We analyzed Ore; production of reacfive oxygen species, the acctrmttlalian of salicylic acid (SA}, and peraxiclnsc activity cluving the; ~ n ~ o ~ ~ i p a ~ ~ ~ l e interaction botvueen cotyledans of the coitoti (Gossypftrnr hixsufumf CY Beba ~ ~ ( ~ ~ ~ ~ n ~ ~ ï { ~ ~ ~ ~ ~ r ~ ~ $ catnp?s,srrk pv niíilrr;rceanan IXcni) race 18, SA was detected in petioles af coty- Iedons G h after ítrfcction and 24 h post inoctrlatian in datyledons atid untreated leaves. The first gcak nF SA occurred 3 li after generation of su~ieroxidc (o;-), and was inhibited by infiltratíon of catalase. Peroxidase activity and accumulation of SA increased hi petioIcs of cotyledons and tcavm following H,CIL iafiltrntion of cotyleclans from 0.85 fe I nw. lnfjltration of 2 tiiM SR increased pcroxidasc activity in treated cotyledons and in trie first toavcs, but most of the ínfilirnkrl SA wa5 rapidly canjugarcd within the caty- hfons. When increasing coricentratiuns of SA werc irililtrated 2.9 h past in~culiltiati al tho bcginnfng af the axidative burst, the activity or the agoplastic C J ~ ~ Q R ~ C C2,'-Mgenerating peroxidase docreased in a dosc~depmdent mannet, We have shown that during f h ~ ~ cotton hypersensitive response to Xcrrr, Et#, is raquired far local. and systernic accumulation of SA, which may locally contra1 the goner. diori of O;-. netaching cotyledons at intervats &cr inoculation demrmtrated that the signal leading to systoinic accumutation aF SA was ernitted around 3 h post inoculation, and was associated wifh the oxidativc burst. SA produced C h pasf infection a l [IR sites was not. the primary mobile signal diffusing systcmieally from infected cotykdatts,

l'lie hypersensitive response (FIR) in plants is a mecha- nism of resislance to ptztliugenic ndcrobes, and is charac- terized by a rapid and locdizcd tissue colhpse resulting in necrotizaiion and inimahjliza&m uf the inkuding pathoe gen at. sitos of attack (Goodman and Novacky, '19941. Drrr- ing incompa tible inkcrackians followirzt; pathogenic stress, generation of reactive oxygen species (ROS) i.5 an evezit activated at the onset of &e H R (Lwiiie et: al., 1994; Lmib and Dixon, 199; Tiedemsnn et al., i.9971. Luca1 delensr:

genes to the invading p[ho[;cn are dso triggered md uuay esttmd EO the uninfectcd BSSLXBS surrounding the T-IR and

1997, I%%; Sticlicr et al., 1997). X,ocel i*esistance (I&) and systemic acquired resistance

($AR) are generally accompanicd by elevated levels of endogcnous salicylic acid (SA) (hifalsmy a i c l Klessig, 15292; Darey ct ai., 199%). Thew is strong ~videncc that ÇA plays il centrnl mIc in LR and SAR sipling (Malamy et at,, 1990; M&tram el al., 1998; Rasinussen el al., 19

it has been demonstrated thut infiltration of Adidopsis with SA induced the !ìfirne set: of $AR ganes as pathogen infection (Ward et al., 1391: Uknes et al., 1992). The induc- tion of U and SAR by SA might occur kh~m~glz generation of SA radicals, a likely by-product of the interaction uf SA tvith caialase and ~ ~ ? Y Q X ~ ~ E S @umer aiid KI&sjgj 1996). Purthernioxe, pian ts extgineaed for cons titutíve expression, of a bacterial salicylate hydrot;ylcme gene, which failed - to accumula kc SA at nurrnal levels, have severe prabll?ms in establisking I-11% axid SAR (Gaffmy et al, 1993; Dekaney kt

\flieklier SA is the plilaem-txanstocated signal that me- diales SAI? is still a matter uf dcbak. l?asmussen et al. (1'931) demon~tr$tted tlml SA is most likely not thc long- distance signal that Ieads tu Lhe induction of SAR, but instcad íti required faor hransducfion of the perceived long- distance signal leading to the onset of SAIL Similarly, wkik experiments clearly deunonstrated a correkttion hetwen 'che detection of SA in the phIoai and SAIt expression in pathogen-infected plants (Haaimond-Kosak and Jones, '1996; RynIs et. al,$ L986), they did nut prove that SA is thc long-diskmce mobile signal. Nevertheless, evideiicc of transport has cume from a dmmmtmtiori in tvhich the tmnslocaliofl of 'l"O-labeic?d SA was evidenced in tobacco mosaic virus-infected tobacco (Shulaev et al., 2995)* 11 ~t 'as shown titat methyl-SA, produced from SA trpon tobacco

the ~1101e p'la~t (ROM, 1901; Ityak ct d., 1996; DOXY e l al.,

Klessig, 16912; Dorey et al., 1997; Burx1lrr et ar., 1997)' silice

al., 1994). -

75 8 Martinet et al. ~ PlalIl Physiol. Vol. 122, 2000

mosaic virus infection of tobacco, may function as ali air- borne signal (Shulaev et al., 2997; Seskar et al,, 3998).

Resistance of cotton (Gossyph Ifirsutinn) plants to the bacteria1 pathogen Xunilro/rrom~~ crr~riyesfris pv. n d t m w z r i t i i i (Xcm) i 5 mediated by a gene-for-gene interaction (De Peyter et al., 1993). In the incompatible interaction between cv Reba 050 and thc avirufezlt race I S of XcniJ a slxirp production of superoxide (O,-.) was characterized at I-IR sites 3 11 after cotyledon infection, followed by an accumu- lation of H,O, between 4 and fi 11 post inoculation (Mar- tinez et al., 1998). Ganeration of was demonstrated to be mediated by an a;~oplastic cationic NARH-p-peroxidase, wJiîle the constituti'i'c NADPH-oxidase remained inactivc (Martinez et: al., 1998). Resistance of cotton plants was associated wirk a skong iircrease in anionic-peroxidase activiti.ies botlx at HR sites in cotyltrdotls 12 1x after ,kfectiOn and systemically i n Icilves 24 h after cotJkdon infection (Martinez et at., 1996). The activity of perosidase is a uSefui marker for LR and SAXI, in cotton plants clxailcngcd by ail avirulent isolate of Xcm.

We studied Lhe relationships betwcen the oxidative burst and the actixlties of SA and peroxidase. SA has been pre- viously cvidcnced to act upstream 1997) ar down- siream (Leon et al., 19%) to the production of ROS. Riese;, fore, i t was of interest to fuirrther investigate the cole of both SA and l-I2Cl2 inoleculcs during an incoinpatible interaction between cotton plants and Xcm race 18 Lo better under- stand the time sequence of events leading to cotton LI3 and SAR. J

MATENAL5 AND METHODS

Plant Material and Bacteria1 Strains

Two cotton (Gossypitrnz Irirsrrtuitr) varieties were used in this skudy, The susceptible Acala-44 variety posscsss no known major genes for resistalice to Xcm (Hunter et al., 1968; De Feyter et al,, '1993). T h cv Reba U50 (A1lt;rx X Stoneville 2E), similar to aie Lutlier Bird's 101-102B h e , contains the B2E3'bligl1t resistance key genes. Associated with resistant determinaits introduced from Einplre WR and MVW (Brinbrhoff et: al,, 198$), tliosc genes confer imiunity to all Xcm races, except. race 20 {Innes, 1983; Hillocks, 1992). Ten-da y-old cotylcdmrs of both varieties were inoctilated

with Xcm race 18 or race 20, collected in cotton fields ín Burkina Faso, by idilkration of the bacterial suspension (IO7 colony forming units [cfu]/mL) (Daï et al., 3996). This gives an initial bacterial density of about 4.W cfu/cm2. So far, thrce interactions have been investigated: the incom- patible one (cv Reba B50IXcm race 18) <and the two com- patible ones (cv Reba 85O/~cni race 20 and cv Sicala-&/ Xcm race 18). Controls consisted In p)ank fiam each variety that were infiltratcd with sterile water. Plants tverc grown in a greenhouse at 3J'C 2 1°C under 80% humidity.

Collection of Petiole Exudates

After Xcm inoc&tion of cv Reba I350 or cv Ada-44 cotykdons, apoplastíc wasMg fluids (AWF) were pre-

pared by vaciium infiltration of petioles of fresh cotyledons (Rasmussen et: al,, 1992). Petioles were cut a t bath their stem and cotykdon ends and were washed with distillcd water. They were iiiirnerged in a Petri didi for 15 min in 50 ~IIM sodium acetate, pl-l &O4 containing 0.25 RI NaCI. Vac- uuin $vas applied and slowly released, F'etioles were then introduced verficaJIy i n Eppendorf tubcs and centrifuged (~5,OOflg per min); 20 to 30 pL per g of petiole of A M T were 0bt.ahied. An ~ L M I volume of methanol was then acldcd to the AWF. Exudates izferc collectcd U, L 3, (i, and 12 h and I, 2,3,4,5,7, and 9 cl after inoculation. 111 parallel, activity of the cytoplasmic ciuyme Glr-6-P dchydrogcnase (ECI.1.J A V ) was itssn>d in thc AWP to detcct cytoplasmic conkm~inn Eion.

HPLC Analysis of SA

Fifty micrditrts of nietlwiolic wtract-s of petiole exirdate were injeclcd onto a C,, COTUTW (250 X 4.6 mm; 5 Ihm; Lidvaspher 100 Rp I$, Alltech, Dcrrfield, IL) cquilibrnted wit11 5% (v/v) biiffcred acetonitrile (50 miv sodium acetate buffer, pX-J. 4.5). SA was clrilad isucratioally 15 min follow- ing injection, and cktected by fluorwenee (excitation, 290 nm; cmissicm, $02 nm). Concentration was determincd us- ing a Iinear range o€ calibration standards cottsisting in 0 to 1.3 ps/50 pl, of SA (Signia-Aldrich, St. 1,ouJs). SA concen- tratiorc was expressed in micrograms por grain fresh weight.

Cheniical hydrdysis of residues ww performud ut H O T in 500 pL of 2 M NaDl1. After 2.5 h, the hydrolysis mixture was acidified with 13Gl (to obtain a 4 M N[Cl solution) and Incubated for an additional 1 11 a t 80°C. The hydrolysis mixture was tlien centrlhgated at l,aOOg, and the supenxa- tant was partitioned end prepared fox HPLC (Enyedi ut al., 2992),

Assay of O,--Generatiríg Activity of Cotyledo? Discs

The Oi--generating activity of cotyledori discs \MS as- sayed s~ectroyhorometrìca~iy by measui-ing the reduction of esogeneously supplied cytochrome c a t 550 nm as pre viously described {Martinez et: al., 1998).

lsoelectris Focusing flEF} atrd Assay af Peroxidases

Foq IEF, cotyledons of each ppup of plants were mixed hi liquid nitrogen alid homogeriizcd 51 0.05 N sodium acetate buffer, pI-1 B,0, containing 25 mh1 p-niercapta- eilianol and 5% poIyvinylpolypyrolídonr (vlg fresh weight), After centrifugation (15 min a t X2sQOOg), the SU- pernatant liquid was filtered on polysulfone membrane (0.45 pm, Gelman, Pall FJ"LC~?, St. Germain en Laye, France). XEP was pcrfotmed atcording to the method of Robertson et al. (1987) on vestical plates (70 X SO mm, Dia-Rad Laboratofies, Hercules, CA). For the analysis of cationic peroxidase isoenzymes, the pld gradient of the gel ranged from 3.0 Eo 11, with a larger amount of pT-I 9.0/11 amplxolytes (0.6% anphalytes 3.0/10; 2.5% ampliolytes 9.0/11, from Scxva, Heide.lberg)< The anode solution con- sisted in 20 f y t ~ acetic acid and the cathode solution in 2.5

" . i ' . . '1

. . __ . . . .. _. ..

Re'eactivc Oxygen Specics and Sal icy1 ic Acid in Cotton HypcisensEtive Respnnse tu Xmhmonas 759

mhf NaOX I. The lane corresponding to the pl. markers was cut and staincd with Coomttssie Blue R-250 (Ncuhoff et al., 1988). After migration of proteins (40 p g of proteins per lam), peroxidase activity was revealed using 0.2% (CVJV) guaiacol, 0.01% @/v) 3-ainîno-g-etl~y~carb~~ole, and 0.03% (w/v) EI,O, in 0.05 M sodium pliospltate buffer, plf 6.0. Total peroxidase activity of crudc extracts was spectropho- . tometrically assessed a t 470 nm using only guaiacol as tlie hydrogen donor. Peroxidase activity was calculated using the molar cxtiiiction coefficient of tch;7guaiacol (26.0 x I O 3 mol-' mi"^") and spccsifk activity was expremed in nano- katals per milligram of total proteins.

lnfiltratlon of H2CJ2, 5A, Catalase, or Aminalriatolc

Cotyiedons were syringr-ixzfiltriited with MzOz (1 mat), SA (2 ni^), ralalasc (500 units/mL), or with the aminolria- zole (5 m) catalase inhibitor$ The 2 - m ~ SA solutiox~ was prepued by titration with 0,l iii NeOH to íi p1-I value aroutid 7.0. The effecis of SA on 0;- production and on thc activity of cationic poruxidases was observed 3 11 posk infection hy Xcm during the uxidative burst after infiltra- tion of SA realized 2S h post: inoculation. To deternine ihe effect of H,O2 on plant defmse responses, catalase and aminatriazule were also infiltrated 3 h after infectfan. In dose-dependent experimcnts, H202 was infiltrated with 5 m~ aminotriazole in concentrations of 0.5, 0.7, 085, 1.0, 5.0, 10, 50, 100, 150, 200, ancl 250 mbi, while SA wos injected at cmcentrations ul 20, SO, and 100 FM and 1.0, 2.U, and 3.0 n'iw,

Cotyleclon Excision Experiments

To determine the time of s i p a l h.ansniission leading to the expression of SAR, infeclcd cotyledons were excised from plmis O, 0.5, 1, 13, 2, 3, 6, 12, 24, 48, and 72 h alter inoculation, and SA coizkent was measured in pet-iolc cxu- date. The mxiinoculated leaves of the first: rank un S~IIIE

plants were excised 72 h after inoculation of coiyleduns, and exudates were collected kom pctiolcs €OK assessìng SA content and peroxidase wtivity, Estimation was performed on six seprated plants pw infected line and per time.

Bacterial Growth Determitiation

Bc7cterIal growth was detcrmirted by triturating infected cotyledons or leaves in sterile dcionizcd water. After serial dilutions, bacterial concentration were determincd by plate cotin.ts and cxpsessed a6 cfu per square'cwrtimctet. Estima- tion was perfomicd 4 d after inoculation, based on 10 reglicates per infectcd line and per snmplc.

-

RESULTS

Tirrte Course of SA Production during Infecfion

In the incompatible interaction m R6zba*@5U/Xcm race 18, SA was first detected 5 h aftcr jnfection in petide exudate from itlocuiated cotyledons with a peak at Gh (Fis;. IA). The Ievel ol SA then increased in both &e emdatcs of untreated

Ieaves and infected cotyledons 24 11 post inoctxlatinn, with a higher content in coqledons {Pig. 1B). 'In the compatiMe ititcractions cv AcalaalXcm race 18 and ctr .Reba BSOlXcm facc 20 (not shown), no significant increase in SA contexTt Wias detcctcd in petiole exudatm. PIants i ~ o m bath cultivars infiltrated with vat ter coiitained no detectable levd of SA. Xcm race t8-jnfected cotyledora co-infiltrated with catalase did not induce any significant production of SA.

Effect of SA on the Oxidative Burst

Thlr relations between $14 md tltc oxidative burst occur- ring in cobyyledons during the incompatible interaction cv Reba B50/Xcm race 18 were investigated 3 fi after inocu- Iatjon. Af ta Xcm inoculatiun (2.5 1.1) at -ille beginning of the oxidative burst, cotyledons were ixifiltxatett tvith increasing cancentmtinns uE SA; gemration of C.?,..- was assmessed via reduction of cytocht.ome c, Pigure 2 shows khat axfire reduc- tion decreased ia R dose-dependent m~tmer follotvkg SA treatments. The use of SOD {1,000 uni& mL-rf fnlribited the reaction, as previously shownby Martifiei! et d. (1948),

Three fraurs afkr irtfection, cationic peroxidases have previousiy been demonstrated to be responsible for the production of 0;- during the incompatible interaction cv Reba BSO/Xcm race 16 (Martinez ri al, 1998). A dase- response axperimmtl OR IEF gel was carried ou1 fo h e i + ti@e !he effect on the cationic peroxiclme activity of in- cteasing qtinnt.jtrit?s of SA inFiltrated 2.5 11 after XC~I inoculation. Guaiacol activities of the cationic pc~oxidases decreased in a dose-dependcnni nxmncr following SA infil- tration in cotyledons (Fig. 3). Furthermore, in non-infected plantsz the oxidative burst was nuver induced whatever the SA concentratioil (1-250 m ~ ) .

Effect of Mi@, on the Accumutation af SA

Prom previotrs cxpcriments, ít appeared that in t h in- compatible interaction cv Reba B50jXcm racc 18, a strong productlon of occmred 3 h aHcr infe'ecliaiz, foliowecl by accurnulatiori of: l-f2C12 britvcen 4 and 6 h post infection (Marinez et al., 1998). Rte het: that ROS in infectcd cotyledons prece suggested tlial F12Q nttd/rtr O,.- could induce SA accu- mulation. 11-1 this respect., noninfa!tcd co~ledoRts af cv R&ba E-50 zycre tscatcd wid1 increasing CQIXWII~XX~~~M'~ of 1-1,02 with aniinotriazole or not, and subsaqjtluntly assessed fus lhe accumuiation of h e SA. Free $A wa5 dctcctecl after inAltmtion a€ cntylcdms with 0.85 m~ H,O, plus aminatrlazale ur more. (Fig. 4). W i t h u t thc addition of aminofriazolc, H,O, induccd detectable SA from 150 mh-s.

Effect of SA on Perolidase Activities

A si@;nificant increase in thP total peroxidase activiiy essayed gects~ghotornetrically 72 h €onllowing ilnfiltratioii of increasing qoantities of SA in noninfected cv Reba 1350 cotyledoris and lea ve^, \.vas induced by 2 m~ SA aird more (Pig. 51, Infiltration of 2 ni^ SA in rntyleduns of both nanin€ecected cv Rebrt. B50 and cy Acnln-E4 resulted in an increase in the lord peroxidase activity in cotyledo~s 3 11

7 60

Figure 1. Effect of Xcni ríicc i O ori the cndoge- IlouS level af free SA in relationship tu timc (A, 0-12 li; 13, 0-240 h) in the pctiolcs of infected calton catyledons (0; 0 1 and the upper un- treated lcaves (1; U1 of the cv Reba B50 (O: .) and cv Acala-44 (O; O) 1inc.s. Thc! cotylcdons of each variety were inacuJatcd, and samples of petinlc phloctii cxudatc tvei'c collecicd ni vari- ous tiinos foilowing inkction. SA was separated by HI'LC and mcasurcd as clcscribcd in "Mate-

of t 0 repticales iron1 different plants. The arrow in A inclicalcs thc tirnu, at which thc oxidativc burst QCCU~S, SA cantcnl is cxpresscd as p g g"' frc51i matter (FMI.

rials and Methods." Each VtllUC is thC nlCi%n 2 SE

4

Martinez et al,

o 1:

dfer treahiiuits ancl. in the systemic peroxidase activity 12 11 a f t w infiltration (Fig. 6). No significant change was detected in plants injected with water.

Is SA Free or Conjugated followitig Infiltratiori in Cotton Cotyledons?

When cotyledon extracts froin rmlreatcd cott~li plants wexe analyzed, most of the SA was conjugated arid 'tvas found in hydrolyzed extract (time O-) (Pig. 7), JmnediateXy ahcr infiltration of 2 mhi SA, 85% of free SA was recovered from cotyledons {time O+). &If an hour later, only 40% w a ~ found in uiiliyclrolyzed extracks, while 25% was ex- tracted in the hydmlyzed past. Conjugation af 5A in- creased with timc, and 3 li after trcakfiicnt, 60% was fotind in the hydrolyzed extracts.

SA Accumulation and Peroxidase Activity following Infiltration of 1 ~ I M W,Q, Plus Aminotriaiale

The time course of 9A accumulation in cotyledons and leaves of both cultivars was annlyzd following infiltra tion of cotyledons with 1 ni^ €f,Or pluç amiriutriazaie hours after inatration, the level of SA increased it1 the two

cultivars, hut a t a lower í~ikqsiky in the susceptible Acala-44 variety (Fig, 8, A and D). An increase in SA contcnt: was detected in pctiolo exudate of untreated leaves fro111 12 li post inoculation. In water-infilti4atcd cotylectons and untreated lcavcs, na hicrease in SA was dcteclcd (not sl~own). The time coursu of total peroxidase activity after jn f i l tdon of cotylcdons with 3. 1 1 1 ~ H20, revealcd a sig- nificant increase bctween 12 and 24 11 post trcatmcnt in cotyledons of the two cultivars, while j n leaves, stimuiatiori of this activity was observed 24 h post irioculalion (Fig. 8, A and U).

Led Detachment Exfieriment in cv Reba B50 Plants Infectcd with Xcrn 'xh determine the time oft signal emission leading to S.AIt8

infectcd cotyledons were excised from plarils after irioru- lation; the content of SA was measured in petiole exudate ol leaves and cutyledons, and peroxidase activity was mea- sured in leaves.

Following hifiltraticai with race 15, infected cv Reba €350 cotyledons were detached at different times to detemine

time sequence of SA apparition in petioles of chtyle- dons and ieaves (Table 1). No SA was detected in petiole

761 Reactive Oxygen Species and Salicylic Add in Cottan Hypersensitive Response 'CO Xmttiomonnas

0.6 I" i 11

O 2 4 ci G 2012141611320222426

Time (min) Figure 2. Influence of $A on Od'-. production. cv Rebd B50 cotton cotyledons were infiltratcd tvith various contentratloriç of SA 2.5 ti after inoculation with Xcm race 1 O. Cotyledon discs were incubated in cytochromcrmedium, and OL'- production wa5 monitored by the reduction of cytochrome c 3 II post Intection. Each V ~ U E is thc mean SE of six replicates from driiemnt plants. E, cv Reba f350ficm race 18; A, 0.05 m~ SA; 0, 0.1 IY~M SA; O, 1 MM SA; a, 2 mh4 SA,

exudate of coty%cdoxxs before 3 h post inoculation. 'In leaves, SA was detect&l only when inoculated cotyledons were detached after 3 h post-inec~ilaH~n. This indiratrd khat ihe i;ignal khat induced accumulation of SA h i We first fionin- vculated leaves was translocated between 2 and 3 h nftecr inoculation4 AmIysls of tufal peroxidase activity in leaves showed a? increase oïdy if SA accxtmdated in petioles of lt7aves.

Similar experiments were conducted on cv Reba 850 cotyledons inoculated with Xcm race 20. ~'revjous works demonstrated that in tixis compntilsrle interactionf the Enfec- tion caused an hicreage in peroxidase activity in non-

'

I a b c d

Figure 3. Dose-dependont effect af SA on the aaivity of cationic pcroxidasc (pl 9-9.5) a$sessed following IFE, SA WS infiltratcd 2.5 It following inoculation with Xcm race 18 at cunc 0.05, O.?, 1 .O, 2.0, 3.0, and 5 O mr~i (lanes b h l , Bild peroxidase activity was assayed 3 II post infection, Lene with Xcm Face 18 only.

Figure 4, Uosc-dependent e (lone; 0; cv Reba El50 plants. different concentrations of bars) or with tlaQ, alane 0% after infiltration, Leveis of flf%C. Each value is the mmn plants.

infected Icnves (Martinez et al, l99G}< Mo- content was detwted ín htected catjdedans nor in the fht: leaves (not sltown). 'She sigLiificnnt increase in peroxidase activity occurred in leaves only if inlected cotyledons re- mained attached on plant 48 h after inoculation. This îndi- cated that the sipal txant"tted 2 d after infection of cv &ha 1350 Cotyledons tvlth Xcm race 20 induced an increase in peroxidase activity but did not trigger SA accmuhtion.

Effect of SAR on Bacterial Growth

sc Q

Tn cv Reba B50, pre-inoculation of cotyledons with Xcm race 18, followed by leaf post ificiCulati011 with the same

1200 - ..4 fi

1000

E 4

1 0 0.62 11.65 0-1 1 2 3

Salicylic Acid (mM) Figure 5, Duse-dependent efFcct of SA on peroxidase activity in coiyledons and lenves of ilic cotton cv Reba B5D. Cotyledons were infiltrated with vc\rlacts cancenkratíons rrf SA, and peroxidase acfivity was ineasuretl 72 11 later in extracts of catyleclorrs (black bars) and untreatccl leaves (white barst. Each duci i5 the fnean 2 SE of '10 replicates from different. pimp,.

762

O 2 4 6 8 1012141618202224

Time (hour) ,

Figure 6. Time course effect of 2 mM SA on peroxidase activity in cotyledons and leaves of the cotton cv Reba 850 and cv Acala-44. SA was infiltrated into cotyledons, and cotyledons and untreated leaves were harvested at the indicated times on the x axis and analyzed for peroxidase activity. M, cv Acala-44 cotyledon; U, cv Reba 850 cotyledon; O, cv Acala-44 leaves; O, cv Reba 850 leaves.

race, induced 50% inhibition of the bacterial growth com- pared with post inoculation of water-infiltrated cotyledons (Tables II and III); when leaves were post-treated with Xcm race 20, inhibition reakhed 70%. In plants whose cotyledons were pre-inoculated with Xcm race 20, about 30% of growth inhibition was observed whatever the race leaves were post-inoculated with. In similar experiments per- formed on cv Acala-44, about 20% inhibition of the bacte- rial growth was estimated.

Martinez et al. Plant Physiol. Vol. 122, 2000

a m l .- u

O O- O+ 0.5 1 3

Time (hour)

Figure 7. Free SA in cotton cotyledon extracts following hydrolysis. Cotyledons were sampled before infiltration of 2 mM SA (O-), and O (O+), 0.5, 1, and 3 h-after infiltration. Black bars, Free SA; white bars, conjugated SA. Each value is the mean C SE of six replicates.

- 3 3000 .$

Y

ii ?

$ 2 2000 4 9 Y 5 - 1 .r( 1000 3 .r( P 9

E L4

t? U

U

l .4

Kl al W

3 O 0 . 2 o

k O 1 2 3 6 1 2 2 4 4 8

Time (hour)

i

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bo 3

u .r( - .r( I

Kl

O

-.. T

O 1 2 3 G 1 2 2 4 4 8

Time (hour)

bo

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1000 5 Y

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0 5 X

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Figure 8. Time-dependent effect of H,O, (1 mM) on SA accumula- tion (hatched bars, cotyledons; white bars, leaves) and peroxidase activity in infiltrated cotyledons (E) and.upper untreated leaves (O) of cv Reba.. 850 plants (A) or cv Acala-44 (BI. Petioles of infiltrated cotyledons were harvested with the upper untreated leaves at the indicated times, and analyzed for SA content by HPLC. Each value is the mean i SE of 10 replicates from different plants.

DISCUSSION

The hypersensitive response of cotton to Xcm appears to follow the gene-for-gene concept leading to specific host cell death (De Feyter et al., 1993; 1998). In the incompatible interaction cv Reba B50/Xcm race 18, the production of 0;- 3 h after infection, followed by the accumulation of H202 (Martinez et al., 1998),.is an event that precedes two accumulations of free SA. The first one is locally produced in cotyledons at HR sites 6 h post inoculation, while the second occurs systemically from 24 h post inoculation. A strong stimulation of the total peroxidase activity was ob- served in cotyledons and leaves, and bacterial growth sig- nificantly decreased in post-inoculated leaves of plants whose cotyledons were pre-inoculated. As predicted, con- trols consisting of Xcm race 20-infected plants or infiltra- tion of Xcm race 18-infected cotyledons with catalase did not reveal any sigruficant production of SA or increase in peroxidase activity. TO better understand the roles of H202 and SA in cotton m, we further investigated effects of exogenous H202 and SA on events of the HR. The fact that the production of 0;- and the activity of the 0;--generating cationic per- oxidase were inhibited by increasing quantities of SA sug-

_I

i

Reactive Oxygen Species and Salicylic Acid in Cotton Hypersensitive Response to Xanfhomonas 763

Table I. Determination of signal emission in CY Reba 650 plants challenged by the Xcm avirulent race 18

of catalase-dependent degradation of H202 into molecular oxygen. But Co-infiltration with the catalase inhibitor ami- notriazole indicated that a concentration around 1 mM was able to induce significant effects in cotton cotyledons, in accordance with previous works indicating that the action of SA in SAR is mediated by elevated amounts of H202 (Wu et al., 1997).

h pglg fresh material nkavmg protein Infiltration of SA into cv Reba B50 or cv Acala-44 coty- ledons stimulated total peroxidase activity in cotyledons t o O O 7 0 2 18

72 2 20 and leaves (Figs. 5 and 6), in a manner similar to that in 0.5 O O 1 O O 75 2 15 1.5 O O 60 2 12 pathogen-induced resistance. Compared with SA content 2 O O 7 4 2 11 produced in cotton during infection, the relatively large 3 0.08 2 0.001 0.67 2 0.13 245 2 13 quantity of infiltrated SA (2 m) required to stimulate 6 0.3 2 0.015 0.65 2 0.1 237 -i- 20 peroxidase activity is explained by the possibility that SA

12 0.05 t 0.01 0.68 2 0.1 241 2 15 could be conjugated to Glc to detoxify plant tissues from 24 0.64 5 0.012 0.68 rf: 0.2 248 t 25 free SA (Enyedi et al., 1992; Hennig et al., 1993). In our

experiments, 30 min after SA infiltration, about 25% were 48 1.4 2 0.2 0.65 2 0.1 238 2 20 1.6 2 0.2 0.65 2 0.14 245 2 12 found to be conjugated. A cell wall-associated ß-glucosi- 72

72 h Post dase that releases SA from Glc has been identified in tobacco, suggesting that SA-ß-glucoside serves as an in- active storage form of SA (Chen et al., 1995). It is likely that in cotton a part of infiltrated SA escaped glycosyla-

The leaf detachment experiment conducted in our study

'Om cv Reba B50 plants prior post

plants challenged by X c m race 18. It is thus possible that the at the Onset Of sAR was generated around following cotyledon inoculation in parallel to (or resulting

the Oxidative burst. The higher level of SA frmnd in noninfected leaves compared With that in the inoculated cotyledons before ablation (Table 1) SbOnglY indicates that the systemically accumulated free SA did not originate from the inoculated cotyledons but, rather, was induced by another putative systemically translocated signal. Al- though our data revealed that cotyledon SA is likely not the primary S i P d that triggers SAR in cotton, they are Consis- tent with the involvement of SA in the cascade of down- stream events that are associated with HR and culminate with the manifestation of SAR. This is consistent with the observation that in the compatible interactions Reba 50/ Xcm race 20 and cv Acala-&/Xcm race 18, no increase in SA or peroxidase activity was detected and no limitation of the bacterial growth was recorded in post-inoculated leaves of plants whose cotyledons were pre-inoculated.

Data are the means of six replicates per time. SA Content

exudatea exudateb

Peroxidase Activity Time of Cotyledon Detachment after Cotyledon

inoculation Leaf in Leavesb

a At the time of cotyledon detaChment- inoculation.

gests that SA accumulating in cotyledons 6 h post infection may be involved in the control of the production of O;-. Previous studies have demonstrated that SA inhibits ascor-

enging H202, by serving as an electron-donating substrate

a better than an effective inhibitor (Kvaratskhelia et al., 1997). This role is supported by the redox deactivation by SA of the iron Faton reaction centers (Cheng et al,, 1996). Our observations (Figs. 2 and 3) indicate that in xcm race 18-infected cotton cotyledons, SA may cause perturbations of the cellular redox state and block the O;--generakg peros- dase activity. h addition to this putative re@ahg role for SA, the

marked activity of SOD observed just after generation of Oi- also likely contributed to local detoxication by 0;- dismutation into H202 (c. Martinez, E. Bresson, and M. Nicole, unpublished data). The application of 1 - H202 in aminotriazole-treated cv Reba B50 or cv Ac&-44 coty- ledons induced SA accumulation (Fig, 4) and caused an increase in total peroxidase activity (Fig. 8), as in the in- compatible interaction cv Reba B50/Xcm race 18 (Martinez et al., 1996). The physiological concentration at which H202 causes these effects in planta is difficult to evaluate because

and may as a .

bate peroxidase and catalase, w o key enzymes for scav-

(Dumer and mesSig, 1995), although SA was shown to be

I) 'lem'y that removing

the Of Reba B50

'

I

Table II. Effect of SAR on bacterial growth in cv Reba 650 Cotyledon Pre-Inoculation

Race 18 Race 20 Water

Leaf post-inoculationa Race 18 Race 20 Race 18 Race 20 Race 18 Race 20 Bacterial densityb 63 2 5 900 2 52 87 2 11 1,965 ? 110 130 + 15 3,024 2 110 Growth inhibition (%Y 51.5 70.3 33 35 - -

Estimation of the bacterial growth in leaves was performed 4 d after leaf infiltration; bacterial density is expressed as d u x io5 cm+; 10 infected plants were analyzed. Percentage.of inhibition of bacterial growth was estimated using the bacterial density of infected leaves from plants whose cotyledons were previously water-infiltrated; 1 O plants were analyzed.

a Leaves of rank 1 were infiltrated 72 h following cotyledon inoculation.

764 Martinez et al. Plant Physiol. Vol. 122, 2000

Table 111. Effect of SAR on bacterial growth in cv Acala 44 Cotyledon Pre-lnoculation

Race 18 Race 20 Water

Leaf post-inoculationa Race 18 Race 20 Race 18 Race 20 Race 18 Race 20 Bacterial densityb 4,102 4- 85 3,995 Z 80 4,195 2 101 4,327 5,002 2 230 5,311 2 80 Growth inhibition (%)' 18 20.2 21 18.5 a Leaves of rank 7 were infiltrated 72 h following cotyledon inoculation.

- - Estimation of the bacterial growth in leaves was performed 4 d

after leaf infiltration; bacterial density is expressed as cfu x i05/cm-2; 10 infected plants were analyzed. Percentage of inhibition of bacterial growth was estimated using the bacterial density of infected leaves from plants whose cotyledons were previously water-infiltrated; 1 O plants were analyzed.

Our observations strengthen previous suggestions that H202 accumulation is required for SA-dependent re- sponses (Leon et al., 1995; Neuenschwander et al., 1995; Alvarez et al., 1998) and led us to propose the_following model for the H202- and SA-mediated LR and SAR of cotton to Xcm (Fig. 9). Three hours following plant infec- tion, apoplastic cationic peroxidase generated Oi-, which could result in the accumulation of H202 between 4 and 6 h, likely from dismutation by SOD, although other sources of H202 are suspected (Martinez et al., 1998). Ac-

-cumulation of SA 6 h post infection in cotyledons occurs downstream of pathogen-dependent ROS production, likely under H202 control. In parallel, and perhaps in response to SA, an increase in total peroxidase activity and the inhibition of hcterial growth were demonstrated. In this model, SA may inhibit the O,'--generatiig system. The translocated signal both induced a systemic accumulation of SA and the activation of peroxidase activity. Since plants responded to H202 (Figs. 4 and 8) in a way similar to the way they respond to pathogen infection-systemic produc- tion of SA and activation of peroxidase activity (Fig. 1; Martinez et al., 1996)-it is expected that H202 promotes establishment of cotton SAX to Xcm.

In contrast to the proposed model, Draper (1997) indi- cated that the accumulation of SA within developing le- sions on tobacco leaves begins to accumulate within 1 to 2 h after inoculation, prior to the sustained oxidative burst. In addition, several authors (Kauss and Jeblick, 1995; Mur et al., 1996; Rao et al., 1997) demonstrated an early role for SA

' that may cause the generation of the oxidative burst in incompatible interactions. Accordingly, the addition of SA to a tobacco suspension culture immediately induced a rapid transient generation of O;-, followed by a transient increase in the cytosolic free calcium ion concentration (Kawano et al., 1998). These observations may suggest that SA and oxidative burst pathways occur independently in some host-pathogen systems.

Similar responses of both cotton cultivars following in- filtration with H202 (Fig. 8), SA (Fig. 6), or in the incom- patible interaction indicate that ROS in the cotton/Xcm . system play the role of an internal emergency signal for the induction of the hypersensitive cell death, as previously reported with other plants (Chen et al., 1993; Levine et al., 1994; Teqhaken et al, 1995; Jabs et al., 1996; Alvarez et al., 1998).

It should be noted that plants of the cv Reba B50 chal- lenged by the virulent Xcm race 20, did not display my HR

symptoms, nor did they accumulate SA in cotyledons or in leaves (Fig. 8), but showed symptoms of bacterial blight. Surprisingly, a systemic stimulation of peroxidase activity was detected 48 h after infection and was associated with a relative inhibition of the bacterial population in cotyledons (Table II). In the excised cotyledon experiment (Table I), we demonstrated that in the compatible interaction cv Reba B5O/Xcm race 20, the signal inducing the systemic re- sponse was only generated after 48 h following inoculation. Mechanisms underlying the systemic stimulation of perox- idase activity in the cv Reba B50 infected with incompatible Xcm race 18 seem different from those implicated in the compatible interaction cv Reba B50/Xcm race 20, in which no SA was detected. This strongly suggests that a signal different from SA could be responsible for the activation of

Recognition: R geneskm race 18 7 Oxidative burst

Cationic Pox generated 02.-

LR/HR v

(6 hours) Salicylic acid (24 hours)

SAR Figure 9. Schematic diagram illustrating relationships between the oxidative burst and salicylic acid in LR and SAR to Xcm in cotton. PoxA, activity of total peroxidase.

Reactive Oxygen Species and Salicylic Acid in Cotton Hypersensitive Response to Xanthomonas 765

a delayed systemic response in this interaction. In contrast to pathogen-induced SAR, a signaling pathway controlling induced systemic resistance has been recently reported (Van Wees et al., 1997; Pieterse et al., 19981, and is inde- pendent of SA accumulation. The plant growth regulators jasmonic acid and ethylene have been shown to be impli- cated in this plant defense response (Wastemack and Parthier, 1997).

Our data demonstrated that incompatible recognition of Xcm by cotton triggers the oxidative burst that precedes the production of SA in cells undergoing the HR ta race 18. Our data emphasized the upstream role of HzC& as the initiating signal of LR and SAR in cotton, which was con- firmed by the inhibition of SA production and the HR phenotype after Co-infiltration of catalase with Xcm (data not shown). We provide evidence that treatment with H202 positively influences the local and systemic accumulation of SA, which is correlated with the enhancement of perox- idase activity. This strengthens the hypothesis that H202 is a key molecule in plant resistance to pathogenic microbes (Levine et al., 1994; Léon et al., 1995; Tenhaken et al., 1995; Wu et al., 1997; Alvarez et al., 1998). In the sequence of events following pathogen recognition in Xcm-infected plants, SA acts after the oxidative burst but plays a central signaling role for LR and SAR, since pre-exposure of cotton tissues to SA never induced generation of O;'-. Further- more, SA could be involved in feedback regulation of the 0,'--generating peroxidase activity in these plants. It should be now of interest to determine whether the ROS in Xcm-infected cotton plants could be involved in gene acti- vation, including the Phe ammonia-lyase-encoding gene. Phe ammonia-lyase could be involved, not only in phenol synthesis by hypersensitive responding cells (Daï et al., 1996) including SA, but also transiently stimulated in stem vascular fluids (Smith-Becker et al., 1998). This may con- firm that in Xcm-infected cotton, SA is not the signal trans- ported from cells undergoing the HR to the whole plant, but accumulates in stems as a transient molecule involved in SAR signaling.

ACKNOWLEDGMENT

The authors acknowledge Dr. J. Dumer (University of New Jersey) for kindly revising the manuscript.

Received August 10,1999; accepted November 4,1999.

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