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

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1 Moluls 2014, 19, ; oi: /moluls Artil OPEN ACCESS moluls ISSN Hyrogn Proxi n Nitri Oxi r Involv in Sliyli Ai-Inu Slvinoli Ai B Proution in Slvi miltiorrhiz Cll Culturs Hongo Guo, Xiolin Dng n Jun Dong * Stt Ky Lortory of Crop Strss Biology for Ari Ars, Collg of Lif Sins, Northwst A&F Univrsity, Yngling , Chin; E-Mils: hguo@nwsuf.u.n (H.G.); ongj@nwsuf.u.n (X.D.) Ths uthors ontriut qully to this work. * Author to whom orrsponn shoul rss; E-Mil: zsys@nwsuf.u.n; Tl.: ; Fx: Riv: 27 Mrh 2014; in rvis form: 24 April 2014 / Apt: 4 My 2014 / Pulish: 8 My 2014 Astrt: Hyrogn proxi (H 2 O 2 ) n nitri oxi (NO) r ky signling moluls in lls whos lvls r inrs in rspons to vrious stimuli n r involv in plnt sonry mtolit synthsis. In this ppr, th rols of H 2 O 2 n NO on slvinoli i B (Sl B) proution in sliyli i (SA)-inu Slvi miltiorrhiz ll ulturs wr invstigt. Th rsults show tht H 2 O 2 oul signifintly liit y SA, vn though IMD (n inhiitor of NADPH oxis) or DMTU ( qunhr of H 2 O 2 ) wr mploy to inhiit or qunh intrllulr H 2 O 2. Ths liit H 2 O 2 lvls signifintly inrs NO proution y 1.6- n 1.46 fol in IMD+SA n DMTU+SA trtmnts, rsptivly, n inu n 4.85-fol Sl B umultion, rsptivly. NO ws lso mrkly liit y SA, in whih L-NNA (n inhiitor of NO synths) n PTIO ( qunhr of NO) wr us to inhiit or qunh NO within lls, n th inu NO oul signifintly nhn H 2 O 2 proution y n 1.37-fol in L-NNA+SA n PTIO+SA trtmnts, rsptivly, n n 1.50-fol for Sl B umultion, rsptivly. Ths rsults init tht liittion of SA for ithr H 2 O 2 or NO ws inpnnt, n th liit H 2 O 2 or NO oul t inpnntly or synrgistilly to inu Sl B umultion in SA-liit lls.

2 Moluls 2014, Kywors: Slvi miltiorrhiz; sliyli i; hyrogn proxi; nitri oxi; slvinoli i B; ll ultur 1. Introution Plnts provi wi vrity of sonry mtolits usful to mnkin, suh s phrmutils, foo itivs, flvors, n othr inustril mtrils. Oftn high-vlu sonry mtolits r foun in low unn in Ntur [1]. Bus of th high ost n unrliility of hrvsting prouts from nturl rsours to fulfill th ns of phrmutil inustry, strtgis suh s trting with liitors to inrs thir proution hv n us in plnt ll ulturs [2,3]. Mny sonry mtolits hv n osrv in muh highr onntrtions in liit ultur lls thn in whol plnts of th sm spis [1,2]. Sliyli i (SA), phnoli ompoun roly istriut in plnts, hs n rogniz s rgultory signl miting plnt rspons to ioti strsss suh s rought, hilling, hvy mtl tolrn, ht n osmoti strss [1]. Most of rsrh on this hormon fouss on its rol in lol n systmi rspons ginst miroil pthogns, n on fining th trnsution pthwy ling to gn xprssion inu y SA [4]. Howvr, knowlg out its rol in nhning th proution of sonry mtolits s n liitor is rthr limit. Hyrogn proxi (H 2 O 2 ) n nitri oxi (NO) r ky signling moluls prou in lls in rspons to vrious stimuli n involv in ivrs rng of plnt signl trnsution prosss [5]. On of th rlist rsponss of plnts to vrious stimuli is th proution of tiv oxygn spis (AOS) suh s H 2 O 2, n suproxi nion. It hs n foun tht th H 2 O 2 -svnging nzym tls hs n isolt s SA-ining protin, whih n intivt y SA n its tiv nlogs [6], ut trtmnt with SA n inrs H 2 O 2 lvls, n thus H 2 O 2 is propos to th ownstrm signl of SA [7]. Howvr, this ft nnot hrmoniz with th osrvtion tht SA is rquir for inution of systmi quir rsistn (SAR) protins y H 2 O 2, n tht tls intivtion hs ltrious ffts on plnt hlth [8]. In ft, th rltion twn H 2 O 2 n SA in oth tivtion of fns ginst pthogns n umultion of sonry mtolits is urrntly not lr. In plnts, NO hs n intifi s son mssngr uring hyprsnsitiv rspons (HR), n n nhn plnt growth, ly snsn n tivt phytolxin proution. Som stuis hv highlight th puttiv links twn NO n oxitiv strss, whih suggst tht NO plys prominnt rol uring HR, n omplx funtions tht r oth omplmntry n omptitiv to thos of H 2 O 2 [9,10]. Although onsirl vns hv n m in unrstning th rol of rox signls uring oth HR n SAR, ritil spt rquiring furthr invstigtion is how signls suh s H 2 O 2 n NO n spifi [7]. Th rlt prolm is how plnts rspon to ths signls whn using SA-inu signling systm. Th omplx intrtions twn H 2 O 2, NO n SA my volv from th n for pthogn fns pthwys n proution of sonry mtolits.

3 Moluls 2014, Slvinoli i B (Sl B) is on of fftiv ompouns list in qulity ontrol of Dnshn, th root n rhizom of Slvi miltiorrhiz Bung, whih is us s tritionl Chins hrl rug for rmoving loo stsis, llviting pin, promoting th irultion of loo, promoting mnstrution, trnquilizing th rin, n trting riovsulr n rrovsulr iss [11,12]. Phnoli ompouns hv n provn to hv signifint iotivitis suh s ntioxint, nti-ishmi rprfusion, n ntithromoti ffts, n Sl B, on of thm, shows grt fr ril svnging n ntioxint tivity [13,14]. Th im of this work is to rvl th rol of H 2 O 2 n NO in SA-inu Sl B proution in S. miltiorrhiz ll ulturs. For this purpos, oth imthylthiour (DMTU) n roxy-2-phnyl- 4,4,5,5-ttrmthylimizolin-1-oxyl-3- oxi (PTIO) wr mploy s svngrs of H 2 O 2 n NO, rsptivly, n imizol (IMD) ws us to inhiit th nzym tivity of NADPH oxis. 2. Rsults n Disussion 2.1. H 2 O 2 Proution in SA-Inu Clls n Its Efft on Sl B Aumultion Diffrnt trtmnts using SA, IMD, CAT n DMTU wr rri out in orr to invstigt th proution of H 2 O 2 in SA-inu lls n its fft on Sl B proution. Th ontnts of H 2 O 2 n Sl B in SA-inu Slvi miltiorrhiz ll ulturs wr n 8.37-fol highr thn tht of H 2 O, rsptivly (Figur 1). By ontrst, th xognous H 2 O 2 lso signifintly inrs th ontnts of H 2 O 2 n Sl B (3.25- n 6.05-fol, rsptivly), ut oth wr lowr thn thos of SA-inu lls. Figur 1. Effts of sliyli i (SA), H 2 O 2, imizol (IMD), tls (CAT), imthylthiour (DMTU) n omplx trtmnts on th proution of Sl B n H 2 O 2 in Slvi miltiorrhiz ll ulturs. Th onntrtions for SA, H 2 O 2, IMD, CAT n DMTU wr 22 mg L 1, 10 mmol L 1, 100 μmol L 1, 100 U n 500 μmol L 1, rsptivly. SA ws to suultur Slvi miltiorrhiz lls ftr 8 ultur, n oth ontnts of H 2 O 2 n Sl B wr trmin in th susqunt 8 h n 2, rsptivly, with thr rplitions. Diffrnt lowrs lttrs rprsnt signifin t 5. Sl B( mg/g) Sl B H 2 O f f f f H 2 O SA H 2 O 2 IMD IMD+SA CAT CAT+SA DMTU DMTU+SA H 2 O 2 (umol/l. gfw)

4 Moluls 2014, IMD is n inhiitor of NADPH oxis in th plsm mmrn, whih n inhiit th proution of H 2 O 2 y intivting NPDPH oxis. DMTU is H 2 O 2 qunhr tht n fftivly rmov H 2 O 2 within lls. Nithr IMD nor DMTU trtmnt fft th ontnts of Sl B n H 2 O 2 whn ompr with H 2 O, initing oth hmils with suh onntrtions hv no ltrious fft on ll growth n thir ility to prou H 2 O 2 n Sl B. Although oth IMD+SA n DMTU+SA trtmnts signifintly rs th ontnts of H 2 O 2 n Sl B, whn thy wr ompr with SA-liit lls, oth ontnts wr signifintly highr thn tht of H 2 O. This inits tht H 2 O 2 n still mrkly prou in SA-liit lls, vn though oth IMD n DMTU r to svng or qunh intrllulr H 2 O 2. At th sm tim, this H 2 O 2 liit y SA signifintly inrss th umultion of Sl B (Figur 1). CAT is svngr of H 2 O 2, whih nnot pss through th ll mmrn, n thus th xognous CAT nnot svng intrllulr H 2 O 2. In this ppr, th ontnts of Sl B n H 2 O 2 in CAT trtmnt wr roughly quivlnt to H 2 O (Figur 1), initing CAT t suh onntrtion hs no hrmful fft on lls. Similrly, thr ws no iffrn twn CAT+SA trtmnt n SA-liit lls in trms of H 2 O 2 proution, whih my orrlt with th ft tht th xognous CAT nnot svng H 2 O 2 within lls. Th Sl B umultion in CAT+SA trtmnt, mnwhil, ws signifintly lowr thn tht of SA-liit lls, whih my u to th ft tht CAT n in som of th SA n thus iminish its liittion fft on Sl B proution. Figur 2. Effts of SA, soium nitroprussi (SNP), N ω -nitro-l-rginin (L-NNA), roxy-2-phnyl-4,4,5,5-ttrmthylimizolin-1-oxyl-3-oxi (PTIO) n omplx trtmnts on th proution of Sl B n NO in Slvi miltiorrhiz ll ulturs. Th onntrtions for SA, SNP, L-NNA n PTIO wr 22 mg L 1, 0.5 mmol L 1, 2 mmol L 1 n 50 μmol L 1, rsptivly. SA ws to suultur Slvi miltiorrhiz lls ftr 8- ultur, n in th susqunt 4 h n 2, oth ontnts of NO n Sl B wr trmin, rsptivly, with thr rplitions. Diffrnt lowrs lttrs rprsnt signifin t 5. Sl B (mg/g) Sl B NO NO ( umol/l) H 2 0 SA SNP L-NNA L-NNA+SA PTIO PTIO+SA

5 Moluls 2014, NO Proution in SA-Inu Clls n Its Efft on Sl B Aumultion To invstigt th proution of NO in SA-inu lls n its fft on Sl B umultion, trtmnts inluing SA, SNP (NO onor), L-NNA n PTIO wr mploy. In SA-liit lls, th ontnts of NO n Sl B wr inrs y n 8.29-fol, rsptivly, ompr with H 2 O. In omprison, th xognous SNP inrs oth ontnts y n 6.80-fol, whih wr lowr thn in oth SA-liit lls (Figur 2). L-NNA is n inhiitor of NO synths (NOS), whih n fftivly inhiit th proution of NO y intivting NOS, n PTIO is qunhr of NO, whih n fftivly svng NO within lls. Th rsults show tht no iffrn in ithr Sl B n NO ontnts ws foun twn L-NNA trtmnt n H 2 O, or twn PTIO trtmnt n H 2 O, whih inits tht nithr L-NNA or PTIO trtmnt uss hrmful ffts on NO proution n Sl B umultion uring ll growth. Whn ompr with SA-liit lls, nithr omin L-NNA+SA or PTIO+SA trtmnt signifintly rs th ontnts of Sl B n NO, ut oth ontnts wr mrkly highr thn H 2 O with ny of th omin trtmnts, showing tht NO still n ffiintly prou in SA-inu lls spit th ft tht L-NNA n PTIO r to inhiit or qunh intrllulr NO, n th NO liit y SA inus lrg mount of Sl B umultion Proution of NO Eliit y SA n Its Efft on th Aumultion of H 2 O 2 n Sl B In orr to isovr th omplx intrtions twn H 2 O 2, NO n SA, th proution of NO liit y SA n its fft on th umultion of H 2 O 2 n Sl B wr invstigt y using SNP, L-NNA n PTIO trtmnts. Figur 3. Effts of SA, SNP, L-NNA, PTIO n omplx trtmnts on th proution of Sl B n H 2 O 2 in Slvi miltiorrhiz ll ulturs. Th onntrtions for SA, SNP, L-NNA n PTIO wr th sm s Figur 2. Th ttion tim for trmining oth ontnts of H 2 O 2 n Sl B ws th sm s Figur 2. Diffrnt lowrs lttrs rprsnt signifin t 5. SlB( mg/g) SlB H 2 O H2O 2 (umol/l.g FW) H 2 O SA SNP L-NNA L-NNA+SA PTIO PTIO+SA

6 Moluls 2014, On on hn, th ontnts of H 2 O 2 n Sl B wr slightly rs in oth L-NNA n PTIO trtmnts, whn ompr with H 2 O, whih shows tht th nwly prou NO within lls in ithr L-NNA or PTIO trtmnt (Figur 2) nnot prou mor H 2 O 2 n Sl B thn H 2 O (Figur 3). On th othr hn, s mntion ov, NO oul signifintly liit y SA in ithr L-NNA+SA or PTIO+SA trtmnt (Figur 1), n H 2 O 2 proution ws lso inrs n 1.37-fol, rsptivly, n Sl B ontnt ws nhn n 1.50-fol, rsptivly (Figur 3). Ths rsults init tht NO liit y SA signifintly gnrts H 2 O 2 n thus mrkly promots th umultion of Sl B Proution of H 2 O 2 Eliit y SA n Its Efft on th Aumultion of NO n Sl B Th proution of H 2 O 2 liit y SA on th umultion of NO n Sl B wr lso invstigt y using IMD n DMTU in orr to illustrt th rltionships twn H 2 O 2, NO n SA. Firstly, th ontnts of NO n Sl B in oth IMD n DMTU trtmnts wr slightly inrs, ut not signifintly, showing tht th nwly prou H 2 O 2 in IMD n DMTU trtmnts (Figur 1) is not l to liit mor NO n Sl B thn H 2 O (Figur 4). Sonly, s xhiit in Figur 2, H 2 O 2 oul signifintly inu y SA in oth IMD+SA n DMTU+SA trtmnts, n ths liit H 2 O 2 mrkly inrs NO proution y n 1.46-fol, rsptivly, n nhn Sl B to n 4.85-fol, rsptivly (Figur 4). Ths rsults show tht H 2 O 2 liit y SA signifintly prous NO n thry stimulting th umultion of Sl B. Figur 4. Effts of SA, xognous H 2 O 2, IMD, DMTU n omplx trtmnts on th proution of Sl B n NO in Slvi miltiorrhiz ll ulturs. Th onntrtions for SA, H 2 O 2, IMD, CAT n DMTU wr th sm s Figur 1. Th ttion tim for trmining oth ontnts of NO n Sl B ws th sm s Figur 3. Diffrnt lowrs lttrs rprsnt signifin t SlB NO Sl B (mg/g) NO (umol/l) H 2 O SA H 2 O 2 DMTU DMTU+SA IMD IMD+SA 2.5. Both H 2 O 2 n NO Mit th SA-Eliit Sl B Proution As xhiit in Figurs 1 n 2, oth H 2 O 2 n NO oul liit in SA-inu lls, n h of thm oul promot th umultion of Sl B inpnntly. Th nwly prou NO liit y SA oul inu H 2 O 2 proution n vi vrs, n th umultion of Sl B wr ll signifintly

7 Moluls 2014, nhn in oth situtions (Figurs 3 n 4). In Figur 5, th ontnt of Sl B ws signifintly highr ftr SNP + H 2 O 2 + DMTU + IMD trtmnt thn tht in H 2 O, whih lso shows tht NO n t inpnntly, n so n H 2 O 2, us th Sl B prou from SNP+H 2 O 2 +L-NNA+PTIO trtmnt ws signifintly highr thn tht of H 2 O (Figur 5). Figur 5. Effts of iffrnt trtmnts on th proution of Sl B in Slvi miltiorrhiz ll ulturs. 1: ontrol (H 2 O); 2: SA; 3: SNP + H 2 O 2 ; 4: SNP + H 2 O 2 + SA; 5: SNP + H 2 O 2 + DMYU + IMD; 6: H 2 O 2 + SNP + PTIO + L-NNA. Th ttion tim for trmining Sl B proution ws th sm s Figur 1. Diffrnt lowrs lttrs rprsnt signifin t 5. SlB ( mg/g) On th othr hn, th Sl B ontnt in ithr SNP + H 2 O 2 + DMTU + IMD or SNP + H 2 O 2 + L-NNA + PTIO trtmnt ws signifintly lowr thn tht of SNP + H 2 O 2, whih inits tht NO n H 2 O 2 n t synrgistilly. This synrgisti tion ws lso xhiit twn SA n SA+SNP+H 2 O 2 trtmnts, in whih Sl B proution in th formr ws signifintly lowr thn tht of th lttr (Figur 5) Disussion SA hs n intifi s ky omponnt of th SAR signl trnsution pthwy [15] n n inu gn xprssion rlt to iosynthsis n proution of sonry mtolits in plnts [2,16]. H 2 O 2 oms to light s son mssngr involv in SA-liit pthwy [8,17]. In this ppr, umultion of H 2 O 2 n Sl B inrss n 4.38-fol, rsptivly, in SA-liit S. miltiorrhiz ll ulturs (Figurs 1 n 2), whih is onsistnt with th rport tht SA n inrs H 2 O 2 lvls [6]. At th sm tim, xognous H 2 O 2 n lso nhn oth nzym tivitis n Sl B proution [2], n 6.05-fol inrs of Sl B proution ws foun in this ppr. In ft, thr r mny rports highlighting th ft tht H 2 O 2 mits th liitor-inu umultion of sonry mtolits [2]. On th othr hn, if H 2 O 2 proution ws inhiit y IMD or qunh y DMTU, th ontnt of H 2 O 2 i not rs signifintly, whih my ttriut to propos mhnism of proution (Figur 6). In this proution mhnism, C 2+, NO, tivtion of trnsriptionl ftors (TFs) n TF gns, SA n ROS signling my ll involv to prou rtin mount of H 2 O 2 for th sl lvl of ll ns [2]. This intrply twn SA n ROS signling lso ours in too n Ariopsis, in whih pplition of H 2 O 2 n SA inus

8 Moluls 2014, h othr [18 20]. Notly, ll inhiition us y IMD, CAT n DMTU n rvrs in SA-liit lls, of whih intivtion of CAT is th most prominnt (Figur 1). Rsrhs hv rvl tht CAT n othr H 2 O 2 -svnging nzyms n oun n intivt y SA n its tiv nlogs [6,10]. This my th most importnt rson why H 2 O 2 still n lrgly prou in SA-liit lls, vn though CAT hs n (Figurs 1 n 6). Figur 6. A propos shmti illustrtion of H 2 O 2 n NO rols in SA inu Slvi miltiorrhiz ll ulturs. Th irl shows ngtiv rgultion. SA: sliyli i; CAT: tls; NO: nitri oxi; Sl B: slvinoli i B. In plnts, NO hs n rport s son mssngr uring HR, xhiiting funtions tht r oth omplmntry n omptitiv to thos of H 2 O 2 [9,21]. It n lso rvrsily inhiits ytohrom oxis n O 2 uptk in oth niml n plnt mitohonri [18], n inhiition of ytohrom oxis tivity my inrs th ltron flow from uiquinon towrs oxygn, thry stimulting suproxi n H 2 O 2 formtion [10]. This is onsistnt with our osrvtion tht SNP trtmnt (NO onor) inrs H 2 O 2 y 1.75-fol (Figur 3). On th othr hn, NO ws foun to inu xprssion of PAL n hlon synths (CHS) [22], n to nhn Sl B proution tht ws 6.86-fol highr in SNP-trt lls thn H 2 O (Figur 3). Trnsriptionl profiling lso show tht NO trtmnt inu som strss- n iss-rlt signl trnsution omponnt gns n fns gns, implying tht th NO signl pthwy(s) my rlt to sonry mtolism [23]. Togthr, NO my triggr gn xprssion vi t lst two pthwys, on ls to phnylpropnoi n flvonoi iosynthsis through tivting oth TF n sonry mtolism gns, n son tht ls to NO signling pthwy, inluing NOS, GMP, ADP rios, C 2+ influx, protin kins n tivtion of TFs (Figur 6) [2]. If NO ws inhiit y L-NNA (NOS inhiitor) or rmov y PTIO (NO svngr), th ontnts of NO within lls rs slightly ut not signifintly whn ompr with H 2 O (Figur 2). This phnomnon inits tht thr is mhnism for NO proution, in whih NR my plys n importnt rol involv in H 2 O 2 - n/or ABA-mit NO proution [5,24 26]. It hs n foun tht NO lvls in th root of wil-typ Ariopsis plnts hv n shown to inrs 8-fol ftr H 2 O 2 trtmnt [27]. In this ppr, n inrs of 60% for NO proution ws osrv ftr tht xognous H 2 O 2 ws to lls. Intrstingly, NO proution woul not inhiit ut lvt whn IMD n DMTU wr to inhiit th tivity of NADPH oxis n to svng H 2 O 2, rsptivly (Figur 4). As propos in Figur 6, whn H 2 O 2 proution ws inhiit, NO

9 Moluls 2014, umultion woul nhn to rls H 2 O 2 y tivting NOS n rgulting O 2 uptk [28], whih minly pns on th son NO signling pthwy mntion ov. Th othr optionl rout my rlt to SA n ROS signling, ut this ns to pn on th NO signling pthwy to prou NO too. 3. Exprimntl Stion 3.1. Cll Cultur n Trtmnt Th til protools of S. miltiorrhiz ll ultur n SA liittion trtmnt wr provi y Dong t l. [13]. Stok solutions of H 2 O 2, DMTU (H 2 O 2 svngr, Sigm, Shnghi, Chin), CAT (Sigm), IMD (NADPH svngr, Sigm), soium nitroprussi (SNP, NO onor, Sigm), N ω -nitro- L-rginin (L-NNA, inhiitor of NO synths, Sigm) n PTIO (NO svngr, Sigm) wr prpr in istill wtr n thn striliz ftr filtrtion through 0.22 μm mmrn. To svng H 2 O 2 n NO in S. miltiorrhiz ll ultur, finl onntrtions of 500 μmol L 1 DMTU n 50 μmol L 1 PTIO wr sprtly to th ll ultur 30 min for SA trtmnt. All xprimnts wr prform with thr rplits Dtrmintion of H 2 O 2 n NO Th lli ( g) wr homogniz with ton (5 ml) prool low 4 C n thn groun to homognt on i. Th mixtur ws ntrifug t 10,000 rpm for 10 min t 4 C, n 5% titnous sulft (0.5 ml) n onntrt mmoni wtr (2 ml) wr to 2 ml of suprntnt. Aftr mixing, it ws ntrifug t 10,000 rpm for 10 min t 4 C n th suprntnt ws rmov. Th pllt ws issolv y 2 mol L 1 sulphuri i (5 ml) ftr rmoving pigmnts y using ton. This solution ws us for H 2 O 2 trmintion n th sorn ws msur t 415 nm. Th ttion ws trmin ftr 8 h SA-inution n this point of tim ws trmin y xprimnt h of ttion. Th ontnt of NO ws trmin oring to th proution of nitrit with Griss rgnt tht oul oxiiz NO to nitrit unr ii onitions [29]. Th S. miltiorrhiz ll ultur ws lh with 0.22 μm mmrn n Griss rgnt (1 ml) ws to 1 ml of filtrt. Th mixtur ws inut for 30 min t room tmprtur to onvrt nitrit into purpl zo-y n th sorn ws trmin t 550 nm. Th ttion ws trmin ftr 4 h SA-inution n this point of tim ws lso trmin y xprimnt h of ttion Sl B Extrtion n HPLC Anlysis Th S. miltiorrhiz lls wr ollt from ll ulturs y ntrifugtion t 1,200 rpm, n thn ri t 47.5 C in n ovn to onstnt wight. Th ri lls (5 g) wr xtrt ultrsonilly with mthnol-wtr solution (2 ml, 7:3 v/v) for 45 min. Th xtrt ws filtr through 0.22 μm mmrn n th filtrt ws otin for ttion. Th ontnt of Sl B ws quntifi y HPLC nlysis tht ws prform using Shimzu (Kyoto, Jpn) systm quipp with UV/visil sorn ttor (DAD). A Shim-pk VP-ODS olumn (250 mm 4.6 mm, 5 μm) ws us t olumn tmprtur of 30 C. Th flow rt ws

10 Moluls 2014, ml min 1 n th injtion volum ws 10 μl. Th DAD ttion wvlngth ws 281 nm. Th xtrtion yil of Sl B ws lult s on th intgrtion of th hromtogrphi pk rs. Mor tils wr sri in Dong t l. [30]. 4. Conlusions Th prsnt work shows SA is n fftiv liitor inuing umultion of Sl B in S. miltiorrhiz ll ulturs. H 2 O 2 oul signifintly liit in SA-inu lls, vn though H 2 O 2 ws inhiit or qunh. Ths liit H 2 O 2 signifintly inrs NO proution n Sl B umultion. NO ws lso mrkly liit y SA unr th irumstns whr NO ws inhiit or qunh, n th inu NO oul signifintly nhn H 2 O 2 proution n Sl B umultion. Th liittion of SA for ithr H 2 O 2 or NO ws inpnnt, n th liit H 2 O 2 or NO oul t inpnntly or synrgistilly to inu Sl B umultion in SA-liit lls. Aknowlgmnts Th work ws support y Ntionl Nturl Sin Fountion of Chin (No ), n Ami Bkon Youth of Northwst A&F Univrsity (No. Z ). Author Contriutions Jun Dong oniv th rsrh projt. Xiolin Dng omplt th xprimntl stion n prt of t nlysis. Hongo Guo nlyz som t n wrot th mnusript. Conflits of Intrst Th uthors lr no onflit of intrst. Rfrns 1. Chnr, S.; Chnr, R. Enginring sonry mtolit proution in hiry roots. Phytohm. Rv. 2011, 10, Zho, J.; Dvis, L.C.; Vrpoort, R. Eliitor signl trnsution ling to proution of plnt sonry mtolits. Biothnol. Av. 2005, 23, Zhng, C.; Yn, Q.; Chuk, W.; Wu, J. Enhnmnt of tnshinon proution in Slvi miltiorrhiz hiry root ultur y Ag + liittion n nutrint fing. Plnt M. 2004, 70, Vint, M.R.S.; Plsni, J. Sliyli i yon fn: Its rol in plnt growth n vlopmnt. J. Exp. Bot. 2011, 62, Bright, J.; Dsikn, R.; Tnok, J.T.; Wir, I.S.; Nill, S.J. ABA-inu NO gnrtion n stomtl losur in Ariopsis r pnnt on H 2 O 2 synthsis. Plnt J. 2006, 45, Chn, Z.; Silv, H.; Klssig, D.F. Ativ oxygn spis in th inution of plnt systmi quir rsistn y sliyli i. Sin 1993, 262, Vn Cmp, W.; vn Montgu, M.; Inzé, D. H 2 O 2 n NO: Rox signls in iss rsistn. Trn Plnt. Si. 1998, 3,

11 Moluls 2014, Chmnongpol, S.; Willkns, H.; Mor, W.; Lngrtls, C.; Snrmnn, H., Jr.; vn Montgu, M.; Inz, D.; vn Cmp, W. Dfns tivtion n nhn pthogn tolrn inu y H 2 O 2 in trnsgni plnts. Pro. Ntl. A. Si. USA 1998, 95, Dllonn, M.; Xi, Y.J.; Dixon, R.A.; Lm, C. Nitri oxi s signl in plnt iss rsistn. Ntur 1998, 394, Durnr, J.; Shh, J.; Klssig, D.F. Sliyli i n iss rsistn in plnts. Trn Plnt Si. 1997, 2, Ntionl Committ of Chin Phrmopoi. Chin Phrmopoi; Chmil Inustry Prss: Bijing, Chin, 2010; p Shu, Z.; Wng, Z.; Mu, X.; Ling, Z.; Guo, H. A ominnt gn for ml strility in Slvi miltiorrhiz Bung. PLoS On 2012, 7, Dong, J.; Wn, G.; Ling, Z. Aumultion of sliyli i-inu phnoli ompouns n ris tivitis of sonry mtoli n ntioxitiv nzyms in Slvi miltiorrhiz ll ultur. J. Biothnol. 2010, 148, Zho, G.R.; Zhng, H.M.; Y, T.X.; Xing, Z.J.; Yun, Y.J.; Guo, Z.X.; Zho, L.B. Chrtriztion of th ril svnging n ntioxint tivitis of nshnsu n slvinoli i B. Foo Chm. Toxiol. 2008, 46, Stihr, L.; Muh-Mni, B.; Métrux, J.P. Systmi quir rsistn. Ann. Rv. Phytopthol. 1997, 35, Tguhi, G.; Yzw, T.; Hyshi, N.; Okzki, M. Molulr loning n htrologous xprssion of novl gluosyltrnsfrss from too ultur lls tht hv ro sustrts spifiity n r inu y sliyli i n uxin. Eur. J. Biohm. 2001, 268, Alvrz, M.E.; Pnnll, R.I.; Mijr, P.J.; Ishikw, A.; Dixon, R.A.; Lm, C. Rtiv oxygn intrmits mit systmi signl ntwork in th stlishmnt of plnt immunity. Cll 1998, 92, Lon, J.; Lwton, M.A.; Rskin, I. Hyrogn proxi stimults sliyli i iosynthsis in too. Plnt Physiol. 1995, 108, Ro, M.V.; Pliyth, G.; Ormro, D.P.; Murr, D.P.; Wtkins, C.B. Influn of sliyli i on H 2 O 2 proution, oxitiv strss, n H 2 O 2 -mtolizing nzyms. Plnt Physiol. 1997, 115, Shrsu, K.; Nkjim, H.; Rjskhr, V.K.; Dixon, R.A.; Lm, C. Sliyli i potntits n gonist-pnnt gin ontrol tht mplifis pthogn signls in th tivtion of fns mhnisms. Plnt Cll 1997, 9, Durnr, J.; Wnhnn, D.; Klssig, D.F. Dfns gn inution in too y nitri oxi, yli GMP n yli ADP rios. Pro. Ntl. A. Si. USA 1998, 95, Lvin, A.; Tnhkn, R.; Dixon, R.; Lm, C. H 2 O 2 from th oxitiv urst orhstrts th plnt hyprsnsitiv iss rsistn rspons. Cll 1994, 79, Aziz, A.; Poinssot, B.; Dir, X.; Arin, M.; Bzir, A. Lmrt, B.; Jourt, J.M.; Pugin, A. Lminrin liits fns rsponss in grpvin n inus prottion ginst Botrytis inr n Plsmopr vitiol. Mol. Plnt Miro. Intrt. 2003, 16,

12 Moluls 2014, Dsikn, R.; Griffiths, R.; Hnok, J.T.; Nill, S. A nw rol for n ol nzym: Nitrt ruts-mit nitri oxi gnrtion is rquir for sisi i-inu stomtl losur in Ariopsis thlin. Pro. Ntl. A. Si. USA 2002, 99, Gri-Mt, L.; Lmttin, L. Nitri oxi n sisi i ross-tlk in gur lls. Plnt Physiol. 2002, 128, Lu, S.; Su, W.; Li, H.; Guo, Z. Asisi i improvs rought tolrn of triploi rmugrss n involvs H 2 O 2 - n NO-inu ntioxint nzym tivitis. Plnt Physiol. Biohm. 2009, 47, Wng, Y.; Ris, A.; Wu, K.; Yng, A.; Crwfor, N.M. Th Ariopsis prohiition gn PHB3 funtions in nitri oxi-mit rspons n in hyrogn proxi-inu nitri oxi umultion. Plnt Cll 2010, 22, Poroso, J.J.; Prlt, J.G.; Lisro, C.L.; Crrrs, M.C.; Risi, M.; Shöpfr, F.; Cns, E.; Bovris, A. Nitri oxi rgults oxygn uptk n hyrogn proxi rls y th isolt ting rt hrt. Am. J. Physiol. 1998, 274, C112 C Hu, X.; Nill, S.; Ci, W.; Tng, Z. Hyrogn proxi n jsmoni i mit oligoglturoni i-inu sponin umultion in suspnsion-ultur lls of Pnx ginsng. Physiol. Plnt 2003, 118, Dong, J.; Liu, Y.; Ling, Z.; Wng, W. Invstigtion on ultrsoun-ssist xtrtion of slvinoli i B from Slvi miltiorrhiz root. Ultrson. Sonohm. 2010, 17, Smpl Avilility: Pls onnt th uthors y th uthors; lins MDPI, Bsl, Switzrln. This rtil is n opn ss rtil istriut unr th trms n onitions of th Crtiv Commons Attriution lins (