Extracellular ATP is Involved in the Salicylic Acid-Induced Cell Death in Suspension-Cultured Tobacco Cells

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1 Plnt Pro. Si. 18(2): (215) Extrllulr ATP is Involv in th Sliyli Ai-Inu Cll Dth in Suspnsion-Cultur Too Clls Hnqing Fng, Dongong Gun, Kun Sun, Yi Fng, Yo Zho n Lingyun Ji (Collg of Lif Sin, Northwst Norml Univrsity, 737 Lnzhou, Chin) Astrt: Extrllulr ATP (ATP) n funtion s signling molul to rgult wi rng of llulr prosss. W invstigt th rgultory rol of ATP in th ll th inu y sliyli i (SA) in suspnsion-ultur too (Niotin tum L.) lls. Trtmnt of too suspnsion lls with SA inu ll th. Th sm trtmnt lowr th lvls of ATP, ompni y rs of oth th rspirtory O 2 uptk n intrllulr ATP lvls in too suspnsion lls. Trtmnt with β,γ-mthylnnosin 5 -triphospht (AMP-PCP), whih is th non-hyrolysl nlogu of ATP n n xlu ATP from ining sits of ATP rptors, lso inu ll th in too ll ulturs. Trtmnt with xognous ATP prtilly llvit th ll th inu y SA. Ths osrvtions suggst tht ATP is involv in th SAinu ll th in too ll ulturs. Ky wors: Cll th, Extrllulr ATP, Sliyli i. Introution Sliyli i (SA) is n importnt signling molul in plnt lls (Gffny t l., 1993). Th lvl of SA in th plnt inrss with tril n virl inftions, n th inrs of SA is rquir to tivt th xprssion of pthognsis-rlt (PR) gns n fns rsponss of plnts to pthogn inftion (Gffny t l., 1993; Dlny t l., 1995; Shh, 23). Plnts inft with iotrophi fungi or oomyts lso show SA umultion, n th plnts umulting SA show nhn susptiility to fungi n oomyts (Glzrook, 25). Som ioti strsss, suh s rought, hilling, hvy mtl toxiity, n ht, n lso inrs th SA ontnts in th plnt, whih is foun to ply importnt rols in nhning th rsistn to ths strss onitions (Yun n Lin, 28). Anosin 5 -triphospht (ATP) hs n rgr s n intrllulr nrgy urrny molul for mny yrs. Howvr, ATP in xtrllulr miliu of niml lls hs rntly n foun to funtion s signling ompoun rgulting mny llulr prosss through intrtion with mmrn-ssoit rptor protins. Ths prosss inlu nurotrnsmission, immun rsponss, ll growth, n ll th (Khkh n urnstok, 29). ATP hs lso n rport to xist in xtrllulr sps of plnt lls. Mny stuis monstrt tht xtrllulr ATP (ATP) n mit ivrs physiologil prosss of plnts, suh s growth of root-hir, fns rsponss to pthogn inftion, ll viility, n thigmotropism (Roux n Stinrunnr, 27; Tnk t l., 21; Sun t l., 212). Furthrmor, ATP n stimult th proution of mny intrllulr signling moluls, suh s ytosoli fr lium ([C 2+ ] yt ), nitri oxi (NO), n rtiv oxygn spis (ROS) (Roux n Stinrunnr, 27; Tnk t l., 21; Sun t l., 212). Most rntly Choi t l. (214) intifi rptor for plnt ATP from Ariopsis. Chmil trtmnt of ultur too lls with SA inu th xprssion of PR-1 gn n lowr th lvl of ATP. In ition, loking th rution of ATP supprss th SA-inu xprssion of PR-1 gn (Chivs t l., 29). A similr phnomnon ws lso osrv in Ariopsis ll ulturs (Chivs t l., 29). Ths osrvtions suggst tht th SA-inu pthogn fns rspons oul mit y ATP. sis inuing th fns (or rsistn) rsponss to ioti or ioti strsss, hmil trtmnt with SA hs n foun to us ll th of plnts, spilly t high onntrtions or unr long-trm pplition (Qio t l., 23; rors t l., 25). A high onntrtion of SA, typilly foun in th pthogn-inft rs of th plnt, is rquir for mounting th hyprsnsitiv ll th, known s fns rspons to rstrit pthogn Riv 16 April 214. Apt 29 August 214. Corrsponing uthors: H.Q. Fng (hnqing_fng@hotmil.om, fx ). Arvitions: AMP-PCP, β,γ-mthylnnosin 5 -triphospht; iatp, intrllulr ATP; ATP, xtrllulr ATP; FDA, fluorsin itt; ROS, rtiv oxygn spis; SA, sliyli i.

Fng t l. SA-inu Cll Dth n ATP 155 A Exognous SA μmol 1 μmol 3 μmol 5 μmol 7 μmol.7.6 OD 6.5.4.3.2.1. 1 3 Fig. 1. Th SA-inu ll th xmin using FDA n Evns lu stining ssy.

Th mns not y th sm lttr i not signifintly iffr t P <.5. 5 7 growth n iss vlopmnt (Alvrz, 2; Gust n Nürnrgr, 212).

In th prsnt stuy, w xmin th ffts of ATP on th SA-inu ll th. W liv tht this rsrh will furthr vlop n xpn th urrnt knowlg out th physiologil funtion of ATP n its rol in SAmit physiologil vnts.

Jing Liwn (Th Chins Univrsity of Hong Kong, Chin) n grown in suspnsion using Murshig n Skoog (MS) (Murshig n Skoog, 1962) liqui mium (ph 5.8) (Sigm-Alrih) supplmnt with 3% (w/v) suros n.

2 Fng t l. SA-inu Cll Dth n ATP 155 A Exognous SA μmol 1 μmol 3 μmol 5 μmol 7 μmol.7.6 OD Fig. 1. Th SA-inu ll th xmin using FDA n Evns lu stining ssy. A: rprsnttiv imgs pit th SA-inu ll th visuliz y FDA stining. : th lvls of ll th wr msur using Evns lu stining ssy y msuring th sorn of xtrt y t 6 nm. Eh vlu rprsnts th mn ± SD of six inpnnt xprimnts. Th mns not y th sm lttr i not signifintly iffr t P < growth n iss vlopmnt (Alvrz, 2; Gust n Nürnrgr, 212). Unr mium strss, SA n lrt th ll th of mium-strss roots to voi mium uptk y plnts or to prott th strss roots from mium-inu mg (Guo t l., 29). Whthr or not th SA-inu ll th is ssoit with ATP is not lr. In th prsnt stuy, w xmin th ffts of ATP on th SA-inu ll th. W liv tht this rsrh will furthr vlop n xpn th urrnt knowlg out th physiologil funtion of ATP n its rol in SAmit physiologil vnts. Mtril n Mthos 1. Too ll ulturs A too (Niotin tum L. v. right Yllow-2) ll ultur (ll lin Y-2) ws kinly provi y Prof. Jing Liwn (Th Chins Univrsity of Hong Kong, Chin) n grown in suspnsion using Murshig n Skoog (MS) (Murshig n Skoog, 1962) liqui mium (ph 5.8) (Sigm-Alrih) supplmnt with 3% (w/v) suros n.4 mg L 1 2,4-ihlorophnoxy ti i unr onstnt shking t 25ºC in rknss (Ngt t l., 1981). Cll suspnsion ulturs wr suultur t 7- growth yl intrvls y piptting 1 ml of ll ultur into 1 ml of frsh liqui mium. All prours wr on unr spti onitions. 2. Trtmnts of lls All xprimnts wr prform with th too suspnsion lls t 3 ftr suultur. In th first st of th xprimnts, th ll suspnsions wr sujt to, 1, 3, 5, or 7 μm SA, rsptivly, n inut for 1 hr t 25ºC in th rknss. In th son st of xprimnts, th ll suspnsions wr sujt to, 5, 1 or 15 μm AMP-PCP (β,γ-mthylnnosin 5 -triphospht), n inut for 1 hr t 25 ± 1ºC in rknss. In th thir st of xprimnts, th ll suspnsions wr sujt to 5 μm ATP-N 2, 5 μm SA, or 5 μm ATP-N 2 plus 5 μm SA n thn inut for 1 hr t 25ºC in th rk. Th ll suspnsions without ny hmil trtmnt wr us s th ontrol.

3 156 Plnt Proution Sin Vol.18, Cll th ssys Th ll suspnsions wr stin with fluorsin itt (FDA): living lls mtoliz FDA to fluorogni sustrt fluorsin, so thy mit grn light ftr xittion (Guilult n Krmr, 1964). Th ll suspnsions wr trt with 4 mg L 1 fluorsin itt (FDA, Sigm-Alrih) n inut in th rk for 1 min t room tmprtur. Th smpls wr visuliz unr th fluorsnt mirosop (Li, DM5, Wtzlr, Grmny). Cll th ws lso quntifi using th Evns lu stining ssy s sri y Yng t l. (24) with som moifitions. Clls with mg mmrns tk up Evns lu y, whrs vil lls tht rtin intt plsm mmrns n xlu th y (Kwi n Uhimiy, 2; Hung t l., 27). A 1-mL liquot of th ll suspnsions wr stin for 8 min with 5 ml of.25% (w/v) Evns lu solution n thn wsh fiv tims with phospht uffr slin (PS) to rmov th xss n unoun y. Th y oun to lls ws soluiliz in solution ontining 1% (w/v) SDS (soium oyl sulpht) n 5% mthnol (v/v) for 3 min t 5ºC. Th xtrt y ws trmin sptrophotomtrilly t 6 nm. 4. Msurmnts of intrllulr ATP n xtrllulr ATP Th lvls of intrllulr ATP (iatp) n ATP of th ll suspnsions wr ssy using n ATP ioluminsn Dttion Kit (yotim Institut of iothnology, Himn, Chin) y using luifrs to tlyz th rls of light y ATP n luifrin (Stmlr t l., 1987). For th msurmnt of iatp, th ll suspnsions wr ntrifug t 35 g for 4 min t room tmprtur, n th pripitt ws wsh with ATP-fr liqui mium to rmov th rsiul ATP. Th intrllulr miliu ws xtrt n ATP in th intrllulr miliu ws msur following th mnufturrs instrutions. For th msurmnt of ATP, th ll-fr suprntnt from th ll suspnsions ws ollt y th mtho sri y Mills n L (1996), n th msurmnt of ATP in th suprntnt ws prform s sri y th mnufturr s instrutions. 5. Rspirtory O 2 uptk Th lls in th ultur mium wr trnsfrr to 3-mL ir-tight uvtt. Th oxygn uptk of th lls ws monitor using Clrk-typ oxygn ltro (SP-2 typ, onstrut y th Institut of Plnt Physiology n Eology, Chins Amy of Sins, Shnghi, Chin). Th sty rt of rspirtory O 2 uptk ws msur n lult s sri y inghm n Frrr (1989). V t [μmol O 2 g -1 FW min -1 ] Rltiv ATP lvl [1%] Rltiv iatp lvl [1%] C A Fig. 2. Th ffts of xognous SA on th lvls of ATP (A), iatp (), n th rspirtory O 2 uptk (C) of too llsuspnsion ultur. Th lvls of ATP n iatp r xprss s prntg to th initil mount of ATP of th llsuspnsion ultur without SA trtmnt ( μmol). Eh vlu rprsnts th mn ± SD of t lst four inpnnt xprimnts. Th mns not y th sm lttr i not signifintly iffr t P <

Fng t l. SA-inu Cll Dth n ATP 157 A Exognous AMP-PCP μm 5 μm 1 μm 15 μm.6.5.4 OD 6.3.2.1. 5 1 15 Exognous AMP-PCP [μm] Fig. 3. Th ffts of AMP-PCP on th ll viility xmin using FDA n Evns lu stining ssy.

4 Fng t l. SA-inu Cll Dth n ATP 157 A Exognous AMP-PCP μm 5 μm 1 μm 15 μm OD Exognous AMP-PCP [μm] Fig. 3. Th ffts of AMP-PCP on th ll viility xmin using FDA n Evns lu stining ssy. A: rprsnttiv imgs pit th AMP-PCP-inu ll th visuliz y FDA stining. : th lvls of ll th wr msur using Evns lu stining ssy y msuring th sorn of xtrt y t 6 nm. Eh vlu rprsnts th mn ± SD of six inpnnt xprimnts. Th mns not y th sm lttr i not signifintly iffr t P < Sttistil nlysis Th rsults r xprss s th mn ± stnr vition (SD). Th t wr nlys using th Kruskll Wllis on-wy nlysis of vrin tst. P <.5 ws onsir sttistilly signifint. Rsults 1. SA inu ll th in too ll ulturs Th too ll ulturs wr trt with xognous SA t to 7 μm. FDA stining ws us s qulittiv tool to visuliz th SA-inu ll th. Mirosopi osrvtion show tht FDA fluorsn rs with th inrs in xognous SA onntrtions (Fig. 1A). Th lvls of ll th wr lso quntifi y th Evns lu stining mtho. Th rsults show tht th lvls of ll th inrs with th inrs in th onntrtion of xognous SA (Fig. 1). 2. SA rs th lvls of ATP, iatp n th rspirtory O 2 uptk of too ll-suspnsion ultur Trtmnt with xognous SA signifintly rs th lvls of ATP. Trtmnt with 1, 3, 5, n 7 μm SA rs ATP lvls y 2., 37.2, 54.6 n 67.7%, rsptivly (Fig. 2A). Exognous SA lso rs th iatp lvls. Th lvls of iatp lvl wr rs y 5.9, 19.8, 27.7, n 32.8%, ftr th trtmnt with 1, 3, 5, n 7 μm SA, rsptivly (Fig. 2). As shown in Fig. 2C, trtmnt of th ll ulturs with xognous SA rs rspirtory O 2 uptk in ospnnt mnnr, whih is similr to th hng in th lvls of ATP n iatp ftr th trtmnt with xognous SA (Fig. 2A, ). 3. AMP-PCP inu ll th of too ll ulturs In th prsnt work, th too suspnsion lls wr trt with xognous AMP-PCP (β,γ-mthylnnosin 5-triphospht). AMP-PCP is non-hyrolysl nlogu

158 Plnt Proution Sin Vol.18, 215 A Control ATP SA + ATP SA.6.5.4 OD 6.3.2.1. ATP SA+ATP SA Fig. 4. Th ffts of iffrnt trtmnts on th ll viility xmin using FDA n Evns lu stining ssy.

xognous SA. A: rprsnttiv imgs pit th ll th visuliz y FDA stining. : th lvls of ll th msur using Evns lu stining ssy y msuring th sorn of xtrt y t 6 nm.

Its pplition n xlu ATP from ining sits of puttiv ATP rptors/atp-ining protins n thus inhiit th rtions rquiring ATP.

5 158 Plnt Proution Sin Vol.18, 215 A Control ATP SA + ATP SA OD ATP SA+ATP SA Fig. 4. Th ffts of iffrnt trtmnts on th ll viility xmin using FDA n Evns lu stining ssy. Th lls wr trt s follows: ontrol: th lls without hmil trtmnt; ATP: th lls wr xpos to 5 μm xognous ATP; SA + ATP: th lls wr xpos to 5 μm xognous SA plus 5 μm xognous ATP; SA: th lls wr xpos to 5 μm xognous SA. A: rprsnttiv imgs pit th ll th visuliz y FDA stining. : th lvls of ll th msur using Evns lu stining ssy y msuring th sorn of xtrt y t 6 nm. Eh vlu rprsnts th mn ± SD of six inpnnt xprimnts. Th mns not y th sm lttr i not signifintly iffr t P <.5. of ATP. Its pplition n xlu ATP from ining sits of puttiv ATP rptors/atp-ining protins n thus inhiit th rtions rquiring ATP. Thus, AMP-PCP hs ntgonisti ffts on th physiologil prosss rquiring ATP n is wily us to fin ATP funtions in niml n plnt lls (Chivs t l., 29, 21). Th osrvtion otin using FDA stining show tht th trtmnt with AMP-PCP rs th fluorsn intnsity of th too suspnsion lls (Song t l., 26; Chivs t l., 21). Th rs ws most ovious in th lls trt with 15 μm AMP-PCP (Fig. 3A). Quntittiv msurmnt using Evns lu ssy show tht ompr with th lls without AMP-PCP trtmnt, 5 μm AMP-PCP slightly, ut not signifintly, inu ll th (Fig. 3). In ontrst, trtmnt with 1 or 15 μm AMP-PCP signifintly inu ll th. Exposur to 15 μm AMP-PCP inu th highst rt of ll th (Fig. 3). Ths rsults suggst tht th rplmnt of ATP with AMP-PCP initit ll th in too ll ulturs. 4. Aition of ATP llvit th ll th inu y SA To xmin whthr th inution of ll th y SA is link to th pltion of ATP, w xognous ATP (5 μm) to th SA (5 μm)-trt too ll ulturs to limit th rution of ATP u to th pplition of SA. Trtmnt with 5 μm xognous ATP lon h no signifint fft on th ll viility, ompr to th ontrol (lls without ny hmil trtmnt) (Fig. 4). Howvr, th lls sujt to th omin trtmnt with 5 μm SA plus 5 μm ATP h lowr ll th rt, ompr to th lls trt with 5 μm SA lon (Fig. 4). Ths osrvtions init tht th SA-inu ll th oul ssoit with rs in ATP lvl. Disussion In th prsnt work, trtmnt of too suspnsion ll with SA inu ll th (Fig. 1). Th inution of ll th y SA ws ompni y th rs of oth th rspirtory O 2 uptk n iatp lvls (Fig. 2, C). SA

6 Fng t l. SA-inu Cll Dth n ATP 159 is thought to impir th mitohonril ltron trnsport hin (metc) n TCA (triroxyli i) yl (Normn t l., 24; Rüffr t l., 1995), oth of whih r importnt for th rspirtory oxygn rution n th proution of iatp (Mknzi n MIntosh, 1999). In ition, th isruption of metc n th lin of iatp proution r foun to ply importnt rols in triggring ll th rsponss (Lm t l., 21; Rhos t l., 26; Vil t l., 27). Thus, th osrv ll th n th rs of th rspirtory O 2 uptk n iatp oul th rsult of impir mitohonril rspirtory mtolism us y SA. Similr to th hng of iatp lvl, SA signifintly lowr th ATP lvls (Fig. 2A). ATP synths hs not n foun in th ll surf or plsm mmrn of plnt lls. Prvious stuis hv rvl tht plnt lls n rls ATP from th iatp pool ithr vi th ATPining sstt trnsportrs or xoytosis (Kim t l., 26; Thoms t l., 2). Thus, ATP origints from iatp tht is rls into th xtrllulr miliu (Khkh n urnstok, 29; Tnk t l., 21). This mns tht ny hng in iatp lvl woul hv n importnt influn on th lvl of ATP. Mitohonril ATP is th mjor rsour of iatp. Thus, it is rsonl to ssum tht SA rs th mitohonril ATP proution y impiring mitohonril rspirtory mtolism n susquntly l to th lin of ATP lvls. In th lst fw s, th SA-mit physiologil vnts rsponsil for th inution of ll th hv n sri in th intrllulr miliu (Alvrz, 2; Kwi-Ym t l., 24; Gust n Nürnrgr, 212). In th prsnt work, w furthr invstigt whthr th lin of ATP lvl in th prsn of SA, s xtrllulr vnt, is involv in th SA-inu plnt ll th. us of its high hrg, ATP nnot pssivly iffus ross th plsm mmrn. ATP ins to th sit of puttiv ATP rptors/atp-ining protins lot in th plsm mmrn, whr hyrolysis of ATP ours. Th hyrolysis of ATP, following ining, is ruil in triggring signl trnsution n physiologil vnts (Song t l., 26; Chivs t l., 29, 21). In th prsnt work, th fft of ATP on th viility of too suspnsion lls wr stui y using AMP-PCP (β,γmthylnnosin 5-triphospht). As mntion ov, AMP-PCP hs ntgonisti ffts on th physiologil prosss rquiring ATP. Exognous pplition of AMP-PCP inu ll th of too ll ulturs (Fig. 3), suggsting tht th rs in ATP lvl oul initit ll th. Furthrmor, th trtmnt with xognous ATP prtilly llvit th ll th inu y SA (Fig. 4). Ths osrvtions suggst tht SA inus ll th through signling pthwy tht is ssoit with lin of ATP lvl. Th mhnism of th llvitiv fft of ATP on th SA-inu ll th is not lr. In niml lls, ATP n stimult th umultion of [C 2+ ] yt y ining n tivting th mmrn-ssoit P2-typ purinoptor protin (Dihmnn t l., 2; Arhio t l. 26). Trtmnt with xognous ATP n lso rsult in th spifi umultion of [C 2+ ] yt in plnt lls (Dmihik t l. 29). Although gnomi squn s survys to intify plnt ATP rptors homology to niml purinoptors fil to fin ny suitl nit protins, Choi t l. (214) rvl tht th DORN1 protin of Ariopsis ins ATP with high ffinity n is rquir for ATP-inu umultion of [C 2+ ] yt. Prvious stuis rvl tht n inrs in C 2+ lvl n nhn th proution of iatp y inrsing th vilility of mitohonril sustrts (Jouvill t l. 1999; Logn n Knight 23). Thus, if, s suggst ov, th SA-inu ll th is rsult of lin of iatp proution u to th inhiition of th mitohonril rspirtion y SA, it is rsonl to ssum tht th inrs in C 2+ lvl y ATP oul llvit th SAinu inhiition in rspirtion n iatp proution, n thus rsu too suspnsion lls from SA-inu th. Howvr, futur work will n to onfirm th mhnism. Aknowlgmnts n Funing W thnk Prof. Jing Liwn (Th Chins Univrsity of Hong Kong) for th supply of th too Y-2 ll ultur. This work ws support y th Ntionl Nturl Sin Fountion of Chin (NO n 3915), Ky Projt of Chins Ministry of Eution (NO ), th Funmntl Rsrh Funs for th Gnsu Univrsitis of Gnsu Provinil Dprtmnt of Finn, n NWNU-kjxg-3-77&49; NWNU-9-31& NWNU-LKQN Rfrns Arhio, M.P., urnstok, G., oynms, J.M., rnr, E.A., oyr, J.L., Knny, C., Knight, G.E., Fumglli, M., Ght, C., Joson, K.A. n Wismn, G.A. 26. Intrntionl Union of Phrmology LVIII: upt on th P2Y G protin-oupl nuloti rptors: from molulr mhnisms n pthophysiology to thrpy. Phrmol. Rv. 58: Alvrz, M.E. 2. Sliyli i in th mhinry of hyprsnsitiv ll th n iss rsistn. Plnt Mol. iol. 44: inghm, I.J. n Frrr, J.F Ativity n pity of rspirtory pthwys in rly roots priv of inorgni nutrints. Plnt Physiol. iohm. 27: rorsn, P., Mlinovsky, F.G., Hémty K., Nwmn, M.A. n Muny, J. 25. Th rol of sliyli i in th inution of ll th in Ariopsis 11. Plnt Physiol. 138: Chivs, S., Murphy, A.M., Hmilton, J.M., Linsy, K., Crr, J.P. n Sls, A. R. 29. Extrllulr ATP is rgultor of pthogn fn in plnts. Plnt J. 6: Chivs, S., Simon, W.J., Murphy, A.M., Linsy, K., Crr, J.P. n

7 16 Plnt Proution Sin Vol.18, 215 Sls, A.R. 21. Th ffts of xtrllulr nosin 5'-triphospht on th too protom. Protomis 1: Choi, J., Tnk, K., Co, Y., Qi, Y., Qiu, J., Ling, Y. n L, S.Y Intifition of plnt rptor for xtrllulr ATP. Sin 343: Dlny, T.P., Fririh, L. n Ryls, J.A Ariopsis signl trnsution mutnt ftiv in hmilly n iologilly inu iss rsistn. Pro. Ntl. A. Si. U.S.A 92: Dmihik, V., Shng, Z., Shin R., Thompson, E., Ruio, L., Lohvisit, A., n Mortimr, J.C., Chivs, S., Sls A.R., Glovr,. J., Shhtmn, D. P., Shl S. N. n Dvis J. M. 29. Plnt xtrllulr ATP signlling y plsm mmrn NADPH oxis n C 2+ hnnls. Plnt J. 58: Dihmnn, S., Izko, M., Zimpfr, U., Hofmnn, C., Frrri, D., Luttmnn, W., Virhow, C.J., Di Virgilio, F. n Norgur, J. 2. Anosin triphospht-inu oxygn ril proution n CD11 up-rgultion: C ++ moiliztion n tin rorgniztion in humn osinophils. loo 95: Gffny, T., Fririh L., Vrnooij,., Ngrotto, D., Ny G., Ukns, S., Wr, E., Kssmnn, H. n Ryls, J Rquirmnt of sliyli i for th inution of systmi quir-rsistn. Sin 261: Glzrook, J. 25. Contrsting mhnisms of fns ginst iotrophi n nrotrophi pthogns. Annu. Rv. Phytopthol. 43: Guilult, G.G. n Krmr, D.N Fluoromtri trmintion of lips, yls, lph-, n gmm-hymotrypsin n inhiitors of ths nzyms. Anl. Chm. 36: Guo,., Ling, Y. n Zhu, Y. 29. Dos sliyli i rgult ntioxint fns systm, ll th, mium uptk n prtitioning to quir mium tolrn in ri? J. Plnt Physiol. 166: Gust, A. A. n Nürnrgr, T Plnt immunology: A lif or th swith. Ntur 486: Hung, W.C., Hung, D.D., Chin, P.S., Yh, C.M., Chn, P.Y., Chi, W. C. n Hung, H.J. 27. Protin tyrosin phosphoryltion uring oppr-inu ll th in ri roots. Chmosphr 69: Jouvill, L.S., Pinton, P., stinutto, C., Ruttr, G.A. n Rizzuto, R Rgultion of mitohonril ATP synthsis y lium: vin for long-trm mtoli priming. Pro. Ntl. A. Si. U.S.A. 96: Kwi, M. n Uhimiy, H. 2, Coloptil snsn in ri (Oryz stiv L.). Ann ot. 86: Kwi-Ym, M., Ohori, Y. n Uhimiy, H. 24. Disstion of Ariopsis x inhiitor-1 supprssing x-, hyrogn proxi-, n sliyli i-inu ll th. Plnt Cll 16: Khkh,.S. n urnstok, G. 29. Th oul lif of ATP. Si. Am. 31: Kim, S.Y., Sivguru, M. n Sty, G. 26. Extrllulr ATP in plnts. Visuliztion, loliztion, n nlysis of physiologil signifin in growth n signling. Plnt Physiol. 142: Lm, E., Kto, N. n Lwton, M. 21. Progrmm ll th, mitohonri n th plnt hyprsnsitiv rspons. Ntur 411: Logn, D.C. n Knight, M.R. 23. Mitohonril n ytosoli lium ynmis r iffrntilly rgult in plnts. Plnt Physiol. 133: Mknzi, S. n MIntosh, L Highr plnt mitohonri. Plnt Cll 11: Mills, D.R. n L, J.M A simpl, urt mtho for trmining wt n ry wight onntrtions of plnt ll suspnsion ulturs using mirontrifug tus. Plnt Cll Rp. 15: Murshig, T. n Skoog, F A rvis mium for rpi growth n iossys with too tissu ulturs. Physiol. Plnt 15: Ngt, T., Ok, K., Tk, I. n Mtsui, C Dlivry of too mosi virus RNA into plnt protoplsts mit y rvrs-phs vportion vsils (liposoms). Mol. Gn. Gnt. 184: Normn, C., Howll, K.A., Millr, A.H., Whln, J.M. n Dy, D.A. 24. Sliyli i is n unouplr n inhiitor of mitohonril ltron trnsport. Plnt Physiol. 134: Qio, J.J., Yun Y. J., Zho, H., Wu, J.C. n Zng, A.P. 23. Apoptoti ll th in suspnsion ulturs of Txus uspit otrt with sliyli i n hyrogn proxi. iothnol. Ltt. 25: Rhos, D.M., Umh, A.L., Suih, C.C. n Siow, J.N. 26. Mitohonril rtiv oxygn spis. Contriution to oxitiv strss n introrgnllr signling. Plnt Physiol. 141: Roux, S.J. n Stinrunnr, I. 27. Extrllulr ATP: n unxpt rol s signlr in plnts. Trns Plnt Si. 12: Rüffr, M., Stip,. n Znk, M.H Evin ginst spifi ining of sliyli i to plnt tls. FES Ltt. 377: Shh, J. 23. Th sliyli i loop in plnt fns. Curr. Opin. Plnt iol. 6: Song, C.J., Stinrunnr, I., Wng, X., Stout, S.C. n Roux, S.J. 26. Extrllulr ATP inus th umultion of suproxi vi NADPH oxiss in Ariopsis. Plnt Physiol. 14: Stmlr, M.E., Stmk, G.W. n Rortson, J.A ATP msurmnts otin y luminomtry provi rpi stimtion of Urplsm urlytium growth. J. Clin. Miroiol. 25: Sun, J., Zhng, C., Zhng, X., Dng, S., Zho, R., Shn, X. n Chn, S Extrllulr ATP signling n homostsis in plnt lls. Plnt Signl hv. 7: Tnk, K., Gilroy, S., Jons, A.M. n Sty, G. 21. Extrllulr ATP signling in plnts. Trns Cll iol. 2: Thoms, C., Rjgopl, A., Winsor,., Dulr, R., Lloy, A. n Roux, S.J. 2. A rol for tophosphts in xnoioti rsistn. Plnt Cll 12: Vil, G., Ris-Cro, M., Grmir, M., Durtrt, G., Rsmusson, A. G., Mthiu, C., Foyr, C. H. n Pp, R. 27. Lk of rspirtory hin omplx I impirs ltrntiv oxis nggmnt n moults rox signling uring liitor-inu ll th in too. Plnt Cll 19: Yng, S.W., Kim, S.K. n Kim W.T. 24. Prturtion of NgTRF1 xprssion inus poptosis-lik ll th in too Y-2 lls n implits NgTRF1 in th ontrol of tlomr lngth n stility. Plnt Cll 16: Yun, S. n Lin H.H. 28. Rol of sliyli i in plnt ioti strss. Z. Nturforsh. C. 63:

Hydrogen Peroxide and Nitric Oxide are Involved in Salicylic Acid-Induced Salvianolic Acid B Production in Salvia miltiorrhiza Cell Cultures

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