Fluoranthene fumigation and exogenous scavenging of reactive oxygen intermediates

Transcription

1 1 2 3 Fluornthene fumigtion nd exogenous scvenging of rective oxygen intermedites (ROI) in evergreen Jpnese red pine seedlings (Pinus densiflor Sieb. et. Zucc.). 4 5 Scvenging rective oxygen intermedites in stressed Jpnese red pine seedlings. 6 7 Ilemobyo Oguntimehin nd Hiroshi Skugw* Deprtment of Environmentl Dynmics nd Mngement, Grdute School of Biosphere Science, Hiroshim University, Kgmiym, Higshi-Hiroshim, , Jpn *Corresponding uthor 17 Address:1-7-1 Kgmiym, Higshi-Hiroshim, , Jpn. 19 Tel & Fx: E-mil: hskug@hiroshim-u.c.jp 21 1

2 Abstrct Genertion of rective oxygen intermedites (ROI) such s O 2, H 2 O 2, nd. OH is known to be mjor mechnism of dmge in biologicl systems. This study investigted nd compred effectiveness of scvenging ROI generted in fluornthene (FLU) pre-fumigted Jpnese red pine seedlings. Three kinds of eco-physiologicl ssessments were used to express the impct of the different fumignts used inside the green house. Gs exchnge mesurements showed negtive chnges induced by 10 μm FLU on Jpnese pine seedlings during 10 d exposure period whilst no negtive chnge ws found during 5 d exposure period. Moreover, during 14 d FLU exposure incorporting ROI scvengers, results reveled tht chlorophyll fluorescence, needle chemicl contents nd needle dry mss per unit re of the seedlings were ffected. The negtive effects of FLU on the conifer were dependent on both the dose nd period of FLU fumigtion. Peroxidse (PERO), superoxide dismutse (SOD) nd mnnitol (MANN) were ll effective scvengers of ROI. MANN scvenged. OH, the most lethl of the ROI. For prcticble use, MANN is more economicl, nd my be the best ROI scvenger mong the three considered. It cn be concluded tht efficient scvenging of ROI in biologicl systems is importnt to mitigte the negtive effects of FLU on Jpnese red pine trees Keywords: Fluornthene, rective oxygen intermedites, pine needles, chlorophyll fluorescence, eco-physiology, enzymes. 2

3 Introduction Fluornthene (FLU) belongs to group of orgnic compounds known s polycyclic romtic hydrocrbons (PAHs) tht comprise t lest two fused condensed romtic rings. It is lso one of the 17 US Environmentl Protection Authority (USEPA) priority PAHs. These compounds re generlly formed by pyrolysis nd incomplete combustion processes t tempertures of pproximtely 700 C (ATSDR, 2006). Anthropogenic sources of PAHs include combustion of fuels, refining, coke production, bush burning, utomobile exhusts nd cigrette smoke. Plnts needles nd leves re importnt sinks for tmospheric PAHs. The structures of these plnt orgns re complex, nd the route by which PAHs move through them, nd become stored or processed by them dicttes their environmentl fte nd plys role in their nnul cycling (Wild et l., 2005; Wild et l., 2006). A common mechnism of contminnts toxic ction is inhibition of biologicl pthwys such s photosynthesis nd mitochondril electron trnsport (Bbu et l., 2001). Photosynthesis is very sensitive indictor of plnt disorders. Its mesurement enbles the detection of erly reversible chnges in plnt metbolism tht re difficult to detect otherwise (Blck nd Unsworth, 1980). Therefore, mesuring inhibition of photosynthesis ws found to be useful in ssessing the potentil toxic effects of xenobiotic contminnts (including PAHs) on plnts (Hung et l., 1997). Studies hve shown tht plnt responses to PAHs depend on the intensity nd durtion of fumigtion, the developmentl stge of the plnt, nd the concentrtion of fumigting solutions (Wieczorek nd Wieczorek, 2007). Lipophilicity (in terms of log K ow ) nd vilbility re two fctors found to control the biovilbility of PAHs in inner needle comprtments, nd thus, tht influence the uptke efficiency of PAHs into plnts (Wenzel et l., 1997). Photo-induced toxicity of PAHs could be bsed in the formtion of intrcellulr singlet oxygen nd other rective oxygen intermedites (ROI) tht led to biologicl dmge 3

4 (Eisenberg nd Cunninghm, 1985; El-Alwi et l., 2002). Phytotoxicities pper to vry, depending on the prticulr PAH nd plnt species (Hwng et l., 2003). The production of reduced nd excited species of ROI in chloroplsts hs been reviewed (Asd, 2006). The rection centers of photosystem I (PSI) nd photosystem II (PSII) in chloroplst thylkoids re the mjor sites of ROI genertion. Superoxide dismutse (SOD) constitutes the first line of defense ginst ROI within cell (Alscher et l., 2002). SODs re mong the fstest enzymes known (V mx of M 1 s 1 ; Krlson, 2003). Peroxidses (PERO, hydrogen peroxide oxidoreductse) re widely found in plnts, nd oxidize vst rry of compounds in the presence of hydrogen peroxide (H 2 O 2 ) (Chen nd Schopfer, 1999). Mnnitol (MANN) is produced in some plnts nd is recognized s potent ROI quencher. It ws shown to scvenge hydroxyl rdicls (. OH) generted by cell-free oxidnt systems (Uphm nd Jhnke, 1986). The rte constnt of MANN with. OH t ph = 7 is (1 8 ± 0 4) 10 9 M 1 s 1 (Goldstein nd Czpski, 1984). FLU toxicity hs been studied in higher plnts (Hung et l., 1996; Kummerov nd Kmentov, 2004; Kummerov et l., 2006). Previously, we investigted the fumigtion effects of FLU nd phennthrene over long exposure period (~3 months) on the needles of Jpnese red pine (Oguntimehin et l., 2007) using open-top chmbers. The negtive effects of FLU were more pronounced thn those of phennthrene. In the present study, our ims re s follows: () to investigte the fumigtion effects t two different molr concentrtions of FLU on Jpnese red pine over 5 nd 10 d exposure periods; (b) to relte the genertion of ROI induced by FLU with the resulting physiologicl or morphologicl chnges in 2-yer-old Jpnese red pine; nd (c) to compre the efficiency of exogenous ROI scvengers in reducing the negtive eco-physiologicl effects of FLU on Jpnese red pine needles. Generlly, three kinds of eco-physiologicl ssessment criteri were considered: (i) gs exchnge prmeters including net photosynthesis rte t sturted irrdince, A mx ; stomt 4

5 conductnce, g s ; nd intercellulr CO 2 concentrtion, C i ; (ii) chlorophyll fluorescence prmeters including initil chlorophyll fluorescence, F o ; nd mximl photochemicl efficiency of PSII, F v /F m ; nd (iii) physiologicl prmeters of the needles, including contents of chlorophyll, (Chl ); chlorophyll b, (Chl b); nd totl chlorophyll, (Chl +b ); nd needle dry mss per unit re (NMA). In considering (b) nd (c) bove, we ssumed tht the ctivities of the ntioxidnt enzymes would provide n eco-physiologicl mesure of Jpnese red pine s reltive resistnce to FLU toxicity. Previous studies relted to enzyme ctivities hve used biologicl nd biochemicl ssys. In our study, however, we fumigted MANN, PERO, SOD, nd n equl-units mixture of PERO nd SOD directly onto needle surfces of Jpnese red pine over 14 d exposure period with 10 μm FLU. This method is similr to horticulturl nd griculturl folir feeding prctices, nd is modified for the ppliction of non-toxic formultions of orgnic enzymes (Gorton nd Sollinger, 2005) Mterils nd Methods Greenhouse growth conditions Experiments were crried out from My to June 2007 in the green house (metl-frmed shelters) built inside the Hiroshim University cmpus (34 24 N, E). Shelters were constructed in wy to prevent rin nd dew from flling on the seedlings. The shelters were semi-cylindriclly shped on the horizontl xis, well ventilted, nd covered n re of bout 8 m 12 m on the ground. The upper hlves of the frmed structures (excluding the two ends) were covered with 0.06 mm thick Tefzel film. The film is trnsprent to visible light nd UV-A (DuPont, Wilmington, DE, USA). The men photosynthetic photon flux density (PPFD), (LI-190SA Quntum Sensor, Licor, USA) incident on the green house between My nd June, 2007 in the first, second nd third btches of the experiments were 5

6 (0 1665), 533 (0 1707) nd 536 (0 1719) μmolm -2 s -1 (respectively. The men ir temperture nd men reltive ir humidity (Thermo recorder TR-72S, T&D Corp., Jpn) mesured in mbient environment of the green house from My to June, 2007 in the first, second nd third btches of the experiments were 14.7 (1-27) o C, 14.8 (1-30) o C, 14.8 (3-31) o C nd 69 (13-99)%, 68 (13-99)%, 68 (12-96)% respectively. Although we hve not mesured these prmeters inside the greenhouse, but we re of the opinion tht the differences between the mbient conditions (outside) nd the inside conditions of the greenhouse during the exposure period were negligible bsed on the design of the green house. Also, the difference in vlues of these prmeters during the three different btches of the experiments cn be considered negligibly smll to effects ny vritions in the results of the experiments Plnt nd soil mterils Two-yer-old Jpnese red pine seedlings grown in nursery in Fukuok prefecture were purchsed nd trnsplnted into 0.35 m 0.3 m deep pots (1 seedling per pot) on Mrch 4, The pots were filled with 21 L soil, which ws mixture of yellow sndy soil (wethered grnite), perlite (white lom 4 20 mm, Toho-Leo Co.), isolite (CG2, Isolite Insulting Products Co.) nd humus soil (Midori-Sngyo Inc.) t 11:2:2:4 volumetric rtios, respectively. Net litters composed of helthy pine woodlnd from the university cmpus were collected nd spred over the soil surfce in ech pot (pproximtely 50 g pot 1 ). Inside the green house, the pots were wtered with de-ionized wter once dily by 7:00 AM using n uto-irrigtion system

7 Fumigtion systems A stock solution ws prepred by dissolving FLU (Sigm-Aldrich, USA) in 50% cetone (ACT, Wko pure chem. Ind., Jpn) nd MilliQ (MQ) wter (Millipore Co., Jpn) djusted to 1 mm (202 mg L 1 ). The stock ws diluted s pproprite to finl concentrtions of 5 nd 10 μm with MilliQ wter. This mde the finl concentrtion of ACT in solutions 0.5%. The highest concentrtion of FLU used is comprble to miniml concentrtions used elsewhere (Hung et l., 1996; Kummerov nd Kmentov, 2004). 1 mm MANN (Ncli, Kyoto, Jpn) ws prepred nd used s n. OH scvenger. The fumigtion system in the first exposure experiment consisted of MQ, ACT, MANN, FLU nd FLU+MANN. The solutions were pplied to the folige of pine seedlings using n electronic spry mchine with nozzle (BS-4000, Fujiwr Sngyo, Miki, Jpn) twice dily (6:00 7:00 AM nd 6:00 7:00 PM), over 5 nd 10 d period. On the verge ech seedling ws fumigted with 50 ml of the fumignt per one sprying period. The soil surfce ws covered with wterproof sheet during fumignt ppliction to prevent solutions from entering the roots of the seedlings. The coverings were removed bout 2 hrs fter the fumigtion. Three experiments were conducted in btches. In the first experiment (5 d fumigtion), MQ, ACT, nd 5 or 10 μm FLU were used. Secondly, the 10 d experiment involved ll the five components of the fumigtion system mentioned bove with FLU concentrtions t10 μm. In ddition, mnnitol ws introduced s prt of the fumigtion system. 165 In the third exposure experiment, 6 units ml 1 SOD (S KU; Bovine erythrocytes; Sigm) nd 6 units ml 1 PERO (P8250; Type II Horserdish; Sigm) were prepred from 30 KU ml 1 SOD nd 30 KU ml 1 PERO stocks tht were stored t 20 C nd used within 2 weeks. In ll, the totl of ech enzyme fumigted ws 1200 units. FLU solutions were pplied 7

8 to the folige of pine seedlings using n electronic spry mchine with nozzle (BS-4000, Fujiwr Sngyo) from 6:00 8:00 AM nd 5:30 7:30 PM 4 d per week. On verge, ech seedling ws fumigted with pproximtely 100 ml FLU dily. Also 100ml ech of MANN nd enzymes were pplied twice weekly, between 6:00 8:00 AM. Here, the fumignt system consisted of FLU + MANN, FLU + PERO, FLU, FLU + SOD, nd FLU + SOD + PERO. In the cse of ech fumignt, the components were fumigted singly, one t time. In most cses, the FLU ws fumigted first nd fter mking sure tht the wet needles of the pine seedlings were lmost dried, the next component (either mnnitol or enzymes) ws then pplied Photosynthesis nd chlorophyll fluorescence mesurements Gs exchnge mesurements were conducted on one-yer-old needles of pine seedlings. Strting from 7:00 10:30 AM, net photosynthesis t ner sturting irrdince (A mx ), stomt conductnce (g s ) nd intercellulr CO 2 concentrtion (C i ) were mesured for helthy needles in ech tretment. A mx, g s, nd C i were mesured t ner-sturting irrdince of 1500 μmol m 2 s 1 photosynthetic photon flux density (PPFD) nd t needle temperture of 25 ± 2 C. Lef to ir vpour pressure deficit (VpdL) ws mintined between 0.8 nd 1.3 kp, ir into lef chmber CO 2 concentrtion ws kept t 370 μmol CO 2 mol 1 t flow rte of 500 μmol s 1 by n open-flow infrred gs nlyzer with light nd temperture control systems (LI-6400, Li-cor Inc., Lincoln, NE, USA). After ech mesurement, the needles used were hrvested nd their width nd length mesured with digitl cliper (CD-15, Mitutoyo Co., Kngw, Jpn). The cross-section of the needle ws pproximted s semi-circle hving dimeter equl to the mesured width; hlf of the lef surfce re of the needle ws used s the effective lef re for A mx, g s nd needle dry mss per unit re (NMA) determintions (Kume et l., 2001; Nktni et l., 2007). In generl, photosynthetic cpcity 8

9 is influenced by re-bsed chlorophyll content in needles. Therefore, needle eco- physiologicl trits re expressed bsed on the effective lef re Chlorophyll fluorescence ws mesured t night (7:00 8:00 PM) using portble chlorophyll fluorometer (MINI-PAM, Heinz Wlz GmbH, Effeltrich, Germny) with lef-clip holder 2030B (Heinz Wlz GmBH). The needles were rrnged compctly in prllel rry nd clmped with the holder, then miniml fluorescence vlues (F o ) nd the mxim photochemicl efficiency of PS II (F v /F m ) were mesured Needle chlorophyll content Chl nd Chl b contents were determined by extrcting 100 mg needles (the sme needles used for A mx g s nd C i ) with N, N-dimethylformmide (DMF). Absorption of the extrct ws mesured in the scnning mode ( nm) t nd nm wvelengths with spectrophotometer (UV-2400, Shimdzu Co., Jpn.). Concentrtions of Chl, Chl b, Chl (+b) were clculted with equtions given by Porr et l. (1989) Sttisticl nlysis Sttisticl evlution of results ws crried out using SPSS 13 (SPSS, USA). Results re verge determintions from five seedlings in ech tretment group ± stndrd error of the men (S.E). Significnces of the differences in verge vlues between ech tretment were evluted by one-wy ANOVA nd Tukey nlysis (p < 0.05). Person s correltion coefficient (r, p < 0.05) ws used to test correltions mong the needle eco-physiologicl trits

10 Results nd Discussion Fluornthene concentrtion effects We exmined the effect of two FLU concentrtions (5 nd 10 μm) on the eco-physiologicl sttus of the seedlings. Fig. 1(A-C) shows the gs exchnge ecophysicologicl trits of Jpnese red pine fter 5 d exposure. There were no effects of 5 or 10 μm FLU on A mx, g s, nd C i, Compred with the MQ nd ACT tretments vlues, there were no sttisticlly significnt differences mong ll the trits exmined. It is probble tht fter 5 d of exposure, the FLU dosges were insufficient to cuse ny significnt eco-physiologicl chnges in Jpnese red pine. More likely, the concentrtion of FLU used on seedlings during the 5 d period could not produce ny significnt chnge. However, considering the totl reduction in vlues of the trits over the 5 d exposure period, it is evident tht 10 μm hd greter negtive effect thn 5 μm FLU. A mx vlues decresed by 12% in response to 10 μm FLU, nd by 2% with 5 μm FLU (from initil vlues of 16.2 nd 14.3 μmol CO 2 m 2 s 1, respectively). These results suggest tht longer ppliction period of 10 μm FLU solution on the Jpnese red pine might produce sttisticlly significnt chnge in the ecophysiologicl trits Effect of incresed ppliction period Jpnese red pine seedlings were fumigted with 10 μm FLU for 10 d. After the 10 d fumigtion, A mx, g s nd C i were significntly reduced in the FLU-treted pine seedlings s compred with MQ, ACT nd MANN tretments (Fig. 1D-F). However, F o, F v /F m, NMA nd Chl / Chl b were not sttisticlly different in ll the tretments (F o, F v /F m, NMA nd chlorophyll dt not shown). The much decresed A mx in this study might hve been cused by the limittion in stomtl conductnce nd the reduced internl CO 2 intke. Decresed 10

11 A mx of Jpnese red pine needles by the FLU in this study is similr to tht reported by Kobyshi et l. (2002), using. OH generting solutions s fumignts. This suggests tht gs exchnge mesurement is sensitive in detecting the negtive chnges inflicted on the seedlings in 10 d. In ddition, correltion coefficients of the eco-physiologicl trits exmined in this study indicted tht only g s nd C i showed positive correltions with A mx (r, p < 0.05; = 0.85 nd 0.63, respectively). There ws no sttisticlly significnt difference in the Chl, Chl b nd Chl (+b) contents of the pine needles in the 10 d period. Also, Chl /Chl b, chrcteristic stress indictor in plnts (Shn et l., 1997), ws unchnged in FLU-treted seedlings. The decresed A mx by FLU in the present study might be due to the genertion of ROI such s O 2 nd. OH (Krylov et l., 1997). This hypothesis is similr to tht proposed by Wng et l. (2005) on the photolysis of PAH in wter. The wx lyers in the needles of the Jpnese red pine my ccumulte sufficient FLU for photolysis rection to tke plce (Wng et l., 2005; Dolinov et l., 2004). In this study, MANN displyed mitigting ction ginst the negtive effects of FLU. MANN t 1 mm could be used s n. OH scvenger, even though some studies reported osmotic stress t concentrtions bove 90 mm (Lin nd Ko, 2002; Gill et l., 2002) Scvenging of ROI in FLU pre-fumigted seedlings Before fumigtion, seedlings showed no significnt differences mong ll determined ecophysiologicl prmeters. However, mesurements mde 2 weeks (14 d) fter fumigtion indicted tht A mx incresed in ll tretment groups except FLU-treted pine seedlings. Fig. 2A shows lower A mx vlue in FLU-treted seedlings when compred with control MANN nd the seedlings treted with enzymes. The decresed A mx in FLU-treted seedlings in this study showed the inhibition of photosynthesis by FLU. However, the similr photosynthetic 11

12 rte found mong MANN+FLU, SOD+FLU, PERO+FLU nd SOD+PERO+FLU tretments implied tht MANN, SOD nd PERO mitigted the negtive effect of ROI. Previous studies on the effects of sulphur dioxide (SO 2 ) on plnts showed tht high superoxide dismutse ctivity confers incresed resistnce to pollution (Tnk nd Sughr, 1980). In ddition, studies crried out using the green lg Spermophilus rmtus cultured with nthrcene provided evidence for strong oxidtive stress under light, nd in response, incresed SOD ctivity (Aksmnn nd Tukj, 2004). Stomtl conductnce (g s ) is n importnt index for the trnsport of ir pollutnts into plnts. Differentil sensitivity in pine species my be relted to the number nd size of stomt. Unlike the re-bsed A mx, there ws no significnt difference fter 14 d in FLU-treted seedlings (Fig. 2B). This seems inconsistent with our previous results in the 10 d. However, it is remrkble to note tht FLU treted seedlings hd the lowest vlue compred with other tretments. F o vlues of the exposed pine seedlings slightly decresed in ll tretments during 14 d fumigtion. Vlues for PERO+FLU-treted pine seedlings were significntly lowered compred with FLU-treted seedlings (Fig. 2C). F v /F m ws reltively stble for ll tretments during fumigtion except for FLU-treted seedlings, where the vlue ws significntly different compred with SOD+FLU-treted seedlings (Fig. 2D). F o nd F v /F m re chlorophyll fluorescence prmeters relting to PS II sites, nd indicte light hrvesting nd utiliztion system efficiency. In this study we observed little F o increse nd slightly decresed F v /F m in seedlings fumigted with FLU, this my imply slightly lowered mximl photochemicl efficiency in PS II. As previously reported in Oguntimehin et l. (2007), MANN mitigted the negtive effect of FLU on Jpnese red pine. Greter mitigting ctions on the F o increse nd F v /F m depression were observed in the present study (Fig 2C, D). However, SOD+PERO did not pper to hve ny dditive (synergistic) effect, but rther, showed similr effects to MANN. This might imply tht H 2 O 2 nd O 2 ply similr roles in effecting chlorophyll fluorescence chnges in the FLU-treted 12

13 plnts. The mechnisms of. OH formtion my comprise vrious pthwys nd combintions of compounds nd pthwys in these species. This my then necessitte the different cellulr trgets inside the plnt (Hlliwell, 2006). Needle dry mss per unit re generlly reduced within the tretment period. NMA in FLU-treted seedlings is lowered compred with NMA in SOD+FLU-treted seedlings (Fig. 2E). Nktni et l. (2007) found strong positive correltion between A mx nd NMA. Decresed NMA (pprox. 10% decreses) t the end of the 14 d fumigtion period in response to FLU tretment in the present study my grees closely with the findings of Nktni et l. (2007). Also, it supports the findings of relted study on B. npus nd Cucumis stivus (Hung et l., 1996). In C. stivus, FLU hd reduction effect on the fresh weight of shoots nd roots but in the present study, reduction in NMA of FLU treted pine seedlings ws recorded. Chlorophyll /chlorophyll b rtio in Jpnese red pine needles fter 14 d fumigtion is shown in Fig. 2F. FLU-treted pine seedlings hd the lowest rtio, the highest being found eqully in SOD+FLU nd SOD+PERO+FLU tretments. The lower Chl /Chl b rtio in mesophyll thylkoids of FLUtreted pine seedlings might be ssocited with lower photosystem II ctivity. The substntilly decresed chlorophyll content in FLU-treted pine seedlings s compred with other tretments is consistent with previous findings on plnt responses to FLU (Hung et l., 1996; Kummerov et l., 2006b). However, seedlings treted with SOD+FLU, PERO+FLU, SOD+PERO+FLU nd MANN+FLU showed stble chlorophyll contents during the fumigtion experiment Reltionship between eco-physiologicl trits nd fumigted FLU dosge per seedling Totl dosges of fumigted FLU per seedling during 5, 10 nd 14 d exposure periods were clculted. The highest dosge of 10 nmol FLU per seedling ws used for the 10 d exposure, followed by the 14 d exposure of 8 nmol FLU per seedling. The lest ws the 5 d exposure 13

14 of 5 nmol FLU per seedling. The reltionships mong A mx, g s, C i, F o, F v /F m, nd NMA nd dosge of FLU pplied per seedling re shown in Fig. 3. In this study, the high negtive reltionships (r vlues; 0.99, 0.97, 0.71 nd 0.98) between A mx, g s, C i nd NMA with FLU dosge respectively, suggests high dependence of these prmeters on FLU dosge. This strongly indicted tht the FLU dosge determined the extent of negtive effects on these prmeters (Fig. 3 A, B, C nd F). However, there seems to be no direct reltionship between F o nd F v /F m vlues in this present work (Fig. 3 D nd E). These my be imply tht PSII nd other light rection systems were not seriously dmge, the primry decrese in g s nd the following physiologicl down regultion processes my likely be the cuse of the decresed A mx Conclusion FLU fumigtion on the surfce of the evergreen conifer Jpnese red pine produced significnt negtive effects in the needles in short time. Both the dosge nd period of fumigtion were importnt fctors in the extent of FLU-induced negtive effects. We studied the scvenging mechnisms of ROI by fumigting FLU pre-fumigted seedlings with MANN, PERO, SOD, nd n equl mixture of SOD nd PERO. Scvengers of ROI used in this study mitigted the negtive effects of FLU on the needles. MANN is more economicl nd hs greter reltive stbility compred with the enzymes. This dvntge might imply tht MANN is the best ROI scvenger mong the three considered. Also, tht one of the negtive effects of FLU on Jpnese red pine is the production of. OH. A field-to-forest scle-up experiment my be used to estblish dditionl results on the negtive effects of FLU on Jpnese red pine trees. Fluornthene in the ir, dew, rin or snow is potentil thret to the photosynthetic pprtus nd tissues of Jpnese red pine

15 Acknowledgements We thnk Dr. K. Tked nd Dr. N. Nktni for their technicl ssistnce during the course of this work. Also, the uthors gretly pprecite Dr. T. Kobyshi (Fculty of Agriculture, Kgw University, Jpn) for his dvice on this mnuscript. Finlly, we thnk ll members of Skugw nd Tked Lbortory for their support nd encourgements. : References 15

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22 LIST OF FIGURES Fig. 1: (A-C) Photosynthesis rte, stomtl conductnce, nd internl CO 2 concentrtion respectively, of Jpnese red pine (Pinus densiflor) seedlings exposed to fumigtion with 5 nd 10 μm fluornthene solutions for 5 d. (D-E) Photosynthesis rte, stomtl conductnce, nd internl CO 2 concentrtion respectively, of Jpnese red pine (Pinus densiflor) seedlings exposed to fumigtion with 10 μm fluornthene solutions for 10 d. Dt re mens of determintions from five pine seedlings, error brs re ± stndrd error (S.E.). Identicl superscript letters indicte the sme homogenous groups; different letters indicte significnt differences t p < 0.05 (Tukey nlysis). Fig. 2: A) Photosynthesis rte mesured t ner-sturting irrdince (A mx ). B) Stomtl conductnce to wter vpors (g s ). C) Miniml fluorescence vlue (F o ). D) Mximl photochemicl efficiency of PSII (F v /F m ). E) Needle dry mss per unit re (NMA). F) Chlorophyll content of pine seedlings mesured fter 14 d exposure with fluornthene nd ROI scvengers. Dt re mens of determintions from five pine seedlings, error brs re ± stndrd error (S.E.). Identicl superscript letters indicte the sme homogenous groups; different letters indicte significnt difference t p < 0.05 (Tukey nlysis). Fig. 3: Reltionships between totl dosges of FLU fumigted/seedling in the 5, 10 nd 14 d (5, 10 nd 8 nmol, respectively) fumigtion periods with eco-physiologicl trits of Jpnese red pine needles. Dt re mens of determintions from five pine seedlings, error brs re ± stndrd error (S.E.). 1

23 27 A A mx ( μmolco 2 m 2 s 1 ) B g s (mmol H 2 O m 2 s 2 ) C C i (mmol CO 2 m 2 s 1 ) 0 10 μm FLU 5 μm FLU ACT MQ D A mx (μmol CO 2 m 2 s 1 ) μm FLU 5 μm FLU ACT MQ b b μm FLU 5 μm FLU ACT MQ 0 10μM FLU 10μM FLU + MANN MANN ACT MQ E g s (mmol H 2 O m 2 s 2 ) b b b F C i (mmol CO 2 m 2 s 1 ) b b b b μM FLU 10μM FLU + MANN MANN ACT MQ μM FLU 10μM FLU + MANN MANN ACT MQ 31 Fig. 1 2

24 A A mx (μmolco 2 m 2 s 1 ) b B g s (mmolh 2 Om 2 s 2 ) MANN+FLU PERO+FLU FLU SOD+FLU SOD+PERO+FLU 0 MANN+FLU PERO+FLU FLU SOD+FLU SOD+PERO+FLU C F o (mv) b b b b D F v /F m b b b b MANN+FLU PERO+FLU FLU SOD+FLU SOD+PERO+FLU 0.78 MANN+FLU PERO+FLU FLU SOD+FLU SOD+PERO+FLU E NMA (g.d.w.m 2 ) b b b b F Chl :Chl b b b b MANN+FLU PERO+FLU FLU SOD+FLU SOD+PERO+FLU 2.8 MANN+FLU PERO+FLU FLU SOD+FLU SOD+PERO+FLU F 35 ig. 2 3

25 A A mx (μmolco 2 m 2 s 1 ) r = B g s (mmolh 2 Om 2 s 2 ) r = FLU Dosge/seedling (nmol) FLU Dosge/seedling (nmol) 37 C C i (mmolco 2 m 2 s 1 ) r = FLU Dosge/seedling (nmol) D F o (mv) r= FLU Dosge/seedling (nmol) E F v/ F m Fig r = FLU Dosge/seedling (nmol) F NMA (gdw m 2 ) r = FLU Dosge/seedling (nmol) 4