Signaling molecules and cell death in Melissa officinalis plants exposed to ozone

Transcription

1 lnt Cell Rep DOI /s ORIGINAL AER Signling moleules n ell eth in Meliss offiinlis plnts expose to ozone Elis ellegrini Alie Trivellini Alessnr Cmpnell Alessnr Frnini Giomo Lorenzini Cristin Nli olo Vernieri Reeive: 30 July 2013 / Revise: 13 Septemer 2013 / Aepte: 1 Septemer 2013 Ó Springer-Verlg Berlin Heielerg 2013 Astrt Key messge The stuy fouses on the intertion etween retive oxygen speies n hormones tht regulte the progrmme ell eth in plnts of Meliss offiinlis expose to ozone. Astrt Intertion etween hormone n reox signling pthwys hs een investigte in ozone-stresse (200 pp, 5 h) lemon lm to verify if the response resemles the ioti efense retions. In omprison to ontrols, plnts exhiite folir injury n the ell eth ws inue y (1) iphsi proution of hyrogen peroxie n superoxie ril; (2) hormonl regultion of ozone-inue lesion formtion with signifint proution of ethylene, sliyli, jsmoni n sisi i; (3) ozone egrtion to retive oxygen speies n their etoxifition y some enzymti (suh s superoxie ismutse) n non-enzymti ntioxint systems (suh s sori i, glutthione n rotenois), tht worke in oopertion without proviing efense ginst free rils (suh s onfirme y the moifition of the ntioxint properties of lef tissue). This integrte view showe tht retive oxygen speies intert with hormonl signling pthwy regulting ell eth n the sensitivity of lemon lm to ozone. Keywors Retive oxygen speies hytohormones Oxitive stress Antioxint systems Meiinl plnts Communite y A. Feher. E. ellegrini A. Trivellini A. Cmpnell A. Frnini G. Lorenzini C. Nli (&). Vernieri Deprtment of Agriulture, Foo n Environment, University of is, Vi el Borghetto 80, 5124 is, Itly e-mil: ristin.nli@unipi.it Introution During the 20th entury the rpi inreses in nthropogeni tivities hve ontriute signifint mounts of toxi gseous pollutnts to the tropospheri environment, using extensive mge for the survivl n proutivity of wil n ultivte plnts. Among vrious ontminnts, ozone (O 3 ) is onsiere the most uiquitous n phytotoxi ir pollutnt in inustrilize n in eveloping ountries (Wgg et l. 2012). lnt response to O 3 resemles the ioti efense retions (Ro et l. 2000) n inlues two steps: the first is iphsi oxitive urst with mssive, rpi n trnsient inrese in poplsti retive oxygen speies (ROS) proution; the seon is the inution of the hypersensitive response (HR) n systemi quire resistne (SAR). Although the eleterious effets of ROS, in prtiulr of hyrogen peroxie (H 2 O 2 ) n superoxie ril (O 2 - ), hve long een known (Romero-uerts et l. 2002; Cooke et l. 2003; Montillet et l. 2005), the reognition of their role(s) in ell signling n regultion of gene expression is reltively reent n still poorly unerstoo (Cheng et l. 2013). In reltion to their onentrtion, ROS hve ul funtions n must e utilize n/or integrte with other signling pthwys or moleules to regulte evelopmentl proesses n to ffet plnt growth n ell metolism. There is the eviene tht plnt hormones suh s ethylene (ET) n sliyli (SA), jsmoni (JA) n sisi (ABA) is re positione ownstrem of the ROS signl n tht these toxi oxygen (O 2 ) intermeites themselves re lso seonry messengers in mny hormone signling pthwys (ei et l. 2000). Therefore, fee-k or feeforwr intertions my our etween ROS n hormones. Aoring to Kngsjärvi et l. (2005), following poplsti ROS inrese over threshol limit, omplex

2 lnt Cell Rep sequene of events tke ples in the gur ells, leing to enogenous ROS proution n tivtion of mitogentivte protein kinse se. This proess seems to e involve in the up-regultion of the synthesis of ifferent hormones tht ontrol the extent of HR n O 3 -lesion propgtion. In prtiulr, SA n ROS hve een propose to e on positive feek loop tht mplifies signls leing to efense responses n ell eth t the sites of lesion initition. This SA-epenent signl is require to mintin the ellulr reox stte n to inue n potentite the tivtion of ellulr efenses, tht is neessry to minimize the oxitive stress uring plnt pthogen intertions (Durner et l. 1997) n uring O 3 symptom evelopment (squlini et l. 2002). From these initil sites, urst of ET proution is require for the ontinution of ROS umultion, whih results in positive feek yle tht (1) promotes lesion spre n propgtion to surrouning ells, n (2) rives n inues ompetene for progrmme ell eth (CD). The roles of ET pereption n iosynthesis in ontrolling or promoting the spre of ell eth uring symptom formtion fter the pthogen infetion (Bent et l. 1992) n O 3 -inue lef injury (Di Bio et l. 2012) hve een well-oumente (Wng et l. 2013). JA n (1) overome the promoting effet of ET for ROS genertion, resulting in the ontinment of lesion propgtion n spre (Sntino et l. 2013), n (2) t s negtive regultor of the oxitive ell eth, promoting hypersensitive ell eth n onsequently senesene. The ul ility of JA, tht influenes not only responses to severl stress ftors (inluing pthogens n wouning), ut lso growth n evelopment, hs een reporte in plnts unergoing HR (Thler et l. 2012) n O 3 -inue oxitive stress (Overmyer et l. 2000). Despite this well-known link etween ABA n ROS, little ttention hs een rwn so fr on the reltionship etween these two signling moleules n their pthwys. There is eviene tht ABA is positione ownstrem of the ROS signl; this hormone is le to regulte the stomtl losure preventing exessive trnspirtion (Kohler et l. 2003). As onsequene, the vilility of ron ioxie (CO 2 ) for fixtion of the C3 yle is limite n the proution of ROS (in prtiulr of H 2 O 2 ) in stomtl gur ells enhnes. ROS themselves re lso seonry messengers in ABA signling pthwys. It hs een emonstrte tht the genertion of ROS n ffet stomt ting s n rtifiil replement for the physiologil funtion of ABA with n inrese of the ytosoli C 2? level tht regulte stomtl losure or perture (Royhouhury et l. 2013). A rosstlk etween ROS n ABA is esrie in plnts tht intert with pthogens or pest (Xiong n Yng 2003) n with O 3 -stress (Kngsjärvi et l. 2005). The eviene on the hormonl ontrol of plnt responses to unfvorle onitions is strong n the plnt pility to tivte the efense system(s) ginst oxitive estrution my e key link in the mehnism of plnt sensitivity/resistne. To remove ROS n to protet ellulr memrnes n orgnelles from oxitive stress, plnt ells re enowe with importnt enzymti n non-enzymti ntioxint systems. These ply n importnt role in efining the lef ell fte (1) keeping ROS level uner ontrol n (2) ting s entrl omponent of the ell reox lne n of the signling moultion. The first line of efense ginst overproution of these toxi O 2 intermeites is the poplst, where sorte (AsA) is elieve to prevent or minimize the mge use y ROS in reltion to its ility to onte eletrons in numer of retions. However, hnges in the AsA reox stte might e expete to lter stomtl movement, prtiulrly uner onitions in whih ABA n H 2 O 2 signling tivte stomtl losure (Chen n Gllie 2004). The ul role plye y AsA (i.e. ntioxint n signling ompoun) is stritly epenent on the ell pility to mintin it in reue stte tht usully ours t the expense of reue glutthione (GSH), vi Hlliwell As yle. This yle is onsiere s the most importnt intrellulr efense ginst free rils in plnts uner oxitive stress onitions (Meyer 2008) n is involve in the omplex enzymti mhinery tht ontrols the intrellulr levels of H 2 O 2 n singlet oxygen ( 1 O 2 ) (My et l. 1998). GSH is prtiulrly importnt in hloroplsts where it helps -rotene to (1) protet the photosyntheti pprtus from oxitive mge e-exiting hlorophyll n to remove 1 O 2 (Collins 2001) n (2) suppress lipi peroxition. These moleules with -toopherol n phenoli ompouns ply key role in svenging free rils in plnts y onting eletrons or hyrogen ions. Within plnt ells, the superoxie ismutse (SOD) onstitutes the first enzymti line of efense ginst ROS, euse it removes superoxie y tlyzing its ismuttion (Kngsjärvi et l. 1994). SOD, tlse, peroxise, sorte peroxise n other enzymes of Hlliwell As yle re involve in the etoxifition n in the mitigtion of free intermeites. It is importnt for plnt survivl n growth uner stress onitions tht ntioxints n work in oopertion, thus, proviing etter efense n regenertion of tive reue forms. lnt response to ioti n ioti stress is omplex n, to te, little is known out whether high etoxifition potentil ginst ROS or stress-inue etoxifition proesses is responsile for resistne/sensitivity (Heth 2008). The im of the present work ws to investigte the time ourse reltionships mong oxitive urst, iosynthesis of signling moleules (suh s SA, JA, ET n ABA) n tissue mge in Meliss offiinlis L. (lemon lm)

3 lnt Cell Rep expose to O 3. Lemon lm is known s her of long trition with lrge vriety of uses in ooking n in meiine. Our previous stuies onute on this speies showe how the ltertions of photosyntheti funtions, the memrne mge n the visile injury suggest n evient suseptiility to elevte O 3 onentrtion (ellegrini et l. 2011). In this pper, we ttempt to nswer to some key questions: first, M. offiinlis plnts expose to O 3 tivte CD? Seon, whih re the ftors tht regulte CD in this speies? Thir, wht out the mehnisms unerlying initition, propgtion n ontinment of O 3 - inue ell eth? Mterils n methos Culturl prties, plnt mteril n ozone exposure Homogeneous 4-month-ol uttings of M. offiinlis, grown in plsti pots ontining mix of stem-sterilize soil n pet (1:1), were ple for 2 weeks in ontrolle environment fility t temperture of 20 ± 1 C, RH of 85 ± 5 % n photon flux ensity t plnt height of 530 lmol photon m -2 s -1 provie y innesent lmps, uring 14 h photoperio. Uniform-size plnts were ple in ontrolle environment fumigtion fility uner the sme limti onitions s the growth hmer. The entire methoology hs een performe oring to Frnini et l. (2008). lnts were trete with O 3 in terms of single squre wve of 200 pp for 5 h etween n Anlyses were performe t 0, 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Smpling Young fully expne leves were ollete t eh time point, weighe n kept t -80 C. Leves were frozen in liqui N 2, groun with ol mortr n pestle for lter use. The homogentes were entrifuge t 12,000g for 20 min t 4 C, n the superntnts were use for the etermintions. ROS nlysis Lef H 2 O 2 proution ws mesure fluorometrilly using the Amplex Re Hyrogen eroxie/eroxise Assy Kit (Moleulr roes, Invitrogen, Crls, CA), oring to Shin et l. (2005). The retion mixture ontine 20 mm potssium-phosphte uffer ph.5, 50 lm of 10-etyl- 3,7-ihyroxyphenoxzine, 0.1 U ml -1 horserish peroxise n 50 ll of the superntnt. Smples were inute t 25 C for 30 min in the rk n the resorufin fluoresene (E x /E m = 530/590 nm) ws quntifie with fluoresene/sorne miroplte reer (Vitor Multilel Counter, erkin Elmer, USA), fter sutrting the kgroun fluoresene ue to the uffer solution n to the ssy regents. Eh result ws plotte ginst H 2 O 2 stnr urve (from 0 to 3.5 lm). The O 2 - etermintion is se on the reution of tetrzolium ye soium, 3 0 -(1-[phenylmino-ronyl]-3,4- tetrzolium)-is(4-methoxy--nitro) enzene-sulfoni i hyrte (XTT) y O 2 to solule XTT formzn, oring to Ale et l. (1998). The retion mixture ontine 50 mm Tris HCl uffer ph 7.5, 0.5 mm XTT n 100 ll of the superntnt. Smples were inute t room temperture for 15 min n sorne t 470 nm ws re with spetrophotometer (505 UV Vis, Jenwy, UK), fter sutrting the kgroun sorne ue to the uffer solution n to the ssy regents. The quntity of O 2 - proue ws etermine using the molr extintion oeffiient M -1 m -1. Hormone iossys 15 min fter exision, ET proution ws mesure y enlosing one lef (ollete t the proper vegettive stge n ut few millimeters elow the petiole) in ir-tight ontiners (250 ml). Gs smples (2 ml) were tken from the hespe of ontiners fter 1 h inution t 22 C. ET onentrtion in the smple ws mesure y gs hromtogrph (H5890, Hewlett-kr, USA) using flme ioniztion etetor, stinless steel olumn ( m internl imeter pke with Hysep T) n etetor tempertures of 70 n 350 C, respetively, n N 2 rrier gs t flow rte of 30 ml min -1 (Mensuli Soi et l. 1992). Quntifition ws performe ginst n externl stnr. ABA ws etermine y n iniret ELISA se on the use of DBA1 monolonl ntioy, rise ginst S(?)- ABA s esrie y Trivellini et l. (2011). The ELISA ws performe following Wlker-Simmons (1987), with minor moifitions. ABA ws mesure fter extrtion in istille wter (wter: tissue rtio = 10:1, v/w) overnight t 4 C n quntifie t 415 nm with n sorne miroplte reer (MDL 80, erkin-elmer, USA). Conjugte n free SA ws etermine oring to Zwoznik et l. (2007) with some moifitions. Frozen folige smples (150 mg) were e to 1 ml 90 % (v/v) methnol, vortexe n sonite for 3 min. After entrifugtion t 10,000g for 10 min t room temperture, the superntnt ws trnsferre n the pellet ws re-extrte in 0.5 ml 100 % (v/v) methnol following the sme proeure. Superntnts from oth extrtions were omine n evporte t C uner vuum. The resiue ws resuspene in 0.25 ml of 5 % (w/v) TCA n prtitione twie using 0.8 ml of 1:1 (v/v) mixture of ethyl

4 lnt Cell Rep ette/ylohexne. The upper phse ontining free SA ws onentrte t C uner vuum n the lower queous phse (with onjugte SA) ws hyrolyze y ing 0.3 ml 8 N HCl n inuting for 0 min t 80 C. Both of the SA ollete from the upper phse n reovere from the lower phse were omine n issolve in 00 ll of the moile phse, ontining 0.2 M soium ette uffer, ph 5.5 (90 %) n methnol (10 %). HLC seprtion ws performe t room temperture with Dionex olumn (Alim 120, C18, 5 lm prtile size, 4. mm internl imeter mm length). SA ws quntifie fluorometrilly (RF 2000 Fluoresene Detetor, Dionex, USA), with exittion t 305 nm n emission t 407 nm n it ws elute using the moile phse esrie ove. The flow rte ws 0.8 ml min -1. To quntify the SA ontent, known mounts of pure stnr were injete into the HLC system n n eqution, orrelting pek re to SA onentrtion, ws formulte. Frozen folige smples (150 mg) were e to 3 ml methnol n inute overnight t 4 C. The extrt ws entrifuge t 10,000g for 10 min t room temperture n the superntnt ws filtere n evporte t room temperture uner vuum. After ing mixture of 0.2 % (v/v) queous eti i, the superntnt ws filtere n immeitely nlyze with HLC using the Dionex olumn esrie ove. JA ws etete y its sorne t 210 nm. The flow rte ws 1 ml min -1 (Krmell et l. 1999). To quntify the JA ontent, known mounts of pure stnr were injete into the HLC system n n eqution, orrelting pek re to JA onentrtion, ws formulte. Antioxint mesurements Totl SOD tivity ws ssye in terms of its ility to inhiit the photohemil reution of Nitro Blue Tetrzolium (NBT) oring to Zhng n Kirkhm (1994). The retion mixture ontine 100 mm potssium-phosphte uffer ph 7.8, 0.1 mm EDTA (soium slt), 13 mm methionine, 75 lm NBT, 2 lm rioflvin n 5 40 ll of the enzyme extrt. Smples were illuminte for 10 min y fluoresent lmp (Osrm R80) t 150 W n sorne t 50 nm ws re ginst unilluminte smples. Frozen folige smples (500 mg) were e to ml 5 % (w/v) trihloroeti i n entrifuge. The superntnt ws use for the AsA n AsA? DHA etermintion oring to Wng et l. (1991). This ssy is se on the reution of ferri ions (Fe 3? ) to ferrous ions (Fe 2? ) with sori i in i solution followe y the formtion of the re helte etween ferrous ions n 4,7-iphenyl-1,10-phennthroline (thophennthroline) tht sors t 534 nm. Totl sorte (AsA? DHA) ws etermine through reution of ehyrosorte to sorte y ithiothreitol. DHA onentrtions were estimte from the ifferene etween mounts of totl sorte n AsA. A stnr lirtion urve overing 0 1 mm of AsA or DHA rnge ws use. Frozen folige smples (125 mg) were e to 2 ml 5 % (w/v) trihloroeti i n entrifuge. The superntnt ws use for the GSH? GSSG n GSSG etermintion oring to Sgherri n Nvri-Izzo (1995). This ssy is se on n enzymti reyling proeure in whih glutthione ws sequentilly oxiize y 5,5 0 -ithiois-2-nitroenzoi i n reue y NADH in the presene of glutthione reutse. For speifi ssy of GSSG, GSH ws mske y erivtiztion with 4-vinylpyriine in the presene of triethnolmine. Chnges in sorne of the retion mixtures were mesure t 412 nm for 1 min t 25 C. The mount of GSH ws lulte y sutrting the GSSG mount, s GSH equivlents, from the totl glutthione mount. A stnr lirtion urve, where GSH equivlents (0 10 mm) were plotte ginst the slope of hnge in sorne t 412 nm, ws use. igment nlysis ws performe oring to Ciompi et l. (1997). Thirty milligrms of leves ws homogenize in 3 ml of 100 % methnol overnight. The superntnt ws filtere n nlyze with HLC using the Dionex olumn esrie ove. The pigments were elute y 2-solution grient: solvent A (etonitrile/methnol, 75/25, v/v) n solvent B (methnol/ethyl ette, 8/32, v/v). The flow rte ws 1 ml min -1. The pigments were etete y their sorne t 445 nm. To quntify the pigment ontent, known mounts of pure stnr were injete into the HLC system n n eqution, orrelting pek re to pigment onentrtion, ws formulte. Frozen folige smples (400 mg) were e to 4 ml of ol methnol, in the rk. The ntioxint tivity of the extrt ws etermine s hyroxyl ril ntioxint pity (HORAC) n oxygen ril sorne pity (ORAC) using the OxiSelet HORAC n ORAC Ativity Assy (Cell Biols, USA), oring to the mnufturer s guielines. The HORAC tivity metho is se on the oxition-meite quenhing of fluoresent proe y hyroxyl rils proue y hyroxyl ril inititor n Fenton regent. The ORAC tivity metho is se on the oxition of fluoresent proe y peroxyl rils proue y free ril inititor, 2,2 0 -zois(2- miino-propne) ihyrohlorie. The ntioxint tivity ws quntifie with the fluoresene/sorne miroplte reer ove reporte, with exittion t 480 nm n emission t 530 nm n lulte using the ifferenes of res uner the fluoresene ey urve etween lnk n the smple n ompre with n ntioxint stnr urve otine with glli i for HORAC n the wter-solule vitmin E nlog, Trolox (T) for ORAC.

5 lnt Cell Rep Fig. 1 Meliss offiinlis leves mintine in filtere ir () n expose to ozone (200 pp, 5 h), with visile injury oserve on xil surfe fter 48 h from the eginning of exposure (r mrker 1 mm) () Sttistil nlysis A minimum of four plnts per tretment were use in eh of the three repete experiments. Following performne of the Shpiro Wilk W test, t were nlyze using twowy nlysis of vrine (ANOVA) n omprison mong mens ws etermine y Fisher s LSD post-test ( \ 0.05). Anlyses were performe y NCSS 2000 Sttistil Anlysis System Softwre. Results Forty-eight hours from eginning of exposure, plnts showe severe minute (Ø 1 2 mm), rounish, well-efine, rk-lkish nerosis lote in the interveinl xil res of the young ompletely expne leves (Fig. 1). Cross setions (not shown) revele tht injure ells were onfine to plise tissue. The injure re ws out 15 % of the totl (rnge %). No symptoms were etete in oler leves of trete plnts n in ontrol ones mintine in filtere ir. Results of ROS folir etermintion inite tht there ws iphsi H 2 O 2 proution in response to O 3 exposure (Fig. 2): the first smller inrese trnsiently peke in the first 2 h uring the fumigtion (out twofol higher in omprison to untrete plnts) n then eline (phse I). The seon mssive umultion peke t 8 h from eginning of exposure (fourfol tht of filtere-ir ounterprts), followe y shrp eline to the onstitutive levels t 48 h. A grul erese in O 2 - levels is oserve throughout the entire perio of tretment in ozonte leves (-8, -3, n -3 %, respetively, fter 1, 2 n 5 h from eginning of exposure in omprison to those of plnts grown uner ontrolle mient, Fig. 2), rehing minimum vlue fter 8 h (12-fol lower thn ontrol); only then, they showe mrke n ontinuous inrese t 24 n 48 h from eginning of exposure (two- n threefol thn filtere-ir mteril). In trete plnts, totl SOD tivity lso exhiite iphsi time ourse (Fig. 2), eing higher thn ontrol lrey fter 1 n 2 h uring the fumigtion (out twofol), showing no ifferenes t 5 n 8 h from eginning of exposure, n inresing gin fter 24 h (?9 % when ompre to untrete mteril). At the en of the experiment, the tivity of this enzyme ereses to ner onstitutive vlues. A iphsi response to O 3 hs lso een oserve for ET evolution (Fig. 3). In trete mteril, there ws first high pek fter just 1 h uring the tretment, remining high throughout the entire perio of exposure (overll three times higher thn the ontrol) n then, seon pek lter, 48 h from eginning of exposure (more thn twofol). Conversely, mesurements of ABA ontent 1 n 2 h of exposure revele no signifint ifferene etween ozonte n ontrol plnts (Fig. 3). Strting from 5 h onwrs, the levels of this hormone were lwys signifintly higher in trete iniviuls thn in filtere-ir ounterprts, rehing mximum vlue t the en of tretment (?31 %). During the post-fumigtion perio, vlues remine higher (?27,?3 n?27 %, respetively t 8, 24 n 48 h from the eginning of exposure) thn those of ontrols. Chnges in onjugte n free SA onentrtions of lef tissues uring the entire perio of the experiment re shown in Fig. 4,. Time ourse nlysis revele tht enogenous totl SA levels inrese immeitely fter the en of fumigtion, rehing mximum vlue fter 8 h

6 lnt Cell Rep 5 Ozone *** Time *** 10 8 Ozone ** Time * H 2 O 2 (nmol g -1 FW) Ethylene (nl g -1 FW h -1 ) O 2 - (nmol g -1 FW) SOD tivity (Units mg -1 protein) from eginning of exposure: onjugte n free SA umultion ws out twofol higher thn in the ontrol. A similr tren ws oserve for the JA ontent tht i not e f Ozone Time Ozone x Time Ozone Time Ozone x Time *** ** ** ** ** *** Fig. 2 Time ourse of hyrogen peroxie (H 2 O 2, ) ontent, superoxie nion (O 2 -, ) generting rte n superoxie ismutse tivity (SOD, )inmeliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Different letters inite signifint ifferenes ( B 0.05). In the oxes, results of two-wy ANOVA re reporte, sterisks showing the signifine of ftors/intertion for: *** B 0.001; ** B The thik line inites the time (5 h) of ozone exposure ABA (ng g -1 FW) hnge when ompre to ontrols until the en of O 3 tretment n tht showe mrke inrese uring the post-fumigtion perio (Fig. 4). The gretest inution of this metolite ws oserve fter 8 h from eginning of exposure (18-fol higher thn ontrol). Strting from 1 h onwrs, AsA onentrtions were lwys signifintly higher in trete plnts (out twofol higher t ll time points, Fig. 5); in these leves, the lrgest solute AsA ontent ws mesure fter 24 h from eginning of exposure. DHA levels rmtilly erese elow ontrol (more thn twofol lower t ll time points, Fig. 5) n the lowest ontent of this metolite ws mesure in ozonte plnts ollete t 1 h uring the tretment n t the en of the exposure. The onentrtion of totl sorte (AsA? DHA) i not show ler tren (Fig. 5): it ws lower thn ontrol (-20 %) lrey fter 1 h of fumigtion, showing no ifferenes fter 2 h n Ozone Time Ozone x Time *** Fig. 3 Time ourse of ethylene () emission n sisi i (ABA), ontent in Meliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Different letters inite signifint ifferenes ( B 0.05). In the oxes, results of twowy ANOVA re reporte, sterisks showing the signifine of ftors/ intertion for: *** B 0.001; ** B 0.01; * B The thik line inites the time (5 h) of ozone exposure * **

7 lnt Cell Rep ConjugteSA ( µg g -1 FW) Free SA (µg g -1 FW) JA (µg g -1 FW) Ozone Time Ozone x Time *** * ** Ozone *** Time *** Ozone *** Time * inresing t the en of tretment n uring the postfumigtion perio, rehing mximum vlue fter 24 h (onefol higher). Oxitive stress le to n evient inrese Fig. 4 Time ourse of onjugte () n free () sliyli i (SA) n jsmoni i (JA, ) ontents in Meliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Different letters inite signifint ifferenes ( B 0.05). In the oxes, results of two-wy ANOVA re reporte, sterisks showing the signifine of ftors/intertion for: *** B 0.001; ** B 0.01; * B The thik line inites the time (5 h) of ozone exposure in AsA/(AsA? DHA) rtio within the 48 h of the experiment (out twofol thn ontrol t ll time points, Fig. 5); in these leves, the lrgest solute rtio ws oserve t 5 n 24 h from eginning of exposure (?87 n?81 %, respetively). GSH levels exhiite ler triphsi time ourse (Fig. ), eing higher thn ontrol lrey fter 1 h of fumigtion (twofol), showing no ifferenes uring n t the en of the exposure, inresing gin t 8 h (twofol), remining unhnge t 24 h n inresing gin t 48 h (twofol). A similr tren ws oserve for GSH/GSSG rtio, s shown in Fig. : phse I peke fter 1 n 2 h of exposure (out threefol), phse II t 8 h (twofol) n phse III t 48 h (3.0-fol). Trete plnts showe signifint erese in GSSG ontent only t 2 n 5 h from eginning of exposure (three- n twofol, Fig. ); uring the post-fumigtion perio, no sttistil ifferenes were shown in omprison to untrete leves. During the time ourse, vlues of totl glutthione of trete plnts remine unhnge (t not shown) when ompre to their filtere-ir ounterprts. After the tretment, generlize signifint erese of -rotene n lutein (-48 n -19 %, respetively) ws oserve n this reution ws prolonge uring the postfumigtion perio (out two- n onefol, respetively) (Fig. 7, ). Totl rotenois followe the sme pttern: strting from 5 h onwr, the ontent of essory pigments in ozonte leves remine signifintly lower thn in their filtere-ir ounterprts (-30, -25, -2 n -31 %, respetively, fter 5, 8, 24 n 48 h from eginning of exposure) (Fig. 7). Violxnthin ws signifintly inrese t the en of the tretment n uring the postfumigtion perio, the mximum vlue eing rehe t 5 n 8 h from eginning of exposure (threefol higher thn ontrols) (Fig. 7). The ntioxint pity (expresse s the HORAC n ORAC vlues) of trete n untrete leves is shown in Fig. 8,. With the HORAC ssy methos, O 3 inrese the ntioxint tivity of M. offiinlis plnts t the en of exposure (17-fol more thn ontrols), lthough their hyroxyl ril verting pity erese to ner onstitutive vlues uring the post-fumigtion perio (Fig. 8). After 5 n 24 h from eginning of exposure, the O 2 ril sorption pity (Fig. 8) i not hnge when ompre to ontrols; only t the en of the post-fumigtion perio, the ORAC vlues signifintly erese (-1 % in omprison to filtere-ir ounterprts). Disussion Reltively little reserh hs een fouse on the impt of O 3 on hers (Fuhrer n Booker 2003) n until now, few

8 lnt Cell Rep AsA( µmol g -1 FW) 8 Ozone *** Time *** e DHA (µmol g -1 FW) Ozone *** Time *** AsA+DHA( µmol g -1 FW) Ozone ** Time *** e AsA/AsA+DHA Ozone ** Time ** Fig. 5 Time ourse of sorte (AsA, ), ehyrosorte (DHA, ), totl sorte (AsA? DHA, ) onentrtions n sorte/ ehyrosorte rtio (AsA/AsA? DHA, ) in Meliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h stuies hve een performe onerning their sensitivity to this pollutnt (Dvison n Brnes 1998). Most of them re se on the effets on growth n reproutive effort (Krup et l. 2000), ut these mrkers (1) my e use to etet verse tion only of long-term exposures to reltively low onentrtions n lso (2) they re not lwys esy to e unerstoo. On the ontrry, the oservtions of symptom evelopment n the nlysis of moleulr, iohemil n physiologil events inue y shortterm n reltively high-level exposure oul e regre s iomrker. In ft, this pproh my help in eteting O 3 mge t very erly stge n, in prtiulr, to etter unerstn the mehnisms involve in the overll plnt response to oxitive stress, s reporte in ellegrini et l. (2011). From 2 to 4 h fter the en of the tretment, the young ompletely expne leves showe the typil injury from the eginning of exposure. Different letters inite signifint ifferenes ( B 0.05). In the oxes, results of two-wy ANOVA re reporte, sterisks showing the signifine of ftors/intertion for: *** B 0.001; ** B The thik line inites the time (5 h) of ozone exposure represente y wter-logging; these initil hloroti spots evelope into rounish rk-lkish nerosis 48 h from the eginning of exposure. No symptoms were etete in oler leves of trete plnts euse, in reltion to their metoli n physiologi hrteristis, they were le to voi the oxitive stress (in prtiulr with stomtl regultion) to prevent n/or reue the erly events of senesene. In irh, it ws similrly shown tht the plnts hving expne n fst enlrging leves were most suseptile to O 3 exposure (O 3 oses higher thn the mient uner fiel onitions uring growing seson ) thn the mture leves (ääkkönen et l. 1995). Our t onfirm previous finings tht O 3 n e onsiere highly toxi gent whih uses folir neroti lesions, the pperne of whih resemles HR, tht ours s result of inomptile plnt pthogen intertions (Kngsjärvi et l. 2005). Reserh uring

9 lnt Cell Rep GSH (µmol g -1 FW) GSSG (µmol g -1 FW) GSH/GSSG reent yers hs shown tht the similrity of O 3 -, pthogenn other stress-inue response is not only externl, s reviewe y Ro et l. (2000). These phenomen shre Ozone ** Time * Ozone Time Ozone x Time *** * ** Ozone ** Time ** Ozone x Time ** Fig. Time ourse of reue (GSH, ) n oxiize glutthione onentrtions (GSSG, ) n reue/oxiize glutthione rtio (GSH/GSSG, ) in Meliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Different letters inite signifint ifferenes ( B 0.05). In the oxes, results of two-wy ANOVA re reporte, sterisks showing the signifine of ftors/intertion for: *** B 0.001; ** B 0.01; * B The thik line inites the time (5 h) of ozone exposure mny physiologil n moleulr fetures strting from the exposure of the plnt n ening with the evelopment of symptoms. This sequene of retions is not ompletely unerstoo n only uring the pst 10 yers, n integrte view hs een propose y Di Bio et l. (2012) n reviewe y Brtoli et l. (2013). In the present work, we esrie the si mehnisms of O 3 -inue ell eth isseting it into the following four key omponents: (1) O 3 -inue tive proution of ROS; (2) hormonl regultion of O 3 -inue lesion formtion; (3) O 3 egrtion to ROS n etoxifition y some enzymti n non-enzymti ntioxint systems n (4) O 3 sensing/pereption. Given the ft tht O 3 inues n enogenous, tive n self-propgting ROS genertion in the poplst n susequent oxitive urst, some uthors propose tht short-term O 3 exposure mimis pthologil gent (Kngsjärvi et l. 2005). We foun tht plnts exhiite iphsi tren of H 2 O 2 ontent tht might reflet the ourrene of iphsi tive H 2 O 2 proution s reporte lrey in response to O 3 (squlini et l. 2002; Di Bio et l. 2012) n uring HR (Tománková et l. 200; Mnl et l. 2011). O - 2 hs ul funtion: uring the exposure, the onentrtion signifintly eline, suggesting tht it ts s preursor of H 2 O 2 n even more toxi oxyril erivtives. On the other hn, in trete leves O - 2 ontent mrkely inrese in the postfumigtion perio, emonstrting tht this ril itself is involve in O 3 -inue lef injury. Diret eviene for O - 2 proution in tissues expose to oxitive stress is still lking. Within ell, SOD n e onsiere the primry svenger for O - 2 generte oth from norml physiologil tivities n from exposure to oxitive stress (Alsher et l. 2002). This enzyme shows iphsi time ourse in response to O 3 tretment, with the highest levels rehe just 2 h from eginning of exposure, resulting in n inrese ismuttion of O - 2 into H 2 O 2 to prevent the toxiity of this ril. After 24 h, the equilirium etween O - 2 rils n H 2 O 2 is not mintine: it is presumle tht the SOD tivity is unle to remove O - 2 n to limit the ROS formtion, ontriuting to lipi peroxition (s reporte y ellegrini et l. 2011) n to the onsequent ourrene of mrosopi effets. In irh, it ws similrly shown tht the inrese in SOD tivity t 24 h fter the en of O 3 fumigtion (150 pp, 8 h) might e more tissue mge-relte thn protetive from the ROS regenerte iretly from oxitive stress (Tuominen et l. 199). Further investigtions in reltion to the lne etween SOD n other H 2 O 2 -svenging enzymes re require to etermine the stey stte level of O - 2 n H 2 O 2. hytohormones ply key roles in the moleulr events relte to lesion evelopment, leing to O 3 -inue

10 lnt Cell Rep -Crotene (µg g -1 FW) Ozone *** Time *** Lutein (µg g -1 FW) Ozone *** Time *** Totl rotenois (µgg -1 FW) e Ozone * Time ** Ozone x Time ** Violxnthin (µg g -1 FW) g f e Ozone ** Time ** Fig. 7 Time ourse of lef pigment ontents in Meliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Different letters inite signifint HR-mimiking folir symptom. The role of ET in plnt response to ioti n ioti stress is omplex: the reserh uring the lst 40 yers hs shown tht O 3 inues the emission of this gseous plnt hormone in severl speies (Grntz n Vu 2012). The mount of ET emitte is well-orrelte with the egree of lesion propgtion (Ro et l. 2002; Tuominen et l. 2004) s well s with the extent of ell eth (Lngertels et l. 1991; Kngsjärvi et l. 2005). ET, s H 2 O 2, shows ler iphsi time ourse with the highest levels uring the tretment (mximum fter just 1 h from eginning of exposure) n fter 48 h in the post-fumigtion perio. On one hn, this seems to onfirm tht ET proution is (1) one of the fstest n tive responses of plnts to pereption of O 3 n (2) mjor regultor of O 3 -inue efenses, s suggest y Kngsjärvi et l. (2005). On the other hn, the inrese ET synthesis t 48 h from eginning of exposure is ssoite with the lesion propgtion (s onfirme y the visul survey of O 3 ifferenes ( B 0.05). In the oxes, results of two-wy ANOVA re reporte, sterisks showing the signifine of ftors/intertion for: *** B 0.001; ** B 0.01; * B The thik line inites the time (5 h) of ozone exposure injury) initing tht ET is promoter of ell eth in ROS-epenent CD (Vhl et l. 2003). Similr iphsi pttern in the genertion of this hormone hs een oserve y Lngertels et l. (1991) in n O 3 - sensitive ultivr of too expose to single O 3 tretment (150 pp, 5 h). Reent stuies on the mehnism of the HR in Ariopsis hve shown tht iphsi ET genertion uring stress n meite pthogen-inue mge y moulting the spre of ell eth n y ontriuting to stress llevition (Mur et l. 2009). It is worth noting tht (1) the mximl ET emission ws oinient with the first H 2 O 2 pek n tht (2) the seon pek of this hyroron is onomitnt with the mximum O 2 - level, whih is reminisent of the tight sptil n funtionl orreltion etween ET proution n ROS umultion, oserve in other experimentl stuies (Di Bio et l. 2012). Aoring to Overmyer et l. (2005), ET iosynthesis is n erly O 3 response, n lter, ABA, SA n JA re proue. In our stuy, fter the

11 lnt Cell Rep ORAC (µmol TE g -1 FW) HORAC (µmol GAE g -1 FW) initil n noteworthy inrese of ET synthesis, the enogenous ontent of ABA enhne t the en of exposure. O 3 enters through the stomt n ABA plys key role in regulting their losure. Thus, the ROS generte from O 3 egrtion in the poplst re lso involve in the regultion of the losure of stomt y n ABA-epenent mnner (ei et l. 2000; Zhng et l. 2001). ABA hs long een reognize s plnt stress hormone; ABA synthesis is one of the fstest responses of plnts to ioti stress, triggering ABA-inuile gene expression (Ymguhi-Shinozki n Shinozki 200; Royhouhury et l. 2013). Currently, there re few stuies eling with ABA n O 3 n the inrese of ABA ontent O 3 -inue hs een lrgely neglete Ozone ** Time ** Ozone x Time * Ozone ** Time ** Fig. 8 Time ourse of ntioxint pity expresse s the hyroxyl ril ntioxint pity (HORAC, ) n oxygen ril sorne pity (ORAC, ) vlues in Meliss offiinlis mintine in filtere ir (open irle) n expose to ozone (200 pp, 5 h, lose irle). Dt re shown s men ± stnr evition. The mesurements re rrie out t 1, 2, 5, 8, 24 n 48 h from the eginning of exposure. Different letters inite signifint ifferenes ( B 0.05). In the oxes, results of two-wy ANOVA re reporte, sterisks showing the signifine of ftors/intertion for: *** B 0.001; ** B 0.01; * B The thik line inites the time (5 h) of ozone exposure. GAE glli i equivlents, TE Trolox equivlents (Overmyer et l. 2008; Wilkinson n Dvies 2009; Blomster et l. 2011). In our system, O 3 exposure les to inrese ABA onentrtion, prtiulrly t the en of the tretment, still following the erly pik of ET proution, ut preeing the seon phse of H 2 O 2 enhnement n the shrp inrese of O 2 - uring the post-fumigtion perio. These t suggest tht O 3 - inue ABA umultion might lso e involve in the inrese genertion of ROS. Similr results were foun in leves of ethe mize plnts expose to wter stress: time ourse nlyses of ABA ontent n the proution of ROS of wter-stresse leves showe tht signifint inrese in the levels of this hormone preee tht of ROS (Jing n Zhng 2002). Overll, this suggests tht the responses to ROS genertion oul e regulte through inrese ABA onentrtion. Moreover, iosyntheti pthwy of this hormone n intert with the sorte glutthione yle (Ton et l. 2009). The step for the onversion of violxnthin into zexnthin requires the oxition of AsA into DHA n it is tlyze y violxnthin e-epoxise (VDE), s reporte y Smirnoff (199). In Ariopsis vt1 (vitmin C1) mutnt, the levels of sorte re low s ompre to the wil type n this onition stimultes ABA proution. Inste, when the tivity of VDE is enhne, the proution of ABA is ntgonize, the levels of sorte re reue n the onversion of GSH into GSSH is promote n overll this les to higher ROS umultion (stori et l. 2003). In ontrst, M. offiinlis showe n inrese vilility of AsA within 5 h of fumigtion n y the wy the levels remine high n the rise in ABA t 5 h ws oinient with the first inrement of sori i. It is ler tht first line of efense ginst O 3 - inue poplsti ROS umultion is sori i overproution (Conklin n Brth 2004), n in our stuy the inrese ABA ontent might not erive from the intertion etween the xnthophyll yle n the ROS-uffering sorte glutthione yle. It is possile tht the ABA umultion oserve t the en of fumigtion perio erives just from the onversion of violxnthin into xnthoxin (whih is onsiere to e the immeite preursor of ABA; Buren n Tylor 1970) through series of retions sine the ontent of violxnthin ws enhne t 5 h from eginning of exposure. Thus, violxnthin eing n ABA preursor, it serves s ABA-soure n its inrese ontent oul e ssoite to the rise in ABA mnifeste t the sme time. In ition to ET n ABA, SA hs lso een shown to hve entrl role in plnt response to pthogen infetion (Durner et l. 1997), ioti stress (Brtoli et l. 2013) n in plnt growth (Wng et l. 2013). Results of the present stuy inite tht O 3 triggers mrke inreses in SA (onjugte n free forms) in leves fter 8 h from

12 lnt Cell Rep eginning of exposure. Similrly, squlini et l. (2002) reporte tht, in n O 3 -sensitive ultivr of too expose to short-term O 3 tretment (200 pp, 3 h), the mximum inution of this metolite ws oserve fter 7 h of reovery. Di Bio et l. (2012) oumente n nlogous pttern of SA umultion uring post-o 3 (200 pp, 5 h) reovery in ultivr of tomto onfirming tht O 3 exposure, suh s pthogen infetion, inues SA synthesis within few hours from the eginning of the exposure, s suggeste y Kngsjärvi et l. (2005). At 8 h from eginning of exposure, the O 3 -inue umultion of free SA ws higher when ompre with tht of onjugte SA; mong sliyltes, the free SA is onsiere the most iologilly tive form s meitor of plnt stress responses, inluing isese n SAR (Lee et l. 1995). In the present work, it is worth noting tht the mrke inrese in SA ontent ws oinient with the seon H 2 O 2 pek, suggesting H 2 O 2 SA intertion pttern. Qun et l. (2008) propose tht these ompouns onstitute self-mplifying system where H 2 O 2 inues SA umultion (tlyzing the onversion of enzoi i into SA) n SA enhnes H 2 O 2 levels (inhiiting tlse tivity through speifi ining to the enzyme or y inuing the formtion of this moleule y phenolepenent peroxise; Dir et l. 2005). This grees with the stuies tht provie eviene tht SA pereption hs n essentil role in SAR, in the tivtion of n hypersensitive ell eth pthwy (Koh et l. 2000) n in the lesion initition n progression in response to O 3 (Ro et l. 2002). It hs een reporte tht the retion of O 3 with unsturte lipis of the plsm memrne les to the proution of peroxitive proesses n, onsequently, of the sustrtes for otenoi pthwys, suh s linolei i (Mu 1997). This ompoun is preursor of JA iosynthesis tht prtiiptes in the ontinment of the ROSepenent lesion propgtion in response to ioti (Devoto n Turner 2005; Kngsjärvi et l. 2005) n ioti (Vijyn et l. 1998; Sntino et l. 2013) stress ftors. In reltion to the overwhelming similrities etween O 3 - n pthogen-inue responses, it ws not surprising to oserve tht in our se short-term O 3 tretment triggers mrke umultion not only of SA, ut lso of JA levels. Similrly, Koh et l. (2000) reporte tht n O 3 -sensitive hyri poplr lone umultes SA n JA in response to single pulse of this ontminnt (300 pp, 3 h). In our trete leves, JA peke t 8 h from eginning of exposure n remine high throughout the entire post-fumigtion perio. This ppers to onfirm tht (1) this ompoun is involve in limiting n in moulting the lesion spreing in the lter stges, oring to Cstgn et l. (2007), n (2) the egrtive proesses of memrnes inrese (s onfirme y the inution of lipi peroxition reporte y previous stuy, ellegrini et l. 2011, 2012). As lrey forementione, the levels of ROS proue following short-term O 3 tretment hve to e finely tune to hieve positive ell retion through the hormonl signling se regulting the O 3 -inue lesion formtion. To protet themselves ginst these toxi O 2 intermeites, plnts hve evolve effiient enzymti n nonenzymti ntioxint systems, ple of etoxifying sustntil mount of them. AsA is onsiere s powerful ROS svenger in reltion to its ility to onte eletrons in numer of retions. We foun n inrese in AsA uring the whole time ourse. Similrly, Rnieri et l. (199) reporte tht, in the intrellulr flui of pumpkin leves expose to short-term O 3 tretment (150 pp, 5 h y -1, 5 ys), mrke inrese in AsA ontent ws oserve t the en of the exposure. ukette et l. (2007) oumente n nlogous pttern of this metolite in Meigo truntul trete with this ontminnt (300 pp, h). Aoring to our results, it is possile tht in the first step this metolite n provie protetion to memrnes y iretly svenging the O 2 - until 8 h from eginning of exposure (s onfirme y the signifint eline of this oxyril). Also it ts s oftor of VDE sustining issiption of exess exittion energy (oring to the higher vlue of non-photohemil quenhing reporte y ellegrini et l. 2011). Moreover, uring n fter O 3 exposure AsA/DHA rtio (n importnt inex of the ell reox sttus) lso inrese ontinuously. In the literture, it is ssume tht AsA plys ul role, suh s ntioxint n signling ompoun: to mintin high norml reue stte of ells (s AsA/DHA rtio), to limit the inhiitory effets of ROS inue oxitive stress (Meyer 2008) n to t s signl moleule in the reltive ptive response (Lomonte et l. 2010). Chnges in this prmeter might e expete to lter stomtl movement, prtiulrly uner onitions in whih ABA or H 2 O 2 signling tivtes stomtl losure (Chen n Gllie 2004). It is lrgely known tht reue stte of AsA ours t the expense of GSH, vi Hlliwell As yle. GSH ontent, s GSH/GSSG rtio (n initor of generl ellulr reox lne), showe ler triphsi time ourse in response to O 3, with the highest levels fter the first hours of fumigtion n uring the post-fumigtion perio. This my onfirm tht GSH plys role in meiting the efense responses of plnt ells to O 3 : higher mounts of this metolite were ville to (1) prtiipte in the tivtion of Hlliwell As yle n (2) to svenge free rils, s propose y Gill n Tutej (2010). However, evient inrese in ROS levels (espeilly in O 2 - ) uring the entire perio of the exposure suggests the ineffiieny of this yle to ounter the O 3 -inue oxitive stress. For this reson, other non-enzymti

13 lnt Cell Rep ntioxint mehnisms re involve in the etoxifition n mitigtion of ROS. Crotenois re pigments tht ply multitue of funtions in plnt metolism inluing (1) issiption of exess exittion energy s het (euse they n t s essory light-hrvesting pigments), (2) svenging of ROS (in reltion to the ility of their onjugte oule-oun to quenh hlorophyll triplet stte) n (3) suppression of lipi peroxition. After O 3 tretment, generlize signifint erese of totl rotenois ws oserve n this reution ws prolonge uring the post-fumigtion perio; - rotene (involve in lef protetion ginst 1 O 2 ) n lutein (importnt in the protetion of the photosyntheti pprtus) followe the sme pttern. It is worth noting tht there ws n inility of these pigments to quenh the free rils n to inhiit their peroxition tion (suh s lrey onfirme y the inution of lipi peroxition ove reporte). Similrly, Rnieri et l. (2000) reporte tht, in n O 3 -sensitive poplr lone expose to short-term O 3 tretment (150 pp, 5 h), mrke erese in -rotene levels following exposure ws oserve, suggesting tht this ompoun n e one of the possile ftors responsile for the high O 3 -sensitivity of this lone. It is importnt for plnt survivl n growth uner stress onitions tht ntioxints n work in oopertion, thus proviing etter efense n regenertion of tive reue forms. For this reson, we nlyze not only the levels of some phytohemils (sori i, totl rotenois, - rotene n lutein), ut lso the totl ntioxint pity. With HORAC ssy, in the trete plnts, the ntioxint pity is signifintly high n limite t the en of exposure, suggesting mximum pity of the mteril just fumigte. With ORAC metho, the vlue initilly i not hnge n then it erese within 48 h from eginning of exposure. This is in greement with the ft tht HORAC n ORAC ssys mesure two ifferent, ut eqully importnt, spets of ntioxint properties (ril hin reking n ril prevention, respetively). It is expete tht the smples with high HORAC vlues o not neessrily hve high ORAC vlues, n vie vers (Ou et l. 2002). The ntioxint ontents here nlyze i not show ny orreltion with the totl ntioxint pity, suggesting tht further investigtions re require to efine other ntioxint omponents, suh s totl phenols. Similrly, Corrl-Aguyo et l. (2008) reporte tht in severl hortiulturl rops, only totl solule phenols were orrelte with the totl ntioxint pity mesure with ORAC ssy. ET, ABA, SA n JA re importnt phytoregultors of O 3 responses n the omplex intertion of these signls is extremely importnt for n effetive efense response n to etermine the extent of lesion evelopment (Overmyer et l. 2003; Kohli et l. 2013). These hormone Fig. 9 Temporl hnges in O 3 -inue tivtion or umultion signling intermeites in Meliss offiinlis expose to ozone (200 pp, 5 h) [rwn fter Kngsjärvi et l. (2005) with our t]. The lk thik line inites the 5 h of ozone exposure signling pthwys o not operte inepenently ut rther re interrelte y synergisti or ntgonisti ross-tlks so tht they moulte eh other s iosynthesis or responses (Cui n Lun 2012). The genertion of these omplex we intertions plys ruil roles in the response to ioti n ioti stresses (eleg n Blumwl 2011). Uner short-term O 3 exposure, oring to Di Bio et l. (2012), SA tion is tightly epenent on ET synthesis. Our ozonte plnts showe high erly ET proution just fter 1 h from eginning of exposure n lte SA umultion (t 8 h from eginning of exposure) uring the post-fumigtion phse when ET levels were not signifintly ifferent from those mesure in ontrols (Fig. 9, sensu Kngsjärvi et l. 2005). Susequently, uring the lte post-fumigtion phse (24 n 48 h), SA rehes the onstitutive vlues, while ET inrese its proution. Tken together, our results might suggest tht n O 3 - inue ET my ntgonize the SA umultion. A similr mutul ntgonism of ET n JA regultes O 3 -inue ell eth n ws previously reporte in the O 3 -sensitive, JA-insensitive jsmonte resistnt 1 (jr1) n the O 3 - tolernt, ET-insensitive ethylene insensitive 2 (ein2) Ariopsis mutnts (Tuominen et l. 2004). Similrly, the erly n rpi umultion of ET n suppress the proteting funtion of JA n the susequent spreing of ell eth les to lte umultion of JA (fter 8 h from the strt of the fumigtion; Fig. 9), whih possily slows own the propgtion of ell eth until the seon pek of ET fter 48 h. ET synthesis n lso trigger ABA iosynthesis (Grossmnn 2003) or ABA n ntgonize the proution of ET (Trivellini et l. 2011). In our stuy, the rise of ET proution is never simultneous to the inrese in ABA ontent, suggesting tht their lne is se on mutul ntgonisti intertion etween the signling of two hormones (Ahlfors et l. 2004).

14 lnt Cell Rep In response to initil key questions, we n onlue tht: first, M. offiinlis plnts re le to tivte CD in response to short-term O 3 tretment. Seon, severl hormonl signling ses n, in prtiulr, the lne etween SA, JA, ET n ABA regultes the ell eth progrm tivte in this speies. Thir, the ntgonisti reltions etween SA, JA n ET re more importnt to etermine the initition, propgtion n ontinment of O 3 -inue ell eth n, onsequently, the egree of the sensitivity of lemon lm to this ontminnt. Aknowlegments We grtefully knowlege Dr. Giuli Fini n Dr. Mrí José Díz for their support uring the iohemil nlyses. 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