Resistance to cyanide by salicylate pretreatment in Salix babylonica L.

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1 RESEARCH ARTICLE Resistne to ynie y sliylte pretretment in Slix yloni L. Rsoul Ghsemi 1*, Rzieh Mokhtri 2 1 Deprtment of Biology, Pyme Noor University, Tehrn, Irn. 2 Deprtment of Biology, Pyme Noor University, Isfhn, Irn. *Corresponing uthor: ghsemi@nj.isfpnu..ir Reeive: 11 July 213; Aepte: 14 Novemer 213 ABSTRACT: Cynie is unontrollly proue in some inustril opertions n hs lethl effets on humns n the environment. Sine removing proesses of ynie re omplite n ostly, the phytoremeition hs reently een extensively onsiere. To hieve n inrese ynie resistne, whih is neee for n effiient phytoremeition of the meiums with high onentrtions of ynie, the effets of sliylte s phytohormone were stuie. Cuttings of Slix yloni, s moel plnt, were use in ompletely rnomize esign with three replites in hyroponis using moifie Hogln nutrient solution. Plnts were pretrete with sulfosliylte (,, 1 n 1 mg L -1 ) for 21 ys n then were trete with toxi onentrtion of ynie (9 mg CN - L -1 ); some of the physiologil inies whih show ynie toxiity/resistne were mesure. Fvorle responses to sliylte towr the inrese in resistne to ynie were onentrtion epenent whih were oserve t 1 mg L -1 sulfosliylte n it ws ompnie with n inrese in superoxie ismutse tivity n reuing the pity of root extrt. Dehyrogense tivity n eletrolyte lekge from roots were erese relting to ontrol plnts. It lso prevente the ynie inhiitory effet on oxygen onsumption. The oserve effets oul e ttriute to reox sttus n ltertion of proution n svenging of retive oxygen speies y sliylte n ynie. The results inite tht proper onentrtion of sliylte oul e use s ynie resistne stimultor in willows. Keywors: reox sttus, respirtion, ROS, SOD, tolerne. Arevitions: AOX: lterntive oxise, CN - : ynie, ROS: retive oxygen speies, IV omplex: IV omplex in mitohonril respirtory hin, SOD: superoxie ismutse. INTRODUCTION Cynie (CN - ) is n nion ompose of ron triply one to nitrogen tom. This nion is one of the most lethl hemils n highly use in ifferent inustries suh s gol, le n zin extrtion n lso in the proution of nylon n plstis. In ition to the regulr usges of CN -, it is yprout of some inustril proesses in whih high onentrtions of ron n nitrogen re put together in hot n reuing onition, suh s hemil mnufturing, iron n steel mking, petroleum refining, n luminum smelting. The free CN -, whtever its soures, finlly enters into the wstewter or releses to the environment whih, if possile, must e refine. Different methos re usully use for CN - etoxifition inluing nturlly svenging of CN -, lkline hlorintion n oxition using hyrogen peroxie. Mngement of CN - using hemil proesses is ostly, omplite n usully some new hrmful yprouts re proue. Inee, the phytoremeition of CN - hs reently een pi ttention to. Phytoremeition is the use of vsulr plnts, lge, n fungi to metolize or sequester ontminnts, or to inue ontminnt rekown y miroorgnisms in soil (MCutheon n Shnoor 23). Although CN - is highly toxi sustne n hs high potentil to prevent ifferent metoli pthwys, there re lmost 2, speies known s ynogeni plnts whih proue high mounts of CN - (minly s gluosies) for Theoretil n Experimentl Plnt Physiology, 2(4): ,

2 Ghsemi n Mokhtri efense purpose (Hlkier n Møller 199). In ition, in ll vsulr plnts n in some of the lge n fungi, it is proue s yprout uring the synthesis of ethylene. Sine plnts, similrly to the other orgnisms, re sensitive to CN -, they must hol ifferent strtegies for svenging the proue CN -. CN - ffets generl metolism of the ells through loking of eletron trnsport hin in mitohonri n hloroplsts, eresing oxygen onsumption n inhiiting the tivity of some of the metl ontining enzymes suh s Cu n Zn isoform of superoxie ismutse (Cu/Zn SOD). The hnge in the ells energy sttus n the mlfuntion of ellulr protetive enzymes oul inue isintegrtion in memrnes n eletrolyte lekge from the ells. Plnt mitohonri ontins CN - resistnt eletron trnsport hin lle lterntive oxise (AOX) y whih they re le to tolerte inrese onentrtions of CN -. In ition, ifferent metoli pthwys, suh s ynolnine pthwy (Tittle et l. 199), mke it possile to metolize CN - y plnts. Inee, the plnts oul hve signifint potentil for the phytoremeition of CN -. Willow (Slix spp.) hs een onsiere promising moel plnt in the stuies of CN - phytoremeition (Es et l. 26). Desirle properties of willows for phytoremeition re: fst growth n umultion of high iomss, high ilities to uptke wter n hemils from the environment, n high wter evportion rtes. Bushey et l. (26) showe tht the estintion of CN - fter uptke y Slix erioephl is the ssimiltion into some of the ellulr metolites, s its evportion from the plnt is negligile. Different stuies hve een performe with the im of moeling prtil CN - phytoremeition (Bushey et l. 26, 26). For instne, uptke n trnslotion of CN - in free n other forms hve een oserve in ifferent plnt speies suh s Horeum vulgr, Sorghum iolor, Eulyptus spp., Aven stiv n Slix erioephl (Es et l. 23, 28, Smiotkis n Es 24). It hs lso shown tht some of the physiologil onitions, suh s nitrogen nutrition sttus n environmentl stresses like wter efiieny, inue the ssimiltion pthwys of CN - (Es et l. 21, Mhingur n Es 21, Mhingur et l. 213). The inrese of the AOX ility n metolizing pthwys hve speil importne in the phytoremeition of CN -. Sliylte s plnt hormone strongly stimultes AOX (Vn er Streten et l. 199). In ition, it stimultes retive oxygen speies (ROS) proution n svenging systems in plnts (Hyt et l. 27). In this stuy, the effets of sliylte on the resistne of Slix yloni to CN - were investigte. Some of the potentil or known responses of plnts to sliylte n CN - were teste to onlue out the responsile mehnisms of quire resistne. The metoli n other response pthwys overlppe in the responses to oth sliylte n CN - re trgets of this stuy. We lso mesure the responses simultneously in orer to ttin more omprehensive view on these intertions. MATERIAL AND METHODS Prepring the uttings n ulture meium: Cuttings with 1 to 2 mm long were prepre from semi-wooy two-yer ol rnhes of Slix yloni tree in lte spring of 211. Cuttings were ple in perlite y hlf of the length from se, wtere with tp wter n kept in growth hmer with light regime of 16:8 (light:rk) onstnt temperture of 24 C. The rooting of the uttings ourre fter 2. A hyroponi ulture ws use for the tretment of plnts. The uttings were then trnsferre to the greenhouse in pots ontining 1 L of moifie Hogln solution ph.8, ontining. mm CNO 3,. mm MgSO 4,.1 mm KH 2 PO 4,. mm KNO 3,. mm FeEDDHA (Ferri ethyleneimine i-2- hyroxyphenylette),.1 mm H 3 BO 3, 2 µm MnCl 2,.2 µm ZnSO 4,.2 µm CuSO 4 n.1 µm N 2 MoO 4. The solutions were erte perioilly every 3 h n exhnge every 1. Evportion ws ompenste ily y ing istille wter to the solutions. The plnts were kept in this sitution until omplete pttion to the greenhouse onitions. Then, the experiments were performe in the greenhouse uring the lte spring n summer. The rnge of temperture uring the y ws 2 36 C n in the night ws 22 2 C. Although the ph of the Hogln solution h een juste to.8, fter the ition of CN - the solution ph ws juste to 7.8 to reue evportion of CN -, whih ours t lower ph. Sine CN - omplexes with ifferent metls n the omplexes re not esily sore y plnts, the onentrtion of ulture solutions were reue 1/1 to iminish omplex formtion fter the tretment of plnts with CN -. Pre-tretment y sliylte n tretment of plnts y ynie: Two plnts were trnsferre into pots ontining 1. L ulture solution efore the tretments. Plnts with even weights were selete n the totl weight of eh plnt ws 12±1 g. The plnts were pre-trete with ifferent onentrtions of sulfosliylte inluing,, 1 n 1 mg L -1 for 2 weeks. The tretment with CN - ws performe y using NCN t toxi onentrtion of 9 mg CN - for 24 h. The toxiity threshol of CN - onentrtion ws etermine y sumitting the plnts to ifferent onentrtions of CN - t 276 Theoretil n Experimentl Plnt Physiology, 2(4): , 213

3 Sliylte n resistne to ynie in Slix, 3,, 9, 1 n 2 mg CN - L -1. After the tretments, ifferent prmeters inluing oxygen onsumption y roots, lekge of eletrolytes from roots, reuing pity of root extrt, ehyrogense n superoxie ismutse tivities in roots were mesure. Mesurement of oxygen onsumption: The roots were ut (1 g FW, fresh weight), wshe twie with istille wter n then trnsferre into test tues ontining 4 ml of istille wter. Dissolve oxygen ws mesure every 2 h using n oxygen meter (WTW with n eletroe moel Cellox 32, Germny). The oxygen meter ws lirte efore use se on the evie mnul. Mesurement of the eletrolyte lekge: After hrvesting, 1 g FW of root from eh tretment ws sujete to esorption using n ie oole esorption solution ontining mm CSO 4 n mm N 2 EDTA, ph.8, for 1 min, n then wshe twie with iistille wter. The roots were then trnsferre into test tues ontining 4 ml of iistille wter n shken very gently t rpm. The eletril onutivity (EC) of the wter ws mesure every 2 h until 6 h, using n EC-meter (Elmetron, Poln). Reuing pity test: The reuing pity of the extrt ws mesure se on the ferri reuing ntioxint power (FRAP) test (Benzie n Strin 1999). A setion of root (1 g FW) ws grine with liqui nitrogen using n ie oole mortr n pestle. Then, 3 ml of mm phosphte uffer, ph 6, were e n fter more grining the extrts were entrifuge t 12, x g t 4 C for 1 min. In ll steps, the temperture ws kept elow 4 C. The reuing pity ws performe using of TPTZ (2,4,6-tri(2-pyriyl)-1,3,- trizine). At low ph, reuing Fe 3+ to Fe 2+ y reuing gents of the extrt uses n intense lue olor fter the formtion of TPTZ-Fe 2+ omplex, with n sorption pek t 93 nm. A stnr urve ws otine using ifferent onentrtions of FeSO 4 whih ws liner etween 1 n 1 µm FeSO 4. Mesurement of ehyrogense tivity: Dehyrogense tivity ws mesure oring to Kittok n Lw (1968) y using 2 g FW of root sumerge in 1 ml of 1% TTC (2,3, three-phenyltetrzolium-hlorie) solution for 2 h. The roots were then sumerge into 3 ml of methyl ellosolve (2-methoxyethnol) solvent n gently shken for 6 h until the roots eme white. The solution ws then seprte n entrifuge t 1, x g for 1 min. Finlly, the sorption ws mesure t 48 nm n the results were expresse s perentge of the ontrol plnts. Mesurement of the superoxie ismutse tivity: The tivity of SOD ( ) ws mesure oring to Ginnopolitis n Ries (1997). The retion solution ontine mm uffer phosphte, ph 7.8, 13 mm methionine, 7 µm NBT (ρ-nitroluetetrzolium hlorie), 2 µm rioflvin n.1 mm EDTA. The retion ws strte y ing 1 µl of root extrt (from the preprtion ove mentione) to 1, µl of retion solution, mixe n put uner floresene light with the intensity of 3 µmol photon m -2 s -1 for 1 min. Using rk smples s lnk, the sorption ws mesure t 6 nm. Bse on efinition, hlf of the retion of onverting of NBT (yellowish) to Formzn (gry-luish) is efine s unit of SOD tivity. Mesurement of ynie in meium: A olorimetri metho ws use to mesure CN - in meium (Goulen et l. 1972) y ing 1 ml of 1 M NOH n 1 ml of.4% of hlormine T to ml of eh smple. The mixture ws kept for 2 min n then 1 ml of pyriinerituri i ws e to eh smple. The mixture ws kept for min uring whih time purple olor ppere. Light sorption t 78 nm ws mesure. Using stnr urve (t the rnge onentrtion of.2.2 mg CN - L -1 ), the onentrtion of CN - in meium ws etermine. Sttistil nlyses: The experiments were performe in omplete rnomize esign. To etermine the sttistilly signifint ifferenes etween the mens, multiple omprisons were performe y one wy ANOVA n Tukey s HSD test. The Sttistil Pkge for the Soil Sienes (SPSS) softwre (version 16 for Winows; SPSS In., Chigo, IL, USA) ws use for sttistil nlyses. The reporte t re the mens for 3 replites ± SD. RESULTS Resistne ws stuie from two viewpoints. Firstly, from the ourrene of toxiity symptoms in plnts n seon from the urtion etween the tretment n the ourrene of the symptoms. In high onentrtions of CN -, the roots turne rownish n the shoots were rie without nerosis, estrution of hlorophyll or sheing of leves. In moerte onentrtions of CN -, growth epression of the pil meristems, sheing of leves n yellowing of lef interveins were oserve. At non-toxi onentrtions, none of the symptoms were oserve. Aute symptoms ppere t the onentrtions of 1 n 2 mg CN - L -1 fter 24 h. Moerte symptoms of toxiity t 9 mg L -1 were oserve fter 48 h. At the onentrtions of 3 n mg CN - L -1, no toxiity ws oserve. The results lso showe tht pretretment of Theoretil n Experimentl Plnt Physiology, 2(4): ,

4 Ghsemi n Mokhtri plnts with sulfosliylte reue the symptoms t 9 mg L -1 CN -. After 24 h, some moerte symptoms were oserve ut they i not persist n the plnts returne to norml growth. Tle 1 shows the viility of pretrete plnts fter eing trete y toxi onentrtion of CN - (9 mg CN - L -1 ). Pretretment y sulfosliylte use sttistilly signifint inrese in plnt viility fter the tretment y lethl onentrtion of CN - (9 mg CN - L -1 ) in 48 h. Among the ifferent onentrtions of sulfosliylte use, 1 mg L -1 showe the highest viility. The mounts of onsume CN - y pretrete plnts re shown in Figure 1. In ll tretments, more thn 9% of the e CN - ws sore y roots. The highest onsumption ws oserve y ontrol n t mg L -1 sulfosliylte pretrete plnts. By inresing the onentrtion of sulfosliylte, less uptke of CN - ws oserve (p<.). Figure 2 shows the oxygen onsumption y roots trete with ifferent onentrtions of sulfosliylte. In CN - trete plnts whih were not trete with Tle 1. Viility of the sulfosliylte pretrete plnts (Slix yloni) trete y 9 mg CN - L -1 in 48 n 72 h. Amounts re expresse s perentge of vile plnts Sulfosliylte onentrtion Viility (%) (mg L -1 ) 48 h 72 h Cynie onsumption (mg L -1 ) Sulfosliylte onentrtion in meium (mgl -1 ) Figure 1. Consumption of ynie y sliylte pretrete plnts uring 24 h. Brs represent rithmeti mens±sd of n=3 replite smples, eh ontining 2 plnts. Different letters show sttistilly signifint ifferenes (p<.) etween ifferent onentrtions of sulfosliylte se on ANOVA (Tukey s HSD test). sulfosliylte, signifint erese in oxygen onsumption ws oserve (p<.1) while no erese ws oserve in sulfosliylte trete plnts. The tretment with CN - inue eletrolyte lekge from roots (p<.). In omprison to the ontrol plnts with no pretretment of sliylte, less lekge of eletrolytes ws oserve from the roots tht were pretrete with 1 mg L -1 sulfosliylte (Figure 3). Pretretment y n 1 mg CN - L -1 use no hnge or even inue n inrese in eletrolyte lekge. The ehyrogense tivity in roots ws erese with CN - (p<.1). By inresing the onentrtion of sulfosliylte from to 1 n 1 mg L -1, sustntil erese in ehyrogense tivity ws oserve (Figure 4). The results showe tht the SOD tivity ws signifintly inrese (p<.) in CN - trete plnts. Pretretment of plnts y sulfosliylte lso h more stimultory effet whih ws sttistilly signifint t 1 mg CN - L -1 e to the meium (p<.; Figure ). The tretment y CN - in non-pretrete plnts ws ompnie y signifint erese in reuing pity, while t 1 mg L -1 sulfosliylte no hnge ws oserve. At the pretretments of n 1 mg L -1 sulfosliylte, signifint inrese ws oserve s ompre with the ontrol non-pretrete plnts (p<.; Figure 6). Oxygen onentrtion in meium (mg L -1 ) Time (h) Figure 2. Effet of pretretment of ifferent onentrtions of sulfosliylte n tretment of toxi onentrtion of ynie (9 mg CN - L -1 ) on the oxygen onsumption y Slix yloni roots. Brs represent rithmeti mens±sd of n=3 replite smples, eh ontining 2 plnts. Different letters in eh series of time (norml letters for the time of, itliize letters for 2 h, ol letters for 4 h n itliize ol letters for 6 h) show sttistilly signifint ifferenes (p<.) etween ifferent onentrtions of sulfosliylte n ontrol plnts se on ANOVA (Tukey s HSD test). W: wter without root; *: roots without neither pretretment of sulfosliylte nor tretment of ynie;,, 1 n 1: pretretment of to 1 mg L -1 sulfosliylte. 4 6 W * Theoretil n Experimentl Plnt Physiology, 2(4): , 213

5 Sliylte n resistne to ynie in Slix EC (µs m -1 ) Dehyrogense tivity (%) Figure 3. Effet of pretretment of ifferent onentrtions of sulfosliylte n tretment of toxi onentrtion of ynie (9 mg CN - L -1 ) on the lekge of eletrolytes from Slix yloni roots. Brs represent rithmeti mens±sd of n=3 replite smples, eh ontining 2 plnts. Different letters in eh series of time (norml letters for the time of, itliize letters for 2 h, ol letters for 4 h n itliize ol letters for 6 h) show sttistilly signifint ifferenes (p<.) etween ifferent onentrtions of sulfosliylte n ontrol plnts se on ANOVA (Tukey s HSD test). EC: eletril onutivity; W: wter without root; *: roots without neither pretretment of sulfosliylte nor tretment of ynie;,, 1 n 1: pretretment of to 1 mg L -1 sulfosliylte. * 2 Figure 4. Dehyrogense tivity of the Slix yloni roots pretrete y ifferent onentrtions of sulfosliylte n trete with 9 mg L -1 ynie. Brs represent rithmeti mens±sd of n=3 replite smples, eh ontining 2 plnts. Different letters show sttistilly signifint ifferenes (p<.1) etween ifferent onentrtions of sulfosliylte se on ANOVA (Tukey s HSD test). *: roots without neither pretretment of sulfosliylte nor tretment of ynie. Time (h) 1 Sulfosliylte onentrtion in meium (mg L -1 ) 4 6 W * 1 1 DISCUSSION Plnts oul e onsiere s CN - onsumers sine, uring the metolism of the ethylene, it is proue with the rtio of 1:1 (Tiz n Zeiger 22) n the proue CN - shoul e metolize or relese from tissues. In stuies fousing 1 SOD tivity (U g -1 FW) Reuing pity (µmol Fe(II) g -1 FW) Figure. Superoxie ismutse tivity of the Slix yloni roots pretrete y ifferent onentrtions of sulfosliylte n trete with 9 mg L -1 ynie. Brs represent rithmeti mens±sd of n=3 replite smples, eh ontining 2 plnts. Different letters show sttistilly signifint ifferenes (p<.1) etween ifferent onentrtions of sulfoliylte se on ANOVA (Tukey s HSD test). *: roots without neither pretretment of sulfosliylte nor tretment of ynie; SOD: superoxie ismutse * * Figure 6. Reuing pity of the Slix yloni root extrts pretrete y ifferent onentrtions of sulfosliylte n trete with 9 mg L -1 ynie. Brs represent rithmeti mens±sd of n=3 replite smples, eh ontining 2 plnts. Different letters show sttistilly signifint ifferenes (p<.1) etween ifferent onentrtions of sulfosliylte se on ANOVA (Tukey s HSD test). the resistne of plnts to the unfvorle hemil gents, n importnt question is whether the resistnt plnt is le to metolize the gent. In other wors, o plnts onvert the toxi gent to non-toxi form? Some stuies hve shown tht no ler orreltion oul e foun etween resistne ginst the unfvorle gent n its etoxifition (Muriio 2, Rusher 1992). Resistne is mehnism tht moerte the mge on the plnt n tolerne is the ility of plnt to 1 Sulfosliylte onentrtion in meium (mg L -1 ) 1 Sulfosliylte onentrtion in meium (mg L -1 ) 1 1 Theoretil n Experimentl Plnt Physiology, 2(4): ,

6 Ghsemi n Mokhtri experiene the mge evoi of reution in the vigor (Bker 1987, Rusher 1992). To hieve this onition, plnt metolism interferes with struture n properties of enzymti systems n hnges the physio-hemil properties of ell memrnes n trnsporters. Resistne to CN - my e ue to the metoliztion n estroying of the CN - n, in plnts whih hve evolve mehnism(s) of meliorting, the effets of CN -, the tivities of metoli pthwys suh s sulfur trnsferse n ynolnine hve positive orreltion with CN - resistne (Blumenthl et l. 1968, Cstri et l. 1972). Immeite metoliztion of CN - in ifferent ellulr metolizing pthwys prevents CN - from rehing sensitive sites suh s metls in metlloproteins. More tive metolizing pthwys mens less onentrtion of CN - in the ells n more resistne to higher onentrtions of externl CN -. Sine the tivity of ifferent metolizing pthwys strongly is ffete y the sttus of nutrition, energy, hormones n environmentl onitions (Gouey et l. 1989; Tittle et l. 199), resistne to ynie hnges y hnge in remrke ftors. One of the phytohormones whih strongly ffets the energy sttus n respirtory tivities of the ells is sliylte (Hyt et l. 27). This hormone strongly ffets eletron trnsport hin in mitohonri. The effet of sliylte on ellulr respirtion is onentrtionepenent (Xie n Chen 1999). However, in the urrent stuy, no ifferene ws oserve in oxygen onsumption t ifferent onentrtions of sliylte. It hs ul role in the proution of ROS in the ells. If sliylte promotes isturing in the respirtion hin in mitohonri, more ROS re proue (Mxwell et l. 22). The reson oul e mehnism like CN - n ntimyine A oth inhiit the oxition of ytohrome (Chen et l. 1993). On the other hn, sliylte is one of the most importnt stimultors of CN - resistnt respirtory pthwy or AOX pthwy (Rhos n MIntosh 1992). Therefore, sliylte is n inhiitor of eletron trnsport tht inreses ROS proution n lso tivtes AOX, whih y onsuming reuing gents prevents ROS proution. In this stuy, we oserve tht the tivity of SOD inrese t the presene of sliylte. This fining oul e ue to n inrese in the proution of ROS y sliylte n CN - n onsequently stimultion of ntioxint system of the ells (suh s SOD). CN - inhiits not only eletron trnsport hin t the IV omplex in mitohonril respirtory hin (IV omplex) ut lso some of the metlloenzymes suh s Cu/Zn ontining form of superoxie ismutse (Cu/Zn SOD), whih is the most importnt isoform of SOD in ytosol (Tsng et l. 1991). In this stuy, the tretment y CN - erese the reuing power of the root extrts, whih were not pretrete y sliylte. This result is possily ue to high proution of oxiizing gents (suh s ROS). The pretretment of sliylte inrese the reuing pity, ut it ws not ifferent of the ontrol plnts. The effet of sliylte oul e ue to preventing ROS proution. Enzymti ehyrogense tivity in the root ells ws erese y CN -. We hypothesize tht the long time pretretment with sliylte n its inhiitory effet on eletron trnsport hin, whih ours t the site of reution of uiquinone (Normn et l. 24), oul reue the eletron trnsport. By ompring with ifferent stuies, Glis n Mtsuok (27) onlue tht severl hunres of genes oul e moulte y to sliyli i inluing ehyrogenses. Therefore, it is ponere tht the reox sttus of the ells is ffete y sliylte y hnging the tivity of ehyrogenses. ROS hs entrl role in mintining the homeostsis of reox stte of ells n the inrese or erese in their onentrtions ffets the tivity of prouing or svenging pthwys (Mittler 22). We lso oserve tht sliylte inrese ion lekge from the roots (exept t the onentrtion of 1 mg L -1 sulfosliylte). Ion lekge is result of high onentrtions of ROS in the ells, s it uses lipi oxition n impirs ellulr memrnes integrity, whose physiologil onsequene is ion lekge (Dietz et l. 1999). It oul e hypothesize tht sliylte ffete the ellulr homeostsis y inresing the ROS proution, whih susequently inrese the tivity of SOD n the tivtion of AOX. Inrese tivity of AOX prevents the erese in oxygen onsumption t the presene of CN - n fvors the ells with ATP proution t low rtes. The most importnt finings of this stuy re summrize in Tle 2. The results suggest tht, to inrese resistne of plnts ginst CN -, onentrtion epenent pretretment of sliylte might e use. In ition, mesure resistne prmeters will e useful to ssess the tivity of the CN - onsuming or metolizing pthwys with the sliylte pretretment sine the tivity of these pthwys is onsiere s resistne inex. 28 Theoretil n Experimentl Plnt Physiology, 2(4): , 213

7 Sliylte n resistne to ynie in Slix Tle 2. Summrize results of the urrent stuy n short esription on the most possile resons of the oserve results. The results re omprison etween pretrete (1 mg L -1 ) or other onentrtions ( n 1 mg L -1 ) of sulfosliylte n ontrol plnts. Proper onentrtion of sulfosliylte ws efine se on the ourrene of notiele resistne Mesure Proper onentrtion of sulfosliylte (1 mg L -1 ) Result Other use onentrtions of sulfosliylte SOD tivity Inrese Equl or inrese Oxygen onsumption Inrese Inrese Eletrolyte lekge Derese Equl or inrese Dehyrogense tivity Derese Derese Reuing pity Inrese Inrese Uptke of ynie from meium Derese Equl or erese Resistne to lethl onentrtion of ynie Best result Less suitle SOD: superoxie ismutse; ROS: retive oxygen speies; AOX: lterntive oxise. Desription Proper onentrtion showe the most stimultion resulte in pproprite ROS svenge Possily ue to tivtion of AOX pthwy y sliylte A lne ellulr reox sttus n ROS proution/svenge t proper onentrtion of sliylte n keeping memrne integrity Inhiition y ynie fter tretment or hormonl effet of sliylte on gene expression Aumultion of reuing gent(s) ue to inhiition of eletron trnsport hin In the ses of erese, still more thn 9% of e ynie into the meium Sliylte n e use to inrese resistne in onentrtion epenent moe Referenes Bker AJM (1987) Metl tolerne. New Phytologist 16: Benzie FF, Strin JJ (1999) Ferri reution/ntioxint power ssy: iret mesure of totl ntioxint tivity of iologil fluis n moifie version for simultneous mesurement of totl ntioxint power n sori i onentrtion. Methos in Enzymology 299:1-23. Blumenthl SG, Henrikson HR, Arol YP, Conn EE (1968) Cynie metolism in higher plnts. III. The iosynthesis of ynolnine. Biologil Chemistry 243: Bushey JT, Es SD, Dzomk DA (26) Development of plnt uptke moel for ynie. Interntionl Journl of Phytoremeition 8:2-43. Bushey JT, Smll MJ, Dzomk DA, Es SD (26) Prmeter estimtion of plnt uptke moel for ynie: pplition to hyroponi t. Interntionl Journl of Phytoremeition 8:4-62. Cstri PA, Frnen KJF, Conn EE (1972) Cynie metolism in higher plnts. V. The formtion of sprgine from β-ynolnine. Arhives of Biohemistry n Biophysis 12: Chen Z, Silv H, Klessig DF (1993) Ative oxygen speies in the inution of plnt systemi quire resistne y sliyli i. Siene 262: oi: /siene Dietz KJ, Bier M, Krämer U (1999) Free rils n retive oxygen speies s meitors of hevy metl toxiity in plnts. In: Prs MNV, Hgemeyer J (es), Hevy metl stress in plnts, pp Springer, Berlin, Germny. Es SD, Bushey JT, Bon BS, Ghosh RS, Dzomk DA (26) Cynie phytoremeition. In: Dzomk DA, Ghosh RS, Wong- Chong GM (es), Cynie in wter n soil, pp Tylor n Frnis, Bo Rton, USA. Es SD, Bushey J, Poston S, Kosm D, Smiotkis M, Dzomk D (23) Trnsport n metolism of free ynie n iron ynie omplexes y willow. Plnt, Cell n Environment 26: Es SD, Kosm DK, Nielson EH, Mhingur M, Bker AJM, Woorow IE (21) Nitrogen supply n ynie onentrtion influene the enrihment of nitrogen from ynie in whet (Tritium estivum L.) n sorghum (Sorghum iolor L.). Plnt, Cell n Environment 33: Es SD, Piinin RC, Googer JQD, Kolev SD, Woorow IE, Bker AJM (28) Trnsport of ferroynie y two eulypt speies n sorghum. Interntionl Journl of Phytoremeition 1: Glis I, Mtsuok K (27) Trnsriptomi nlysis of sliyli i-responsive genes in too y-2 ells. In: Hyt S, Ahm A (es), Sliyli i: plnt hormone, pp Springer, The Netherlns. Ginnopolitis CN, Ries SK (1997) Superoxie ismutse. I. Ourene in higher plnts. Plnt Physiology 9: oi: 1.114/pp Gouey JS, Tittle FL, Spener MSA (1989) A role for ethylene in the metolism of ynie y higher plnts. Plnt Physiology 89: Goulen PD, Afghn BK, Brooksnk P (1972) Determintion of mnogrm quntities of simple n omplex ynies in wter. Anlytil Chemistry 44: Hlkier BA, Møller BL (199) The iosynthesis of ynogeni gluosies in higher plnts. Ientifition of three hyroxyltion steps in the iosynthesis of hurrin in Sorghum iolor (L.) Moenh n the involvement of 1-ACI-nitro-2-(p-hyroxyphenyl)ethne s n intermeite. Journl of Biologil Chemistry 26: Theoretil n Experimentl Plnt Physiology, 2(4): ,

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