Salicylic Acid lnhibits Synthesis of Proteinase - Plant Physiology

Plant Physiol. (1 995) 108: 1741-1 746

Rapid Communication

Salicylic Acid lnhibits Synthesis of Proteinase lnhibitors in Tomato Leaves lnduced by Systemin and Jasmonic Acid'

Steven H. Doares, Javier Narváez-VPsquez2, Antonio Conconi, and Clarence A. Ryan*

lnstitute of Biological Chemistry, Washington State University, Pullman, Washington 991 64-6340

Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wounding, oligouronides (H.M. Doherty, R.R. Selvendran, D.J. Bowles [1988] Physiol MOI Plant Pathol33: 377-384), and linolenic acid (H. Peda-Cortés, T. Albrecht, S. Prat, E.W. Weiler, 1. Willmitzer [19931 Planta 191: 123-128), are shown here to be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer mole- cules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 1 3s-hydroperoxy linolenic acid to 12-0x0-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step i n the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes.

Wounding of leaves of several plant families by chewing insects or other mechanical damage induces the synthesis of defensive proteinase inhibitor proteins in both wounded leaves and dista1 unwounded leaves (Green and Ryan, 1972; Brown and Ryan, 1984; Roby et al., 1987; Bradshaw et al., 1989). Severa1 chemical signals have been identified in plant leaves that regulate this response, including oligosac- charides derived from polygalacturonic acid (Bishop et al., 1984), the plant hormones ABA (PeÍía-Cortés et al., 1993) and auxin (Kernan and Thornburg, 19891, the octadecapep- tide systemin (Pearce et al., 19911, and several components of the octadecanoid pathway (Vick and Zimmerman, 1983),

' This work was supported by Washington State University College of Agriculture and Home Economics Project 1991 and National Science Foundation grants IBN-9184542 and IBN- 91 17795.

Present address: Programs Nacional Investigación en Biotec- nología Agrícola, Corporación Colombiana de Investigación Agro- pecuaria, Apartado Aéreo 151123, E1 Dorado, Bogotá, DC, Colombia.

* Corresponding author; fax 1-509-335-7643.

including linolenic acid, 13S-HPLA, 12-oxy-PDA, and JA (Farmer and Ryan, 1992).

ASA and related hydroxybenzoic acids that are signals for systemic acquired resistance to pathogens have been shown to inhibit the accumulation of proteinase inhibitors in tomato (Lycopersicon esculentum) leaves induced by wounding or by supplying excised plants with oligosaccharide elicitors (Doherty et al., 1988). ASA blocks the octadecanoid pathway in leaves of excised tomato plants induced in the dark after supplying them with linolenic acid or 13s-HPLA (Penã-Cortés et al., 1993), thereby inhibiting the synthesis of JA. The inhibition of the pathway by SA was shown to occur at the site of conversion of 13s- HPLA to 12-oxy-PDA, where it likely inhibits the enzyme 13s-hydroperoxide dehydrase (Pefia-Cortes et al., 1993). We have further investigated the role of SA in inhibiting the induction of proteinase inhibitor genes in tomato in response to systemin, a proposed systemic wound signal, and to JA, a key intermediate in the octadecanoid pathway downstream from 12-oxy-PDA. SA is shown to inhibit proteinase inhibitor synthesis induced by systemin and JA, as well as MJ. The cumulative results indicate that SA inhibits the signaling pathway at two sites, one site previously reported between the synthesis of 13s-HPLA and 12-oxy-PDA, and the second site reported here, between JA synthesis and transcriptional activation of the genes.

MATERIALS A N D METHODS

Plants and Bioassays

Lycopersicon esculentum var Castlemart plants were grown under 17-h days at 28°C with >300 mE m-2 s-' of light and 7-h nights at 18°C. A11 data points for levels of inhibitor I or I1 proteins are the means of at least six assays.

To assay the effects of ASA on the induction of proteinase inhibitors by systemin, excised plants (12-15 d after planting) were supplied with water or with 1, 5, or 10 mM SA for 30 min through their cut stems and then supplied with 90 pL of 28 nM systemin (Pearce et al., 1991) in 15 mM sodium phosphate, pH 6.5. After imbibing the solutions for approximately 45 min, the plants were transferred to vials of water in a sealed transparent acrylic box

Abbreviations: ASA, acetylsalicylic acid; 13S-HPLA, 13s-hydroperoxy linolenic acid; JA, jasmonic acid; MJ, methyl jasmonate; 12-oxy-PDA, 12-0x0-phytodienoic acid; PR, pathogenesis related; SA, sodium salicylate.

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1742 Doares et al. Plant Physiol. Vol. 108, 1995

containing a CO, trap (Ryan, 1977) and maintained under constant light for 24 h. Another group of systemin-induced plants was incubated with the cut stems in 1 mM SA, as described by Doherty et al. (1988), for the duration of the 24-h period. Juice from the epicotyl tissue of individual plants was expressed using a small mortar and pestle and assayed for proteinase inhibitor content by a radial diffusion immunoassay (Ryan, 1967; Trautman et al., 1971).

The induction of proteinase inhibitors by MJ was assayed using excised plants (12-15 d after planting) that were supplied through their cut stems with either water or 1 mM ASA for 30 min, followed by water or 1 mM ASA for 24 h. The treated plants were placed in sealed 1-L glass jars containing 1 p L of 10-fold diluted (into ethanol) MJ (Be- doukian Research, Danbury, CT) immediately after treatment with ASA or water. After 8 h of exposure to MJ vapor, the plants were transferred to sealed acrylic boxes and incubated for the duration of the experiments, when proteinase inhibitors were quantified.

To assess the effects of SA on the induction of proteinase inhibitors by JA, excised plants (9-11 d after planting) were pretreated with either water or 1 mM SA for 60 min and then transferred to small vials containing 90 pL of 400 p~ JA with or without 1 mM SA dissolved in 15 mM sodium phosphate, pH 6.5. After the plants had soaked in these solutions for approximately 45 min, the plants were transferred to glass vials containing water or 1 mM SA as ap- propriate. The plants were then placed in an acrylic box as described above and incubated for 24 h, when the leaf juice was assayed for proteinase inhibitor content.

The effects of SA on the abilities of tomato plants to accumulate proteinase inhibitors in leaves in response to wounding, JA, systemin, or oligouronides in 9- and 14-d- old plants was assessed by pretreating excised plants in either water or 1 mM SA for 1 h and subsequently wounding the leaves or supplying the plants with 400 PM JA, 28 nM systemin, or 250 pg/mL oligouronides (Bishop et al., 1984) for 45 min and incubating the plants for 24 h with the stems immersed in either water or 1 mM SA. Juice was expressed from the cotyledons and each small first leaf of the 9-d-old plants and from the leaf tissues of the 14-d-old seedlings and assayed immunologically for proteinase inhibitor content as described above.

The effects of SA on the induction of proteinase inhibitor mRNAs in light and darkness were determined in leaves of 10-d-old plants supplied with 400 p~ JA in the presence or absence of SA (for 45 min). Control plants were treated as above except that they were pretreated with water or 1 mM SA. The plants were incubated in either constant light or darkness for 24 h with their stems immersed in solutions of water or 1 mM SA and then assayed for inhibitor I and I1 mRNA contents.

RNA Isolation, Denaturing RNA Cel Electrophoresis, and Northern Analysis

One set of plants was supplied with 400 p~ JA for 45 min in the presence or absence of 1 mM SA and then incubated for 7 h in light. A second set of plants was exposed to MJ vapors with their cut stems in either water or 1 mM ASA for

8 h in a closed plexiglass box. Each set of plants was then ground to a fine powder in liquid nitrogen, and total RNA was extracted as described by Wingate et al. (1989). Sam- ples of RNA (10 pg) were separated by electrophoresis through formaldehyde-1.4% agarose gels and blotted onto HyBond-N+ (Amersham). The filters were hybridized with 20 to 30 ng of 32P-labeled (random priming) tomato proteinase inhibitor I and I1 cDNAs (Graham et al., 1985; Lee et al., 1986; Thornburg et al., 1987), Antirrhinum ubiquitin cDNA (courtesy of Dr. Cathie Martin, John Innes Institute, Norwich, UK), and cDNAs coding for tobacco acidic PR3a protein and tobacco basic PR3 protein (courtesy of Dr. John Bol, University of Leiden, The Netherlands). Hybridization and washing conditions have been described previously (Conconi et al., 1989).

Uptake of [35SlMet by Young Tomato Plants

Sets (three plants/set) of excised tomato plants (9 d after planting) were supplied with either water or 1 mM SA through their cut stems for 45 min, followed by 400 p~ JA for 45 min. The plants were then provided with a 15-min pulse of 250 pCi/mL [35S]Met (New England Nuclear; specific activity 1390 Ci/mmol) and were transferred to water or 1 mM SA. The plants were collected 15 min following the pulse and assayed for [35S]Met uptake as fol- lows. The stems of the plants were cut just below the cotyledons and discarded, and the plants were placed in glass scintillation vials, bleached by adding 670 pL of 30% hydrogen peroxide and 200 pL of 70% perchloric acid, and incubated at 70°C for 2 h (Sun et al., 1988). Scintillation cocktail (15 mL of BSC, Amersham) was added, and the radioactivity of the samples was determined.

Autoradiography of [35S]Met-Labeled Proteins from Leaves of Plants Treated with JA and SA

Sets of 9-d-old plants were supplied with 400 p~ JA, 1 mM SA, or water for 45 min and then incubated under light for 7 h. Another set of plants was supplied with 400 p~ JA plus 1 mM SA. AI1 sets of plants were then supplied with a 15-min pulse of [35S]Met and were collected 105 min later. The stems were discarded, and the plants were homoge- nized using a Sorva11 OmniMixer for 1 min in 1.5 mL of Tris buffer-saturated phenol and 2 mL of a buffer containing 0.75 M SUC, 0.5 M Tris-HC1, 50 mM EDTA, 100 mM KCI, 1 mM PMSF, and 2% (v/v) 2-mercaptoethanol, pH 7.3. The mixture was centrifuged at 2500g for 5 min, and the aque- ous phase was re-extracted with 1 mL of equilibrated phenol. The extracted proteins were precipitated from the combined phenol phases by adding 12 mL of 100 mM ammonium acetate in methanol and incubating at -20°C overnight. Precipitated proteins were pelleted by centrifugation at 25008 for 20 min, and the pellets were sonicated twice in ice-cold 80% acetone for 10 min, followed by centrifugation to remove pigments. The pellets were dissolved in 500 p L of 2% Nonidet P-40, 2% 2-mercaptoethanol. Protein was quantified by the Bradford assay (Brad- ford, 1976), and the specific radioactivity of each sample of extracted protein was determined.

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Salicylic Acid lnhibits Signaling by Systemin and Jasmonic Acid 1743

Equivalent amounts of radiolabel from each protein sample were separated using denaturing SDS-PAGE through a 12% gel. The gels were fixed in 40% (v/v) ethanol, 10% (v/v) acetic acid, treated with a fluorographic enhancer (En3Hance, New England Nuclear), dried, and exposed to preflashed film (Kodak XAR-5) to visualize the labeled proteins.

RESULTS AND DISCUSSION

Studies of wound-inducible proteinase inhibitor protein and mRNA synthesis were performed previously with tomato plants of the two- to three-leaf size (14 d after planting). To conserve time and greenhouse space in some experiments, we used cotyledons of both 9-d-old plants (having expanding cotyledons and one small apical leaf) and 14-d-old plants (having two primary leaves and a small expanding apical leaf) to study the effects of SA and ASA on the induction of proteinase inhibitor I by wounding, JA, systemin, and oligouronides. A comparison of the inducibility of 9- and 14-d-old excised plants by wounding and various elicitors is shown in Table I. Also shown is the effect of supplying 1 mM SA to both ages of plants on the wound- and elicitor inducibility of proteinase inhibitor I. Although the inducible levels of proteinase inhibitor I in response to the various elicitors and wounding were slightly lower in the cotyledons and primary leaf of 9-d-old seedlings t h a n the leve1 in the primary leaves of 14-d-old seedlings, the relative effectiveness of the various inducers was similar within the two groups of plants (Table I), and the effect of SA on the response was similar in both groups (Table I), confirming the validity of comparing young cotyledons to primary leaves where reported.

Young, unwounded tomato plants do not normally accumulate proteinase inhibitors in leaves, and the excision of plants at the base of the stem with a razor blade elicited only a weak wound response. Excised plants, supplied in light with water through their cut petioles for 24 h, usually accumulate from 10 to 20 pg of inhibitor I and II/g tissue because of the excision event. Severe wounding of leaves

Table 1. Comparison o f the effects of I mM SA on the accumulation of proteinase inhibitor I in 9- and 74-d-old tomato seed/ings

Percentage Accumulation” (?SE)

Treatmentd 9-d-old seedlingsb 14-d-old seedlings‘

-SA +SA -SA + SA

Control 1 6 2 2 4 2 1 1 3 2 3 5 2 1 Wound i ng 2 9 5 3 3 2 1 6 7 2 7 1 1 2 1 JA (400 p ~ ) 7 7 2 2 4 6 2 2 1 0 0 2 4 5 3 2 4 Systemin (28 nM) 4 6 2 5 1 2 2 1 9 2 2 5 1 8 2 2 Oligouronides (250 pg/mL) 31 2 3 8 2 O 44 2 13 8 2 3

“Values are reported as percentages of the highest inhibitor levels recorded. bSeedlings 9 d after planting. Plants had two expanding cotyledons and one small apical leaf. ‘Seedlings 14 d after planting. Plants had two expanding primary leaves and one small apical leaf. dCut stems of young tomato plants were supplied with water (-SA) or 1 mM SA (+SA).

0 Inh. I I

CONTROL SYS S Y S + SYS+ S Y S + SYS+ 1mM ASA 5mM ASA lOmM ASA 1mM ASA (30 min) (30 min) (30 mm) (24 h)

100 p 3 80 g!c

3: 2; 60

40 t m 5 20

O CONlRoL ASA ASA M J MJIASA MJIASA

(30 mi l ) (24 h) (30 min) (24 h)

1 O0

80

60

40

20

O

1 O0

80

60

40

20

O m m SA J A JA + CONTROL SA JA JA c m m SA J A JA +

Inh. I

Inh.11

. . . . CONTROL SA JA JA c

SA SA LlGHT D A R K

Figure 1. lnhibition of induction of accumulation of proteinase inhibitor I (Inh I) and I I (Inh Ir) proteins in cotyledons of 9-d-old tomato plants by ASA and SA, induced by systemin, MJ, and JA. A, Young tomato plants were excised and supplied with 1, 5, or 1 O mM ASA for 30 min, followed by 2.5 pmol of systemin (SYS) or supplied with 2.5 pmol of systemin and then incubated in 1 mM ASA for 24 h. Bars on the left represent data for excised plants that were supplied with water alone (CONTROL) and systemin alone (SYS). lnhibitor I and II proteins in the cotyledons were quantified immunologically as described in ”Methods and Materials.” B, Tomato plants were excised and supplied with water or 1 mM ASA for 30 min or 24 h. At the same time, the seedlings were placed in an airtight acrylic chamber in the presence of a wick containing 0.1 pL of Mj. Exposure to MJ was for 8 h, followed by incubation in either water or ASA for 16 h, when inhibitor I (Inh I) and I I (Inh Ir) levels in the leaf juice were quantified. C, Effects of SA on the JA-induced accumulation of proteinase inhibitor I (Inh I) and I I (Inh 11) proteins in constant light or in total darkness. CONTROL, Excised plants supplied with water; SA, plants supplied with 1 mM SA; JA, plants supplied with 400 mM JA; JA + SA, plants treated with 400 mM JA and 1 mM SA for 45 min, followed by 1 mM SA for 24 h.

with a hemostat, or by supplying plants with picomole levels of systemin, induces about 80 to 100 pg of each inhibitor/g tissue. Therefore, for simplicity of presentation, the inhibitor inductions in Table I and Figures 1 to 3 are reported as percentages of the highest levels recorded during a given set of experiments.

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Effects of ASA on the Induction of ProteinaseInhibitors by Systemin

ASA, when supplied to 9-d-old excised tomato plantsthrough their cut stems at 1 HIM for 30 min, followed by 2.5pmol of systemin, reduced the accumulation of proteinaseinhibitors I and II in leaves during the next 24 h by approx-imately 25% (Fig. 1). Supplying the excised plants with 5 or10 mM ASA for 30 min before supplying systemin inhibitedthe accumulation of proteinase inhibitors by approximately75% (Fig. 1A). ASA is known to be metabolized rapidly inleaves of tobacco plants (Metraux et al., 1990; Enyedi et al.,1992a). Thus, supplying the plants with 1 mM ASA duringthe 24-h period following induction by systemin was aseffective as supplying 10 mM ASA for only 30 min (Fig.1A). ASA supplied continuously to tomato plants at 1 mMhad previously been shown (Doherty et al., 1988) tostrongly inhibit proteinase inhibitor synthesis in leaves inresponse to wounding and to exogenously supplied oli-gouronides. The similar inhibiting effects of ASA on theinduction of proteinase inhibitor synthesis by systeminsupports the hypothesis (Farmer and Ryan, 1992) that thesame signal transduction pathway is involved in all threeinduction processes.

Effects of ASA or SA on the Induction of ProteinaseInhibitors by JA and M)

MJ and JA are potent inducers of proteinase inhibitor Iand II genes, with JA being a proposed key component ofthe octadecanoid-signaling pathway (Farmer and Ryan,1992). ASA was recently reported to inhibit JA-inducedproteinase inhibitor II mRNA synthesis in leaves of tomatoplants incubated in the dark but not to inhibit the inductionof inhibitor II mRNAs by JA (Pena-Cortes et al., 1993). Wereport here that the synthesis of proteinase inhibitor I andII proteins induced in cotyledons of 9-d-old plants by ex-posure to MJ vapors in constant light is strongly inhibitedby 1 mM ASA (Fig. IB). As with the inhibition of systemininduction (Fig. 1), incubation with ASA for the entire 24 hof incubation with 1 mM ASA was most effective. Similarresults were obtained with plants induced in either thelight or the dark with a solution containing 400 /U.M JA for45 min, followed by 1 mM SA for 24 h (Fig. 1C). Althoughthe levels of proteinase inhibitors induced in the dark are

only about one-fourth of those induced in the light, SAreduced the induction of the inhibitors to less than 50% ofthe levels induced by JA alone (Fig. 1C).

The inhibitory action of SA was confirmed at the mRNAlevels using both inhibitor I and II cDNAs as probes innorthern analyses (Fig. 2A). In these experiments, SA andASA inhibited the induction of inhibitor I and II transcriptsin light by JA and MJ, respectively (Fig. 2A). Two tobaccoPR protein cDNAs were also used to monitor the levels ofPR3 and PR3a transcripts. These transcripts are known tobe inducible by SA (Bol et al., 1990; Malamy and Klessig,1992). Both PR protein transcripts showed substantial in-creases in response to ASA or SA, as expected (Fig. 2A), butthe expression of these genes was not affected by MJ or JA.Transcript levels of the constitutive ubiquitin gene werenot induced by MJ or JA and not inhibited by ASA or SA(Fig. 2). In Figure 2B, an identical experiment was per-formed with SA and JA in the dark. It is clear that JAinduction of inhibitor II mRNA is strongly inhibited by SAunder dark conditions, as in light.

Effects of SA on the Overall Uptake of [35S]Met and on inVivo Translation in Tomato Leaves

The possibility was assessed that the inhibitory effects ofSA might have caused a differential imbibition of inducersolutions or have caused a general reduction of translationof all mRNAs by SA. Nine-day-old plants were suppliedwith JA and/or SA for 30 min as described previously andthen pulsed for 15 min with a solution of [35S]Met, and theuptake of label was determined. Plants pulsed at the be-ginning of the induction (i.e. the label was added to theinducer solutions) took up identical amounts of labelwhether they had been pretreated with water or 1 mM SA(data not shown). These results indicated that SA did notaffect the ability of the plants to imbibe solutions of induc-ers through their cut stems and therefore most likely didnot influence the results in which SA was supplied toplants through the cut petioles.

In a separate experiment, plants supplied with water,SA, JA, and JA plus SA for 45 min were incubated for 7 hwith water and were supplied with a 15-min pulse of[35S]Met, and the proteins were extracted 105 min later. Thelabeled proteins were analyzed by denaturing SDS-PAGE

Figure 2. Northern analysis of RNAisolated from tomato seedlings incu-bated in light. A, left, 7 h after induc-tion with water (C), 1 mM SA (SA), 400JUM JA (JA), and 400 /LIM JA in the pres-ence of 1 mM SA (JA + SA). A, right, 8h after induction with water (C), 1 mmASA (ASA), MJ vapors (MJ), or MJ in thepresence of 1 mm ASA (MJ + ASA). B,Conditions as in A except that incuba-tion was entirely in the dark and onlyinhibitor II and ubiquitin probes wereused. Electrophoresis conditions,probes, and hybridization conditionsare given in "Materials and Methods." •t»t

Inhibitor I

ubiquitin

B

Inhibitor

ubiquitin

DARK

C SA JA JA+SA

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Salicylic Acid Inhibits Signaling by Systemin and Jasmonic Acid 1745

and fluorography. No gross effects of SA on overall proteinsynthesis were observed (Fig. 3). Although a number ofproteins appeared to be specifically induced by SA, onlysome of those corresponding to the molecular masses ofpreviously reported PR proteins from tomato (Granell etal., 1987; Fischer et al., 1989; Joosten and De Wit, 1989) aremarked with arrows in Figure 3. Several proteins accumu-lated specifically in response to JA (e.g. at approximately84, 60, 45, 38, and 28 kD), but these proteins did notaccumulate in response to JA in the presence of SA (Fig. 3).

The results presented in this paper demonstrate that SAand ASA have a specific, pronounced inhibitory effect onthe accumulation of proteinase inhibitors in tomato plantsin response to systemin, similar to the inhibition of inhib-itor synthesis induced by wounding or oligouronide elici-tors (Doherty et al., 1988), JA, or linolenic acid (Pena-Corteset al., 1993). The inhibition by SA or ASA of proteinaseinhibitor synthesis appears to be transient, and the inhibi-tory effects require a constant presence of the inhibitorduring prolonged incubation periods. Our results agreewith previous evidence that SA inhibits the octadecanoid-signaling pathway (Pena-Cortes et al., 1993), but in contrastto those data, the data presented here indicate that SA caninhibit an additional step in the signaling pathway forproteinase inhibitor induction (Farmer and Ryan, 1992)that is downstream from the synthesis of JA. The reasonsfor the discrepancy between our data and those of Pena-Cortes et al. (1993) are not clear, but the difference may be

kfi

-106

-80

-27.5

Figure 3. Autofluorogram of proteins in cotyledons of tomato seed-lings supplied with water (C), 1 HIM SA for 7 h (SA), 400 .̂M JA for 45min and then water for 7 h (JA), and 400 IJ.M JA for 45 min and then1 HIM SA for 7 h (JA + SA). In each case, at 7 h the plants weresupplied with [15S]Met for 15 min and then water for 105 min. Totalprotein was then extracted from the pulse-labeled plants, and the leafproteins were subjected to denaturing SDS-PAGE and autofluorog-raphy. Some of the proteins specifically induced by either JA or SAare indicated on the left. Migration distances of molecular massmarkers and previously reported PR proteins (arrows) are indicated.

due to the low levels of inhibitor II that were synthesized inthe dark in response to JA in those experiments.

SA has been proposed as a systemic signal for PR pro-teins to activate systemic acquired resistance in tomato andpotato plants in response to viruses and microbial patho-gens (Yalpani et al., 1991; Raskin, 1992). The data ofDoherty et al. (1988) and Pena-Cortes et al. (1993) and thedata presented here all indicate that SA inhibits the octa-decanoid pathway that regulates defense signaling in re-sponse to predator attacks. Thus, as shown in Figure 2A,SA can simultaneously regulate two independent plantdefense-signaling pathways, one (PR protein synthesis)positively and the other (proteinase inhibitor synthesis)negatively. We are currently investigating whether the syn-thesis of SA in tomato plants in response to pathogenattacks will inhibit the synthesis of proteinase inhibitorsand other defensive proteins in response to insect attacks toprovide "cross-talk" between these two systemic signalingsystems.

ACKNOWLEDGMENTSThe authors thank Greg Wichelns and Sue Vogtman for growing

the plants used in this study. We thank Dr. John Bol (University ofLeiden, The Netherlands) for the gifts of PR protein cDNAs andDr. Cathie Martin (John Innes Institute, Norwich, UK) for the giftof the ubiquitin cDNA.

Received January 13, 1995; accepted May 5, 1995.Copyright Clearance Center: 0032-0889/95/108/1741/06.

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Salicylic Acid lnhibits Synthesis of Proteinase - Plant Physiology