hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. l h e SA increase in the ...
Plant Physiol. (1993) 101: 1375-1380
lnduction of UDP-Glucose:Salicylic Acid Clucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves’ Alexander J. Enyedi and llya Raskin* Center for Agricultural Molecular Biology, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231
During SAR, pathogen-free plant tissues become more resistant to subsequent attack by a wide range of pathogens (Ross, 1961a, 1961b). Following TMV inoculation, SA levels in Xanthi-nc tobacco increase systemically (Malamy et al., 1990; Enyedi et al., 1992). The first increase in the leve1 of SA occurs 36 h after inoculation, coincidentally with the appearance of HR (Malamy et al., 1990). The highest levels of SA are found in and around TMV-induced necrotic lesions (Enyedi et al., 1992). Tobacco leaves rapidly metabolize exogenously supplied or endogenously produced SA to GSA (Enyedi et al., 1992). Therefore, most of the SA in TMV-inoculated leaves of tobacco is present in the form of GSA (Enyedi et al., 1992; Malamy et al., 1992). GSA was found only in leaves that exhibited HR. Phloem sap and pathogen-free leaves of TMVinoculated tobacco did not contain significant levels of GSA (Enyedi et al., 1992). Various hydroxybenzoic acid glucosides have been reported to occur in higher plants (Griffiths, 1959; Ibrahim and Towers, 1959; Klambt, 1962; Umetani et al., 1990). Hypocotyls of sunflower incubated with carboxy-labeled benzoic acid formed trace amounts of SA and larger amounts of GSA (Klambt, 1962). SA-inducible glucosyltransferases, which catalyze the glucosylation of SA to SA-glucosides, have been partially purified from cell-suspension cultures of Mallotus japonicus (Tanaka et al., 1990) and from oat roots (Yalpani et al., 1992b). This paper describes how the local and systemic induction of tobacco 0-GTase during HR to TMV relates to the temporal and spatial distribution of SA and GSA. It demonstrates that (a) P-GTase activity increases following TMV inoculation; (b) the increase in /3-GTase activity coincides with the accumulation of GSA; (c) the SA levels present in the inoculated leaves are sufficient for the induction of P-GTase activity; (d) systemic increases in SA are not sufficient for the induction of P-GTase activity, which may explain the lack of significant amounts of GSA in the uninoculated leaves; and (e) P-GTase induction is specific for SA.
Salicylic acid (SA) i s a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. l h e SA increase in the inoculated leaf coincided with the appearance of a 8-glucosidase-hydrolyzable SA conjugate identified as 8O-o-glucosylsalicylic acid (CSA). SA and CSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-g1ucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) (8-CTase) capable of converting SA to CSA. Healthy tissues had constitutive B-GTase activity of 0.076 milliunits g-’ fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated 8-CTase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. l h e effect of TMV inoculation on the 8CTase and CSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMVinoculated leaves (2.7-27.0 fig g-’ fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of 8-CTase by SA and prevented the formation of CSA. O f 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced B-CTase activity.
Tobacco (Nicotiana tabacum L. cv Xanthi-nc) with the N genotype is resistant to TMV (Holmes, 1938). The incompatible interaction between tobacco and this pathogen results in tissue necrosis that prevents the spread of TMV throughout the plant. This coordinated cell death is termed the HR and is associated with the intra- and intercellular accumulation of nine distinct classes of pathogenesis-related proteins (Ward et al., 1991) and the appearance of elevated levels of SA (Malamy et al., 1990). Currently, SA is hypothesized to act as a phloem-mobile, endogenous signal molecule that triggers the induction of defense-related proteins and SAR throughout the plant (Malamy et al., 1990; Metraux et al., 1990). This research was supported in part by the U.S. Department of Agriculture/Competitive Research Grants Office grant No. 9037261-5659, Division of Energy Biosciences of the U.S.Department of Energy grant No. DE-FG05-91ER20049, New Jersey Commission for Science and Technology grant No. 93-240380-1, and the New Jersey Agricultura1Experimental Station (NJAES). * Corresponding author; fax 1-908-932-6535.
Abbreviations: CHX, cycloheximide;GSA, (3-O-D-glucosylsalicylic acid; (3-GTase, UDP-glucosesalicylic acid 3-O-glucosyltransferase (EC 2.4.1.35);HR, hypersensitive response; SA, salicylic acid; SAR, systemic acquired resistance; TMV, tobacco mosaic virus. 1375
Enyedi and Raskin
1376 MATERIALS AND METHODS Plant Material
A11 experiments were performed with leaves of tobacco
(Nicotiana tabacum L. cv Xanthi-nc NN genotype) grown as described previously (Enyedi et al., 1992). The uppermost, near-fully expanded leaves of 6- to 8-week-old plants were dusted with dry carborundum (240 grit) and inoculated by gently rubbing the upper leaf surface with 200 pL of a suspension of U1 strain TMV (25 Fg mL-' in 0.1 M potassium phosphate buffer, pH 6.7). Leaves were rinsed with deionized water immediately following inoculation. Control plants were dusted with carborundum and mock inoculated with 200 pL of 0.1 M potassium phosphate buffer (pH 6.7). Following inoculation, plants were maintained at 24OC under continuous illumination provided by cool-white fluorescent lamps (200 pmol m-2 s-I). For SA infiltration experiments, 22-mm-diameter leaf discs were infiltrated through the abaxial surface with SA solutions ranging in concentration from O to 1 mM using a 1-cc syringe. Following infiltration, leaf discs were kept on wet filter paper inside 14-cm Petri dishes at 24OC under cool-white fluorescent lamps (200 pmol ,-2 s-~).Leaf discs were harvested at various times, frozen in liquid nitrogen, and stored at -800c for future determinations of 0-GTase activity and SA and GSA levels.
Plant Physiol. Vol. 101, 1993
was determined with the Bio-Rad protein assay using BSA fraction V (1 mg mL-I) as the standard. Extraction and Quantitation of SA
SA was extracted from tobacco leaf tissue (0.5-g sample) and quantified by HPLC as described (Enyedi et al., 1992). A11 data were corrected for SA recovery, which ranged from 44 to 52%. Hydrolysis of Leaf Extracts
To determine the GSA content, methanolic leaf extract was dried and resuspended in 500 p L of hydrolysis buffer (100 mM sodium acetate buffer, pH 5.5) containing 20 units of Pglucosidase (EC 3.2.1.21; almond). After 1.5 h of incubation at 37OC, extracts were acidified to pH 1.0 with TCA acid and subjected to SA extraction and quantitation (Enyedi et al., 1992). CHX Treatment
Leaf discs were infiltrated with CHX (40 pg mL-I) and allowed to incubate for 30 min. Control discs were infiltrated with deionized H20. Following incubation, leaf discs were infiltrated with 1 mM SA (35 pg g-' fresh weight) or deionized H20,incubated for an additional6 h, harvested, and assayed for (3-GTase activity, GSA, and SA as described above.
Extraction and Analysis of 8-GTase Activity
Effects of SA Analogs
P-GTase was extracted from tobacco leaf tissue using a modified method of Yalpani et al. (1992b). Frozen tissue (700 mg) was pulverized to a fine powder in liquid nitrogen using mortar and pestle and transferred to a chilled 15-mL Corex tube. One milliliter of extraction buffer (25 mM Tris-Mes, 10% [v/v] glycerol, 20 mM P-mercaptoethanol, 1 PM leupeptin, pH 6.5) containing 1% (w/w leaf) polyvinylpolypyrrolidone was added to the frozen tissue and the contents of the tube were allowed to thaw on ice. The resulting slurry was vigorously mixed and then centrifuged at 10,500g for 20 min at 4OC to pellet the debris. An 850-pL aliquot of the supernatant was transferred to a 1.5-mL microfuge tube and centrifuged at 15,500g for 5 min at 4OC to pellet any remaining leaf tissue. For P-GTase activity, the standard assay mixture (200 pL final volume) contained 16 p L of assay buffer (25 mM TrisMes, 0.4 mM SA, 1.2 kBq [7-I4C]SA[2.1 Gbq "01-'1, 10 mM UDP-Glc, pH 6.5). The reaction was started by adding 184 pL of crude extract (15,500g supematant) to the assay buffer and briefly mixing the sample. The reaction was carried out at 3OoC and terminated after 30 min with the addition of 200 p L of methanol. To separate [14C]GSA from ['4C]SA, the reaction mixture was passed through a Polyamide-6 (polycaprolactam/Perlon/Nylon-6, Serva Biochemicals, Paramus, NJ) ion-exchange column (10-mL volume) equilibrated in 10 mM Tris-Mes (pH 6.5) (Yalpani et al., 1992a). [I4C]GSAwas eluted with 5 mL of deionized H 2 0 and radioactivity was determined via scintillation counting. A11 P-GTase activities were expressed as milliunits g-' fresh weight and milliunits mg-' of protein (1 milliunit = 1 pmol min-I). Protein content
The induction of P-GTase activity in tobacco leaf discs was assessed using 12 structural analogs of SA (see Fig. 5). Discs were infiltrated with 20 pL of a 1.0-mM solution of each test compound as described for SA. P-GTase was extracted from frozen discs and its activity assayed as described above. To test for possible interference of SA analogs with the P-GTase assay, standard enzyme assays were performed in which each analog was added to a final concentration of 0.4 mM. Experimental Design and Statistical Analysis
A11 experiments were performed with a minimum of three tissue sample replicates per treatment or sampling time. Each experiment was repeated at least twice with similar results. Data from each experiment are expressed as the mean SE, and paired t tests were performed to determine significant differences between treatments at the P 5 0.05 leve1 (Steel and Torie, 1980).
*
RESULTS lnduction of j3-CTase Activity in TMV-lnoculated Leaves
The time course of the induction of P-GTase activity in TMV-inoculated tobacco leaves was established and related to the tissue levels of free and total SA (SA plus GSA) (Fig. 1).Basal levels of 0-GTase activity (approximately 0.08 milliunits g-' fresh weight or 0.01 milliunits mg-' of protein) were present at the time of inoculation. The highest levels of @-GTaseactivity were observed 72 h after TMV inoculation, when P-GTase activity in the infected leaves increased 6.7-
lnduction of 0-Clucosyltransferase by Tobacco Mosaic Virus 0.6 -
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Exogenous SA induced P-GTase activity in tobacco leaf discs in a dose-dependent manner (Fig. 3A). No significant induction was observed in leaf discs exposed to SA below 0.27 fig g-' fresh weight. At 2.7 pg g-' fresh weight, SA produced the first significant increase in P-GTase activity. This dose corresponded to the lower range of SA levels naturally produced in the TMV-inoculated leaf (Fig. 1B). Increasing the SA dose to 13.5 and 27.0 pg g-' fresh weight
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time course of transcriptional activation of PR-1 genes in TMV-inoculated tobacco leaves (Malamy et al., 1990). No increase in P-GTase activity was observed in the leaf immediately above the TMV-inoculated leaf (Fig. 2, A and B). Reflecting the constitutively low P-GTase activity, uninoculated leaves did not accumulate GSA (Fig. 2C). However, the levels of free SA in the uninoculated leaves increased from 50 to 175 ng g-' fresh weight 72 h after TMV inoculation of the lower leaf (Fig. 2C). The fact that for some time points the level of total SA was less than the level of free SA may be explained by experimental error.
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0-GTase activity expressed as (m) milliunits (mU) g-' fresh weight and (A)mU mg-' of protein (A), free SA content (B),and total SA (SA plus CSA) content (C) in TMV-inoculated leaves of Xanthi-nc tobacco at various times after inoculation. Vertical bars denote -t SE. A n asterisk (') denotes a significant level of P-GTase induction at the P 5 0.05 level. CSA content is derived by subtraction of B from C; for example, GSA is 67% of the total at 84 h after inoculation.
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fold over the basal levels when expressed on a milliunit g-' fresh weight basis (Fig. 1A). The first significant increase in P-GTase activity was observed 48 h after inoculation. This increase corresponded to the appearance of necrotic lesions and an increase in the level of both free and conjugated SA (Fig. 1, B and C). Twenty-four and 36 h after inoculation, leaf tissues contained only basal levels of SA (approximately 75 ng g-' fresh weight). Also, little if any SA conjugate was present in the leaves for 36 h after TMV inoculation. Paralleling the increase in P-GTase activity, SA and GSA levels in the inoculated leaves began to rise 48 h after inoculation. Eighty-four hours after TMV inoculation, leaves contained 26.8 fig g-' fresh weight of total SA, 67% of which was present in the form of GSA. It is interesting that the time course of P-GTase induction paralleled the earlier established
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Plant Physiol. Vol. 101, 1993
inhibitor CHX (Fig. 4, A and B). When leaf discs were pretreated with CHX and infiltrated with 1 mM SA, the level of extractable /3-GTase activity did not significantly differ from those in leaf discs treated with H 2 0 or CHX alone (Fig. 4, A and B). The abiIity of CHX to inhibit /3-GTase induction was reflected by the greater level of free SA and by the absence of GSA 6 h after SA infiltration (Fig. 4C). As expected, control leaf discs not exposed to CHX incorporated most of the infiltrated SA into GSA (Fig. 4C). lnduction of B-GTase Activity by SA Analogs
To test the specificity of P-GTase induction by SA, leaf discs were infiltrated with a 1-mM solution of 12 different analogs of SA (Fig. 5 ) . Only 2,6-dihydroxybenzoic acid induced P-GTase activity to a level comparable with that induced with SA. Benzoic and o-coumaric acid also increased
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Figure 3. P-GTase activity expressed as (m) milliunits (mU) g-' fresh weight and (A)mU mg-' of protein (A), free SA content (B), and total SA (SA plus GSA) content (C) in Xanthi-nc tobacco leaf discs
infiltrated with various amounts of SA and incubated for 6 h. Vertical bars denote & SE. A n asterisk (*) denotes a significant level of 0GTase induction at the P 5 0.05 level.
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The induction of 0-GTase activity was effectively blocked by pretreatment of leaf discs with the protein synthesis
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C caused a proportionally greater induction of P-GTase activity. No visible phytotoxicity was observed following infiltration of leaf tissue with any tested level of SA. The amounts of SA and GSA in the infiltrated leaf discs 6 h after SA treatment were positively correlated with the SA dose (Fig. 3, B and C). The first appearance of GSA was observed at 2.7 p g g-' fresh weight of SA. At greater endogenous levels of SA, GSA accumulation paralleled increases in P-GTase activity. Most of the infiltrated SA could be recovered after hydrolysis with P-glucosidase (Fig. 3C). The first significant increase in PGTase activity in tobacco leaves infiltrated with 50 p g g-' fresh weight of SA was observed 2 h after treatment (data not shown).
CHX
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Free SA Total S I
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Treatment Figure 4. Effect of CHX on P-GTase activity expressed as milliunits ( m U ) mg-' of protein (A) and mU g-' fresh weight (B), and free and
total SA content (C) in Xanthi-nc tobacco leaf discs treated with SA. Leaf discs were infiltrated with SA (35 pg g-' fresh weight) 30 min after CHX treatment and incubated for an additional 6 h. Vertical bars denote & SE.
lnduction of P-Glucosyltransferase by Tobacco Mosaic Virus COOH
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Figure 5. Effect of SA analogs on P-GTase activity (expressed as milliunits mg-' of protein) in Xanthi-nc tobacco leaf discs. Com-
pounds utilized were SA (A), 2,b-dihydroxybenzoic acid (B), ocoumaric acid (C), benzoic acid (D), 2,3-dihydroxybenzoic acid (E), p-hydroxybenzoic acid (F), gentisic acid (G), Z,6-dichlorobenzoic acid (H), 2-fluorobenzoic acid (I),2,3-difluorobenzoic acid (I), 2,4difluorobenzoic acid (K), 2,5-difluorobenzoic acid (L), and 2,6difluorobenzoic acid (M).Number in center of ring indicates x-fold induction of P-CTase activity compared with water control. An asterisk (*) denotes a significant level of P-CTase induction at the P 5 0.05 level.
P-GTase activity 1.8 and 1.9-fold (when expressed as milliunits g-' of protein) compared with those in H20-treated leaf discs, but this level of induction was not statistically significant. The remaining 9 analogs had no biological activity. The same 12 analogs were tested at a concentration of 0.4 mM for their ability to inhibit P-GTase-catalyzed formation of GSA from SA. Only 2,6-dihydroxybenzoic acid was capable of inhibiting the enzyme (data not shown). DlSCUSSlON
The results indicate that elevated P-GTase activity in Xanthi-nc tobacco leaves can be induced by TMV inoculation or by infiltration with SA. In the case of TMV inoculation, the induction of P-GTase activity correlated with the tissue accumulation of SA (Fig. 1). Exogenously supplied SA induced 0-GTase activity in tobacco leaves (Fig. 3), cell-suspension cultures of M. japonicus (Tanaka et al., 1990), and oat roots (Yalpani et al., 1992a). Induction of P-GTase and GSA accumulation observed during tobacco HR could be reproduced by leaf infiltration with SA (compare Figs. 1 and 3). The effects of exogenously applied and endogenously produced SA on P-GTase activity and GSA accumulation were quali-
1379
tatively and quantitatively similar. This suggests that tobacco does not substantially differentiate between the endogenously produced and exogenously supplied SA in terms of compartmentalization and biological activity. The minimal dose of SA capable of inducing tobacco P-GTase (2.7 pg g-' fresh weight) was similar to that observed in the TMVinoculated leaves at the time of GSA appearance (compare Figs. 1 and 3), indicating that the amounts of SA present in the inoculated leaf are sufficient for the induction of 6-GTase activity. In both TMV-inoculated and SA-infiltrated leaves, greater levels of tissue SA were associated with higher PGTase activity and greater amounts of GSA. Induction of PGTase activity by SA was sensitive to the protein synthesis inhibitor CHX (Fig. 4, A and B). Induction of P-GTase activity by SA was also inhibited by RNA synthesis inhibitors applied to cell-suspension cultures of M. japonicus (Tanaka et al., 1990) and to oat roots (Yalpani et al., 1992a). This indicates that SA triggers the de novo synthesis of P-GTase. The data presented indicate that SA accumulation in the TMV-inoculated leaves is responsible for the observed increases in P-GTase activity that is caused by the de novo synthesis of P-GTase protein. Greater levels of P-GTase are, in turn, responsible for the observed accumulation of GSA in the TMV-inoculated leaves (Fig. 1C). TMV-free leaves of the inoculated plant also contained elevated levels of SA compared with the levels present before the inoculation. However, these increases were not sufficient for the induction of P-GTase as demonstrated by SA infiltration studies (compare Fig. 2, B and C, to Fig. 3, A and 9). The lack of 0-GTase induction was reflected by the absence of GSA in TMV-free leaves of the inoculated plant (Fig. 2C). However, SA at the level of 0.27 pg g-' fresh weight is sufficient to induce the PR-1 family of pathogenesis-related proteins that are associated with SAR (Yalpani et al., 1991). Healthy tobacco plants contained basal levels of SA and P-GTase activity (Fig. 1). However, virtually no GSA was detected in the foliage of healthy tobacco plants. This may be explained by the fact that basal P-GTase activity is insufficient for GSA accumulation or that P-GTase and SA are located in different cellular compartments. Induction of P-GTase activity is specific toward SA and its close analog, 2,6-dihydroxybenzoic acid. In addition to being able to induce P-GTase, 2,6-dihydroxybenzoic acid can substitute for SA as an inducer of pathogenesis-related proteins (Van Loon, 1983) and the thermogenic response in Arum lilies (Raskin et al., 1989). In oat roots, however, 2,6-dihydroxybenzoic acid did not induce P-GTase activity (Yalpani et al., 1992a). The physiological role of GSA in TMV-inoculated leaves is unknown. The SA-to-GSA conversion may lead to sequestration of the SA glucoside into the vacuole (Ben-Tal and Cleland, 1982). The ability of SA to induce P-GTase activity may serve as an effective mechanism for the feedback regulation of SA levels in plant tissues. This regulation of tissue SA may be an important control point in the signal transduction pathway leading to the activation of disease-resistance mechanisms in plants. Altematively, GSA may function as a storage pool for SA. Our data demonstrate that P-GTase is one of the many proteins induced during the HR response. Therefore, it is
Enyedi and Raskin
1380
Plant Physiol. Vol. 101, 1993
induction of salicylicacid conjugates during the resistance response to tobacco mosaic virus infection. Plant Cell4 359-366
appropriate to refer to this enzyme as a pathogenesis-related protein. However, the particular importance of this protein is its ability to regulate the levels of SA-a putative plant hormone and a signal molecule in pathogen-resistance responses (Raskin, 1992). Future studies of this enzyme could lead to a better understanding of plant-pathogen interactions.
Metraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inverardi B (1990) Increase in salicylic acid at the onset of systemic acquired resistance in cuc-
ACKNOWLEDCMENTS
Raskin I, Turner IM, Melander WR (1989) Regulation of heat production in the inflorescences of an Arum lily by endogenous salicylic acid. Proc Natl Acad Sci USA 8 6 2214-2218 Ross AF (1961a)Localized acquired resistance to plant virus infection
The authors are grateful to Anya Timofeyeva for technical assistance and to Dr. Nasser Yalpani for helpful discussions. Received November 5, 1992; accepted January 23, 1993. Copyright Clearance Center: 0032-0889/93/101/1375/06. LITERATURE ClTED Ben-Tal Y, Cleland CF (1982) Uptake and metabolism of [“CIsalicylic acid in Lemnn gibba G3. Plant Physiol70 291-296 Enyedi AJ, Yalpani N, Silverman P, Raskin I (1992) Localization, conjugation, and function of salicylic acid in tobacco during the
hypersensitive reaction to tobacco mosaic virus. Proc Natl Acad Sci USA 8 9 2480-2484 Griffiths LA (1959) On the distribution of gentisic acid in green plants. J Exp Bot 1 0 437-442 Holmes FO (1938)Inheritance of resistance to tobacco mosaic disease in tobacco. Phytopathology 2 8 553-561 Ibrahim RK, Towers GHN (1959) Conversion of salicylic acid to gentisic acid and o-pyrocatechuicacid, a11 labelled with carbon-14, in plants. Nature 184 1803 Klambt HD (1962) Conversion in plants of benzoic acid to salicylic acid and its /3-d-glucoside. Nature 196: 491 Malamy J, Carr JP, Klessig DF, Raskin I (1990) Salicylic acid: a likely endogenous signal in the resistance response of tobacco to vira1 infection. Science 250 1002-1004 Malamy J, Hennig J, Klessig DF (1992) Temperature-dependent
umber. Science 250 1004-1006 Raskin I (1992) Role of salicylic acid in plants. Annu Rev Plant Mo1
Biol43: 439-463
in hypersensitive hosts. Virology 1 3 329-339 Ross AF (1961b) Systemic acquired resistance induced by localized virus infections in plants. Virology 13: 340-358 Steel RGD, Torrie JH (1980) Principles and Procedures of Statistics.
McGraw-Hill, New York Tanaka S, Hayakawa K, Umetani Y, Tabata M (1990)Glucosylation . of isomeric hydroxybenzoic acids by cell suspension cultures of Mallotus jnponicus. Phytochemistry 29 1555-1558 Umetani Y, Kodakari E, Yamamura T, Tanaka S, Tabata M (1990) Glucosylation of salicylic acid by cell suspension cultures of Mallotus jnponicus. Plant Cell Rep 9 325-327 Van Loon LC (1983) The induction of pathogenesis-related proteins by pathogens and specific chemicals. Neth J Plant Pathol 8 9
265-273 Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Metraux J-P, Ryals JA (1991) Coor-
dinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3: 1085-1094 Yalpani N, Balke NE, Schulz M (1992a) Induction of UDP-glucose:salicylic acid glucosyltransferase in oat roots. Plant Physiol 100 1114-1119 Yalpani N, Schulz M, Davis MP, Balke NE (1992b) Partia1 purification and properties of an inducible uridine 5’-diphosphateg1ucose:salicylic acid glucosyltransferase from oat roots. Plant Physiol 100 457-463 Yalpani N, Silverman P, Wilson TMA, Kleier DA, Raskin I (1991) Salicylic acid is a systemic signal and an inducer of pathogenesisrelated proteins in virus-infected tobacco. Plant Cell 3: 809-818
