hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced...

Accepted Manuscript

Title: Hydrogen peroxide production and mitochondrialdysfunction contribute to the fusaric acid-inducedprogrammed cell death in tobacco cells

Author: Jiao Jiao Ling Sun Benguo Zhou Zhengliang Gao YuHao Xiaoping Zhu Yuancun Liang

PII: S0176-1617(14)00087-XDOI: http://dx.doi.org/doi:10.1016/j.jplph.2014.03.015Reference: JPLPH 51923

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Received date: 18-10-2013Revised date: 8-3-2014Accepted date: 19-3-2014

Please cite this article as: Jiao J, Sun L, Zhou B, Gao Z, Hao Y, Zhu X, Liang Y,Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaricacid-induced programmed cell death in tobacco cells, Journal of Plant Physiology(2014), http://dx.doi.org/10.1016/j.jplph.2014.03.015

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http://dx.doi.org/doi:10.1016/j.jplph.2014.03.015

http://dx.doi.org/10.1016/j.jplph.2014.03.015

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JIAO J et al J Plant Physiol

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Hydrogen peroxide production and mitochondrial dysfunction contribute to the

fusaric acid-induced programmed cell death in tobacco cells

Jiao Jiaoa,1, Ling Suna,1, Benguo Zhoub,1, Zhengliang Gaob, Yu Haoa, Xiaoping Zhua, Yuancun 5

Lianga*

a Department of Plant Pathology,

Shandong Agricultural University, Taian 271018, Shandong Province, China

b Tobacco Research Institute, 10

Anhui Academy of Agricultural Sciences, Hefei 23003, Anhui Province, China

Abbreviations: APX, ascorbate peroxidase; AsA, ascorbic acid; CAT, catalase; CsA, cyclosporine

A; DHR123, dihydrohodamine123; DPI, diphenyl iodonium; FA, fusaric acid; H2O2, hydrogen

peroxide; MDA, malondialdehyde; MPTP, mitochondrial permeability transition pore; NO, nitric 15

oxide; PBS, phosphate buffered saline; ROS, reactive oxygen species; PCD, programmed cell

death.

* Corresponding author. Tel.: +86 538 8242301.

E-mail address: [email protected] (Y. Liang). 20

1 These authors contributed equally to this work.

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SUMMARY Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can

cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the 35

FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen

peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells

were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial

functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production,

lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment 40

of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl

iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like

protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane

potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP

synthase and the mitochondrial permeability transition pore, respectively, could also reduce the 45

rate of FA-induced cell death significantly. Taken together, the results presented in this paper

demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during

the FA-induced PCD in tobacco suspension cells.

Keywords: 50

Fusaric acid

Programmed cell death

Hydrogen peroxide

Mitochondrion

Tobacco suspension cells 55

Introduction

Programmed cell death (PCD) is an active process that regulates plant organ development and

cellular homeostasis (Green and Reed, 1998). Previous studies have indicated that during plant

growth and development, PCD can be induced by multiple biotic and abiotic stress stimuli 60

including salt stress and toxins produced by plant pathogens (Yao et al., 2001; Duval et al., 2005;

Lin et al., 2006; Samadi and Behboodi 2006; Wang et al., 2010; Jiao et al., 2013). A number of

studies have also indicated that mitochondria, chloroplast, and signalling molecules have profound

effects on PCD (Balk and Leaver, 2001; Vacca et al., 2006; Gadjev et al., 2008; Reape and

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McCabe, 2010; Godbole et al., 2013). 65

Fusaric acid (FA, 5-butylpicolinic acid) is a non-specific toxin produced by Fusarium species

(Bacon et al., 1996), a common phytopathogen of many economically important plant species.

Although the role of FA in Fusarium pathogenicity has not been fully characterized (Gapillout et

al., 1996), it is known to function as a virulence factor and its production can affect disease

symptom development. Several studies have shown that higher concentrations of FA (>10–4 M) 70

were toxic to plant and can cause cell growth, change membrane permeability, decrease of

mitochondrial activity, inhibition of ATP synthesis, and change antioxidant enzyme activities

(Marré et al., 1993; Kuźniak, 2001). In contrast, low concentration of FA (<10–6 M) could act as

an elicitor for phytoalexin synthesis and reactive oxygen species (ROS) production (Bouizgarne et

al., 2006). Furthermore, moderate levels of FA were reported to induce cytoplasmic shrinkage, 75

chromatin condensation, DNA fragmentation, cytochrome c release, and activation of

caspase-3-like protease, eventually leading to PCD in saffron and tobacco cells (Samadi and

Behboodi, 2006; Jiao et al., 2013).

During PCD in animal cells, changes of cell morphology and integrity, and dysfunction of

mitochondria were reported (Petit et al., 1995). The role of mitochondria in PCD has also been 80

demonstrated in plant cells treated with victorin or ultraviolet-C (Yao et al., 2002; Yao et al., 2004;

Vianello et al., 2007; Gao et al., 2008). Our previous report has indicated that FA treatment could

cause significant damage to the integrity of mitochondrial membrane in tobacco cells (Jiao et al.,

2013). Alterations of mitochondrial membrane potential (∆Ψm) and mitochondrial permeability

transition pore (MPTP) were shown to play important roles in early events in specific cell death 85

pathways (Diamond and McCabe, 2011; Vianello et al., 2012). To date, the relationship between

the FA-induced PCD and mitochondrial function remains unknown.

Jiao and co-workers have indicated that FA treatment of tobacco suspension cells resulted in the

production of several hallmarks of PCD, and activation of caspase-3-like protease modulated by

nitric oxide (NO) signalling molecule was responsible for the FA-induced PCD (Jiao et al., 2013). 90

Because FA-induced PCD was not completely prevented by the presence of a NO scavenger,

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), it was proposed that

other molecules or signalling pathways may regulate the FA-induced PCD. Moreover, Samadi and

Behboodi (2006) demonstrated that hydrogen peroxide (H2O2) production could be suppressed by

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diphenyl iodonium (DPI), a well-known inhibitor of NADPH oxidase, in the FA-treated saffron 95

cells.

Here we provide evidence showing that the H2O2 production and mitochondrial dysfunction can

affect the FA-induced PCD.

Materials and Methods 100

Cell culture and treatments

All experiments were conducted using 4-day-old Nicotiana tabacum cv. NC89 suspension cells.

The suspension cells were cultured in the liquid Murashige–Skoog medium, supplemented with 30

g L–1 sucrose, 2 mg L–1 α-naphthalene acetic acid, and 0.2 mg L–1 6-benzyladenine as previously

described (Liu et al., 2010). The cultured suspension cells were then sub-cultured once every 7 105

days by transferring the cells into (1:10, v/v) fresh media. During each experiment, the cultured

suspension cells were treated with 100 μM FA (Sigma-Aldrich, USA). The cultured suspension

cells treated with sterilized water were used as controls for the study. Oligomycin and DPI (both

from Sigma-Aldrich, USA) were individually dissolved in dimethyl sulfoxide to make 20 mM

stock solutions. Cyclosporin A (CsA; Sigma-Aldrich, USA) was dissolved in 90% ethanol to make 110

the 50 mM stock solution. All stock solutions were stored at –20 °C until use. Both scavengers and

inhibitors were added to the culture medium, when required, for 10 min (DPI) or 30 min (AsA,

CsA, and oligomycin) prior to the FA treatment.

Assay of cell death 115

Cells showing death were determined through Evans blue staining as described (Baker and

Mock, 1994) followed by examination under a C-35AD-2 light microscope (Olympus). Cells

stained with Evans blue dye were considered as dead cells and recorded. Over 500 cells were

counted for each treatment. The experiment was repeated at least three times.

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Assay of hydrogen peroxide

Extracellular H2O2 production was measured according to the method described previously by

Bellincampi et al. (2000). Briefly, 1 mL cell culture was collected from each treatment and

pelleted through 20 second centrifugation at 10,000 × g. Concentrations of H2O2 in individual

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samples were measured using the resulting supernatants. An aliquot of supernatant (500 µL) was 125

mixed with 500 µL assay reagent containing 500 µM ferrous ammonium sulfate, 50 mM H2SO4,

200 µM xylenol orange, and 200 mM sorbitol. After 45 min of incubation, the peroxide-mediated

oxidation of Fe2+ to Fe3+ was determined by measuring the samples at A560 for the Fe3+ xylenol

orange complex. The amount of H2O2 was then calculated using the standard curve generated by

using specific dilutions of H2O2 in the culture medium. 130

Intracellular H2O2 accumulation was measured using a fluorescent probe, dihydrohodamine

(DHR) 123 (Sigma-Aldrich, USA), as described (Royall and Ischiropoulos, 1993). The cultured

cells were treated with 5 μM DHR123 for 30 min, rinsed, and re-suspended in fresh medium. The

cells were then treated with FA for an additional 60 min and the resulting fluorescence was

monitored under a confocal laser scanning microscope (Zeiss, LSM 510 META). Fluorescence 135

intensity quantification was performed on over 100 cells per treatment.

Activities of ascorbate peroxidase and catalase

Ascorbate peroxidase (APX) activity was determined as previously describe by Nakano and

Asada (1981). Briefly, cell samples were ground in liquid nitrogen and then homogenized in an 140

extraction buffer [50 mM PBS (pH 7.0), 0.1 mM EDTA, 1% polyvinylpolypyrrolidone (PVP), 0.5

mM AsA] followed by 5 min incubation on ice. After centrifugation at 12,000 × g for 20 min, the

supernatants were collected and used for the APX activity assay. Oxidization of ascorbate should

result in a decrease of absorbance at 290 nm based on the extinction coefficient of 2.8 mM–1 cm–1.

The reaction mixture contained 50 mM PBS (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA Na2, and 145

1.2 mM H2O2. One enzyme unit was defined as μM oxidized ascorbate per min.

Catalase (CAT) activity assay was done as described by Aebi (1984) by measuring the decline

of the extinction of H2O2 at 240 nm for 3 min. Cell samples were extracted in an extraction buffer

containing 50 mM PBS (pH 7.0), 1% PVP, 10 mM β-mercaptoethanol for 5 min on ice. The

reaction mixture (3 mL per sample) contained 50 mM PBS (pH 7.0), 10 mM H2O2, and 200 μL 150

enzyme solution. The absorption of each sample was monitored for 3 min and μM H2O2 destroyed

per min was defined as one unit of CAT.

Assay of lipid peroxidation

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The content of malondialdehyde (MDA) in the assayed suspension cells was used to determine 155

the lipid peroxidation via the thiobarbituric acid (TBA) reaction as described (Dhindsa et al.,

1981). Tobacco cells (500 mg per sample) were harvested from various samples and homogenized

in 0.1% trichloroacetic acid buffer (5 mL buffer per sample). The homogenates were pelleted by

10 min centrifugation at 10,000 × g. The supernatant was diluted 1:5 (v/v) in 20% trichloroacetic

acid and 0.5% TBA. After 30 min incubation at 95 °C, the samples were centrifuged at 20,000 × g 160

for 10 min. The absorbance of each resulting supernatant was then measured at 532 nm.

Assay of caspase-3-like activity

The suspension cells were harvested at 12 h post treatment with FA and then ground in liquid

nitrogen followed by 5 min lysing in an extraction buffer (Beyotime, China) on ice. The lysates 165

were centrifuged for 20 min at 12,000 × g and the resulting supernatants were used for the

caspase-3-like activity analysis. The caspase-3-like activity was determined using the Caspase-3

Activity Assay Kit as instructed (Beyotime, China). Hydrolysis of the peptide substrate

acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3 resulted in the release of

p-nitroaniline (pNA) moiety. The sample absorbance at 405 nm was measured using a microplate 170

reader as instructed (Molecular Devices, SpectraMax M2). Protein concentration of each sample

was determined using the Bradford assay as described (1976). The experiment was repeated three

times.

Measurement of FA-induced mitochondrial membrane potential and ATP content 175

Mitochondria were isolated from the assayed cells using the cell mitochondria isolation kit as

instructed (Beyotime, China), and used for mitochondrial membrane potential (∆Ψm) and ATP

content assays. The protein content in each mitochondrial preparation was determined using the

Bradford assay (1976). Mitochondrial ∆Ψm was determined using the mitochondrial membrane

potential assay kit as described (Beyotime, China). 180

Total ATP content was determined using the luciferase-based assay kit (Beyotime, China), and

the luminescence was measured with a fluorescence spectrometer (Hitachi, F-4600). The ATP

content was estimated based on the detected luminescence and comparison with the ATP

standards provided in the kit.

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185

Statistical analysis

All data were presented as the mean ± standard deviation. For statistical analysis shown in Fig.

3, results were considered to be statistically significant when P < 0.05 as determined by the

Student’s t-tests. For other statistical analyses, the Duncant’s multiple range test was applied.

190

Results

H2O2 production and its role in the FA-treated cells

To determine whether FA induces H2O2 production in the tobacco suspension cells, we

measured both extra- and intracellular H2O2 accumulation. Results shown in Fig. 1A indicate that

the cells showed two peaks of H2O2 production in the extracellular medium within 240 min post 195

FA treatment. The first peak was smaller and occurred at 30 min post the FA treatment and the

second peak occurred at 120 min post the treatment. The highest production level of H2O2 in the

FA-treated cells was about 10 fold higher than that observed in the control cells. At 240 min post

FA treatment, the H2O2 level in the FA-treated cells decreased to a similar level as that observed in

the control cells. Interestingly the H2O2 level in the FA treated cells did not increase significantly 200

after two peaks and remained at a level similar to that observed in the control cells for up to 24 h.

Intracellular H2O2 accumulation was also measured using a permeable fluorescent probe, DHR123,

and confocal analysis showed a significant fluorescence increase in the FA-treated cells compared

with the control cells (Fig. 1B and C).

To further investigate the role of H2O2 in the FA-induced PCD, AsA (a H2O2 scavenger) or DPI 205

(a NADPH oxidase inhibitor) was added to the culture medium before the FA treatment. The

treated cells were measured at 24 h post the FA treatment. Results showed that pre-treatment of

cells with AsA or DPI significantly reduced the rates of the FA-induced cell death (Fig. 1D),

indicating that the H2O2 production is indeed involved in the FA-induced cell death.

210

Changes of APX and CAT activities in the FA-treated cells

Activities of two antioxidant enzymes were monitored in this study to elucidate the function of

H2O2 accumulation in the FA-treated cells. Results show that the activities of APX and CAT were

steadily decreased in the cells treated with 100 μM FA compared with the control cells. At 6 h

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post FA treatment, the activities of APX and CAT were both reduced to about 50% of the control 215

cells and at 24 h post the FA treatment, the CAT and APX activities were 7 and 8-fold lower than

that in the control cells, respectively (Fig. 2A and B).

Increase of lipid peroxidation in the FA-treated cells

To investigate the alteration of redox status upon FA treatment, suspension cells were analyzed 220

for lipid peroxidation level through measuring the MDA content. The MDA content in 100 μM

FA treated cells increased gradually up to 24 h post treatment. The significant change in lipid

peroxidation level was observed initially at 6 h post the FA treatment. The differences of the MDA

content between the FA-treated and control cells reached approximately three fold at 24 h post the

FA treatment (Fig. 3). 225

Effect of H2O2 on caspase-3-like activity in the FA-treated cells

We previously reported that the activity of caspase-3-like protease was induced by the FA

treatment and it reached its maximum at 12 h post the treatment (Jiao et al., 2013). To investigate

the effect of H2O2 on caspase-3-like activity, we pre-treated the cells with an antioxidant molecule 230

AsA or a NADPH oxidase inhibitor DPI prior to the FA treatment. The inhibition of caspase-3-like

activity by AsA or DPI was measured at 12 h post the FA treatment. As shown in Fig. 4, the

pre-treatment of cells with AsA or DPI significantly reduced the caspase-3-like activity in cells.

The caspase-3-like protease activities in cells treated with AsA or DPI were similar to that shown

in the control cells. These results indicate that the activation of caspase-3-like protease during 235

FA-induced PCD was regulated by the H2O2 signalling.

Mitochondrial membrane potential and ATP level in the FA-treated cells

To monitor the changes of mitochondrial function in the FA-treated cells, we analyzed the

mitochondrial membrane potential (∆Ψm) and the ATP content. Mitochondrial ∆Ψm was 240

decreased significantly upon the FA treatment. When cells were analyzed at 24 h post the FA

treatment, the mitochondrial ∆Ψm showed a 65.8% decrease when compared with the control cells

(Fig. 5A). Similarly, the ATP contents in the FA-treated cells were significantly decreased. At 24

h post FA treatment, the cells showed a 82.4% decrease in the ATP content compared with the

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control cells (Fig. 5B). 245

Role of mitochondrial ATP synthase and mitochondrial permeability transition pore in the

FA-induced PCD

To investigate the role of mitochondrial ATP synthase and mitochondrial permeability

transition pore (MPTP) in the FA-induced cell death, we pre-treated cells with oligomycin or 250

cyclosporine A (CsA). Oligomycin and CsA were previously reported as the inhibitors of

mitochondrial ATP synthase (Krömer et al., 1998) and MPTP (Tiwari et al., 2002; Gao et al.,

2008), respectively. Results in Fig. 6 show that the rate of FA-induced cell death was significantly

reduced in the presence of oligomycin or CsA, indicating that the mitochondrial ATP synthase and

MPTP play roles in the FA-induced PCD. 255

Discussion

In our previous study, we demonstrated that the FA treatment could induce hallmarks of PCD.

We also presented evidence showing that the FA-induced PCD was modulated by the NO

signalling pathway via activation of caspase-3-like protease activity (Jiao et al., 2013). However, 260

the roles of other signalling molecules, as well as mitochondrial function in the FA-induced

PCD,remained unknown.

H2O2 is a crucial signalling molecule during plant PCD (Yao et al., 2002; Lin et al., 2006; Gao

et al., 2008; Rodríguez-Serrano et al., 2012). Previous reports have indicated that H2O2 production

was elevated in the FA-treated Arabidopsis thaliana and saffron cells, and the elevation of H2O2 265

production could be inhibited by the NADPH oxidase inhibitor DPI (Bouizgarne et al., 2006;

Samadi and Behboodi, 2006). This suggests that FA invokes the H2O2 accumulation caused by the

membrane-bounded NADPH oxidase. Results presented in this paper showed that the H2O2

production was increased in a diphase manner in the FA treated tobacco cells. This observation

was consistent with that observed in the biotic-stressed or abiotic-stressed cells (Lamb and Dixon, 270

1997; Locato et al., 2008). Bolwell et al. (2002) indicating that the biphasic production of ROS

was a typical feature of PCD. To determine if H2O2 played a role in the FA-induced cell death we

also applied H2O2 antioxidant AsA and NADPH oxidase inhibitor DPI to the tobacco suspension

cells followed by analysis of their effects on cell viability. Our results clearly showed that both

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AsA and DPI could greatly reduce the rate of FA-induced cell death. Similar results were also 275

observed in the salt stress-induced tobacco protoplast death and the aluminum-induced tomato cell

death (Lin et al., 2006; Yakimova et al., 2007). Significant increase of NO production in the

FA-treated cells and pre-treatment of cells with NO scavenger cPTIO resulted in an increase of the

ratio of cell viability during the FA-induced cell death were previously shown (Jiao et al., 2013).

The interaction between H2O2 and NO during the FA-induced cell death remains unclear and 280

requires further studies.

APX and CAT, two key antioxidant enzymes for H2O2 in plants (Gill et al., 2010), were

investigated for their roles in H2O2 elevation. Our results show that the activities of these two

antioxidant enzymes were gradually decreased upon the FA treatment (Fig. 2), leading to an

accumulation of H2O2. The negative correlation between H2O2 accumulation and APX activity 285

was also reported in the heat shock-induced PCD in tobacco Bright-Yellow 2 cells (Vacca et al.,

2004; Locato et al., 2008). Kuźniak (2001) demonstrated that the FA treatment did not influence

the level of H2O2 or APX activity in tomato cells derived from a tomato cultivar, which was

resistant to F. oxysporum f. sp. lycopersici. The content of MDA was used as an indicator for the

level of lipid peroxidation resulted from the oxidative stresses (Smirnoff, 1993). In the present 290

study, the MDA content increased steadily after the FA treatment. This finding indicates that FA is

a molecule that can cause membrane lipid peroxidation, and the result of membrane lipid

peroxidation can contribute to a decrease of CAT and APX activities. Lipid peroxidation is known

to promote the release of cytochrome c into cytosol (a hallmarker of PCD). It was shown that

cytochrome c released from mitochondria to cytosol in the saffron root tip cells after the FA 295

treatment (Samadi and Behboodi, 2006). Consequently, we propose that the decrease of

antioxidant enzyme activity and the increase of NADPH oxidase activity lead to the elevation of

H2O2 , followed by lipid peroxidation during the FA-induced cell death.

Recent studies have indicated that the caspase-3-like activity has a critical effect during PCD in

plants (Wang et al., 2010; Han et al., 2012; Iakimova et al., 2013; Ye et al., 2013). Vacca et al. 300

(2006) demonstrated that ROS scavenger enzymes blocked the caspase-3-like activity in the

heat-shocked tobacco cells. Earlier study by Jiao et al showed that the caspase-3-like activity was

induced in the FA-treated cells, and the caspase-3-like activity could be suppressed by application

of NO scavenger cPTIO (Jiao et al., 2013). Results presented in this paper further demonstrate that

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AsA and DPI can regulate the activity of caspase-3-like protease in the FA-treated cells (Fig. 4). 305

This observation suggests that the H2O2 production is involved in the FA-induced PCD through

activation of caspase-3-like protease. This new finding sheds light on the relationship between the

H2O2 accumulation and caspase-3-like protease activity.

Numerous studies have shown that mitochondria function has a role in regulating PCD in plants

(Balk and Leave, 2001; Lam et al., 2001; Yao et al., 2004; Vianello et al., 2007; Diamond and 310

McCabe, 2011). We reported previously that FA-treatment caused ultra-structural changes of

mitochondrial morphology during PCD in tobacco cells (Jiao et al., 2013). We propose that

mitochondrial function has a critical role during the FA-induced PCD. To further confirm the role

of mitochondrial dysfunction in the FA-induced PCD we measured mitochondrial ∆Ψm and ATP

content. Our results indicate that the decrease of ∆Ψm did occur during the FA-induced cell death 315

(Fig. 5A). It was also demonstrated by others that treatment of Arabidopsis suspension cells with

harpin or acetylsalicylic acid led to a decrease of ∆Ψm during PCD (Krause and Durner, 2004;

García-Heredia et al., 2008). Plant ATP is primarily produced in mitochondria (Haferkamp et al.,

2011), especially in the suspension cells which do not have functional chloroplasts. In this study,

the decrease of ATP content was confirmed in the FA-treated cells (Fig. 5B). This finding is 320

similar to that reported in the tobacco BY-2 cells treated with benzyladenosine and the

Arabidopsis cells induced by oxidative stress (Tiwari et al., 2002; Mlejnek et al., 2003). In the

FA-treated tobacco suspension cells we also investigated the role of mitochondrial dysfunction

through pharmacological assays. Mitochondrial ATPase inhibitor oligomycin is known to block

the respiratory chain complex and can function as the crucial enzyme for ATP generation in 325

mitochondria (Krömer et al., 1988). In this study we determined that the ATP synthesis played a

significant role in cell viability during the FA-induced PCD. Similarly, our results show that CsA,

an inhibitor of MPTP, can significantly reduce the FA-induced cell death rate (Fig. 6) and thus the

open state of MPTP is necessary for the development of FA-induced PCD. This finding is similar

to that observed in the oxidative- or salt stress-induced plant cell death (Tiwari et al., 2002; Lin et 330

al., 2006; Vianello et al., 2012). In addition, release of cytochrome c from mitochondria to cytosol

was observed in the FA-treated saffron root-tip cells (Samadi and Behboodi, 2006). All the above

observations suggest that alterations of mitochondrial function in the FA-treated tobacco cells are

the primary reason contributing to the cell death.

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In conclusion, we have presented strong evidence to demonstrate that the FA treatment can 335

elevate the H2O2 accumulation and suppress the activities of two antioxidant enzymes (APX and

CAT) in tobacco cells. The H2O2 accumulation in cells can lead to lipid peroxidation,

caspase-3-like protease activation, and increase of cell death rate. In addition, the FA treatment

promotes mitochondrial dysfunction, a critical causal event in the FA-induced cell death.

340

Acknowledgements This work was supported by the National Natural Science Foundation

(31171806), Shandong Provincial Natural Science Foundation (ZR2012CM032), and Anhui

Province Tobacco Company Project (20100551002, 20100551005) in China.

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Fig. 1. H2O2 production and its role in the FA-induced cell death. (A) H2O2 production in cell

cultures with 100 μM FA treatment. Confocal microscopic observation (B) and fluorescence

intensities (C) of intracellular H2O2 production in tobacco cells at 60 min post FA treatment. Bars

20 μm. (D) Effects of ascorbic acid (AsA) and diphenyl iodonium (DPI) on the FA-induced cell 495

death. Cells were treated with 100 μM AsA or 10 μM DPI at 30 or 10 min before the FA (100 μM)

treatment. Cells were collected at 24 hours post FA treatment and stained with Evans blue dye

(0.05%) prior to microscopy.The data are shown as the means ± SE from three independent

experiments. Different letters above the bars indicate a significant difference by Duncan’s multiple

range test (P < 0.05). 500

Fig. 2. Activities of APX (A) and CAT (B) in the FA-treated tobacco cells. The cells were treated

at different hours post FA (100 μM) treatment. The data are shown as the means ± SE from three

independent experiments.

505

Fig. 3. Lipid peroxidation in the FA-treated tobacco cells. The cells were treated with 100 μM FA

at the indicated time. The data are shown as the means ± SE from three independent experiments.

Asterisk indicates significant differences from the control by Student’s t-tests (p < 0.05).

Fig. 4. Effect of H2O2 on the caspase-3-like protease activity at 12 hours post FA treatment. Cells 510

were treated with 100 μM AsA or 10 μM DPI at 30 or 10 min before the FA (100 μM) treatment.

Different letters above the bars indicate a significant difference by Duncan’s multiple range test (P

< 0.05).

Fig. 5. Mitochondrial membrane potential and mitochondrial ATP content in the FA-treated 515

tobacco cells. The cells were treated with FA at the indicated time.

Fig. 6. Effect of the mitochondrial ATP synthase inhibitor oligomycin and a mitochondrial

permeability transition pore inhibitor cyclosporine A (CsA) on the death of FA-induced cells. Cells

were treated with 10 μM oligomycin or 50 μM CsA at 30 min prior to the FA treatment. The cells 520

were harvested at 24 hours post FA treatment and stained with Evans blue dye (0.05%) before

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microscopy. Different letters above the bars indicate a significant difference by Duncan’s multiple

range test (P < 0.05).

525

Fig. 1 530 535 540 545 550 555 560

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Fig. 3 610 615 620

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Fig. 5 650

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Fig. 6 680 685

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hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced...

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