Research Article Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide

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1 Hindwi Pulishing Corportion Advnes in Toxiology, Artile ID 47368, 9 pges Reserh Artile Dul Role of Hydrogen Peroxide in Aridopsis Gurd Cells in Response to Sulfur Dioxide Huiln Yi, 1 Xin Liu, 1,2 Min Yi, 1,3 nd Gng Chen 2 1 Shool of Life Siene, Shnxi University, Tiyun 36, Chin 2 Shnxi Provinil Guoxin Energy Development Group Co., LTD., Tiyun 36, Chin 3 Deprtment of Sttistis, University of Missouri-Columi, Columi, MO 6211, USA Correspondene should e ddressed to Huiln Yi; yihl@sxu.edu.n Reeived 28 April 214; Aepted 8 Septemer 214; Pulished 3 Septemer 214 Ademi Editor: Mugimne Mnjnth Copyright 214 Huiln Yi et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Sulfur dioxide (SO 2 ) is mjor ir pollutnt nd hs signifint impts on plnt physiology. Plnt n dpt to SO 2 stress y ontrolling stomtl movement, gene expression, nd metoli hnges. Here we show ler evidenes tht SO 2 -triggered hydrogen peroxide (H 2 O 2 ) prodution medited stomtl losure nd ell deth in Aridopsis leves. High levels of SO 2 used irreversile stomtl losure nd deline in gurd ell viility, ut low levels of SO 2 used reversile stomtl losure. Exogenous ntioxidnts sori id (AsA) nd tlse (CAT) or C 2+ ntgonists EGTA nd LCl 3 loked SO 2 -indued stomtl losure nd deline in viility. AsA nd CAT lso loked SO 2 -indued H 2 O 2 nd [C 2+ ] yt elevtion. However, EGTA nd LCl 3 inhiited SO 2 -indued [C 2+ ] yt inrese ut did not suppress SO 2 -indued H 2 O 2 elevtion. These results indite tht H 2 O 2 elevtion triggered stomtl losure nd ell deth vi [C 2+ ] yt signling in SO 2 -stimulted Aridopsis gurd ells. NADPH oxidse inhiitor DPI loked SO 2 -indued ell deth ut not the stomtl losure triggered y low levels of SO 2, inditing tht NADPH oxidsedependent H 2 O 2 prodution plys ritil role in SO 2 toxiity ut is not neessry for SO 2 -indued stomtl losure. Our results suggestthth 2 O 2 prodution nd umultion in SO 2 -stimulted plnts trigger plnt dpttion nd toxiity vi retive oxygen speies mediting C 2+ signling. 1. Introdution Sulfur dioxide (SO 2 ) is hrmful gs tht is emitted lrgely from urning ol, high-sulfur oil, nd fuels. During the pst few dedes, the onentrtion of SO 2 in the tmosphere hs inresed in mny res of the world, espeilly in the developing ountries. High levels of SO 2 n injure mny plnt speies nd vrieties, resulting in photosynthesis deline, growth inhiition, nd even deth [1 4]. Sulfur dioxide enters plnts minly through the open stomt []. One it enters the lef, SO 2 is hydrted to form HSO 3 nd SO 2 3.ThetoxiityofSO 2 is derived from moleulr speies sulfite (SO 2 3 )ndisulfite(hso 3 ) generted fter SO 2 is dissolvedinellulrfluid[6]. Sulfite oxidtion, whih is the detoxifition retion of sulfite to sulfte (SO 2 4 ), leds to the formtion of retive oxygen speies (ROS) in plnt ells [7, 8]. The prodution nd umultion of ROS re one of the key events in plnt response to SO 2 [9 12]. ROS hve een proposed s entrl omponents of plnt response to oth ioti nd ioti stresses. Under suh onditions, ROS my ply two very different roles: exerting dmge or signling the tivtion of defense responses [13, 14]. It hs een reported tht ROS t s signling moleules mediting vriety of physiologil responses, inluding stomtl movement nd gene expression [1 17], lthough they n ttk iomoleules suh s nulei ids, proteins, nd lipids leding to ell dmge nd deth [16, 18, 19]. Results of previous studies hve shown tht plnts n dpt to SO 2 stress y ontrolling stomtl movement nd gene trnsription [11, 12, 2]. Stomtl losure ould protet the leves ginst further entry of the environmentl SO 2, while differentil gene expression ould regulte the metoli routes of plnt ells providing long-term dpttion to environmentl stress. However, up to now, it is not ler if the ROS prodution is losely ssoited with the initil physiologil mehnisms responsile for plnt responses to SO 2 stress.

2 2 Advnes in Toxiology ROS overprodution n trigger plnt dpttion or ell dmge during ioti stress. However, there hs een little overlp etween these two fields of reserh. In the present study, gurd ells of A. thlin leves, whih re welldeveloped model ell system for studying the signl trnsdution in plnt ells [21 23], were employed to investigte theellulrmehnismofplntresponsetoso 2 stress. To the est of our knowledge, this is the first report of H 2 O 2 mediting oth dpttion response (stomtl movement) nd ytotoxiity (ell deth) in plnt response to SO 2 stress. Our results show tht H 2 O 2 elevtion triggered oth stomtl losure nd ell viility loss vi C 2+ signling in SO 2 -treted plnts. 2. Mterils nd Methods 2.1. Plnt Mteril Preprtion. Plnts of Aridopsisthlin (L.) eotype Columi (Col-) were grown in soil (Klsmnn-Deilmnn) in temperture-ontrolled growth rooms t 22 Cwithnvergelightintensityof24μmoL m 1 s 1, 16 h photoperiod per dy, nd 6% reltive humidity. Young fully expnded leves were hrvested from 4- week-old Aridopsis plnts. The xil epidermes were peeled from the underside of eh lef nd ut into smll piees. The isolted epiderml strips were immeditely floted in 1 mm 2-(N-morpholino)ethnesulfoni id (MES; Bio Bsi In.) uffer (1 mm MES-Tris, ph 7., nd mm KCl) Determintion of Stomtl Aperture. The isolted epiderml strips were inuted in 1 mm MES uffer for 2 h, for stomt-opening under ontinuous light of 18 μmol m 2 s 1 t 22 C or for stomt-losuring in the drk, nd then inuted for 2 h in MES uffer ontining ertin mount of SO 2 hydrtes ( mixture of sodium sulfite nd sodium isulfite, 3 : 1 mm/mm, prepred freshly efore use) under ontinuous illumintion of 18 μmol m 2 s 1 t 22 C. Control smples were treted under the sme onditions with 1 mm MES uffer. The isolted epiderml strips were treted with mixture of SO 2 hydrtes nd ertin mount of ntgonists whih inlude NADPH oxidse inhiitor diphenylene iodonium (DPI, Sigm), ntioxidnts tlse (CAT, Sigm), nd sori id (AsA, Sigm) nd C 2+ ntgonists LCl 3 nd ethylene glyol tetreti id (EGTA, Sigm) to exmine the protetive effets. After 2 h of hemil exposure, the epiderml strips were mounted on mirosopy slide, moistened with 1 mm MES uffer, nd overed with slip. Stomtl perture ws mesured y using digitl mirosope mer system (DP72, Olympus) nd n tthed DP2-BSW softwre. At lest three leves nd 3 stomt per lef were mesured in eh tretment nd ll of the experiments were independently repeted t lest three times. Forthereoverygroups,fter2hofhemilexposure,the isolted strips were resuspended in MES uffer for 2 h under ontinuous light followed y stomtl perture mesurement. For time-ourse experiment, the stomtl pertures were exmined every five minutes fter isolted strips were inuted in SO 2 hydrtes Determintion of Cell Viility. Cell viility ws ssessed y using the method of doule stining with fluoresein diette (FDA; Bio Bsi In.) nd propidium iodide (PI; Sigm). After 2 h of hemil exposure, the epiderml strips were simultneously stined with.1 mg L 1 FDA nd 1 μm PI for visulizing ell viility. At lest three leves nd 3 gurd ells per lef were oserved in eh tretment nd ll of the experiments were independently repeted three times Mesurement of Retive Oxygen Speies. Retive oxygen speies in gurd ells of epiderml peels were deteted using 2,7 -dihlorodihydrofluoresein diette (DCFH- DA; Beyotime) ording to the methods desried y Yi et l. [24]. After 2 h of exposure to the hemils, the epiderml strips were inuted in 2 μm DCFH-DA for 3mininthedrk.Themenvlueoffluoreseneintensity, resulting from 6 gurd ells in three independent experiments, represents the intrellulr H 2 O 2 level for eh tretment. 2.. Mesurement of Intrellulr C 2+. The onentrtion of intrellulr C 2+ ([C 2+ ] yt ) ws mesured using fluo-3 etomethoxyester (Fluo-3 AM; Beyotime) s desried in our previous report [24]. The verge fluoresene intensity of Fluo-3 AM otined from three different leves nd 2 gurd ells represents the [C 2+ ] yt level for eh tretment. All experiments were independently repeted three times Sttistil Anlysis. All vlues of men nd stndrd devition (SD) were otined from three independent experiments. Anlysis of vrine (ANOVA) nd Dunnett s t-test were used to determine the signifint differenes mong the ontrol nd series of tretment groups. 3. Results 3.1. SO 2 -Evoked H 2 O 2 Prodution Is Involved in the Regultion of Stomtl Movement. As shown in Figure 1, SO 2 hydrtes promote stomtl losure nd inhiit light-promoted stomtl opening in Aridopsis leves. The width of stomtl perture deresed in onentrtion-dependent mnner ndshowedsignifintdereseftertheisoltedstrips were exposed for 2 h to SO 2 hydrtes t onentrtions of 1 to μm. However, stomtl losure evoked y low SO 2 onentrtions (elow 2 μm)ould e reversed ompletely fter SO 2 removl; otherwise the deline of stomtl perture in high SO 2 onentrtion ( μm) group ould e prtly reversed. Time ourse nlysis of stomtl movement showed tht the dimeter of the stomtl perture deresed mrkedly within the first 3 minutes of exposure to 1 μmso 2 hydrtes nd ontinued to deline t slower rte in the remining 9 minutes of SO 2 exposure (Figure 1()). SO 2 -indued stomtl losure ws ssoited with n elevted H 2 O 2 level in Aridopsis gurd ells. Exposure to SO 2 hydrtes not only used smller stomtl pertures

3 Advnes in Toxiology Stomtl perture (μm) Stomtl perture (μm) Conentrtions of SO 2 hydrtes (μm) Conentrtions of SO 2 hydrtes (μm) Sulfur dioxide tretment Reovery fter sulfur dioxide tretment Pretretment in the drk () () 6 Stomtl perture (μm) Time ourse from 1 μm SO 2 hydrtes (min) () Figure 1: Promotion of stomtl losure y SO 2 nd its reversiility upon SO 2 removl. Stomt of Aridopsis lef epidermis were llowed to open in light [() nd ()] or to lose in the drk () for 2 h nd then were inuted for 2 h in SO 2 hydrtes., A signifint differene t P <. from ontrol., A signifint differene t P <. etween SO 2 tretment nd its reovery group. (Figure 1) ut lso evoked inresed H 2 O 2 level (1.2- to 1.7-fold) in Aridopsis gurd ells (Figure 2). When H 2 O 2 elevtion ws loked y exogenous H 2 O 2 svengers CAT or AsA, SO 2 -indued stomtl losure ws effiiently reversed (Figure 2). These results indite tht H 2 O 2 prodution in SO 2 -stimulted gurd ells medites stomtl movement upon SO 2 stress SO 2 -Evoked H 2 O 2 Elevtion Triggered Cell Deth. To investigte the role of H 2 O 2 in irreversile stomtl losure in response to SO 2, we detet the viility of Aridopsis gurd ells y doule stining with fluoresein diette (FDA) nd propidium iodide (PI). The results showed tht SO 2 indued gurd ell deth in onentrtion-dependent mnner (Figure 3). Cell deth rehed 24% in 6 mm SO 2 hydrtes tretment group, ut no ovious ell deth ould e oserved in 1 to 2 μmso 2 hydrtes tretment groups. The H 2 O 2 level of gurd ells exposed to 2 to 6 mm SO 2 hydrtes showed sttistilly signifint inrese (1.9- to 2.3-fold), whih is higher thn those exposed to 1 to μm SO 2 hydrtes. Moreover, exposure to SO 2 hydrtes simultneously with 2 U ml 1 CAT or.1 mm AsA, SO 2 - indued ell deth ws effiiently loked, ssoited with signifint derese in H 2 O 2 level of gurd ells (Figure 3). These results lerly demonstrte tht n elevted H 2 O 2 level n trigger gurd ell deth leding to stomtl dysfuntion nd irreversile stomtl losure in SO 2 -treted Aridopsis leves. To further onfirm the role of H 2 O 2 in SO 2 toxiity, we investigte the protetive effets of CAT on ell viility

4 4 Advnes in Toxiology A B C D E F Stomtl perture (μm) A B C D E F Reltive intensity of DCFH-DA fluoresene (%) A B C D E F (A) (B) (C) Control 2 μm SO 2 hydrtes 2 μm SO 2 hydrtes +1mM AsA (D) 2 μm SO 2 hydrtes + 2 U ml 1 CAT (E) 2 μm SO 2 hydrtes +.1 mm EGTA (F) 2 μm SO 2 hydrtes +.1 mm LCl 3 Figure 2: Effets of ntioxidnts nd lium ntgonists on SO 2 -indued stomtl losure nd H 2 O 2 elevtion in Aridopsis gurd ells. Different supersript letters indite signifint differenes (P <.1). The sme letters indite no signifint differene. The green fluoresene (DCFH-DA) of gurd ells indites H 2 O 2 ontent. in SO 2 -treted smples. The results showed tht pplition of 1 U ml 1 CAT ould ompletely lok the ell deth evoked y 2 mm nd lower onentrtions of SO 2 hydrtes, ut the ytotoxiity evoked y 6 mm SO 2 hydrtes ws only prtly loked (Figure 4). These results lerly demonstrte tht H 2 O 2 prodution, whih my work together with other moleules, is enough to trigger SO 2 toxiity H 2 O 2 Ation Is Dependent on Its Conentrtions nd Sptil Genertion Ptterns. As shown ove, SO 2 n use stomtl losing nd deline in ell viility, whih is dependent on SO 2 onentrtions nd H 2 O 2 level. However, there ws no ler dividing line etween sfe level nd toxi level. In order to understnd wht onstitutes sfe level, n inhiitor of NADPH oxidse ws used to detet its inhiitory effets. The results show tht pplition of 2 μm NADPH oxidse inhiitor DPI for 2 h mrkedly loked ell dethevokedy2mmso 2 hydrtes. But 2 μm DPIdid not inhiit stomtl losing evoked y 1 μm SO 2 hydrtes (Figure 4). These findings indite tht SO 2 -triggered stomtl losing n e driven y NADPH oxidse-dependent nd -independent H 2 O 2 genertion, ut NADPH oxidsedependent H 2 O 2 genertion is involved in SO 2 -used ytotoxiity H 2 O 2 Ats Upstrem of C 2+ Signling in Response to SO 2 Stress. SO 2 exposure enhned the fluoresene intensity of Fluo-3 AM (C 2+ inditor) in Aridopsis gurd ells. The reltive fluoresene intensity of Fluo-3 AM, resulting from more thn 6 gurd ells per tretment group in three independent experiments, inresed oviously in gurd ells exposed to 1 μmto6mmso 2 hydrtes for 2 h ut deresed mrkedly when isolted strips were treted simultneously with SO 2 hydrtes nd.1 mm lium hnnel loker LCl 3

5 Advnes in Toxiology 3 e 2 Cell deth rtes (%) d Conentrtions of SO 2 hydrtes (mm) Cell deth rtes (%) A B E F G H Reltive intensity of DCFH-DA fluoresene (%) A B H J (A) (B ) (E) (F) Control 6 mm SO 2 hydrtes.1 mm AsA 6 mm SO 2 hydrtes +.1 mm AsA (G) 2 U ml 1 CAT (H) 6 mm SO 2 hydrtes + 2 U ml 1 CAT (J) 6 mm SO 2 hydrtes +.1 mm LCl 3 Figure 3: Sulfur dioxide used viility deline ssoited with H 2 O 2 elevtion in Aridopsis gurd ells. Different supersript letters indite signifint differenes (P <.1). The sme letters indite no signifint differene. (Figure ). These results indite tht SO 2 exposure evokes n elevtion of [C 2+ ] yt level, nd C 2+ influx through C 2+ hnnels in the plsm memrne results in [C 2+ ] yt elevtion. AsshowninFigures2 nd, the ddition of LCl 3 nd lium heltor EGTA to SO 2 hydrtes loked [C 2+ ] yt elevtion, stomtl losure, nd ell deth evoked y SO 2. Stomtl losure nd ell deth ourred with inresed [C 2+ ] yt in SO 2 -treted smples, ut oth of them were signifintly suppressed y.1 mm LCl 3 or EGTA. These results indite tht elevted [C 2+ ] yt level is n importnt stimulus driving stomtl movement nd ytotoxiity. To understnd the pthwys to plnt responses, we study the signling pthwys in Aridopsis gurd ells. As shown in Figures 2, 3, nd, oth ntioxidnts AsA nd CAT nd C 2+ ntgonists LCl 3 nd EGTA n suppress SO 2 -indued stomtl losure nd ell deth, ut pplition of H 2 O 2 svenger CAT (2 U ml 1 ) deresed [C 2+ ] yt elevtion evoked y SO 2, wheres pplition of C 2+ hnnel inhiitor LCl 3 (.1 mm) did not ffet H 2 O 2 elevtion evoked y SO 2. These results onfirm the involvement of C 2+ downstrem of H 2 O 2 prodution, inditing tht H 2 O 2 triggers stomtl movement nd ell deth vi C 2+ signling in plnt response to SO 2 stress. 4. Disussion Plnt ould dpt to environmentl hllenges through vrious mens. Our reent findings showed tht SO 2 fumigtion used n inresed ROS prodution ompnied with differentil gene expression nd stomtl losure in Aridopsis plnts [12, 2]. The results of the present study

6 6 Advnes in Toxiology 3 6 Cell deth rtes (%) d Stomtl perture (μm) A B C B E M A D F (A) (B) (C) (B ) Control 2 mm SO 2 hydrtes 2 mm SO 2 hydrtes + 1 U ml 1 CAT 6 mm SO 2 hydrtes (E) (M) (D) (F) 6 mm SO 2 hydrtes + 1 U ml 1 CAT 6 mm SO 2 hydrtes +2μM DPI 1 μm SO 2 hydrtes 1 μm SO 2 hydrtes +2μM DPI Figure 4: Hydrogen peroxide svenger nd NADPH oxidse inhiitor DPI loked SO 2 -indued ell deth, ut DPI nnot lok SO 2 - indued stomtl losure. Different supersript letters indite signifint differene t P <.1. The sme letters indite no signifint differene. show the evidenes tht H 2 O 2 ts s n importnt signling moleule triggering stomtl movement nd ell deth in plnt response to SO 2. SO 2 -used ell viility deline ould interfere with the norml funtion of stomt to further ffet plnt physiology under environmentl stress. However,theourreneofSO 2 -used gurd ell deth ws ssoited with deresed stomtl perture, suggesting the existene of omplex signling network in plnt responses to environmentl stress. Environmentl hllenges inluding ioti nd ioti stresses ould indue ROS prodution in plnt ells [2]. It hs een doumented tht ROS ply n importnt role in signl trnsdution of stomtl movement regultion nd gene expression tivtion in plnt response to environmentl stresses [26 29]. ROS, whih n e used s rpid long-distne utopropgting signls tht re trnsferred throughout the plnt in response to different environmentl onditions, re widely onsidered to e n importnt plyer in gurd ell signling [26, 3]. The dt presented ove indite requirement for H 2 O 2 prodution in plnt response to SO 2 stress. First, SO 2 indues stomtl losure nd ell dethssoitedwithninresedintrellulrh 2 O 2 level (Figures 2 nd 3). Seond, two types of H 2 O 2 svengers CAT nd AsA ould lok SO 2 -evoked stomtl losure nd ell deth; in prtiulr, the effets of SO 2 t low onentrtions ould e ompletely reversed y CAT (Figures 2 nd 4). Third, H 2 O 2 svenger loks the SO 2 -indued inrese in intrellulr C 2+ required for stomtl losure nd toxiity (Figure ). Our study, vlidting the strong positive orreltion etween intrellulr H 2 O 2 level nd stomtl losure/ell deth, demonstrtes key role of H 2 O 2 s trigger of stomtl losure nd/or gurd ell deth in response to SO 2. Sulfur dioxide inhiited light-promoted stomtl opening nd promoted stomtl losure leding to the smller size of Aridopsis stomt. High onentrtions of SO 2 lso used viility loss of Aridopsis gurd ells. The time ourse experiments show tht H 2 O 2 level of gurd ells inresed grdully during SO 2 exposure (dte not shown); therefore, n inresed H 2 O 2 level in SO 2 -stimulted Aridopsis ells might trigger stomtl losure firstly nd then ell viility loss through ROS-medited ell deth pthwy s shown in V. f ells [24]orthroughthegrdul umultion of free rdil dmge to iomoleules during SO 2 exposure. There re two primry soures of H 2 O 2 in gurd ells: hloroplsts nd plsm memrne-ssoited NADPH oxidse [22, 31, 32]. The results of the time ourse experimentswithsingleellssysusingthefluoresentproe DCFH-DA showed tht H 2 O 2 genertion ws dependent on SO 2 onentrtion nd tht the inrese in fluoresene intensity of hloroplsts ourred signifintly erlier thn within the other regions of gurd ells (dte not shown), demonstrting n enhned H 2 O 2 prodution in hloroplsts of gurd ells. NADPH oxidse in plsm memrne ontriutes to O 2 genertion nd ROS elevtion in plnt responses to ioti stresses [33]. Applition of DPI, whih is widely epted s reltively speifi diret inhiitor of NADPH oxidse, mrkedly loked SO 2 -indued ell deth ut nnot suppress stomtl losure evoked y low onentrtions of SO 2.ThesefindingsinditethtH 2 O 2 - medited stomtl losure in Aridopsis leves exposed to low onentrtions of SO 2 is not dependent on the tivity of plsm memrne NADPH oxidse, ut ell dethevokedyhighonentrtionsofso 2 is NADPH oxidse-dependent.

7 Advnes in Toxiology Cell deth rtes (%) A B I J K N 2 3 Reltive intensity of fluo-3 AM fluoresene (%) Reltive intensity of fluo-3 AM fluoresene (%) A B C D E F A B H J (A) Control (B) 2 μm SO 2 hydrtes (C) 2 μm SO 2 hydrtes +1mM AsA (D) 2 μm SO 2 hydrtes + 2 U ml 1 CAT (E) (F) (B ) (I) 2 μm SO 2 hydrtes +.1 mm EGTA (J) 6 mm SO 2 hydrtes +.1 mm LCl 3 2 μm SO 2 hydrtes +.1 mm LCl 3 6 mm SO 2 hydrtes.1 mm LCl 3 (K) (N) (H).1 mm EGTA 6 mm SO 2 hydrtes +.1 mm EGTA 6 mm SO 2 hydrtes + 2 U ml 1 CAT Figure : Effets of ntioxidnts nd C 2+ ntgonists on SO 2 -indued C 2+ elevtion nd ell deth in Aridopsis gurd ells. Different supersript letters indite signifint differenes (P <.1). The sme letters indite no signifint differene. SO 2 exposureusedstomtllosurendgurdell deth ssoited with [C 2+ ] yt elevtion, wheres pplition of either C 2+ heltor EGTA or C 2+ hnnel inhiitor LCl 3 loking SO 2 -evoked [C 2+ ] yt elevtion, stomtl losure nd ell deth evoked y SO 2 were effetively loked. These results demonstrte tht hnnel-medited C 2+ influx ross the plsm memrne ontriutes to the elevtion of [C 2+ ] yt nd susequent stomtl losure nd ell deth in SO 2 -stimulted gurd ells. However, it is not ler how C 2+ signling reognizes the different situtions of ells to medite pproprite proesses suh s stomtl losure nd ell deth. It hs een found tht H 2 O 2 ould tivte plsm memrne C 2+ hnnels leding to [C 2+ ] yt inrese in plnt ells [34 36]. Therefore, H 2 O 2 tivtion of plsm memrne C 2+ hnnelsmyeentrlstepinso 2 - indued stomtl losure nd/or ell deth. The results of our present study lso showed tht pplition of ntioxidnt CAT nd AsA signifintly deresed SO 2 -evoked [C 2+ ] yt elevtion, ut pplition of C 2+ hnnel loker LCl 3 did not ffet SO 2 -evoked H 2 O 2 inrese, inditing tht H 2 O 2 ts upstrem of C 2+ signling in SO 2 - indued stomtl losure nd/or ell deth. These oservtions were onsistent with other previous reports tht [C 2+ ] yt inrese ws linked to H 2 O 2 prodution nd wsinvolvedinros-meditedstomtllosure/elldeth [37 39]. Therefore, H 2 O 2 elevtion nd susequent tivtion of C 2+ hnnels re events ourring in SO 2 -indued stomtl losure/ell deth. C 2+ influx from extrellulr

8 8 Advnes in Toxiology region results in [C 2+ ] yt inrese, nd then [C 2+ ] yt elevtion medites susequent stomtl losure/ell deth. These results suggested tht H 2 O 2 medites SO 2 -indued stomtl movement/ytotoxiity y trgeting C 2+ hnnels in the plsm memrne. Briefly, environmentl SO 2 hs remrkle effet on the size of the stomtl perture. Exposure to SO 2 indued the overprodution of H 2 O 2 in gurd ells, s shown in other plnt ells exposed to environmentl hllenges [4, 41]. Elevted H 2 O 2 ts in onjuntion with other ftors to tivte stomtl movement nd ell deth under SO 2 stress. Gurd ells re well-developed model system for hrterizing erly signl trnsdution mehnisms in plnts. In this study, the dul role of H 2 O 2 in plnt ells in response to ir pollutnt ws lerly displyed in Aridopsis gurd ells, whih dditionlly roden the role of stomtl gurd ells in ytotoxiity study. Our results suggest tht gurd ells re vlule model system for the study of ytotoxiity in plnt ells.. Conlusion Sulfur dioxide exposure used n elevted H 2 O 2 level in Aridopsis gurd ells. H 2 O 2 elevtion triggered y SO 2 medited oth stomtl losure nd ell deth vi C 2+ signling. Intrellulr C 2+ inrese is neessry for stomtl losure nd ell deth in Aridopsis gurd ells in response to SO 2.H 2 O 2 prodution y NADPH oxidse plys ritil role in SO 2 toxiity; however NADPH oxidse tivtion is suffiient ut not neessry for SO 2 -triggered stomtl losure. Both stomtl losing nd stomtl opening inhiition evoked y SO 2 led to deline in stomtl perture, proteting the lef ginst further entry of the pollutnt ut lso urtiling photosynthesis. Cell deth evoked y high onentrtions of SO 2 indited the ytotoxiity of SO 2, ut might provide pproprite protetion y reduing ROS prodution in Aridopsis plnts. Conflit of Interests The uthors delre tht there is no onflit of interests regrding the pulition of this pper. Aknowledgments This study ws supported y the Ntionl Nturl Siene Foundtion of Chin (Grnt nos , , nd ),ReserhFundfortheDotorlProgrmofHigher Edution of Chin (Grnt no ), nd Shnxi Sholrship Counil of Chin (Grnt no. 2922). Referenes [1] N. M. Drrll, The effet of ir pollutnts on physiologil proesses in plnts, Plnt, Cell & Environment, vol.12,no.1, pp. 1 3, [2] T. Hogetsu nd M. Shishikur, Effets of sulfur dioxide nd ozone on intt leves nd isolted mesophyll ells of groundnut plnts (Arhis hypoge L.), Journl of Plnt Reserh,vol.17, no.3,pp ,1994. [3] M. Noji, M. Sito, M. Nkmur, M. Aono, H. Sji, nd K. Sito, Cysteine synthse overexpression in too onfers tolerne to sulfur-ontining environmentl pollutnts, Plnt Physiology,vol.126,no.3,pp ,21. [4] R. Rkwl, G. K. Agrwl, A. Kuo et l., Defense/stress responses eliited in rie seedlings exposed to the gseous ir pollutnt sulfur dioxide, Environmentl nd Experimentl Botny,vol.49,no.3,pp ,23. [] H. Rennenerg nd C. 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