Proc. Natl. Acad. Sci. USA Vol. 94, pp. 11085–11089, September 1997 Plant Biology Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors JOHANNES W. STRATMANN AND CLARENCE A. RYAN* Institute of Biological Chemistry, Washington State University, Pullman, WA 99134-6340 Contributed by Clarence A. Ryan, August 5, 1997 ABSTRACT In response to wounding, a 48-kDa myelin Little is known of the intracellular signal transduction events basic protein (MBP) kinase is activated within 2 min, both upstream of the release of linolenic acid from membranes in locally and systemically, in leaves of young tomato plants. The response to wounding. Recently, several laboratories have activating signal is able to pass through a steam girdle on the demonstrated the wound-activation of myelin basic protein stem, indicating that it moves through the xylem and does not (MBP) kinases (MBPKs) in a variety of plant species, including require intact phloem tissue. A 48-kDa MBP kinase is also tomato, that are members of the mitogen-activated protein activated by the 18-amino acid polypeptide systemin, a potent kinase (MAPK) family (9–11). To date, no direct relationship wound signal for the synthesis of systemic wound response of these kinases to the activation of wound-response proteins proteins (swrps). The kinase activation by systemin is strongly has been demonstrated. inhibited by a systemin analog having a Thr-17 3 Ala-17 We report here the localized and systemic activation of a substitution, which is a powerful antagonist of systemin 48-kDa MBPK in response to wounding, systemin, and oligo- activation of swrp genes. A 48-kDa MBP kinase activity also saccharide elicitors. MBPK activation appears to be an early increases in response to polygalacturonic acid and chitosan step in the signaling pathway that activates the localized and but not in response to jasmonic acid or phytodienoic acid. In systemic expression of defensive genes in response to herbivory def1, a mutant tomato line having a defective octadecanoid and pathogen attacks. pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP MATERIALS AND METHODS kinase functions between the perception of primary signals Plant Material. Wild-type and mutant (def1) (8) tomato and the DEF1 gene product. In response to wounding, the plants (Lycopersicon esculentum cv Castlemart) were grown for MBP kinase is phosphorylated on phosphotyrosine residues, 14–16 days under a 17 hry7-hr light-dark cycle (28°C, .300 indicating a relationship to the mitogen-activated protein mEzm22zs21y17°C, darkness). At this stage, they display two kinase family of protein kinases. expanded leaves and a small apical leaf. Wounding, Elicitors, and Proteinase Inhibitor Bioassay. Tomato plants respond to insect herbivory and mechanical Tomato plants, 14–16 days old, were wounded with a hemostat damage by inducing a localized and systemic synthesis of around the edges of the terminal leaflet and incubated under several defense-related proteins, called systemic wound re- standard conditions of 300 mEzm22zs21 at 28°C, as reported (8). sponse proteins (swrps) (1). An 18-amino acid polypeptide, After the times indicated in the text, the wounded and the called systemin, has been isolated from tomato leaves that unwounded leaves of four plants were excised at the petiole appears to be a primary wound signal and is derived from a and immediately frozen in liquid nitrogen to assay kinase 200-amino acid precursor, prosystemin (2, 3). Several lines of activities. Six other plants from the same group were further evidence support a role for systemin as a primary systemic incubated for 24 hr under standard conditions and assayed for signal, including its potent activity (fmol range) (2), the proteinase inhibitor accumulation by radial immunodiffusion suppression of the wound response by the expression of a (12, 13). constitutive antisense CaMV-35S-prosystemin cDNA in to- All signaling compounds were supplied to the tomato plants mato leaves (4), and the phloem mobility of systemin that is through their cut stems as described by Howe et al. (8). The compatible with the timing of the transport of the wound signal uptake rate was 90 mlyhr on average unless indicated other- throughout the plants (2, 5, 6). wise. At the times indicated in the text, the plants were either Wounding of leaves of tomato plants or supplying systemin frozen in liquid nitrogen for the in-gel MBP kinase assay (four or oligosaccharide elicitors through their cut stems produces plants at each sampling time) or transferred to 20-ml capacity the oxylipin signaling molecule jasmonic acid (JA), which is a vials containing water and incubated for 24 hr under standard potent activator of swrp genes (7). JA is derived from its conditions and assayed for proteinase inhibitor I accumula- octadecanoid precursor, linolenic acid, which is released from tion. Because of a potential touch activation of MBPKs, an membranes, presumably via the activation of an intracellular appropriate touch control was carried out for each experiment. lipase in response to primary wound signals (7). A tomato 12-oxo-Phytodienoic acid (PDA) was obtained from Cay- mutant, def1, that lacks a functional octadecanoid pathway, is man Chemical (Ann Arbor, MI). Before use, 90% of the severely blocked in the wound induction of defensive genes and ethanol was evaporated and the compound redissolved in is much more susceptible to insect attacks than the wild type phosphate buffer. Aqueous stock solutions of systemin, the systemin analog Ala-17-systemin, salicylic acid, JA, polygalac- (8). turonic acid (degree of polymerisation '20), and nitrous The publication costs of this article were defrayed in part by page charge Abbreviations: ABA, abscisic acid; JA, jasmonic acid; MAPK, mito- payment. This article must therefore be hereby marked ‘‘advertisement’’ in gen-activated protein kinase; MBP, myelin basic protein; MBPK, MBP accordance with 18 U.S.C. §1734 solely to indicate this fact. kinase; PDA, 12-oxo-phytodienoic acid; swrp, systemic wound re- © 1997 by The National Academy of Sciences 0027-8424y97y9411085-5$2.00y0 sponse protein. PNAS is available online at http:yywww.pnas.org. *To whom reprint requests should be addressed. 11085 Downloaded by guest on September 25, 2021 11086 Plant Biology: Stratmann and Ryan Proc. Natl. Acad. Sci. USA 94 (1997) acid-treated chitosan were diluted in phosphate buffer. Ab- scisic acid (ABA) was dissolved in ethanol and diluted in phosphate buffer [in this case the buffer controls contained the same amount of ethanol (0.1%)]. Systemin and Ala-17-systemin were synthesized as described (14). Insect Feeding Experiments. Tobacco hornworm (Manduca sexta) eggs and hornworm artificial diet were obtained from Carolina Biological Supply. The eggs were hatched according to the supplier’s manual. When the larvae had reached a size of 7 6 2 cm, they were prestarved for several hours and then placed close to a leaf of a tomato seedling. The experimental setup allowed the larvae to reach the leaf with its mandibles and chew on it without any other contact. After 3 min, the wounded and the unwounded leaves were cut at the petiole and immediately frozen in liquid nitrogen for the in-gel kinase assay. Steam Girdling. Plants were steam girdled similarly as described by Nelson et al. (6). A hot steam jet was directed toward the stem for 1–2 min. One or 2 days after girdling the plants were cut either above or below the girdle and placed with their cut stem into phosphate buffer or a solution containing systemin in the same buffer. To estimate the influence of stress from handling during excision and transfer, FIG. 1. A 48-kDa MBPK is systemically activated by wounding in plants were severely handled, but not excised. Incubation and both the wounded and in unwounded leaves. The lower leaf of 14- to sampling for the kinase assay and radial immunodiffusion 15-day-old tomato plants (two expanded leaves) was wounded with a assay was as described above. hemostat and incubated under standard conditions as described. At In-Gel Kinase Assay. The frozen leaf tissue was transferred the times indicated, the lower wounded and upper unwounded leaves to a mortar and homogenized in extraction buffer containing were excised at the base of the petiole and assayed in an in-gel MBPK 50 mM HepeszKOH (pH 7.6), 2 mM DTT, 1 mM EDTA, 1 mM assay (A) and quantified using an InstantImager (B) with kinase EGTA, 20 mM b-glycerophosphate, 20% (vol vol) glycerol, 1 activities expressed as the fold activity above the level of untreated y control leaves, which were assigned a value of 1. ■, Wounded leaves; mM Na VO , 1 mM NaF, and 1 Complete proteinase inhibitor 3 4 F, systemic unwounded leaves. mixture tablet (Boehringer Mannheim) per 50 ml. Homogenates were centrifuged for 25 min at 40 000 3 g. Protein concentrations in the supernatants were determined times higher than that in the unwounded leaf and was sustained with the Pierce BCA protein assay using BSA as the standard. for a longer period. All steps were carried out at 4°C. Equal amounts of proteins Systemin Elicits MBPK Activity. Fig. 2 shows that the in the supernatants (50 mg for 10-well gels and 30 mg for 15-well polypeptide wound signal, systemin, when supplied to young gels) were separated by SDSygel electrophoresis on a 10% tomato plants through their cut stems, induces a 48-kDa polyacrylamid minigel. Before polymerization, 0.25 mgyml MBPK activity in leaves. The activity was detected within 5 MBP (Sigma) was added to the polyacrylamid solution. De- min, reaching a maximum within 15 min of the application of naturing and renaturing of the proteins in the gels and the systemin, and the activity was sustained for at least 60 min.