Accepted Manuscript

Title: NADPH oxidase-dependent H2O2 production is requiredfor salt-induced antioxidant defense in Arabidopsis thaliana

Author: Kilani Ben Rejeb Maali Benzarti Ahmed DebezChristophe Bailly Arnould Savoure Chedly Abdelly

PII: S0176-1617(14)00288-0DOI: http://dx.doi.org/doi:10.1016/j.jplph.2014.08.022Reference: JPLPH 52052

To appear in:

Received date: 9-5-2014Revised date: 28-8-2014Accepted date: 29-8-2014

Please cite this article as: Rejeb KB, Benzarti M, Debez A, Bailly C, Savoure A,Abdelly C, NADPH oxidase-dependent H2O2 production is required for salt-inducedantioxidant defense in Arabidopsis thaliana, Journal of Plant Physiology (2014),http://dx.doi.org/10.1016/j.jplph.2014.08.022

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NADPH oxidase-dependent H2O2 production is required for salt-induced antioxidant 1 

defense in Arabidopsis thaliana 2 

Kilani Ben Rejeba,b,1,*, Maâli Benzartia,1, Ahmed Debeza, Christophe Baillyc, Arnould 3 

Savouréb, Chedly Abdellya 4 

aLaboratoire des Plantes Extrêmophiles, Centre de Biotechnologie de Borj-Cedria (CBBC), 5 

BP 901, Hammam-Lif, 2050, Tunisia. 6 

bAdaptation des plantes aux contraintes environnementales, UR5, Université Pierre et Marie 7 

Curie (UPMC), Case 156, 4 Place Jussieu, 75252 Paris cedex 05, France. 8 

cUMR 7622, UPMC Univ. Paris 06, CNRS, Bat C 2ème étage, 4, place Jussieu, 75005 Paris, 9 

France. 10 

1Both authors contributed equally to this work. 11 

*Corresponding author: Kilani Ben Rejeb , email: kilanib@yahoo.com  12 

Tel. (+216) 79 325 848 Fax (+216) 79 325 638 13 

14 

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Abstract 1 

The involvement of H2O2 generated by NADPH oxidase in the antioxidant defense system 2 

was assessed in salt-challenged Arabidopsis thaliana seedlings. In the wild-type, short-term 3 

salt exposure led to a transient and significant increase of H2O2 concentration, followed by a 4 

marked increase in catalase (CAT, EC 1.11.16), ascorbate peroxidase (APX, EC 1.11.1.11) 5 

and glutathione reductase (GR, EC 1.6.4.2) activities. Pre-treatment with either a chemical 6 

trap for H2O2 (dimethylthiourea) or two widely used NADPH oxidase inhibitors (imidazol 7 

and diphenylene iodonium) significantly decreased the above-mentioned enzyme activities 8 

under salinity. Double mutant atrbohd/f plants failed to induce the antioxidant response under 9 

the culture conditions. Under long-term salinity, the wild-type was more salt-tolerant than the 10 

mutant based on the plant biomass production. The better performance of the wild-type was 11 

related to a significantly higher photosynthetic activity, a more efficient K+ selective uptake, 12 

and to the plants’ ability to deal with the salt-induced oxidative stress as compared to 13 

atrbohd/f. Altogether, these data suggest that the early H2O2 generation by NADPH oxidase 14 

under salt stress could be the beginning of a reaction cascade that triggers the antioxidant 15 

response in A. thaliana in order to overcome the subsequent ROS production, thereby 16 

mitigating the salt stress-derived injuries. 17 

Keywords: salinity, antioxidant defense system, atrbohd, atrbohf, H2O2, Arabidopsis 18 

thaliana. 19 

20 

21 

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Abbreviations: ABA, Abscisic Acid; APX, Ascorbate peroxidase; C, control; CAT, Catalase; 1 

Chl, Chlorophyll; DAB, 3,3′Diaminobenzidine; DMTU, Dimethylthiourea; DPI, Diphenylene 2 

iodonium; DW, Dry weight; F0, Minimal fluorescence; Fm, Maximal fluorescence; FV / Fm, 3 

Maximum quantum efficiency of PSII photochemistry; FW, Fresh weight; GR, Glutathione 4 

reductase; H2DCFHA, 2’,7’-Dichlorofluorescin diacetate; H2O2, Hydrogen peroxide; MAPK, 5 

Mitogen-Activated Protein Kinase; MDA, Malonyldialdehyde; NADPH oxidase, 6 

Nicotinamide Adenine Dinucleotide Phosphate-oxidase; NBT, Nitroblue tetrazolium; NO, 7 

Nitric oxide; O2˙¯, Superoxide anion; OH˙, Hydroxyl radicals; PSII, Photosystem II; Rboh, 8 

Respiratory burst oxidase homologues; ROS, Reactive oxygen species; S, Salt stress; SOD, 9 

superoxyde dismutase; 10 

11 

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1 

1. Introduction 2 

Drought and salt are among the most limiting abiotic stresses to crop productivity and yield. 3 

Generally, salinity reduces the plant growth and/or damage the plant through its: (i) osmotic 4 

effect (causing water deficit), (ii) ion-related toxic effect, and (iii) impact on the uptake of 5 

essential nutriments. As a consequence of these primary effects, secondary stresses such as 6 

oxidative damage often occur (Zhu et al., 2001) due to the excessive accumulation of 7 

deleterious chemical compounds called reactive oxygen species (ROS), including hydrogen 8 

peroxide (H2O2), superoxide anion (O2˙¯), and hydroxyl radicals (OH˙), which induce cellular 9 

damage by protein degradation, enzyme inactivation, alterations in the gene and interference 10 

in various pathways of metabolic significance (Choudhury et al., 2013). Still, there is 11 

increasing evidence of ROS involvement as signaling molecules in plant responses to abiotic 12 

and/or biotic stresses. Indeed, at low concentrations, ROS operate as messengers for the 13 

activation of defense genes (Foyer et al., 2009). 14 

Addressing ROS homeostasis is crucial both for mitigating ROS toxicity and to determine 15 

their likely role in signaling pathways. Cellular ROS homeostasis depends on a balance 16 

between production and degradation of the free radicals. In plants, ROS can be produced by 17 

chloroplasts, mitochondria and peroxisomes or by apoplastic cell wall peroxidases, amine 18 

oxidases and plasma membrane NADPH oxidase (Gill and Tuteja, 2010). In order to reduce 19 

ROS-related damages, plants have evolved a complex antioxidant defense system that 20 

scavenges excessively accumulated ROS under stress conditions. This system includes 21 

enzymatic (CAT, APX, GR, and superoxide dismutase, SOD) and non-enzymatic (low 22 

molecular weight antioxidant compounds) components (Li et al., 2010). 23 

Pharmacological and genetic data indicate that the enzymes responsible for most of the ROS 24 

generated during biotic interactions and in early response to abiotic stresses are the plasma 25 

membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues 26 

(Rbohs) (Wu et al., 2013). The rapid generation of ROS during the early stages of plant 27 

defense signaling is known as oxidative burst (Hao et al., 2008). Because H2O2 is relatively 28 

stable and diffusible through membrane, it is now considered the main ROS that triggers 29 

defense mechanisms in plant cells. H2O2 has been shown to play many critical roles in 30 

signaling and in several aspects of plant development, including in plant defense, root hair 31 

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development, stomatal closure, and early responses to salt stress (Torres et al., 2002; Foreman 1 

et al., 2003; Kwak et al., 2003; Leshem et al., 2007). 2 

The Arabidopsis genome contains 10 NADPH oxidase-encoding genes designated as 3 

AtRbohA to J exhibiting different patterns of expression throughout plant development and in 4 

response to environmental factors (Fluhr, 2009). Moreover, H2O2 produced by NADPH 5 

oxidase has been demonstrated to mediate rapid systemic signaling triggered by multiple 6 

abiotic stresses (Miller et al., 2009). A transient increase in the endogenous levels of H2O2 7 

obtained by exogenous application of salicylic acid or heat can lead to subsequent 8 

thermotolerance in mustard seedlings (Dat et al., 1998). In maize, protection against chilling 9 

injury can be achieved by a transient increase in endogenous H2O2 levels during low-10 

temperature acclimation (Prasad et al., 1994). Alternatively, direct exposure of plant tissues to 11 

H2O2 has been shown to activate antioxidant enzymes as well as the expression of antioxidant 12 

enzyme-encoding genes (Mylona and Polidoros, 2010). Both AtRbohD and AtRbohF have 13 

been identified as main isoforms, which are highly expressed under salt stress (Ma et al., 14 

2012; Xie et al., 2011). Leshem et al. (2007) have described that H2O2 produced by NADPH 15 

oxidase are coordinated by phospholipid-regulated signaling pathways and act in the signal 16 

transduction of salt stress responses in Arabidopsis, and to be required for haem oxygenase 17 

mediated salt acclimation signaling in Arabidopsis (Xie et al., 2011). 18 

The present study aims at better understanding the relationship between the early production 19 

of H2O2 by the NADPH oxidase and the antioxidant response of A. thaliana seedlings 20 

exposed to short and long salt treatments. The function and the implication of H2O2 as a 21 

stressor or as a signaling molecule were also studied. First, the effect of short-term salinity on 22 

the H2O2 production and the antioxidants enzyme activities (SOD, CAT, APX and GR) was 23 

investigated. Then, the effect of pre-treatment with dimethylthiourea (DMTU), a chemical 24 

trap for H2O2 and diphenylene iodonium (DPI) and imidazol, an NADPH oxidase inhibitor on 25 

the activities of the salt stress-induced antioxidant enzymes were examined. Moreover, the 26 

antioxidant response was assessed in atrbohd/f double mutant. Finally, the comparative 27 

responses of atrbohd/f plants and the wild-type to long-term salt-stress were addressed. 28 

Several parameters were considered including the plant growth activity, the photosynthetic 29 

activity, leaf Na+/K+ selectivity, and the oxidative stress leaf responses. 30 

31 

2. Materials and methods 32 

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2.1. Plant material and culture conditions 1 

Arabidopsis thaliana transposon insertion mutant lines atrbohd-3 (European Arabidopsis 2 

Stock Centre code N9555), atrbohf-3 (European Arabidopsis Stock Centre code N9557) and 3 

double mutant atrbohd/f (European Arabidopsis Stock Centre code N9558) (Torres et al., 4 

2002) were ordered from the European Arabidopsis Stock Centre. 5 

In a first experiment, the implication of H2O2 in the induction of antioxidant response to 6 

short-term salt stress was assessed. Surface-sterilized seeds of wild-type (ecotype Columbia 7 

(Col-0)) and Arabidopsis mutant plants were sown in square Petri dishes on half-strength 8 

agar-solidified Murashige and Skoog (MS) medium according to (Parre et al., 2007). After 24 9 

h at 4°C to break dormancy, seedlings were grown at 22°C under continuous light (90 µmol 10 

photons m-2 s-1). Twelve day-old Arabidopsis seedlings were transferred to liquid MS/2 11 

medium supplemented or not with 200 mM NaCl and harvested at 3, 6, 9 and 24 h. In order to 12 

study the effects of scavengers and inhibitors, the seedlings were pre-incubated or not for 4 h 13 

with either 40 mM dimethylthiourea (DMTU, a chemical trap for H2O2) or 20 µM 14 

diphenylene iodonium (DPI) or 10 mM imidazol, two NADPH oxidase inhibitors. They were 15 

then grown in liquid MS/2 medium at 200 mM NaCl for 24 h under the same conditions as 16 

described above. All experiments were performed with three independent biological and three 17 

technical repetitions. 18 

In a second experiment, seeds of the wild-type (Col-0) and the mutant atrbohd/f were sown in 19 

pots containing a mixture of vermiculite:sand (1:3). Seedlings were irrigated with one-20 

quarter-strength (Hewitt, 1960) nutrient solution under greenhouse conditions (300 µmol m-2 21 

s-1 photosynthetic active radiation (PAR), 25 ± 5°C temperature, and 60 ± 10% relative 22 

humidity). Four week-old plants at rosette stage were exposed to 100 mM NaCl for 7 d. To 23 

reduce osmotic shock on plants, salt treatments were daily increased by 50 mM NaCl. Pots 24 

were irrigated every 2 d. 25 

26 

2.2. Chlorophyll fluorescence measurements, plant growth, leaf chlorophyll and ion 27 

concentrations 28 

Chlorophyll (Chl) fluorescence was measured using a modulated chlorophyll fluorimeter 29 

(OS1-FL) following the procedure described by (Genty et al., 1989). The minimal (F0) and 30 

maximal (Fm) Chl a fluorescence were assessed in leaves after 20 min of dark adaptation. The 31 

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maximum quantum efficiency of Photosystem II (PSII) photochemistry was calculated as Fv / 1 

Fm = (Fm - F0)/ Fm. 2 

At the harvest, rosette fresh weigh (FW) was immediately estimated and dry weight (DW) 3 

was determined after having being dried at 60°C until constant weight. Chl was extracted 4 

from 100 mg leaf FW in 5 mL 100% acetone. After centrifugation for 5 min at 500×g, 5 

supernatants were measured at 470, 645, and 662 nm. Concentrations of total Chl were 6 

calculated as given by (Lichtenthaler, 1987). After ion extraction from dried ground leaf in 7 

0.5% HNO3, Na+ and K+ was assayed by flame emission photometry (Corning, UK). 8 

9 

2.3. H2O2 concentration 10 

H2O2 concentration was determined spectrophotometrically as described by (Oracz et al., 11 

2009). 300 mg FW plant materials were ground in a mortar on ice in 1 ml perchloric acid (0.2 12 

M). After 15 min of centrifugation at 13,000 × g at 4°C, the resulting supernatant was 13 

neutralized to pH 7.5 with 4 M KOH and then centrifuged at 13,000 × g at 4°C. The 14 

supernatant was immediately used for spectrophotometric determination of H2O2 using a 15 

horseradish peroxidase-based assay with 3-dimethylaminobenzoic acid and 3-methyl-2-16 

benzothiazolidone hydrazone. The increase in absorbance at 590 nm was monitored for 15 17 

min after the addition of peroxidase at 25 °C and analyzed using a calibration curve obtained 18 

with known amounts of fresh H2O2. 19 

20 

21 

2.4. Histochemical detection of H2O2 22 

Arabidopsis roots were collected after 6 h treatment with NaCl and immersed with 25 µM 23 

2’,7’-dichlorofluorescin diacetate (H2DCFDA) for 15 min in the dark and then washed with 24 

20 mM potassium phosphate buffer (pH 6). Fluorescent signals were visualized using a Zeiss 25 

ApoTome microscope (excitation, 488 nm; emission, 525 nm). 3,3′Diaminobenzidine (DAB) 26 

staining was performed according to (Torres et al., 2002). Detached leaves were vacuum-27 

infiltrated with a DAB solution (10 mg mL-1 DAB-HCl, pH 3.8). DAB generates a radish-28 

brown DAB polymer that can be detected at the site of H2O2 formation. After staining, leaves 29 

were cleared in 96% boiling ethanol and observed with a loupe. For both staining methods, 30 

digital images were obtained via AxioCam camera and AxioVision software (Zeiss). 31 

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1 

2.5. Measurement of lipid peroxidation 2 

Lipid peroxidation was estimated by measuring the concentration of malonyldialdehyde 3 

(MDA). Fresh leaf samples (0.1 g) were homogenized in 0.1% (w/v) TCA solution. The 4 

homogenate was centrifuged at 15,000×g for 10 min. An aliquot of the supernatant was added 5 

to 0.5% TBA in 20% TCA. The mixture was heated at 90° C for 30 min in a shaking water 6 

bath, and then cooled in an ice bath. The samples were centrifuged at 10,000 × g for 5 min, 7 

and the absorbance of the supernatant was read at 532 and 600 nm (Ben Amor et al., 2005). 8 

The MDA concentration was calculated according to the molar extinction coefficient of MDA 9 

(155 mM-1cm-1). 10 

11 

2.6. Enzyme extraction and assays 12 

For CAT (EC 1.11.16), GR (EC 1.6.4.2) and SOD (EC 1.15.11) activity, frozen leaf sample 13 

(0.3 g) was homogenized in 5 ml of a potassium phosphate buffer (0.1 M, pH 7.8) containing 14 

2 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.25 mM polyethylene 15 

glycol 4000 and 10% (w/v) of polyvinylpolypyrrolidone (PVP). For APX (EC 1.11.1.11) 16 

activity, leaf material was homogenized in 50 mM potassium phosphate buffer (pH 7.0), 1 17 

mM ascorbate (Asc), 0.1 mM EDTA, and 1% (w/v) PVP. The homogenate was then 18 

centrifuged for 30 min at 14,000 × g and the supernatant was desalted on a PD 10 column 19 

(GE Healthcare). All steps of the extraction procedure were carried out at 4° C. Protein 20 

contents of the extracts were determined using the Bio-Rad protein assay kit with bovine 21 

serum albumin as calibration standard. The total activities of antioxidant enzymes were 22 

determined according to (Bailly et al., 1996). Total SOD activity was assayed by monitoring 23 

the inhibition of photochemical reduction of nitroblue tetrazolium (NBT). One unit of SOD 24 

activity was defined as the amount of enzyme that was required to cause 50% inhibition of the 25 

reduction of NBT as monitored at 560 nm. Total CAT activity was assayed by measuring the 26 

H2O2decomposition rate at 240 nm. GR activity was determined at 25° C as described 27 

previously by (Esterbauer and Grill, 1978), by following the NADPH oxidation rate at 340 28 

nm. Total APX activity was measured by monitoring the decrease in absorbance at 290 nm as 29 

ascorbate was oxidized. 30 

31 

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RT-PCR Analysis 1 

Total RNA was extracted from 100 mg of homogenized tissue using the RNeasy Plant Mini 2 

kit (Qiagen) according to the manufacturer’s protocol. Traces of DNA were removed by 3 

DNase treatment. RNA quantification was done at 260 nm using a Nanovue® 4 

spectrophotometer (GE Healthcare Life Science). First-strand cDNA was performed from 1.5 5 

µg of total RNA using RevertAidTM reverse transcriptase synthesis kit (Fermentas). For RT-6 

PCR, cDNAs were amplified using Taq polymerase and gene-specific primers. The APT1 7 

(adenine phosphoribosyltransferase 1; At1g27450) gene was used as a control. Amplified 8 

PCR fragments were visualized using ethidium bromide stained 2% (w/v) agarose gels. The 9 

gene-specific primers are as follows: APT1 forward (5’- GAGACATTTTGCGTGGGATT-3’) 10 

and reverse (5’- CGGGGATTTTAAGTGGAACA-3’) Atrbohd forward (5’-11 

CTGGACACGTAAGCTCAGGA-3’) and reverse (5’-GCCGAGACCTACGAGGAGTA-3’) 12 

and Atrbohf forward (5’-TCACAAATCAACGACGAGAGTT-3’) and reverse (5’-13 

CCCATCTTCATTCTTGTCCA-3’) 14 

15 

16 

17 

3. Results 18 

3.1. Role of endogenous H2O2 in the antioxidant activity induction under short-term salt 19 

stress 20 

H2O2 concentration in wild-type A. thaliana seedlings exposed for 1 h to 200 mM NaCl 21 

salinity was similar to that of the control (Fig. 1). However, salt exposure for 3 h resulted in a 22 

marked accumulation of H2O2, reaching a maximum at 6 h before progressively decreasing 23 

(especially at 24 h), whereas no significant changes occurred for the control (Fig. 1). The 24 

H2O2 peak accumulation observed at 6 h was associated with significantly higher SOD 25 

activity, the latter however sharply declining thereafter (Fig. 2A). In the 12-24 h time range, 26 

salt stressed plants showed close values to that of the control. Concomitant with the marked 27 

decrease of H2O2 concentration upon salt exposure for 12-24 h, CAT, APX, and GR (Figs. 28 

2C-D) activities increased significantly in this time range (between 3- and 5-fold higher than 29 

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values registered at 1 h of salt treatment). It is noteworthy that the enzyme activities registered 1 

in the control remained unchanged during the experiment. 2 

To assess whether endogenous H2O2 is involved in NaCl-induced antioxidant enzyme 3 

activities, wild-type A. thaliana seedlings were pre-treated or not for 4 h with 40 mM DMTU, 4 

a chemical trap for H2O2, and then exposed to 200 mM NaCl treatment. No significant impact 5 

on the antioxidant enzyme activities in the control was observed, whereas salt-challenged 6 

plants previously treated with DMTU showed significantly lower SOD, CAT, APX, and GR 7 

activities as compared to plants directly exposed to salinity (-14%, -37%, -34% and -49%, 8 

respectively) (Figs. 3A-D). 9 

10 

3.2. NADPH oxidase-dependent H2O2 production is required for NaCl induced 11 

antioxidant enzymes 12 

To investigate the role of Atrbohd and Atrbohf in the early production of H2O2, the expression 13 

patterns of Atrbohd and Atrbohf in wild-type A. thaliana under salt stress and control 14 

conditions were explored using RT-PCR methods. As shown in Fig. 4, treatment with NaCl 15 

clearly increased the expression of Atrbohd and Atrbohf at 6 h. To ascertain the likely role of 16 

H2O2 originating from NADPH oxidase in NaCl-induced SOD, CAT, APX and GR activities 17 

in Arabidopsis, the effect of pre-treatment with DPI or imidazol, two widely used NADPH 18 

oxidase inhibitors, on NaCl-induced H2O2 generation and antioxidant enzyme activities was 19 

first investigated. Incubation in 20 µM DPI or 10 mM imidazol completely prevented the 20 

production of H2O2 in leaves and root tips of wild-type A. thaliana seedlings (Fig. 5) and fully 21 

blocked the enhancement of SOD, CAT, APX and GR activities previously observed under 22 

salt stress (Fig. 3). To further confirm these pharmacological data, we used Arabidopsis single 23 

and double mutants of atrbohd and atrbohf, two highly expressed Atrboh genes under salt 24 

stress (Xie et al., 2011; Ma et al., 2012). NaCl-induced H2O2 generation (Fig. 6) was partially 25 

inhibited in atrbohf but more severely impaired in atrbohd and the double mutant atrbohd/f as 26 

compared to salt-treated wild-type. Since atrbohf acts redundantly with atrbohd in regulating 27 

salt responses (Ma et al., 2012), we used only the atrbohd/f double mutants in our further 28 

investigations. As observed for the wild-type pre-treated with DPI, NaCl-induced CAT, APX 29 

and GR activities were strongly impaired in the double mutant atrbohd/f as compared to the 30 

salt-treated wild-type plants (Fig. 7). To further address the role of H2O2, we tried to reverse 31 

the effect of atrbohd/f mutation by the exogenous application of 10 mM H2O2. This pre-32 

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treatment restored significantly CAT, APX and GR activities in NaCl-treated atrbohd/f 1 

seedlings whereas no impact was observed for the salt-treated wild-type (Fig. 7). 2 

3 

3.3. Responses of wild-type A. thaliana and atrbohd/f mutant plants to long-term 4 

moderate salt stress 5 

Short-term (2 d) salinity had no significant effect on the rosette fresh weight of both 6 

genotypes, whereas long-term (7 d) salt-exposure restricted significantly the plant growth, 7 

especially in the atrbohd/f mutant as compared to the wild-type (-30% and -17% of the 8 

respective control values at 7 d) (Table 1). Rosette Na+ concentration increased significantly 9 

with salt exposure in concomitance with a marked decrease in K+ concentration (Table 1). 10 

This trend was more pronounced in the salt-sensitive genotype (atrbohd/f), resulting in 11 

significantly higher Na+/K+ values in the latter genotype as compared to the wild-type (Table 12 

1). Chl concentrations were also lower in salt-treated plants of both genotypes, especially in 13 

the atrbohd/f mutant, which showed 50% lower Chl concentration than the wild-type after 7 d 14 

of salt treatment (Table 1). The wild-type showed a decline of the maximal photochemical 15 

efficiency of PSII (Fv/Fm) after 2 d of exposure to NaCl stress, whereas the salt-related 16 

decrease of PSII appeared later (after 7 d salt exposure) and was more pronounced in the 17 

atrbohd/f mutant (Table 1). After 2 d of salt treatment, atrbohd/f showed significantly lower 18 

H2O2 concentration than the wild-type (Table 2). Long-term (7 d) salt exposure clearly 19 

increased the levels of H2O2 in both genotypes, though a higher extent in the mutant 20 

atrbohd/f. In addition, MDA concentration, as an indicator of lipid peroxidation, increased in 21 

the wild-type plants after 2 d of NaCl exposure, whereas remaining unchanged in the 22 

atrbohd/f mutant. In contrast, after exposure to 100 mM NaCl for 7 d, oxidative damage was 23 

more marked in the mutant compared to the wild-type as reflected by the higher MDA 24 

concentration in the former genotype (Table 2). Regarding the antioxidant response, wild-type 25 

plants exposed for 2 d with 100 mM NaCl showed significantly higher CAT, APX and GR 26 

activities (up to 3.3-fold increase for CAT). The same trend was observed under long-term 27 

salinity conditions, although it was less pronounced (Table 2). In the case of atrbohd/f mutant, 28 

no significant change in the abovementioned enzyme activities was observed irrespective of 29 

the salt treatment duration (Table 2). 30 

31 

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4. Discussion 1 

Salinity, as many environmental stresses, is known to generate ROS which are toxic but may 2 

act as a signalling molecule when accumulated at low/moderate concentrations (Mittler et al., 3 

2004). To maintain a relatively low ROS concentration, plants have evolved highly regulated 4 

enzymatic and non-enzymatic mechanisms to keep a balance between ROS production and 5 

detoxification so that the cellular redox homeostasis can be maintained (Ahmad et al., 2010). 6 

In the present study, short-term salt stress (24 h) induced a transient increase of H2O2 7 

concentration up to 6 h treatment (Fig. 1). It is well established that the H2O2 accumulation is 8 

an early common response to various environmental stresses factors, such as pathogen attack 9 

(Torres et al., 2002), heat stress (Saidi et al., 2011), drought and salt stress (Miller et al., 10 

2011). There are many potential sources of H2O2 in plant cells, including chloroplasts, 11 

mitochondria, peroxisomes, plasma membrane NADPH oxidase, cell wall peroxidases, 12 

apoplastic oxalate, and amine oxidase (Gill and Tuteja, 2010). In Arabidopsis, it has been 13 

documented that the plasma membrane-located NADPH oxidase is mainly involved in H2O2 14 

production in early response to salt stress (Leshem et al., 2007). Our result showed that 15 

exposure of wild-type A. thaliana to NaCl increased the expression of Atrbohd and Atrbohf at 16 

6h, in agreement with the previous results reported by Xie et al. (2011). These findings imply 17 

that NaCl can rapidly activate Atrbohd and Atrbohf to produce ROS, which trigger some 18 

mechanisms to alleviate the phytotoxic effects of salt stress (Ma et al., 2012). NADPH 19 

oxidase transfers electrons from NADPH to O2 to form O2˙¯, followed by dismutation of O2˙¯ 20 

to H2O2 by SOD. The likely role of H2O2 originating from this enzyme in ROS signaling has 21 

recently received attention. Interestingly, our findings indicate that in the wild-type A. 22 

thaliana, salt treatment induced H2O2 accumulation in concomitance with higher SOD 23 

activity (Fig. 2A), leading to think that the increase in SOD activity may be partially due to 24 

the increased NADPH oxidase activity. In salt-stressed barley, a transient increase in SOD 25 

activity was also observed, strongly suggesting that O2˙¯ radical dismutation into H2O2 during 26 

the initial phases of salt exposure is of high significance with respect to stress signaling and 27 

adaptation (Maksimovic et al., 2013). In A. thaliana mutants deficient in NADPH oxidase, a 28 

significant reduction of SOD activity, mainly Cu/Zn SOD, was recorded in response to 29 

arsenic (As), contrasting with wild-type plants (Gupta et al., 2013). In the same way, we show 30 

that when A. thaliana seedlings were pre-treated either with 20 µM DPI or 10 mM imidazol, 31 

two widely used NADPH oxidase inhibitors, the NaCl-related transient increase of both SOD 32 

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activity and H2O2 accumulation at 6 h was suppressed, which suggests that the salt-induced 1 

H2O2 production originated at least partially from the plasma-membrane NADPH oxidase. 2 

The decrease of H2O2 concentration in salt-treated plants was associated with an increase of 3 

CAT, APX and GR activities (Fig. 2), likely as a consequence of the direct effect of NaCl or 4 

the indirect effect mediated via an increase in levels of H2O2. Therefore, we hypothesized that 5 

in the wild-type A. thaliana seedlings the early H2O2 accumulation during salt stress might 6 

serve as second messenger to induce CAT, APX and GR activities. This hypothesis was 7 

supported by the fact that plant pre-treatment with 40 mM DMTU, a chemical trap for H2O2, 8 

led to significantly lower CAT, APX and GR activities under salinity as compared to non-pre-9 

treated plants growing in the same conditions (Fig. 3). The rapid increase of APX and GR 10 

activities following salt exposure of A. thaliana seedlings is consistent with previous reports 11 

indicating that the activity and transcript levels of these proteins were increased within 8 h in 12 

salt-treated rice roots (Tsai et al., 2005; Nam et al., 2012). When the response of major 13 

antioxidant enzymes transcripts was analyzed for different developmental stages in salt 14 

stresses rice, increased CAT, APX and GR transcripts accumulation was observed at 12 h 15 

exposure, and the transcript levels remained higher than control samples throughout the 16 

experimental period (72 h) (Menezes-Benavente et al., 2004). Lin and Pu (2010) studied 17 

changes in enzymes involved in ROS scavenging in sweet potato plants tolerant and sensitive 18 

to salinity. After exposure to salinity, APX activity increased in plants at 24 h and 48 h, and 19 

this response was higher in a salt-tolerant genotype than in the salt sensitive ones. The 20 

expression of APX in response to salinity was tissue specific and dependent on stress duration 21 

(Caverzan et al., 2012). Morita et al. (1999) reported that the cytosolic APX is regulated by 22 

the H2O2 level within rice cells. In Arabidopsis, a total of five genes coding for APX isoforms 23 

in the chloroplast (APX4 and APX5), cytosol (APX1 and APX2) and microbodies (APX3) 24 

(Santos et al., 1996) have been identified. APX found in organelles scavenges H2O2 produced 25 

within the organelles, whereas cytosolic APX eliminates H2O2 produced in the cytosol, or that 26 

diffused from apoplast or organelles (Mittler and Zilinskas, 1992). H2O2-treated Arabidopsis 27 

leaves also showed increased tolerance to excess light which was associated with induced 28 

APX1 and APX2 expression (Karpinski et al., 1999). Furthermore, the systemic induction of 29 

APX1 and APX2 was shown to be mediated by the endogenous H2O2. According to (Rizhsky 30 

et al., 2004), Zat12 (zinc finger protein) is an important component of the H2O2 signal 31 

transduction network of Arabidopsis required for APX1 expression during oxidative stress. It 32 

was reported that a cAPX (Apx1)-lacking Arabidopsis mutant showed elevated H2O2 levels 33 

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and the induction of several defense genes, indicating the importance of cAPX in controlling 1 

cellular H2O2 signaling (Pnueli et al., 2003). It seems likely that the induction in cAPX 2 

expression during an early stage of oxidative stress plays an important role in removing H2O2 3 

and minimizing photooxidative damage. The transient accumulation of H2O2 following cAPX 4 

expression has been also observed in methyl viologen and high-light exposed Spinach 5 

(Yoshimura et al., 2000). In the latter study the change in the expression of cAPX paralleled 6 

the increase of the corresponding protein activity. 7 

CAT is also a predominant enzyme controlling H2O2 level in plants (Xing et al., 2007). In 8 

Arabidopsis, three encoding genes (CAT1, CAT2 and CAT3) have been identified (Frugoli et 9 

al., 1996). Salt stress-induced CAT1 expression has been shown to be triggered by an ABA-10 

dependent mitogen-activated protein kinase (MAPK) kinase AtMEK1 and to depend on H2O2 11 

production (Xing et al., 2007). Ye et al. (2011) have shown that endogenous ABA regulates 12 

both H2O2 production and CAT gene expression in rice leaves under water stress, which in 13 

turn keep H2O2 as a signaling molecule rather than a cytotoxic chemical. Several studies also 14 

showed that ABA-can induce the expression of antioxidant genes and enhance the capacity of 15 

antioxidant defense system. In the ABA signal transduction, several signal molecules such as 16 

calcium ion (Ca2+), ROS and proteins kinases such as MAPK and Ca2+-dependent protein 17 

kinase (CDPK) have been shown to play important roles in the regulation of antioxidant 18 

defense systems. Genetic evidence shows that there exists a complex relationship between 19 

ABA, CDPK and MAPK in plants response to environmental stress. AtMPK6 in Arabidopsis 20 

and its homologues in rice and maize, OsMPK1 and ZmMPK5, have been shown to be 21 

involved in ABA-induced antioxidant defense (Xing et al., 2008; Lin et al., 2009; Zhang et 22 

al., 2012). In rice, a recent study showed that OsCPK12 can induce the expression of the 23 

antioxidant genes OsAPX2 and OsAPX8 under salt stress, and reduce the salt-induced 24 

accumulation of H2O2 (Asano et al., 2012). However, the detoxification of ROS regulation by 25 

OsCPK12 under salt stress seems to be ABA independent. (Asano et al., 2012). Zhang et al. 26 

(2006) reported that pretreatment of maize plants with the ROS inhibitors or scavengers, such 27 

as DPI, imidazole, Tiron, and DMTU, significantly reduced the activation of the ABA-28 

induced MAPK. Meanwhile, ABA treatment induced the increases in the transcript levels of 29 

antioxidant genes such as CAT1, cAPX, and GR1 and the increase in the total activities of the 30 

antioxidant enzymes CAT, APX, GR, and SOD in leaves of maize plants were substantially 31 

blocked by pretreatment with the MAPK inhibitors and the ROS inhibitors or scavengers. 32 

More recently, Ding et al (2013) showed that ZmCPK11 is required for ABA-induced up-33 

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regulation of the expression and the activity of cAPX in maize leaves under water stress and 1 

functions upstream of ZmMPK5 in ABA-signaling. However, in Arabidopsis, a recent study 2 

demonstrated that, in response to salt stress, CDPK and MAPKs act in parallel and no direct 3 

cross-talk exists between them (Mehlmer et al., 2010). 4 

Our findings indicated that DPI pre-treatment completely suppressed the enhancement in the 5 

activities of CAT, APX and GR observed under salt stress in the wild-type A. thaliana (Fig. 6 

3). Furthermore, neither H2O2 accumulation (Fig. 6) nor stimulated antioxidant enzyme 7 

activities were observed in the salt-challenged KO-atrbohd/f double mutant (Fig. 7). In the 8 

same way, a significant induction of cAPX transcript was observed 6 h after mild salt stress 9 

exposure of wild-type A. thaliana seedlings, whereas only a slight increase in cAPX transcript 10 

level was observed in atrbohd mutants (Xie et al., 2011). Pre-treating maize leaves with DPI 11 

or DMTU also fully prevented the enhancement in the activities of chloroplastic and cytosolic 12 

SOD, APX and GR induced by abscisic acid (ABA) treatment (Hu et al., 2006). Jiang et al. 13 

(2002a,b) has demonstrated the involvement of ROS originated from NADPH oxidase in 14 

ABA-induced antioxidant defense system in maize leaves exposed to water deficit stress. 15 

Kwak et al. (2003) reported that disruptions in Atrbohd and Atrbohf, impair ABA-induced 16 

Ca2+ signaling through preventing apoplastic Ca2+ to enter the guard cells. Elevated cytosolic 17 

Ca2+ levels has been shown to play important roles in ROS signaling and salt tolerance in 18 

plants (Kader and Lindberg, 2010). In a recent study, Ma et al. (2012) reported that NaCl 19 

significantly elevated the Ca2+ influx currents and [Ca2+]cyt in a dose-dependent manner in the 20 

wild-type A. thaliana seedling. However, the elevation in [Ca2+]cyt was markedly suppressed 21 

in atrbohd/f, suggesting that both Atrbohd and Atrbohf are implicated in salt-stimulated 22 

increase in cytosolic Ca2+ of Arabidopsis. As shown in the above mentioned reports, calcium 23 

has been identified as a central regulator in different signal transduction pathways inducing 24 

antioxidant defense. Thus, it is conceivable that Ca2+ may function, at least in part, on 25 

induction of the antioxidant systems against ROS when Arabidopsis were subjected to salt 26 

stress. Hence, our results provide a pharmacological and genetic evidence for the involvement 27 

of NADPH oxidase-dependent H2O2 generation in the coordination of the activities of 28 

antioxidant enzymes in A. thaliana under short-term salt stress treatment. However, it remains 29 

to be clarified how these secondary stress responses are affected during prolonged salt stress. 30 

In the second part of this study, we addressed the responses of wild-type A. thaliana and 31 

atrbohd/f mutant plants to long-term salinity at non-lethal concentration (100 mM NaCl). The 32 

adverse effects of salt stress on plant growth can be mainly due to: toxic effect, osmotic effect 33 

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and nutritional imbalance. These in turn cause an induction of oxidative stress (Hernandez et 1 

al., 2010). In this study, the mutant atrbohd/f growth was more inhibited than the wild-type 2 

under prolonged salt exposure (Table 1). The over salt-sensitivity in the mutant was related 3 

with higher sodium accumulation and higher Na+/K+ values compared to the wild-type (Table 4 

1). Excessive accumulation of Na+ is harmful for cell physiology and biochemistry, and could 5 

be considered as the main reason for salt toxicity. Our data further confirm recent 6 

assumptions about the involvement of AtRbohD and AtRbohF function in ROS-dependent 7 

regulation of Na+/K+ homeostasis in Arabidopsis under salt stress (Ma et al., 2012). These 8 

authors suggest that the disruption in Na+/K+ homeostasis is the main factor involved in the 9 

salt sensitivity of atrbohd/f under short-term salt stress. 10 

Under long-term NaCl treatment, Fv/Fm decreased to a higher extent in atrbohd/f mutant than 11 

in the wild-type (Table 1), suggesting that salt-induced oxidative damage was more severe in 12 

the former genotype. MDA, the product of lipid peroxidation, has been regarded as an 13 

indicator of oxidative damage at cellular level. In this study, NaCl caused an increase in the 14 

concentration of H2O2, but had no marked effect on MDA concentration in wild-type within 2 15 

d of NaCl exposure, which should be related with the increased antioxidant enzymes activities 16 

(CAT, APX and GR) observed under these conditions (Table 2). MDA concentration 17 

increased significantly in the wild-type after exposure to 100 mM NaCl for 7 d, along with 18 

lower CAT, APX and GR activities but it has to be stated that values remained higher than 19 

that of the control (Table 2). Under short-term NaCl treatment, atrbohd/f had markedly lower 20 

H2O2 and MDA concentrations than the wild-type. However, long-term NaCl treatment 21 

increased both compound levels in the former genotype, suggesting that other cellular 22 

mechanisms also contributed to H2O2 accumulation under prolonged salinity conditions. For 23 

instance, the wild-type and mutant plants might have used non enzymatic pathways for 24 

conversion of O2˙¯ to H2O2 such as α-tocopherol, glutathione and ascorbate (Foyer and 25 

Noctor, 2011). It is also well known that peroxisomes provide H2O2 through the glycolate 26 

oxidase reaction or during β-oxidation of fatty acids (Foyer and Noctor, 2009). In addition, 27 

the more pronounced increase of H2O2 and MDA concentrations in atrbohd/f genotype 28 

compared to the wild-type, together with constant CAT, APX and GR activities in the mutant 29 

(Table 2) could be explained by: (i) MDA accumulation due to the fact that cellular 30 

membranes are particularly sensitive to ROS attack and (ii) the oxidative damage resulting 31 

from an excess of ROS production following the inhibition of CAT, APX and GR activities in 32 

atrbohd/f, likely due to post-transcriptional alteration or down regulation of gene expression. 33 

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A recent study Chaouch et al. (2012) reported a lower basal cAPX1 expression level in non-1 

stressed atrbohf mutant as compared to the wild-type. Interestingly, the relative importance of 2 

AtRbohD in cAPX1 and CAT1 expression during light stress treatment was also pointed out 3 

(Davletova et al., 2005). Under high light conditions, the lower expression of cAPX2 in the 4 

atrbohd/f double mutant suggested that NADPH oxidase may be part of a signaling route 5 

controlling cAPX2 expression (Bechtold et al., 2008), similar to that described for cAPX1. 6 

Silencing of RBOH1, the tomato homologue of AtRbohF, abolished the induction of both the 7 

gene expression and the activity of CAT, SOD, APX and GR in response to chilling treatment 8 

(Zhou et al., 2014). In tobacco, the impairment of the RbohD and RbohF genes via antisense 9 

expression effectively decreased the mRNA levels of antioxidant enzymes including cAPX, 10 

catalase (CAT1 and CAT2) and cytosolic Cu/Zn SOD in response to elicitor treatment (Wi et 11 

al., 2012), which further supports our data about the likely relationship between these two 12 

rboh homologues and the induction of antioxidant enzyme activity. The difference between 13 

enzyme activities during NaCl treatment between wild-type and atrbohd/f may also result 14 

from their impairment by H2O2 (Scandalios, 1993) or direct inhibition by Na+ (Hernandez et 15 

al., 1994). Hence, it seems the wild-type is able to induce an efficient antioxidant response 16 

protecting the plant photosynthetic tissues against oxidative damage under short term 17 

exposure to salinity. Yet, long term exposure to salinity caused ROS excessive accumulation, 18 

which could not be scavenged by the antioxidant machinery (CAT, APX and GR activities 19 

declined), thereby leading to oxidative damage (as reflected by the increasing MDA 20 

concentration) and the disruption of cell structure and metabolism (Li and Yi, 2012). In view 21 

of the results observed in this experiment, it seems that significant activation of antioxidant 22 

enzymes system in wild-type A. thaliana undergoing short-term salt treatment might be 23 

responsible for its relative salt-tolerance under long-term treatment. In contrast, atrbohd/f 24 

sensitivity to long-term salinity could be ascribed to the plant failure to early activate the 25 

antioxidant enzymatic system upon salt stress application but also during the experiment. 26 

As a whole, results inferred from this work suggest that the early H2O2 generation by NADPH 27 

oxidase after exposure to salinity could be the beginning of a cascade of events that triggers 28 

A. thaliana response to salt stress at both physiological and biochemical levels. We 29 

hypothesized that the early low oxidative stress level caused by low concentration of H2O2 30 

might act as an acclimation signal that triggers a preconditioning response by inducing 31 

antioxidant enzyme activities to efficiently cope with the subsequent ROS production, thereby 32 

preventing or minimizing the subsequent salt stress-derived injuries. Considering the 33 

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complexity of signal transduction networks in plants, other possible pathways in the induction 1 

of the antioxidant defense system in response to salt stress cannot be ruled out. Other 2 

experiments will be needed to investigate in more detail the involvement of the NADPH 3 

oxidase-dependent H2O2 production in the up-regulation of the expression of antioxidant 4 

enzyme-encoding genes and possible interaction with other signaling molecules such as ABA, 5 

Ca2+, CDPK, MAPK, and NO. 6 

7 

Acknowledgements 8 

This work was supported by the Tunisian Ministry of Higher Education and Scientific 9 

Research (LR10CBBC02) and the Tunisian-French CMCU (Comité Mixte de Coopération 10 

Universitaire) network (13G0929). Part of this study was supported by European Union 11 

COST program FA0901 “Putting halophytes to work - From genes to ecosystem”. 12 

13 

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18 

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Table 1. Effect of salt stress (100 mM NaCl) on growth (rosette fresh weigh, g plant-1), Na+, K+ 1 

concentrations (mmol g-1 DW), total Chlorophyll content (µg g-1 FW) and on the chlorophyll 2 

fluorescence (Fv/Fm) of leaves of Arabidopsis thaliana wild type and atrbohd/f double mutant. Mean 3 

(n = 6 ± SE) with different letters were significantly different at P < 0.05. C, control; S, salt stress. 4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

FW Na+ K+ Na/K Chl Fv/Fm

C 0.70 ± 0.02b 0.27 ± 0.05a 1.08 ± 0.04b 0.25 ± 0.06a 341 ± 42c 0.81 ± 0.02b 2d

S 0.70 ± 0.01b 1.43 ± 0.04b 0.85 ± 0.03a 1.69 ± 0.12b 268 ± 3b 0.63 ± 0.03a

C 0.71 ± 0.02b 0.20 ± 0.02a 0.98 ± 0.11b 0.22 ± 0.01a 323 ± 24c 0.81 ± 0.01b WT

7d S 0.60 ± 0.03a 1.67 ± 0.04b 0.85 ± 0.08a 1.95 ± 0.21c 167 ± 23a 0.67 ± 0.02a

C 0.72 ± 0.02B 0.24 ± 0.02A 0.9 ± 0.02B 0.27 ± 0.03A 310 ± 12B 0.8 ± 0.03B 2d

S 0.71 ± 0.03B 1.88 ± 0.12B 0.67 ± 0.02A 2.77 ± 0.24B 297 ± 29B 0.76 ± 0.02B

C 0.71 ± 0.01B 0.22 ± 0.01A 1.17 ± 0.14B 0.27 ± 0.01A 291 ± 33B 0.84 ± 0.01B

atrb

ohd/

f

7d S 0.50 ± 0.01A 1.98 ± 0.04B 0.67 ± 0.08A 3.01 ± 0.33C 86 ± 15A 0.58 ± 0.01A

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1 

2 

Table 2. Effect of salt stress (100 mM NaCl) on H2O2 (µmol g-1 FW), malonyldialdehyde (MDA, 3 

nmol g-1 FW) CAT, APX, GR (µmol min-1 mg-1 protein) and SOD (U mg-1 protein) enzyme activities 4 

in rosettes of Arabidopsis thaliana wild type and atrbohd/f double mutant. Mean (n = 6 ± SE) with 5 

different letters were significantly different at P < 0.05. C, control; S, salt stress. 6 

H2O2 MDA CAT APX GR SOD

C 0.29 ± 0.01a 2.2 ± 0.04a 0.021 ± 0.002a 1.31 ± 0.1a 0.61 ± 0.02a 10 ± 2.2a 2d

S 0.72± 0.05b 2.9 ± 0.2b 0.069 ± 0.01c 3.10 ± 0.3c 1.8 ± 0.01c 12 ± 2.1a

C 0.32 ± 0.03a 2.3 ± 0.4a 0.024 ± 0.005a 1.47 ± 0.2a 0.78 ± 0.15a 11 ± 1.3a

WT 7d

S 1.2 ± 0.01c 5.7 ± 0.2c 0.043 ± 0.007b 2.67 ± 0.2b 1.05 ± 0.06b 15 ± 1.7b

C 0.27 ± 0.00A 2.29 ± 0.8A 0.018 ± 0.002A 1.19 ± 0.2A 0.59 ± 0.02A 9 ± 2.6A 2d

S 0.3 ± 0.04A 2.03 ± 0.2A 0.021 ± 0.006A 1.49 ± 0.1A 0.57 ± 0.00A 11 ± 2.2A

C 0.26 ± 0.01A 2.12 ± 0.1A 0.019 ± 0.001A 1.15 ± 0.02A 0.62 ± 0.19A 11 ± 1.8A

atrb

ohd/

f

7d S 1.8 ± 0.09B 7.05 ± 0.4B 0.024 ± 0.003A 1.88 ± 0.05A 0.78 ± 0.27A 14 ± 1.1B

7 

8 

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Figure legends 1 

2 

Figure 1. Time-course changes in H2O2 accumulation of wild-type Arabidopsis thaliana seedlings 3 

exposed or not to 200 mM NaCl for 24 h. Means (± SE) of three independent experiments with 4 

different letters are significantly different at P < 0.05. Each experiment contained three independent 5 

biological repetitions. 6 

7 

Figure 2. Time-course changes in the activities of antioxidant enzymes SOD (A), CAT (B), APX (C), 8 

and GR (D) of wild-type Arabidopsis thaliana seedlings exposed or not to 200 mM NaCl for 24 h. 9 

Means (± SE) of three independent experiments with different letters are significantly different at P < 10 

0.05. Each experiment contained three independent biological repetitions. 11 

12 

Figure 3. Effect of pre-treatment with DMTU, DPI or Imidazol on SOD (A), CAT (B), APX (C) and 13 

GR (D) activities of wild-type Arabidopsis thaliana seedlings exposed or not to 200 mM NaCl. Plants 14 

were pre-incubated with 40 mM DMTU or 20 μM DPI or 10 mM Imidazol for 4 h and then exposed or 15 

not to NaCl. (A) plants exposed to NaCl for 6 h. (B), (C) and (D) plants exposed to NaCl for 24 h. 16 

Means (± SE) of three independent experiments with different letters are significantly different at P < 17 

0.05. Each experiment contained three independent biological repetitions. 18 

19 

Figure 4. Expression patterns of the Atrbohd and Atrbohf genes. RT-PCR were performed with RNAs 20 

extracted at the indicated times from wild-type Arabidopsis thaliana leaves treated without or with 21 

200 mM NaCl. The constitutive APT1 gene is used as a control. Representative data of three 22 

independent RNA extractions are shown. 23 

24 

Figure 5. H2O2 production visualized by using DAB or 2’,7’-dichlorofluorescin diacetate (H2DCFDA) 25 

staining in wild-type Arabidopsis thaliana leaves (A) and roots tips (B) pre-treated with 20 µM DPI or 26 

10 mM Imidazol and grown under normal conditions or in the presence of 200 mM NaCl for 6 h. 27 

28 

Figure 6. H2O2 production, estimated by DAB staining, in leaves of Arabidopsis thaliana wild-type, 29 

atrbohD, and atrbohF single mutant and atrbohD/F double mutant exposed to 200 mM NaCl for 6 h. 30 

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1 

Figure 7. Effect of NaCl stress (200 mM NaCl) on the activities of CAT, APX and GR in wild-type 2 

Arabidopsis thaliana seedlings and atrbohD/F double mutant. Seedling were pre-treated with 10 mM 3 

H2O2 for 4 h and then exposed to 200 mM NaCl for 24 h. Means (± SE) of three independent 4 

experiments with different letters are significantly different at P < 0.05. Each experiment contained 5 

three independent biological repetitions 6 

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H2O

2(µ

mol

g-1FW

)

Duration of treatment (h)

aa a a

a

A

B

C

B B

0.3

0.4

0.5

0.6

1 3 6 12 24

Control

NaCl

  1 

2 

3 

4 

5 

6 

7 

8 

Figure 1 9 

10 

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0

5

10

15

20

25

SOD

(mg-1

prot

ein)

Control

NaCl

0

1

2

3

4

APX

(µm

ol m

in-1

mg-1

prot

ein)

0

0.4

0.8

1.2

1 6 12 24

GR

(µm

ol m

in-1

mg-1

prot

ein)

Duration of treatment (h)

CA

T (µ

mol

min

-1m

g-1pr

otei

n)

0

0.02

0.04

0.06

Duration of treatment (h)

1 6 12 24

a

aa a

aa

a

a

aa

aa

a aa

a

B

A AA

B

B

AA

AA

B

BBB

AA

A

B

C

D

1 

2 

3 

4 

5 

6 

7 

8 

9 

Figure 2 10 

11 

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0

0.02

0.04

0.06

0.08

CA

T(µ

mol

min

-1m

g-1pr

otei

n)

0

1

2

3

4

APX

(µm

olm

in-1

mg-1

pro

tein

)

0

0.3

0.6

0.9

1.2

1.5

GR

(µm

ol m

in-1

mg-1

prot

ein)

0

5

10

15

20

25

SOD

(U m

g-1pr

otei

n)

c

abb

aa

a

b

ab

aaaa

b

aaa

aa

NaCl - - - - + + + + DMTU - + - - - + - -DPI - - + - - - + -Imidazol - - - + - - - +

- - - - + + + + - + - - - + - -- - + - - - + -- - - + - - - +

- - - - + + + + - + - - - + - -- - + - - - + -- - - + - - - +

- - - - + + + + - + - - - + - -- - + - - - + -- - - + - - - +

A B C D

a

ba a

a aa a a a

b

c

a a

1 

2 

3 

4 

5 

6 

7 

8 

Figure 3 9 

10 

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1 3 6 12 24 1 3 6 12 24Time (h)

Control NaCl

Atrobhd

Atrobhf

APT1

1 

2 

3 

4 

5 

6 

7 

Figure 4 8 

9 

10 

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Control DPI NaCl NaCl + DPIImidazol NaCl + ImidazolA.

B.

1 

2 

3 

4 

5 

6 

7 

8 

9 

Figure 510 

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Control NaCl

WT

atrbohD

atrbohF

atrbohD/F

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

Figure 615 

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0

1

2

3

4

5

APX

(µm

olm

in-1

mg-1

prot

ein)

0

0,4

0,8

1,2

1,6

Control H2O2 NaCl NaCl + H2O2

GR

(µm

ol m

in-1

mg-1

prot

ein)

C

0

0,02

0,04

0,06

0,08

CA

T (µ

mol

min

-1m

g-1pr

otei

n)

WT

atrbohD/F

cc

a

b

C

A

B

A

bb

aa

c

B

AB

A

c

b

aA

B

A

  1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Figure 7 21 

22