molecules such as peroxynitrite

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(Corpas et al., 2013). Given that salinity causes oxidative stress in tomato roots (Gapińska et

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al, 2008) and a concomitant increase in NO (Fig. 5), it would be plausible to suggest that the

2 

loss observed in NADP-ICDH activity is partly due to this process.

3 

GSH and nitric oxide connect ROS and RNS metabolisms under salinity-induced

4 

oxidative stress

5 

GSH is considered to be the most abundant low-molecular-weight thiol in plants (Noctor et

6 

al., 2012). The presence of the SH group makes GSH a multifunctional molecule that is

7 

necessary for the functioning of the antioxidative system, and it also contributes to the so-

8 

called ‘redox switch’ of proteins, facilitating their regulation by processes such as S-

9 

glutathionylation and S-nitrosylation (Spadaro et al. 2010; Noctor et al., 2012; Corpas et al.,

10 

2013; Couturier et al., 2013). Under stress conditions, GSH was significantly diminished,

11 

possibly due to its high demand in maintaining the activity levels of GSH-dependent

12 

enzymes. These results are very much in line with data on GSH and GR activity in

13 

Arabidopsis leaves in response to hydrogen peroxide (Mhamdi et al., 2010a).The free radical

14 

NO can also interact with GSH through a process of S-nitrosylation, leading to the formation

15 

of S-nitrosoglutathione (GSNO), which could also interact with SH protein groups that affect

16 

its functionality (Corpas et al., 2013). To the best of our knowledge, few data exist regarding

17 

the quantification of GSNO in plant research, although available information indicates that

18 

the content of GSNO is in a range similar to that of GSSG (Airaki et al., 2011). In tomato

19 

roots under normal conditions, GSNO content was similar to that of GSSG, while GSNO

20 

content declined under salinity stress conditions. However, GSNO content showed a pattern

21 

opposite that of NO content, which increased under salinity stress. These findings are in line

22 

with

23 

Arabidopsis roots (Corpas et al., 2009; Leterrier et al., 2012), sunflower roots (David et al.,

24 

2010), olive leaves (Valderrama et al., 2007), citrus leaves (Tanou et al., 2009, 2012) and

25 

maize leaves (Bai et al., 2011). This increase in NO content under salinity stress could also

Ac ce p

te

d

M

an

us

cr

ip t

1 

data on NO under salinity stress conditions in different plants, for example, in

Page 14 of 32

15   

explain why the exogenous application of NO to specific plants can attenuate the damage

2 

caused by salinity (Kopyra and Gwozdz, 2003; Zhang et al., 2006). In this respect, the

3 

importance of NO under salinity conditions has recently been highlighted. In pea plants

4 

grown under 150 mM NaCl salinity conditions, some authors have observed an increase in

5 

APX and S-nitrosylated APX activity as well as NO and S-nitrosothiol (SNO) content that

6 

supported the induction of the APX activity (Begara-Morales et al., 2014).This indicates an

7 

interplay between the NO metabolism and the antioxidant APX involved in the ROS

8 

metabolism (Corpas and Barroso, 2013).

us

cr

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1 

Given that GSNO is regarded as a reservoir of NO, the equilibrium between these

10 

molecules may act as a mechanism of response to salinity in roots. This is also borne out by

11 

GSNOR activity which behaved in a similar fashion to GSNO content under salinity stress

12 

conditions. Although information on GSNO content is very limited, some data exist on the

13 

behavior of GSNO content under stress conditions. For example, in Arabidopsis seedlings

14 

exposed to toxic concentrations of arsenic (500 µM) causing nitro-oxidative stress, GSNO

15 

content was

16 

(Leterrier et al., 2012).

M

d

te

observed to decrease,

Ac ce p

17 

an

9 

Glutathione

peroxidase

accompanied by a concomitant reduction in

(GPX)

catalyzes

the

reduction

in

H2O2,

GSH

organic

18 

hydroperoxides and lipid peroxides by using GSH as hydrogen donor. GPX is generally

19 

regarded as a major system of enzymatic defense against oxidative membrane damage. Under

20 

our experimental conditions, GPX was stimulated in roots, which could explain the decrease

21 

observed in H2O2 and possibly indicate the presence of a protective mechanism against

22 

membrane damage caused by salinity stress. These findings are in line with reported data on

23 

GPX activity in tomato roots during the application of 50 mM (mild stress) and 150 mM

24 

NaCl (severe stress) (Gapińska et al., 2008). However, the behavior of GPX activity is liable

Page 15 of 32

16   

to vary according to the intensity of salinity stress and the plant species involved. For

2 

example, in Arabidopsis thaliana roots, GPX protein expression decreases under mild salt

3 

stress but increases under severe salt stress conditions (Jian et al., 2007). However, in

4 

Salicornia europaea, a succulent annual euhalophyte regarded as one of the most salt-tolerant

5 

species, GPX protein expression is induced under salinity stress conditions (Wang et al.,

6 

2009).

cr

ip t

1 

Catalase is another key antioxidant enzyme localized in peroxisomes, which are

8 

organelles with very active ROS and RNS metabolisms where the presence of NO, GSNO

9 

and peroxynitrite has been detected (Corpas et al., 2009; Barroso et al., 2013; Corpas and

10 

Barroso, 2014). In tomato, catalase activity decreased drastically, clearly indicating that this

11 

enzyme was affected by salinity stress. Similar effects have been described in pea leaves

12 

exposed to 75 mM NaCl (Corpas et al., 1993), whereas in olive leaves and in Arabidopsis, salt

13 

stress produced an increase in catalase activity (Valderrama et al., 2006; Leterrier et al.,

14 

2011).

te

d

M

an

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7 

These variations in the response capacity of different plant species such as tomato can

16 

be contradictory in many cases. This can be explained by numerous factors, acting separately

17 

or together, such as differences in

18 

(dicotyledon or monocotyledon), genotype, growth substrates (in vitro, hydroponic, soil), the

19 

intensity of salinity stress and the organs analyzed (Stepien and Klobus, 2005; Abogadallah,

20 

2010).

Ac ce p

15 

constitutive antioxidative defenses, plant species

21 

In summary, our findings constitute a new framework showing that the redox state and

22 

NO metabolism in tomato roots are drastically affected by salinity and clearly contribute to

23 

responses to oxidative and nitrosative stress (Corpas and Barroso, 2013). However, certain

24 

aspects of this process remain unexplained, e.g. how the GSH and NADPH pools in different

Page 16 of 32

17    1 

organelles, cells and tissues are generated and maintained under physiological and stress

2 

conditions and how NO interacts with these molecules through a family of related molecules,

3 

such as GSNO, which can contribute to signaling processes.

ip t

4 

Acknowledgements

6 

J. Manai acknowledges a short-term scholarship from Tunisian government. This work was

7 

supported by ERDF-cofinanced grants from the Ministry of Science and Innovation (project

8 

BIO2009-12003-C02-01) and Junta de Andalucía (group BIO 192), Spain. LC-ES/MS

9 

analyses were carried out at the Instrumental Technical Services of the Estación Experimental

an

us

cr

5 

del Zaidín (CSIC) and special thanks are given to Dr. Lourdes Sánchez-Moreno.

The

11 

valuable technical help of Mr. Carmelo Ruíz-Torres is also appreciated. Dr. Juan B. Barroso

12 

is also acknowledged for his critical comments and suggestions and finally Michael O’Shea

13 

for proofreading the article.

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14 

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10 

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14 

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16  17  18  19  20 

te

Ac ce p

15 

d

12 

21 

Page 25 of 32

26   

Manai et al

2 

Titlet: Redox and nitric oxide homeostasis is affected in tomato (Solanum lycopersicum)

3 

roots under salinity-induced oxidative stress

4 

 

5 

Table 1. Nicotinamide adenine dinucleotide phosphate (NADPH and NADP+) content in

6 

roots of tomato plants treated with 0 and 120 mM NaCl in hydroponic cultivation conditions.

7 

Data are the mean ± SEM of at least three different experiments. Asterisks indicate that

8 

differences from control values were statistically significant at P< 0.05.

us

cr

ip t

1 

Roots

Pyridine dinucleotides

an

(nmol · mg-1 FW)

120 mM 194 ± 18*

NADP+

130 ± 13

65 ± 12*

M

NADPH

0 mM 274 ± 61

9 

d

10 

Table 2. Total GSH (reduced, oxidized, and S-nitrosylated) and ascorbate content in roots of

12 

tomato plants treated with 0 and 120 mM NaCl in hydroponic cultivation conditions. Data are

13 

the mean ± SEM of at least three different experiments. Asterisks indicate that differences

14 

from control values were statistically significant at P< 0.05.

Ac ce p

te

11 

(µmol · mg-1 FW)

Roots

GSH

0 mM 0.723 ± 0.05

120 mM 0.350 ± 0.02*

GSSG

1.04 ± 0.14

0.639 ± 0.01*

GSNO

1.13 ± 0.18

0.31 ± 0.13*

0.918 ± 0.002

0.216 ± 0.003*

Ascorbate 15  16 

 

17 

 

18 

 

Page 26 of 32

Figure

(c) 120 FW (g)

0

Leaves

2

1

*

(d)

ip t

NaCl (mM)

(a)

Roots

(e)

(b)

Root lenght

25 20

M

12

*

ed

6

pt

3

120 NaCl (mM)

10 5 0

0 mM NaCl 120 mM NaCl

Ac ce

0

*

15

cm

chlo a chlo b

9

0

cr

an

2 cm

mg chorophyll · g-1 FW

*

0.4

us

FW (g)

0.8

Figure 1. Phenotype and effect of salinity in tomato growth parameters treated with 0 and 120 mM NaCl in hydroponic cultivation. (a) Phenotype. (b) Leaf chlorophyll a and b content. (c) Leaf fresh weight (FW). (d) Roots fresh weight. (e) Root length. FW, fresh way. Asterisks indicate that differences from control values were statistically significant at P< 0.05.

Page 27 of 32

2 1 0 120 mM NaCl

80 40 0

0 mM

120 mM NaCl

ip t cr

Hydrogen peroxide

12 8

*

4 0

0 mM

120 mM NaCl

ed

0 mM

*

120

(c)

us

*

Protein carbonyl

an

3

(b)

M

Lipid peroxidation

nmol carbonyl · mg-1protein

nmol MDA · mg-1protein

(a)

120 mM NaCl

nmol H2O2 · mg-1protein

0 mM NaCl

Ac ce

pt

Figure 2. Oxidative stress parameters in roots of tomato plants treated with 0 and 120 mM NaCl. (a) Lipid peroxidation was determined by the thiobarbituric acid reactive substances (TBARS) method, using malondialdehyde (MDA) as standard. (b) DNPH-reactive protein carbonyl groups on oxidized proteins were determined spectrophotometrically. (c) Hydrogen peroxide content. Asterisks indicate that differences from control values were statistically significant at P< 0.05.

Page 28 of 32

ip t cr

120 mM NaCl

120

*

*

0

ICDH

*

an

60

us

180

M

nmol NADPH · min-1 · mg-1protein

0 mM NaCl

ME

G6PDH 6PGDH

Ac ce

pt

ed

Figure 3. Activity of NADP-dehydrogenases in roots of tomato plants treated with 0 and 120 mM NaCl. ICDH, NADP-isocitrate dehydrogenase. ME, malic enzyme. G6PDH, glucose-6-phosphate dehydrogenase. 6PGDH, 6-phosphogluconate dehydrogenase. Asterisks indicate that differences from control values were statistically significant at P< 0.05.

Page 29 of 32

0 mM NaCl

80 40 0

20

*

10 0

*

60

6

an

40

8

us

(d) Catalase

20

2

*

0

M

0

4

ed

µmol NADPH · min-1 ·mg-1protein

(c) GPX

30

ip t

*

cr

120

nmol NADH · min-1· mg-1protein

(b) GSNOR

µmol H2O2· min-1 · mg-1 protein

nmol NADPH · min-1· mg-1protein

(a) GR

120 mM NaCl

Ac ce

pt

Figure 4. Activity of enzymes involved in glutathione metabolism and catalase in roots of tomato plants treated with 0 and 120 mM NaCl . (a) Glutathione reductase (GR). (b) GSNO reductase (GSNOR). (c) Glutathione peroxidase (GPX). (d) Catalase. Data are the mean ± SEM of at least three different experiments. Asterisks indicate that differences from control values were statistically significant at P< 0.05.

Page 30 of 32

ip t cr

60

20 0

0 120 NaCl (mM)

us

40

M

an

NO (arbitrary units · mg-1 FW)

*

80

Ac ce

pt

ed

Figure 5. NO content in roots of tomato plants treated with 0 and 120 mM NaCl. NO was assayed by as spectrofluorometric method using DAF-2 as fluorescent probe. The fluorescence is expressed as arbitrary units per mg of fresh weight (FW). Data are the mean ± SEM of at least three different experiments. Asterisks indicate that differences from control values were statistically significant at P< 0.05.

Page 31 of 32

Note:This figure is the color version for online J Plant Physiology

Leaves

2

1

*

(d)

Roots

us

0.4

M

2 cm

(e)

*

pt

6

0

*

0

120 NaCl (mM)

15

cm

ed

chlo a chlo b

9

3

Root lenght

25 20

12

Ac ce

mg chorophyll · g-1 FW

*

an

FW (g)

0.8

(b)

ip t

(c) 120 FW (g)

0

cr

NaCl (mM)

(a)

10 5 0

0 mM NaCl 120 mM NaCl

Figure 1. Phenotype and effect of salinity in tomato growth parameters treated with 0 and 120 mM NaCl in hydroponic cultivation. (a) Phenotype. (b) Leaf chlorophyll a and b content. (c) Leaf fresh weight (FW). (d) Roots fresh weight. (e) Root length. FW, fresh way. Asterisks indicate that differences from control values were statistically significant at P< 0.05.

Page 32 of 32