Plnt Physiology nd Biohemistry 52 (2012) 38e51 Contents lists vilble t SiVerse SieneDiret Plnt Physiology nd Biohemistry journl homepge: www.elsevier.om/lote/plphy Reserh rtile Retive oxygen speies from hloroplsts ontribute to 3-etyl-5- isopropyltetrmi id-indued lef nerosis of Arbidopsis thlin Shiguo Chen,1, Chunyn Yin,1, Reto Jörg Strsser,b,, Govindjee d,2, Chunlong Yng, Sheng Qing, * Weed Reserh Lbortory, Nnjing Agriulturl University, Nnjing 210095, Chin b Bioenergetis Lbortory, University of Genev, CH-1254 Jussy/Genev, Switzerlnd North West University of South Afri, South Afri d Deprtment of Plnt Biology, nd Deprtment of Biohemistry, University of Illinois t Urbn-Chmpign, Urbn, IL 61801, USA rtile info bstrt Artile history: Reeived 22 August 2011 Aepted 2 November 2011 Avilble online 11 November 2011 Keywords: (3-etyl-5-isopropyltetrmi id) ROS (retive oxygen speies) Cell deth Photosynthesis inhibitor Chlorophyll fluoresene trnsient OJIP 3-Aetyl-5-isopropyltetrmi id (), derivte of tetrmi id, is responsible for brown lefspot disese in mny plnts nd often kills seedlings of both mono- nd diotyledonous plnts. To further eluidte the mode of tion of, during -indued ell nerosis, series of experiments were performed to ssess the role of retive oxygen speies (ROS) in this proess. When Arbidopsis thlin leves were inubted with, photosystem II (PSII) eletron trnsport beyond Q A (the primry plstoquinone eptor of PSII) nd the redution of the end eptors t the PSI eptor side were inhibited; this ws followed by inrese in hrge reombintion nd eletron lekge to O 2, resulting in hloroplst-derived oxidtive burst. Furthermore, the min ntioxidnt enzymes suh s superoxide dismutse (SOD), tlse (CAT) nd sorbte peroxidse (APX) lost their tivity. Exess ROS moleules diretly ttked vriety of ellulr omponents nd subsequently used eletrolyte lekge, lipid peroxidtion nd ell membrne disruption. Finlly, this led to ell destrution nd lef tissue nerosis. Thus, -triggered lef nerosis of Arbidopsis ws found to be result of diret oxidtive injury from the hloroplst-originted ROS burst initited by the inhibition of norml photosyntheti eletron trnsport. Ó 2011 Elsevier Msson SAS. All rights reserved. Abbrevitions: ABS, photon flux bsorption, by photosystem II ntenn pigments reting exited hlorophyll moleules;, 3-etyl-4-hydroxyl-5- isopropylpyrroline-2-dione; APX, sorbte peroxidse; CAT, tlse; CS, optil ross setion of the photosyntheti smple; DAB, 3,3 0 -diminobenzidine; DCF, dihlorofluoresein; DMTU, dimethylthioure; ET, energy flux tht leds to eletron trnsport beyond Q A ;F O,F V,F M, the initil (minimum), vrible nd mximum hlorophyll fluoresene intensity; F J,F I, hlorophyll fluoresene t the J-step (2 ms) nd t the I-step (30 ms) during fluoresene trnsient; FDA, fluoresein diette; H 2 DCF-DA, dihlorofluoresin diette; MDA, mlondildehyde; NAC, N-etyl-L-ysteine; NBT, nitroblue tetrzolium; PS I, photosystem I; PS II, photosystem II; Q A,Q B, PSII primry nd seondry plstoquinone eletron eptors; R J, number of PSII RCs with Q B -site filled by PSII inhibitor; RC, retion enter; RE, redution of end epters t the PSI eletron epter side; ROS, retive oxygen speies; SOD, superoxide dismutse; TBA, thiobrbituri id; TBD, Trypn Blue D; TR, energy flux trpping (leding to Q A redution) by ll the PSII retion enters; 4 Po, the mximum quntum yield of primry PSII photohemistry; j Eo, the probbility tht trpped exiton moves n eletron into the eletron trnsport hin beyond Q A ; 4 Eo, the mximum quntum yield for PSII eletron trnsport; 4 Ro, the quntum yield for the redution of the end eletron eptors t the PSI eptor side; d Ro, the probbility with whih n eletron from the intersystem eletron rriers is trnsferred to redue the end eletron eptors t the PSI eptor side. * Corresponding uthor. Tel./fx: þ86 25 84395117. E-mil ddress: wrl@nju.edu.n (S. Qing). 1 Authors ontributed eqully to this work. 2 G. ontributed minly in the disussion nd in the writing of the pper, but not in the experiments. 1. Introdution Retive oxygen speies (ROS), hydroxyl rdils ($OH), singlet oxygen ( 1 O 2 ), superoxide nions (O 2 L ) nd hydrogen peroxide (H 2 O 2 ), re known to prtiipte in vrious bioti or bioti stress responses [1]. A rpid inrese in the onentrtion of ROS (lled the oxidtive burst ) is ommon plnt response to vrious dverse environmentl ftors, suh s pthogen ttk nd herbiide tretment. Exess intrellulr ROS rets with lrge vriety of biomoleules, nd uses irreversible ellulr dmge, ultimtely leding to plnt tissue nerosis. On the other hnd, lower level of ROS might t s key meditor of progrmmed ell deth nd be, thus, used s signl moleules leding to diseserelted deth [2,3]. In plnts, oxidtive burst triggered by different stresses re ttributed to different mehnisms [2]. The mjor soures of ROS prodution inlude photosynthesis in hloroplsts, photorespirtion in peroxisomes, mitohondril respirtion, plsm membrne NADPH oxidses nd xnthine oxidse, s well s ell wll oxidses nd peroxidses [1,2]. The retion enters of photosystem II (PSII) nd photosystem I (PSI) in hloroplsts re onsidered to be the mjor genertion sites of ROS sine they possess n environment 0981-9428/$ e see front mtter Ó 2011 Elsevier Msson SAS. All rights reserved. doi:10.1016/j.plphy.2011.11.004
S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 39 rih in oxygen, redutnts, nd high-energy intermedites [4]. A omplex rry of ROS n be generted in hloroplsts resulting from the umultion of 1 O 2 when the bsorption of light energy exeeds the pity for CO 2 ssimiltion or leds to intivtion of PSII retion enters (RCs) nd from the prodution of O 2 L due to the photoredution of oxygen t PSI t high light intensities [5]. Retive Oxygen Speies re formed in hloroplsts when plnts re treted with herbiides, inhibitors of photosynthesis. Bipyridinium herbiides suh s prqut nd diqut, ommonly used in gronomy, exert their herbiidl tivity by repling NADP þ tht is the norml ultimte eletron eptor of PSI. When plnts re exposed to sunlight, these herbiides ept eletrons from PSI nd pss them to oxygen leding to the genertion of O 2 L [6]. The redued photosyntheti eletron eptors of PSI enhne the redution of O 2, whih results in subsequent formtion of O 2 L, H 2 O 2 nd $OH [7,8]. Severl other herbiides, suh s diuron, trzine, bentzon, bromoxynil, nd hexzinone, re PSII herbiides whih use hloroplsti oxidtive burst nd severe oxidtive stress [9]. Additionlly, diphenyl ether (e.g. ltofen) nd protoporphyrinogen oxidse-inhibiting herbiides n indue genertion of 1 O 2 in intt illuminted thylkoids; this is relted to the formtion of protoporphyrin IX, whih results in oxidtive dmge of the extensive membrne system of the thylkoids [10]. In the se of some other photosynthesis-inhibiting herbiides, their primry toxi mehnism is to blok the photosyntheti eletron hin, whih is followed by ROS genertion, oxidtive dmge nd ell deth [9]. Hene, knowledge of the mehnism of ROS genertion nd toxiity is very importnt for reserh nd development of new photosynthesis herbiides. Tetrmi id (pyrrolidine-2,4-diones ring system, Fig. 1 (1)) is reurrent motif mong nturl produts originting from vriety of mrine nd terrestril speies [11]. The fmily of this ompound ontinues to ttrt gret del of interest beuse of brod rnge of desirble biologil nd phrmeutil tivities. Some tetrmi id moieties, suh s 3-benzoyl-pyrrolidin-2,4-dion-derivtes [12] nd 2-ethyl-4,6-dimethyl-phenyl-substituted tetrmi id derivtes [13], hve been found to possess unusul physiologil tivity nd been ptented for herbiidl use. A most frequently used ompound is 3-yl-tetrmi id; it is lso phrmologilly most interesting due to its struturl simpliity nd high biologil tivity [14]. One of the best representtive nturl metbolites of this fmily is tenuzoni id (Fig. 1(2)). A previous study hs reveled tht tenuzoni id is novel type of photosynthesis inhibitor [15], whih uses ell nerosis ttributed to diret oxidtive dmge from the hloroplst-derived ROS burst [16]. It hs the potentil to be developed s new bioherbiide sine it hs wide rnge of herbiidl tivities [16]. 3-Aetyl-5- isopropyltetrmi id () (Fig. 1(3)) is nother very interesting 3-yl-tetrmi id derivte, whih hs been isolted from miroorgnism Alternri tenuis [17]. The side hin of t the 5-position of pyrrolidine-2,4-diones ring is n isopropyl group different from se-butyl group of tenuzoni id. Thus, is nother good tool besides tenuzoni id for the investigtion R 2 HN R 1 N R 3 O OH Tetrmi id (1) H 3 C O CH 3 OH CH 3 O Tenuzoni id (2) H 3 C HN O CH 3 OH CH 3 O (3) Fig. 1. The struture of tetrmi id (1), tenuzoni id (2) nd 3AIPTA (3). of the struturl nd biologil hrteristis of photosyntheti orgnisms, s well s for the mode of tion of tetrmi id fmilies. However, till now there hve been very few reports on 3- AIPTA effets. Our previous studies reveled tht nd some of its nlogues inhibit PSII eletron trnsport rte in lgl ells s well s their growth [15]. Bsed on biossy results, is shown to be phytotoxi in wide rnge of plnts, from weeds to rops (Supplementry Tble 1). Further, it kills seedlings of both mono- nd diotyledonous weeds. ws shown to inhibit both PSII nd PSI eletron trnsport tivity, s well s root nd shoot length of seedlings. strongly interrupts eletron flow beyond Q A by binding to the D1 protein. However, it hs different binding behvior during intertion with the D1 protein s ompred to tht known for lssil PSII inhibitors [18]. At high onentrtions, inhibits the redution of the end eletron eptors on the PSI eptor side [18]. However, the mode of tion of lef nerosis indued by is still unknown. In this pper, we show tht during lef nerosis of Arbidopsis thlin, leds to hloroplst-derived ROS burst under illumintion; subsequently, ROS uses irreversible oxidtive dmge nd ultimtely ell deth. We found tht the inhibition of the PSII eletron trnsport beyond Q A nd the redution of end eletron eptors on the PSI eptor side is n erlier event thn the ROS burst. Bsed on this evidene, it is suggested tht the ROS genertion in the hloroplst results from the blokge of the photosyntheti eletron trnsport, whih plys n importnt role in -indued ell nerosis. 2. Results 2.1. indues lesion formtion in Arbidopsis leves When dethed-intt A. thlin leves re treted with 500 mm for 0e96 h under illumintion, visible lesions re lerly observed (Fig. 2A). Usully, mrosopi wter-soked lesions begin to pper fter 48 h tretment, with the tissues turning brown; then, these lesions spred, nd eventully beome neroti by 96 h (Fig. 2A). The -eliited lesions led to ell deth, s shown by Trypn Blue stining, nd by ion lekge (n inditor of plsm membrne dmge); the ion lekge strted to inrese fter 24 h tretment with, suggesting tht membrne destrution hd ourred (Fig. 2C). At this time, portion of the ells begn to die (dt not shown). After 30 h of tretment, lmost ll the ells hd died, nd ion lekge hd inresed to very high level (Fig. 2B nd C). This pttern is essentilly the sme s for the injury of lef tissues used by photosynthesis-inhibiting herbiide bentzon. 2.2. Histohemil lotion determines -indued oxidtive burst To determine whether retive oxygen speies re involved in ell nerosis of Arbidopsis leves in the bove-desribed experiments, 3,3 0 -diminobenzidine (DAB) nd nitroblue tetrzolium (NBT) were used to stin the smples in order to hek the in situ umultion of H 2 O 2 nd O L 2 in intt illuminted leves treted with 500 mm. DAB polymerizes nd turns deep brown in the presene of H 2 O 2 nd the intensity of olortion nd its lotion n be quntittively ssessed nd photogrphed [19]. The development of the DAB-H 2 O 2 retion produt in intt leves in response to or bentzon tretment is shown in Fig. 3A. No visible H 2 O 2 umultion ws observed in ontrols fter 6 h. However, H 2 O 2 ws deteted s erly s 1 h in leves treted with 3- AIPTA or bentzon, with the olor ontinuing to drken with inubtion over 6 h period. The mirosopi exmintion of lef
Fig. 2. Cell deth-ssoited symptoms indued by or bentzon. A. thlin leves or lef diss were vuum infiltrted with 500 mm or 500 mm bentzon for the indited times under illumintion. (A) Progression of neroti symptoms in dethed-intt leves indued by. Red irles indite lef lesions showing brown lef-spot. (B) ell deth t 30 h fter or bentzon tretment. Leves were stined with Trypn blue D. (C) ion lekge ws determined, s desribed in mterils nd methods, nd reported s the rtio of the vlue mesured in - or bentzon-treted smples to the vlue obtined in distilled wter-treted smples (ontrol) t 3 h; rw vlue of wtertreted smples t 3 h is 6.38 0.93 ms m 1. The rrow indites the time t whih ell deth ws tested. (For interprettion of the referenes to olour in this figure legend, the reder is referred to the web version of this rtile.)
S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 41 Fig. 3. The indution of ROS genertion t the lef level fter or bentzon tretment. The dethed intt-leves of A. thlin were treted with distilled wter (ontrol) or with 500 mm or 500 mm bentzon for the indited time under illumintion. Experiments were repeted t lest four times with similr results. (A) histohemil detetion of H 2 O 2 with DAB stining. The squre box setions of DAB-stined leves were tested under the mirosope. (B) mirosopi observtion of H 2 O 2 prodution. The imges were tken t 600 mgnifition. H 2 O 2 genertion origintes from hloroplsts fter tretment or bentzon (the site observed under mirosope is indited by red squre boxes nd rrows in Fig. 3A). No oxidized DAB deposit ws observed in the hloroplsts of ontrol (the site observed under mirosope is indited by blk squre box nd n rrow in Fig. 3A). (C) histohemil detetion of O 2 L genertion with NBT stining. (For interprettion of the referenes to olour in this figure legend, the reder is referred to the web version of this rtile.)
42 S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 ells showed tht oxidized DAB deposits were mostly loted in the hloroplsts (Fig. 3B). Sine H 2 O 2 ws deteted in hloroplsts, O 2 L prodution must hve tken ple in the hloroplsts sine O 2 L is known to be rpidly onverted to H 2 O 2 by SOD. Intrellulr O 2 L genertion n be deteted using drk-blue insoluble formzen produed by the retion of NBT nd O 2 L [8,20]. A similr result ws observed in leves treted with or bentzon (Fig. 3C). Little NBT stining ws observed in ontrols. Tretment with 3- AIPTA or bentzon for 1 h led to slight umultion of O 2 L in the lef tissues. The olor ws very drk in intt leves treted with or bentzon for 4 h, nd then it beme lighter. These results revel tht lrge mount of ROS is generted in the ells of leves treted with. Thus, we onlude tht hloroplsts re mjor soure of ROS prodution. This ws onfirmed by experiments with dihlorofluores in diette (H 2 DCF-DA) (see below). 2.3. Dihlorofluoresein mesures -indued ROS prodution in mesophyll ells Dihlorofluoresein (DCF) hs been used to visulize oxidtive proesses in plnts in response to vrious stresses [21,22]. In order to visulize intrellulr ROS formtion in ells, H 2 DCF-DA ws used in our studies. After epidermis-less lef segments were treted with 500 mm or bentzon for 0 to 3 h, ROS prodution ws observed in mesophyll ells (see time ourse in Fig. 4A). A rpid inrese of green DCF fluoresene signl in lef segments treted with or bentzon for 15 min ws observed, rehing pek t 60 min. Beyond 1 h fter tretment, the DCF fluoresene intensity begn to deline, nd then deresed to the ontrol level t 3 h (Fig. 4A). Interestingly, DCF fluoresene signls were first found in the hloroplsts of mesophyll ells within 15 min (dt not shown). By 30 min of or bentzon inubtion, DCF signls were still minly lolized in the hloroplsts of mesophyll ells (Fig. 4B). However, by 1 h, DCF signls drmtilly inresed nd dispersed through the ell when mesophyll ells were inubted with for 1 h (Fig. 4B). These results indite tht ROS is generted in the hloroplst of - or bentzon-treted lef segments, whih over time diffuses out of the hloroplsts nd ells into the surrounding ells. ROS svengers N-etyl-L-ysteine (NAC), dimethylthioure (DMTU), CAT nd SOD signifintly suppressed ROS levels in mesophyll ells treted with or bentzon (Fig. 4C). Although exogenously dded CAT only remins in the poplst, intrellulr generted ROS n leve the ells s H 2 O 2 nd enter other ells vi n poplsti route. A similr phenomenon hs been found in the O 3 -indued oxidtive burst [21,22]. Our results lerly demonstrte tht tretment uses hloroplst-derived oxidtive burst in Arbidopsis mesophyll ells. The ides, disussed bove, were further onfirmed by dt obtined from mesophyll protoplst experiments. When ws dded to H 2 DCF-DA preloded protoplsts, DCF fluoresene signl grdully intensified during 10 min tretment of protoplsts with, s observed by onfol mirosopy (see Supplementry Movie 1). In ontrst, DCF signls of ontrol protoplsts lwys remined t very low levels over the sme time period (Supplementry Movie 2). In n ttempt to identify the soure of ROS, both hloroplst utofluoresene (red) nd DCF fluoresene (green) of mesophyll ells, treted with 500 mm for 10 min, were mesured simultneously. The green DCF signls extly mth tht of the red hloroplst utofluoresene signls, showing single yellow imge when the red hloroplst fluoresene ws overlid with the green DCF signls (Fig. 4D). Thus, we onlude tht the primry site of -indued ROS burst in mesophyll ells is the hloroplst. Supplementry dt ssoited with this rtile n be found, in the online version, t doi:10.1016/j.plphy.2011.11.004. 2.4. Effet of on ntioxidnt enzymes In order to exmine the ontribution of ntioxidnt enzymes in ROS prodution nd in the protetion of ells ginst oxidtive stress, we tested the tivities of severl importnt ntioxidnt enzymes suh s SOD, CAT nd APX in epiderml-less Arbidopsis lef segments under tretment (Fig. 5). A signifint derese in the tivities of SOD (Fig. 5A) nd APX (Fig. 5B) ourred within 4 h of tretment. However, the CAT tivity showed mrked deline even fter just 1 h (Fig. 5C). After 6 h of tretment, the tivities of SOD, APX nd CAT deresed by 56%, 65%, nd 73% respetively, ompred to the ontrol. Moreover, the derese of tivities of these ntioxidnt enzymes is lter event reltive to -indued hloroplst-derived ROS burst. However, if the enzyme extrts were treted diretly with, itself hd no signifint effet on the tivities of SOD, CAT nd APX (Supplementry Figure 1). This implies tht in lef tissues, the -indued ROS burst hd destroyed the ntioxidnt enzyme system. 2.5. -triggered ellulr dmge is ssoited with hloroplst-derived ROS burst To further investigte the influene of -indued ROS on ellulr struture nd funtion, we monitored ell vibility of mesophyll ells following tretment. The loss of ell vibility is refleted in loss of fluoresein diette (FDA) fluoresene. As shown s Fig. 6A, the FDA fluoresene signl drmtilly deresed reltive to the ontrol fter mesophyll ells were treted for 3 h with 500 mm bentzon. Within 3 h of 500 mm tretment, only slight derese in FDA signl ws observed. However, fter 6 h of or bentzon inubtion, FDA fluoresene deresed to very low level nd beme negligible (Fig. 6A, B). ROS svengers NAC, DMTU, CAT nd SOD suppressed the -indued dmge to ell vibility (Fig. 6C). Thus, we onlude tht hloroplst-derived oxidtive burst plys n importnt role during -triggered ell deth nd lef tissues neroti lesion. The loss of ell vibility my indite tht -tretment destroys plsm membrnes where lipids exist sine lipids re prtiulrly sensitive to ROS produed during environmentl stress. Thus, the toxiity of ROS hs often been monitored by mesuring lipid peroxidtion s mlondildehyde- (MDA-) retive produts. In order to test the influene of -indued ROS on membrnes nd lipids, we mesured hnges in ion lekge nd lipid peroxidtion in ells treted with, ombining it with ROS svenger tretment. Pretretment with NAC, DMTU, CAT nd SOD signifintly suppressed -used ion lekge (Fig. 6D) nd lipid peroxidtion (Fig. 6E) in lef diss inubted with 500 mm for 30 h, but this did not ompletely prevent the dmge. This indites tht -used dmge of membrne struture nd lipids ws, in ll likelihood, due to -indued hloroplst-derived ROS prodution. 2.6. Inhibition of photosyntheti eletron flow preedes indued ROS burst Bsed on the known tion nd injury symptoms, ppers to be similr to ommeril photosynthesis inhibitor herbiides suh s bentzon, trzine nd diuron. interrupts PSII eletron trnsport from Q A to Q B (where Q A is the primry nd Q B is the seondry plstoquinone eptor of PSII); it is suggested to t
S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 43
44 S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 by binding to the Q B -site on the D1 protein [18]. To exmine the role of photosynthesis inhibition in the formtion of -indued hloroplst-derived ROS, we simultneously mesured, lthough indiretly, the photosyntheti tivity of -treted epidermisless lef segments by the fst Chl fluoresene rise, the OJIP trnsient, nd ROS genertion. It is well known tht Chl fluoresene trnsient is non-invsive, effiient, rpid nd sensitive tool to probe the stte of the struture s well s funtion of the photosyntheti pprtus [23,24, nd referenes therein]. In Fig. 7A nd B, the hlorophyll fluoresene trnsients of the untreted lef segments (ontrol) nd those treted with 500 mm for 5e120 min re shown. With inresing tretment time, the lrgest hnge in the OJIP fluoresene trnsient is in the inrese in the rte of rise from the O to the J-step; the F J (2 ms) beomes equl to the F M (mximum fluoresene). An inrese in the J-level is ttributed to the fster umultion of Q A ; this ours beuse of interruption of eletron flow beyond Q A [23,24]. To evlute the effet of on the IP phse, the therml phse, of the fluoresene indution urve OJIP, two different normliztion proedures were used (Fig. 7C nd D). When the lef segments re treted with for 5e120 min, the mplitude of the IP phse is strongly but not ompletely redued. The IP phse is relted to eletron trnsfer through PSI nd the trffi jm of eletrons used by trnsient blok on the eptor side of PSI possibly due to the intivtion of ferredoxin-nadpþ-redutse (FNR) [25], nd referenes therein]. The OJIP fluoresene trnsients were lso nlyzed by the JIPtest to dedue 6 struturl nd funtionl prmeters. In Fig. 8A, no hnge in the mximum yield of primry photohemistry (4 Po ¼ TR 0 /ABS ¼ F v /F M ; see the list of bbrevitions) ws found until 30 min of tretment. By 60 min, tretment used signifint derese of 4 Po, ompred to wter-treted lef segments (ontrol) (Fig. 8A). Sine PSII eletron flow beyond Q A ws bloked, 4 Eo, whih expresses mximum quntum yield of PSII eletron trnsport further thn Q A, hd shrp derese s erly s 5 min of tretment (Fig. 8B). If the Q A nnot be reoxidized in time, lrge umultion of Q A is expeted to inrese the onentrtion of intive PSII RCs. As the tretment time of ws inresed, PSII RCs were further intivted, exhibiting n pproximtely liner, nd signifint, inrese of the mount of non-q A reduing RCs (Fig. 8C): fter 5 min of inubtion of lef segments, the mount of non-q A reduing RCs hd inresed 130% reltive to the ontrol; by 60 min nd 90 min, bout 40% nd 61% of PSII RCs were intivted. Other evidene hs indeed lso suggested tht PSII inhibitors result in intivtion of PSII RCs when the eletron trnsport from Q A to Q B is bloked by the herbiide oupying the Q B -site [see e.g., 23,24]. Our dt suggest tht 3- AIPTA-used severe losure of PSII RCs should be ttributed to n enhnement of the R J prmeter, whih reflets the number of PSII RCs with the Q B -site filled with (Fig. 8D). The effets of on two prmeters relted to step-i, 4 Ro expressing the quntum yield for the redution of the end eletron eptors t the PSI eptor side, nd RE 0 /CS refleting eletron flux to the end eletron eptors t the PSI eptor side, re shown in Fig. 8E nd F. At shorter tretment time (under 30 min), insignifint hnges in the vlues of 4 Ro nd RE 0 /CS were found. However in the se of tretment for longer time (over 30 min), the 4 Ro level nd the RE 0 /CS level showed distint derese. Suh results indite tht hs visible effet on eletron flow t the PSI eptor side. Obviously, the inhibition of photosyntheti eletron trnsport is n erlier event thn hloroplst-derived ROS burst during -triggered ell nerosis. Thus, there must be series of events tht led from the effets on the eletron flow to the prodution of ROS tht dmges the system. 3. Disussion 3.1. -triggered lef lesion is onsequene of oxidtive dmge Our dt on DAB nd NBT stining revels tht tretment leds to umultion of H 2 O 2 nd O 2 L within the leves, nd this leds to lef nerosis (Figs. 2 nd 3). After intt leves were treted with for severl hours, -indued ROS in ells inresed to the highest level (Fig. 3A, C). However, by 30 h of tretment, signifint eletrolyte lekge took ple s result of plsm membrne dmge. At this point in time, lmost ll the lef ells die (Fig. 2B, C). At 48 h, leves showed ler physiologil dmge due to turgor loss nd tissue ollpse (Fig. 2A). Bsed on the nlysis of the time of ROS genertion nd ell nerosis, it is evident tht -indued ROS overprodution ours before ell membrne disruption whih preeeds ell nerosis. This onlusion is orroborted by evidene from DCF stining experiments, whih showed tht under illumintion, tretment uses distint inrese of ROS genertion in mesophyll ells within 1 h (Fig. 4A nd B). This mode of tion nd the injury symptoms re the sme s tht of photosynthesis inhibitor herbiide bentzon ([26], Figs. 2 nd 3). Photosynthesis-inhibiting herbiides n be divided into two distint groups, PSII inhibitors nd PSI inhibitors. Both blok the synthesis of ATP nd NADPH, followed by ROS genertion, oxidtive dmge nd ultimtely ell deth [9]. When photosyntheti systems re exposed to light, PSI inhibitor herbiides beome tive nd form ROS, whih redily use lipid peroxidtion nd hlorophyll brekdown [9]. This destroys ell membrne integrity, so tht the ells nd the orgnelles lek, leding to rpid lef wilting nd desition, nd eventully to plnt deth [7,9,10]. PSII inhibitor herbiides re known to interrupt eletron flow beyond Q A by binding to the D1 protein t the Q B -site in the PSII retion enter. This blokge stops photosynthesis, leding to lrge mount of ROS prodution in hloroplsts, espeilly 1 O 2 [27,28]. These ROS moleules disrupt ell membrnes nd use hloroplst swelling, membrne lekge, nd ultimtely ellulr destrution nd plnt nerosis., whih s photosynthesis inhibitor, indued hloroplst-derived ROS burst (Figures 3B, 4B nd D; Supplementry Movies 1, 2); nd this is responsible for -triggered ell deth nd neroti lef lesions. Retive oxygen speies normlly exist in ll erobi ells in blne with biohemil ntioxidnts. Oxidtive stress, nd s Fig. 4. Prodution nd intrellulr loliztion of ROS in A. thlin mesophyll ells fter or bentzon tretment. The epidermis-less lef segments or mesophyll protoplsts were treted with distilled wter (ontrol), 500 mm - or bentzon for the indited times. (A) time ourse of hnge in DCF fluoresene intensities from imges of - or bentzon-treted illuminted mesophyll ells. (B) DCF fluoresene imges of mesophyll ells t the indited times. Br ¼ 25 mm. (C) effet of ROS svengers on 3- AIPTA-indued ROS genertion in mesophyll ells. Mesophyll ells were pretreted with either NAC (1 mm) or DMTU (1 mm) or CAT (300U/mL) or SOD (400U/mL) for 2 h, subsequently inubted with 500 mm for 1 h under illumintion. Dt re presented s mens SD (n ¼ 5). Mens denoted by the sme letter did not signifintly differ t p < 0.05. (D) intrellulr loliztion of ROS in -treted mesophyll protoplsts. Protoplsts-preloded with H 2 DCF-DA were treted with distilled wter (ontrol) or 500 mm for 10 min, nd observed by onfol mirosope. Green signls indite GFP of DCF. Red signls indite hloroplst utofluoresene. Note tht the loliztion of the green fluoresene (DCF) signls mthed tht of the red fluoresene (utofluoresene) signls, showing single yellow imge. Green signls were not found in the outer membrne of hloroplsts. This experiment ws repeted t lest three times with similr results. Br ¼ 5.76 mm. (For interprettion of the referenes to olour in this figure legend, the reder is referred to the web version of this rtile.)
S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 45 SOD tivity (U mḡ 1 protein) 80 60 40 20 0 0 2 4 6 Tretment time (h) 0.6 A B C APX tivity (µmol min -1 mg -1 protein) 0.4 0.2 0.0 0 2 4 6 Tretment time (h) CAT tivity (µmol min -1 mg -1 protein) 8 6 4 2 0 0 2 4 6 Tretment time (h) Fig. 5. Time ourse of hnges in the tivities of ntioxidnt enzymes SOD (A), APX (B) nd CAT (C) in the epidermis-peeled lef segments of A. thlin exposed to. Dt re presented s mens SD (n ¼ 4). onsequene ellulr dmge, ours when this ritil blne is disrupted due to ROS burst, ntioxidnt depletion, or both [29]. To protet ginst this ellulr dmge used by ROS, it is bsolutely essentil for plnts to mke use of ntioxidnt enzymes. SOD onverts superoxide nion O 2 L into less toxi produt, nmely H 2 O 2 nd O 2. Two of the mjor enzymti svengers of H 2 O 2 re CAT nd APX [30]. Our present dt show tht tretment not only indues ROS overprodution but lso destroys the ntioxidnt enzyme system of mesophyll ells of Arbidopsis (Fig. 5). In the se of the loss of the tivity of ntioxidnt enzymes suh s SOD, CAT nd APX, ROS moleules re expeted to ret with vriety of ellulr omponents nd then use irreversible dmge tht results in the oxidtive destrution of the ells [2]. In ell, the min risk produed by H 2 O 2 nd O 2 L omes from the genertion of $OH rdils, whih is the most toxi of ll ROS. The $OH rdil diretly ttks most biomoleules, suh s mino ids, membrne lipids nd orgni ids; this uses dmge t site-speifi points on ount of very short life of $OH, so tht the ells nd orgnelles lek, leding to irrepirble metboli dysfuntion nd ell deth, nd eventully to lef wilting nd desition [9]. 1 O 2 is more retive thn H 2 O 2 nd O 2 L, whih n lso ret diretly with mny biomoleules ompnied by hlorophyll brekdown nd lipid peroxidtion. Finlly, these events result in the destrution of membrne integrity nd loss of ell funtion. As result, the formtion of other ROS ould be stimulted, whih ould use further severe dmge of ells [31]. Previous investigtions hve suggested tht 1 O 2 is n importnt dmging speies during photoinhibition used by stress suh s high light or PSII herbiide tretment [27]. It is very ler tht 3- AIPTA tretment used severe oxidtive dmge of mesophyll ells, suh s the loss of ell vibility, ell membrne rupture, ion lekge nd lipid peroxidtion (Fig. 6). ROS svengers n signifintly suppress these dmges beuse ROS genertion is n erlier event thn the injury of mesophyll ells. This suggests tht ROS prodution injures ellulr omponents through lipid peroxidtion nd oxidtive dmge. Bsed on the bove results, hloroplst-originted ROS burst plys n importnt role during -triggered ell deth nd lef tissue neroti lesion. 3.2. -indued hloroplst-derived ROS burst is ttributed to the inhibition of eletron trnsport in photosynthesis The experimentl results of histohemil stining showed tht hloroplsts were mjor soure of -indued ROS prodution (Figures 3B, 4B nd D). Like bentzon, in the erly stge of tretment, DCF fluoresene signl produed by ROS ws observed only within the hloroplsts in mesophyll ells, nd then it diffused out of the hloroplst into ytoplsm, nd even into interellulr spes (Fig. 4B). This mens tht the primry site of -indued ROS genertion in mesophyll ells is the hloroplst. In the hloroplsts, the mjor genertion site of ROS is PSI nd PSII [4]. When photosyntheti eletron trnsport hin is overredued, 1 O 2 is ontinuously produed by PSII [2,8]. Moreover, 1 O 2 genertion in photointivted PSII enters is diretly orrelted with photoinhibition [8]. As PSII inhibitor, inhibits eletron flow beyond Q A by binding to the Q B -site ([18]; Fig. 7A nd B nd Fig. 8B nd D). One the forwrd eletron trnsport nnot proeed, Q A will be overredued, using n umultion of Q A nd the ensuing losure of PSII RCs (Figs. 7B nd 8C). The overredution of Q A on the PSII eptor side results in thylkoid overenergiztion nd thus inreses the energy vilble for the hrge reombintion retion. This n led to the formtion of hlorophyll triplet stte ( 3 Chl), whih in turn, rets with O 2 to form 1 O 2 nd genertes other toxi ROS [4,27,28,32]. Prior studies hve suggested tht the redution of O 2 is the strting point for series of retions leding to ROS genertion on the PSII eletron eptor side [33]. O L 2 moleules re generted by PSII vi eletron trnsport to O 2 under reduing onditions. Experiments show tht Pheo nd Q A nd Cyt b 559 nd redued Q B n serve s redutnts for O 2 [28,33]. Further redution of O L 2 within PSII is expeted to produe other two retive oxygen speies suh s H 2 O 2 nd $OH [4]. It hs been proposed tht H 2 O 2 is formed by dismuttion of O L 2 known to our either spontneously or by tlysis using endogenous SOD, s well s by the intertion of O L 2 with PSII metl enter [33]. $OH is formed in illuminted PSII membrnes through the redution of H 2 O 2 vi the Fenton retion [33]. Reently, the prodution of $OH hs been demonstrted in the presene of either phenoli- or ure-type photosyntheti herbiides [28]. However, the mjor mehnism of O L 2 prodution in hloroplsts is by the redution of O 2 by PSI [4,32,34]. Further redution of O L 2 within the ell produes the other two ROS: H 2 O 2 nd $OH [4]. SOD in hloroplst n onvert O L 2 into H 2 O 2 [4]. In the presene of trnsition metl ions suh s Fe nd Cu, H 2 O 2 nd O L 2 n ret nd form $OH [2,9]. Additionlly, highly redued photosyntheti eletron rriers would enhne redution of O 2 by PSI, whih results in subsequent formtion of O L 2,H 2 O 2 nd $OH [5,7,8]. A smller IeP mplitude during hlorophyll fluoresene trnsient
46 S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 Fig. 6. Effet of on ell vibility (AeC) nd ellulr membrne (DeE) during - or bentzon-indued ell deth. Epidermis-less lef segments of A. thlin were treted with distilled wter (ontrol), 500 mm or 500 mm bentzon for the indited times under illumintion. After tretment, segments were olleted nd stined with 0.01% FDA before mirosopi observtion. Similr results were repeted three times. (A) time ourse of hnge in FDA fluoresene intensities from imges of ontrol nd or bentzon-treted mesophyll ells. (B) FDA fluoresene imges of ontrol or 500 mm 3AIPTA- or 500 mm bentzon-treted mesophyll ells t the indited times. Br ¼ 20 mm. (C) mesophyll ells were pretreted with either NAC (1 mm) or DMTU (1 mm) or CAT (300U/mL) or SOD (400U/mL) for 2 h, then treted with 500 mm for 6 h. The loss of ell vibility is refleted in the loss of FDA fluoresene. (DeE) effet of ROS svengers on ion lekge nd lipid peroxidtion indued by. Lef diss of A. thlin were pretreted with NAC (1 mm) or DMTU (1 mm) or CAT (300 U/mL) or SOD (400 U/mL) for 2 h, nd then exposed to 500 mm for 30 h under illumintion. Ion lekge nd MDA were determined s desribed in mterils nd methods. Ion lekge reported s the rtio of -treted/ontrol vlue, rw vlue of ontrol is 113.03 8.85 ms m 1. Dt re presented s mens SD (n ¼ 4). Mens denoted by the sme letter did not signifintly differ t p < 0.05. (Fig. 7C nd D) nd signifint lower 4 Ro nd RE 0 /CS level (Fig. 8E nd F) indite tht inhibits the redution of the end eptors t the PSI eletron eptor side s well s the tivity of FNR. As result, this proess diverts eletrons from the PSI eptor side, tht normlly goes to rbon fixtion pth (ClvineBenson yle), to redue O 2 generting O L 2 nd its deomposition produt H 2 O 2 [32]. Inhibition of FNR tivity bloks eletron trnsport from the redued ferredoxin to NADP þ nd produes rdil whih redues oxygen to O L 2 [9]. Tking into onsidertion the ft tht the onentrtion of 500 mm, used in this study, is the onentrtion required to blok PSII eletron trnsport tivity only by 50% [18] (Fig. 8B), we propose tht -indued O L 2 in the hloroplsts is minly produed by PSI. This different mehnism of oxidtive burst provides resonble explntion for the observtion tht kills plnts with fster rte reltive to lssil PSII hemil herbiides. Fig. 9 is summry sheme showing the sites of tion of 3- AIPTA in the two pigment system nd two light retion model of photosynthesis. It lso shows the sites of prodution of ROS. The inhibition of PSII eletron flow beyond Q A s well s the redution of the end eptors t the PSI eletron eptor side nd the tivity of FNR re suggested to be muh erlier event thn hloroplst-originted ROS burst, whih preedes ell injury. We onlude tht -triggered ell deth nd lef tissue nerosis is diret result of oxidtive dmge by hloroplst-derived ROS prodution. This my be the ommon mode of tion of tetrmi id fmilies. Thus, this study ontributes to the expnsion of our
S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 47 Fluoresene intensity (.u.) 2000 1500 1000 A J 500 O 5 min 15 min 30 min 60 min 120min 0 0.01 0.1 1 10 100 1000 Time (ms) I P (Ft-Fo)/(FM-Fo) 1 0.6 0.2 O B J -0.2-0.2 0.01 0.1 1 10 100 1000 Time (ms) I P 5 min 15 min 30 min 60 min 120 min 1 0.6 0.2 (Ft-Fo)/(FM-Fo) 1.0 C D P 1.2 (Ft-FI)/(FM-FI) 0.5 0.0 30 130 230 330 430 530 Time (ms) 5 min 15 min 30 min 60 min 120 min I 5 min 15 min 30 min 60 min 120 min 30 130 230 330 430 530 Time (ms) 1.1 1.0 (Ft-Fo)/(FI-Fo) Fig. 7. Chlorophyll fluoresene trnsients of drk-dpted epidermis-less lef segments of A. thlin treted with wter (ontrol) or 500 mm in drkness, mesured with HndyPEA fluorimeter. (A) the kineti urves plotted on logrithmi time-sle of the rw fluoresene dt re shown. (B) reltive vrible fluoresene between F O nd F M : V t ¼ (F t F O )/(F M F O ) (the top figures); ΔV t ¼ Δ(F t F O )/(F M F O ) (the bottom figures). (C) reltive vrible fluoresene trnsient from the I to the P level (V IP ¼ (F t F I )/(F M F I )), fter double normliztion between the F I nd F P points. (D) reltive vrible fluoresene between F O nd F I :V t ¼ (F t F O )/(F I F O ). All results re the verges of bout 15 independent mesurements. knowledge on nd even tetrmi id s novel photosynthesis inhibitor. In ddition, it is lso proposed tht might be useful new tool to study ROS in hloroplsts. 4. Mterils nd methods 4.1. Plnt mterils nd hemils Seeds of A. thlin L. (Columbi eotype) were germinted on mixture of soil nd vermiulite (2:1 [V/V]), soked with 1/4 strength Hoglnd nutrient solution. Plnts were grown in ontrolled environment room t 20e25 C under w 200 mmol photons m 2 s 1 white light (dy/night, 8 h/16 h) nd reltive humidity (bout 70%). After 6e8 weeks of growth, the leves of plnts were smpled. The syntheti ompound (>95%) ws provided by C.L. Yng (College of Siene, Nnjing Agriulturl University, Chin). The stok solution (0.5 M) in 100% methnol ws diluted in distilled wter in ll the experiments in this study; the finl onentrtion of methnol ws fr less thn 0.5% (v/v). All other hemils used in this work were obtined from SigmeAldrih. 4.2. Preprtion of lef segments nd tretments The epidermis ws refully peeled from the bxil surfe of the leves following the proedure desribed in referene [35]. The resulting epidermis-less lef segments of (0.5 1 m) were pled in smll Petri dish ontining 10 mm MES-KCl, ph 7.2, in drkness until use. For hemil tretment, smll lef segments were trnsferred to fresh buffer with or without ROS svengers NAC (1 mm), DMTU (1 mm in 0.2% DMSO), SOD (400 U/mL) or tlse (300 U/mL) for 2 h, then floted on solutions with or without with the peeled surfes in ontt with the liquid nd inubted for the time indited in eh experiment, t 25 C, under white light (100 mmol photons m 2 s 1 ) in the growth hmber. After tretment, lef segments were wshed with distilled wter nd used for biohemil nd ytologil ssys. 4.3. Lef tissue dmge ssy To ssess lesion formtion, dethed-intt leves were vuum-infiltrted for 15 min with distilled wter (ontrol) or 500 mm nd treted for 24, 48, nd 72 h t 25 C under pproximtely 100 mmol photons m 2 s 1 white light. After the bove tretment, the smples were exmined for visible lesions nd this ws reorded with digitl mer (Cnon, SX11S, Jpn). As reported in detil by Joo et l. [22], ell deth ws deteted histohemilly. Smples were briefly stined in Trypn Blue D (TBD) mixture (30 ml ethnol, 10 g phenol, 10 ml H 2 O, 10 ml glyerol, 10 ml of 10.8 M lti id, nd 10 mg of TBD), boiled in wter bth for 3 min, nd then equilibrted t room temperture (w25 C) for 1 h. The smples were then trnsferred to hlorl hydrte solution (2.5 g ml 1 ) nd boiled for 10 min for destining. The ltter smples were stored nd exmined in 96% ethnol nd reorded using digitl mer (Cnon, SX11S, Jpn). The experiments were repeted t lest three times. 4.4. Eletrolyte lekge nd lipid peroxidtion To mesure ion lekge [22], lef diss of 7-mm dimeter were prepred nd rinsed with distilled wter, then 20 piees of lef diss (bout 0.1 g) were submerged in 5 ml of distilled wter or
48 S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 1.00 0.80 A B 0.6 0.4 Po b b Eo 0.60 b 0.40 0 30 60 90 120 b b b 0 30 60 90 120 0.2 0.0 Non-QA reduing enters 0.80 0.60 0.40 0.20 0.00 C b d d e e e b e 0 30 60 90 120 e D b b b 0 30 60 90 120 0.8 0.6 0.4 0.2 0.0-0.2 RJ 0.14 b bb b b b 180 Ro 0.07 0.00 b b b E 0 30 60 90 120 Tretment time (min) bb F b b 0 30 60 90 120 Tretment time (min) 120 60 0 RE0 /CS Fig. 8. Prmeters quntifying the struture of the photosyntheti mhinery of drk-dpted epidermis-less lef segments of A. thlin treted with wter or 500 mm for different times. The mximum yield of primry photohemistry (4 Po ) (A), quntum yield for eletron trnsport (4 Eo ) (B), non-q A reduing enters (C), the number of PSII RCs with Q B -site filled with (R J ) (D), quntum yield for the redution of the end eletron eptors t the PSI eptor side (4 Ro ) (E), eletron flux reduing end eletron eptors t the PSI eptor side per CS (RE 0 /CS) (F), re presented. Men vlues of 15 repetitions SE re plotted. Different smll letters indite vlues signifintly different within tretments (p < 0.05) ording to the LSR test. or bentzon solution for the indited time with osionl gittion (25 C, w100 mmol (photons) m 2 s 1 white light). The ondutivity of the wsh solution (ms/m) ws determined using DDS-12A ondutivity meter. The totl ion lekge ws obtined by mesuring the ondutivity of the sme lef diss-ontining solution, fter they re utolved. Ion lekge per grm of wet weight ws lulted by dividing the ondutivity of the solution before utolving by the ondutivity of the solution fter utolving nd then dividing the vlue by smple weight. Reltive ion lekge is the rtio of the vlue obtined in treted smples to the vlue obtined in smples from ontrol (wter) smples t 3 h. The level of lipid peroxidtion ws monitored by spetrophotometri determintion of MDA, using thiobrbituri id (TBA), ording to Shimizu et l. [36] with some modifitions. After tretment with or without, lef diss (bout 0.5 g) were briefly wshed with distilled wter to remove, nd then they were homogenized in 5 ml of distilled wter nd stirred with tube mixer for 1 min t room temperture. After homogentes were entrifuged t 10,000 g t 4 C for 10 min, 0.6-mL superntnt ws mixed with 2.4 ml of 0.5% TBA in 20% trihloroeti id solution nd inubted for 30 min t 95 C. The retion ws stopped by putting the smple tubes in n ie buket. The smples were then entrifuged t 10,000 g for 10 min nd the bsorbne of the superntnt ws determined t 532 nm. The vlue of nonspeifi light dissiption ws mesured t 600 nm nd subtrted from tht mesured t 532 nm; these finl extrted vlues represented the volume of MDAs-retive TBA. The onentrtion of MDA-retive TBA ws lulted from the extintion oeffiient of 155 mm 1 m 1. 4.5. Histohemil detetion of H 2 O 2 nd O 2 H 2 O 2 nd O 2 L levels were determined by in vivo stining with DAB-HCl [19] nd with NBT [20] s substrte. Briefly, A. thlin
S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 49 h P680* P680 Q A Q B PQ Oxidtive dmge Cell deth leves were exised t the bse petioles with rzor blde nd then they were supplied, through the ut petioles, with 0.1% (w/v) solution of DAB, ph 3.8 or 0.1% (w/v) NBT (dissolved in 25 mm HEPES-KOH) for 8 h under white light t 25 C. Petioles were ut gin nd then pled in distilled wter or or bentzon for the indited times. After these tretments, the leves were briefly rinsed with distilled wter twie, nd then deolorized by immersing the leves in boiling ethnol (96%) for 10 min. The ltter smples were stored in ethnol (96%) nd photogrphed with digitl mer (Cnon, SX11S, Jpn). H 2 O 2 nd O 2 L were visulized s reddish-brown nd drk-blue olortion spots. The results were repeted no less thn three times. 4.6. Fluoresene mirosopy observtion h Cyt b 6f P700 H 2O 2 1.- O 2 O 2 3 O. 2 OH 3 O 2 Chloroplst Intrellulr ROS ws mesured by monitoring the fluoresene of DCF, the oxidtion produt of H 2 DCF [21]. This dye is nonfluoresent in its redued form nd redily permetes the membrne. One in the ell, nonspeifi esterses leve ette groups, fter whih the dye beomes membrne impermeble, beoming trpped inside the ells nd ellulr omprtments. H 2 DCF is onverted to the fluoresent form when oxidized by H 2 O 2,O 2 L nd vrious free rdil produts tht re downstrem from H 2 O 2. After exposure to vrious tretments, the epidermis-less lef segments were inubted with 10 mm H 2 DCF-DA t 37 C for 25 min in the drk. Exess H 2 DCF-DA ws removed with loding buffer (10 mm TriseHCl nd 50 mm KCl, ph 7.2). The smples were floted in 1 ml of the sme buffer nd were observed using fluoresene mirosope (Zeiss Axio Imger A1, Germny; 488 nm exittion, 525 nm emission). Mesophyll protoplsts were isolted from the seond expnded leves from 4 to 6 week-old Arbidopsis plnts, using 1% ellulse R10 nd 0.2% merozyme R10 (Ykult Honsh, Tokyo, Jpn). Protoplsts were inubted in 10 mm H 2 DCF-DA in W5 solution [150 mm NCl, 125 mm CCl 2, 5 mm KCl, 2 mm MES (ph5.7), nd 5 mm gluose] for 15 min t 37 C in drkness. After wshing, protoplsts-preloded with H 2 DCF-DA were treted with 500 mm for the indited times, nd were observed by lser snning onfol mirosope (Lei TCS-SPII, Germny) using the PC P700* Fd FNR NADP + NADPH Fig. 9. A proposed model depiting -indued genertion of ROS hloroplsts leding to ell deth. Cyt b 6 f, ytohrome; Fd, ferredoxin; FNR, ferredoxin-nadpþ redutse; H 2 O 2, hydrogen peroxide; 1 O 2, singlet oxygen; O 2 L, superoxide nion; $OH, hydroxyl rdils; P680 nd P700, PSII nd PSI retion enter pigments; PC, plstoynin; PQ, plstoquinone; Q A nd Q B, PSII primry nd seondry plstoquinone eletron eptors. following settings: 5% power, 488 nm exittion, nd 510e560 nm emission. Exmintion of hloroplst utofluoresene ws performed using 45% power for 488 nm exittion, with 725e795 nm emission. 4.7. Assys of enzyme tivity To mesure the tivity of severl ntioxidnt enzymes, the lef segments of A. thlin were treted with distilled wter (ontrol) or 500 mm for 1, 2, 4 or 6 h (25 C, w100 mmol photons m 2 s 1 white light). The smples (bout 0.5 g) were ground in liquid nitrogen (77K) to fine powder nd were homogenized with 50 mm potssium phosphte buffer (ph 7.0) ontining 1 mm ethylene dimine tetreti id (EDTA) nd 1% polyvinylpyrrolidone; in the se of the APX ssy, 1 mm sorbi id ws inluded. The homogente ws entrifuged t 15,000 g for 20 min t 4 C nd the superntnt ws used for the following enzyme ssys. The totl tivity of SOD (EC1.15.1.1) ws tested by monitoring the inhibition of photohemil redution of NBT s desribed in referene [37]. The enzyme extrts (100 ml) were dded to 2.9 ml retion mixture ontining 50 mm potssium phosphte buffer (ph 7.8), 75 mm NBT, 13 mm methionine, 2 mm riboflvin nd 0.1 mm EDTA. The retion mixtures were illuminted for 15 min t 100 mmol photons m 2 s 1 white light. One unit of SOD tivity ws defined s the mount of enzyme required to derese 50% of the redution of NBT, s monitored t 560 nm. CAT (EC1.11.1.6) tivity ws ssyed by following the onsumption of H 2 O 2 (extintion oeffiient, 39.4 mm 1 m 1 )t 240 nm for 3 min [38]. The retion mixture (3 ml) ontined 50 mm potssium phosphte buffer (ph 7.0), 200 ml enzyme extrts nd 10 mm H 2 O 2. APX (EC1.11.1.11) tivity ws mesured by following derese of bsorbne t 290 nm, A 290 (extintion oeffiient, 2.8 mm 1 m 1 ) for the first 3 min in 1-mL retion mixture ontining 50 mm potssium phosphte buffer (ph 7.0), 200 ml enzyme extrt(s), 0.5 mm sorbte (ASC) nd 0.1 mm H 2 O 2. Corretion ws mde for the low, non-enzymti oxidtion of ASC by H 2 O 2 [39]. 4.8. Cell vibility After tretment with distilled wter or or bentzon, lef segments of A. thlin were inubted with 0.01% FDA for 10 min t 20 C in drkness. FDA fluoresene ws observed using fluoresene mirosopy (Zeiss Axio Imger A1, 495 nm exittion nd 500e550 nm emission) [40]. FDA fluoresene dereses s the dye leks from the ded ells. 4.9. Chlorophyll fluoresene trnsient The epidermis-less lef segments were submerged in distilled wter or solution, nd then inubted for the indited time t 25 C in omplete drkness. Chl fluoresene trnsient (indution) ws mesured t room temperture with HndyPEA fluorometer (Hnsteh, UK). The experiment ws repeted t lest three times with five repetitions per tretment. The OJIP fluoresene trnsients were indued by 1 s pulses of red light (650 nm, 3500 mmol photons m 2 s 1 ); this provided vluble informtion on the photosyntheti tivities nd physiologil vitlity. We note tht O refers to the initil miniml fluoresene level; P (500 ms e 1 s) is for the pek nd J (2 ms) nd I (30 ms) re infletion points between the O nd the P levels. The rise in Chl fluoresene, the OJIP trnsient, ws nlysed ording to the so-lled JIP-test [23,24]. The JIP-test defines the mximl (subsript o indites tht the prmeter refers to the onset of
50 S. Chen et l. / Plnt Physiology nd Biohemistry 52 (2012) 38e51 illumintion) energy fluxes in the energy sde for the following events: Absorption (ABS), Trpping (TR 0 ), Eletron Trnsport (ET 0 ), Redution of End eptors of PSI (RE 0 ), nd lef Cross Setion (CS). For lultion of the vlues of the bove prmeters, the following dt were utilized: the initil fluoresene F O, mesured t 20 ms (O-step): t this time ll retion enters (RCs) re open; fluoresene intensity F J t 2 ms (J-step); fluoresene intensity F I t 30 ms (I-step); the mximl fluoresene intensity F M ; this is equl to F P, sine the pulse used ws sturting, i.e., ll the Q A is redued (ll RCs re losed). Other prmeters were: Reltive vrible fluoresene V t,defined s (F t F O )/(F M ef O ); thus, V J ¼ (F J F O )/(F M F O ), V I ¼ (F I F O )/(F M F O ). The mximum quntum yield of primry photohemistry, 4 Po, defined s TR 0 /ABS ¼ 1 (F O /F M ). The probbility tht n eletron moves further thn Q A (j Eo ), defined s ET 0 /TR 0 ¼ (1eV J ). The mximum yield of eletron trnsport (4 Eo ) s ET 0 /ABS ¼ [1 (F O /F M )]. (1 V J ). The mount of Q A -reduing enters of the referene smples re lulted s: Q A -reduing enters ¼ (RC/RC ref. ).(ABS/ ABS ref. ) ¼ [(RC/CS)/(RC/CS) ref. ]. [(ABS/CS)/(ABS/CS) ref. ]; therefore, the frtion of non-q A reduing enters ¼ 1 Q A -reduing enters ¼ 1 (RC/RC ref. ).(ABS/ABS ref. ). The prmeter R J, n expression for the number of RCII with Q B -site filled by PSII inhibitor: R J ¼ [V J (V J ) ontrol ]/ [1 (V J ) ontrol ] ¼ 1 (j Eo /j Eo(ref) ). The quntum yield for the redution of the end eletron eptors t the PSI eptor side (4 Ro ) ¼ RE 0 /ABS ¼ 4 Po. j Eo. d Ro ¼ 4 Po.(1 V I ). The probbility of redutions t the PSI eletron eptor side per exited ross setion (CS): RE 0 /CS ¼ d Ro. (ET 0 /CS) d Ro ¼ RE 0 /ET 0 ¼ (1 V I )/(1 V J ), whih expresses the probbility tht n eletron is trnsported from the redued intersystem eletron eptors to finl eletron eptors of PSI. 4.10. Sttistil nlysis Reltive results were nlyzed with STATGRAPHICS PLUS softwre Ver.2.1 (Mnugistis, Rokville, MD, USA). One-wy ANOVA ws rried out nd mens were seprted using Dunn s lest signifint rnges (LSR) t 95%. Eh experiment ws repeted three times with t lest three replites per tretment. Aknowledgements This work ws supported by the Ntionl Nturl Siene Foundtion of Chin (31000834), New Teher Foundtion of Dotorl Progrm of Edution Ministry of Chin (200803071004), Dotorl Progrm of Edution Ministry of Chin (200900 97110018), Chin 863 Progrm (2011AA10A206), 111 Projet (B07030) nd Projet Funded by the Priority Ademi Progrm Development of Jingsu Higher Edution Institutions. We thnk Brue Auld (Chrles Sturt University, Austrli) for suggestions for improvements in the mnusript. We refer the reders to very reent highly relevnt review by Dnon (2012) on oxidtive stress nd signlling [41]. Appendix. 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