Elicitor-Stimulated Ion Fluxes and O2 from the Oxidative Burst Are Essential Components in Triggering Defense Gene Activation An
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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 4800–4805, April 1997 Plant Biology 2 Elicitor-stimulated ion fluxes and O2 from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley (furanocoumarinsyion channelsypeptide elicitoryreactive oxygen speciesysignal transduction) THORSTEN JABS*†,MARKUS TSCHO¨PE‡,CHRISTIANE COLLING*§,KLAUS HAHLBROCK*, AND DIERK SCHEEL‡¶ *Max-Planck-Institut fu¨r Zu¨chtungsforschung, Abteilung Biochemie, Carl-von-Linne´-Weg10, D-50829 Cologne, Germany; and ‡Institut fu¨r Pflanzenbiochemie, Abteilung Stress- und Entwicklungsbiologie, Weinberg 3, D-06120 Halle (Saale), Germany Contributed by Klaus Hahlbrock, February 26, 1997 ABSTRACT Fungal elicitor stimulates a multicomponent phytoalexins, and production of ethylene (11–13). Binding of defense response in cultured parsley cells (Petroselinum radiolabeled Pep-13 to parsley microsomes or protoplasts was crispum). Early elements of this receptor-mediated response found to be specific, reversible, and saturable (11). A 91-kDa are ion fluxes across the plasma membrane and the produc- plasma membrane protein was specifically labeled by covalent tion of reactive oxygen species (ROS), sequentially followed by crosslinking of Pep-13 to parsley membranes (14). Because defense gene activation and phytoalexin accumulation. Omis- identical structural elements of Pep-13 were required for sion of Ca21 from the culture medium or inhibition of specific binding, crosslinking and activation of defense reac- elicitor-stimulated ion fluxes by ion channel blockers pre- tions (11, 14), this membrane protein appears to represent the vented the latter three reactions, all of which were triggered elicitor receptor through which the entire defense response is in the absence of elicitor by amphotericin B-induced ion initiated. fluxes. Inhibition of elicitor-stimulated ROS production using In parsley as well as other plant cells, pathogen defense diphenylene iodonium blocked defense gene activation and reactions are induced by elicitor only if Ca21 is present in the 2 phytoalexin accumulation. O2 but not H2O2 stimulated phy- culture medium (11, 15–17). In tobacco, soybean, carrot, and toalexin accumulation, without inducing proton fluxes. These parsley, this requirement correlates with rapid elicitor- results demonstrate a causal relationship between early and stimulated Ca21 influx (11, 18–20), suggesting that Ca21 is an late reactions of parsley cells to the elicitor and indicate a important element in elicitor-mediated signal transduction, as sequence of signaling events from receptor-mediated activa- it is in many other signaling processes (21, 22). This and other tion of ion channels via ROS production and defense gene 2 early responses to elicitor treatment, such as various ion fluxes activation to phytoalexin synthesis. Within this sequence, O2 rather than H O appears to trigger the subsequent reactions. across the plasma membrane, synthesis of ROS, and phos- 2 2 phorylation and dephosphorylation of proteins, have fre- quently been discussed as putative components of signal Plants respond to pathogen attack by activating a large variety transduction chain(s) leading to defense gene activation of defense reactions, including transcriptional activation of andyor hypersensitive cell death (4, 15–17, 23–26). However, genes encoding phenylpropanoid-biosynthetic (1) and various unequivocal evidence for a causal relationship and a sequential other enzymes and proteins (2, 3), in addition to direct enzyme order of the signaling elements is still lacking. Here we report activation that leads to both reactive oxygen species (ROS) production (‘‘oxidative burst’’, ref. 4) and reinforcement of the that the elicitor-stimulated oxidative burst in cultured parsley cell wall (5). Parsley leaf buds respond strongly in this manner cells depends on ion fluxes across the plasma membrane and when inoculated with spores of Phytophthora sojae, a fungal is necessary and sufficient for triggering phytoalexin accumu- pathogen of soybean. The overall response comprises forma- lation. tion of small necrotic lesions resulting from hypersensitive cell death, incorporation of phenolic compounds into, and appo- MATERIALS AND METHODS sition of callose onto, cell walls at the infection site, as well as local and systemic activation of defense-related genes, and Cell Culture and Elicitor Treatment. Cell suspension cul- secretion of furanocoumarin phytoalexins into the infection tures of Petroselinum crispum were propagated according to a droplet (6, 7). With the exception of hypersensitive cell death published procedure (27). Protoplast preparation 5 days after and callose formation, all of these reactions are also stimulated subculturing, treatment with elicitor for 24 hr, and quantifi- in cultured parsley cells or protoplasts by treatment either with cation of furanocoumarins were performed as described (8). a crude elicitor preparation from the mycelium of P. sojae (8, Cell viability was determined by double staining with fluores- 9), with a 42-kDa glycoprotein elicitor from the same fungus cein diacetate and propidium iodide (28). Crude cell-wall (10), or with a 13-amino acid oligopeptide fragment (Pep-13) elicitor was prepared from the mycelium of Phytophthora sojae, derived from this protein (11). race 1 (27). The oligopeptide elicitor, Pep-13 (11), was syn- Pep-13 has been shown to be necessary and sufficient to thesized by Kem-En-Tec (Copenhagen). The polyene antibi- stimulate the complete elicitor response in parsley cells, in- otics, amphotericin B and nystatin, diphenylene iodonium cluding ion fluxes across the plasma membrane, the oxidative (DPI), the ion channel blockers, and KO2 were applied in burst, activation of defense-related genes, accumulation of Abbreviations: DDC, sodium diethyldithiocarbamate; DPI, diphe- The publication costs of this article were defrayed in part by page charge nylene iodonium; Pep-13, oligopeptide elicitor; ROS, reactive oxygen payment. This article must therefore be hereby marked ‘‘advertisement’’ in species. † accordance with 18 U.S.C. §1734 solely to indicate this fact. Present address: RWTH Aachen, Institut fu¨r Biologie III, Worringer Weg 1, D-52074 Aachen, Germany. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA §Present address: Marktplatz 12, D-76337 Waldbronn, Germany. 0027-8424y97y944800-6$2.00y0 ¶To whom reprint requests should be addressed. e-mail: PNAS is available online at http:yywww.pnas.org. [email protected]. 4800 Downloaded by guest on September 25, 2021 Plant Biology: Jabs et al. Proc. Natl. Acad. Sci. USA 94 (1997) 4801 dimethyl sulfoxide [final solvent concentration, 0.1% (voly flux commenced within 2–4 min, thus representing one of the vol)]. earliest detectable reactions of the cells. Increases in extracel- Measurement of Ion Fluxes and ROS. Ion concentrations lular Cl2 and K1 concentrations and alkalinization of the were determined using ion-selective electrodes for H1,K1, culture medium were stimulated with similar kinetics, indicat- 2 45 21 2 1 and Cl or by monitoring the uptake of Ca (11). H2O2 ing concomitant effluxes of Cl and K and uptake of protons release into the culture medium was quantified by measuring (Fig. 1 B–D). Because all of these elicitor-stimulated ion fluxes chemiluminescence produced by ferricyanide-catalyzed lumi- occurred along their concentration gradients, various iono- nol oxidation (29). Briefly, 50 ml medium were added to 750 phores were tested for their ability to simulate these fluxes and ml phosphate buffer (50 mM potassium phosphate, pH 7.9) thereby activate defense reactions in the absence of elicitor. prior to automated injection of 200 ml luminol (0.3 mM in Nigericin, A23187, valinomycin, and ionomycin stimulated phosphate buffer) and 100 mlK3[Fe(CN)6] (14 mM in H2O) by Ca21 and K1 fluxes, but only nigericin and A23187 stimulated the luminometer, Lumat LB 9501 (Berthold, Wildbach, Ger- Cl2 fluxes as well. However, all of these compounds failed to many). Chemiluminescence was recorded 3 sec after the last stimulate alkalinization of the culture medium or phytoalexin injection with a signal integration time of 5 sec. accumulation in the absence of elicitor. The saponin, digitonin, 2 For O2 determination (30), 0.5 mM sodium diethyldithio- which induces callose but not phytoalexin synthesis (35), carbamate (DDC) was added to the cell suspension culture to stimulated Ca21 influx (Fig. 1A) but none of the other ion inhibit superoxide dismutases. At different times after addi- fluxes (Fig. 1 B–D). The polyene antibiotics, amphotericin B tion of elicitor, 50-ml aliquots were removed from the culture and nystatin, stimulated ion fluxes qualitatively similar to those medium and added to 750 ml glycine-NaOH buffer (100 mM observed in response to elicitor; however, the relative inten- glycine, pH 9.0y1 mM EDTA), immediately followed by sities and durations of the individual fluxes varied greatly (Fig. automatic injection of 200 ml lucigenin solution (550 mM 1). The changes in ion concentrations caused by amphotericin lucigenin in glycine-NaOH buffer) and 100 ml glycine-NaOH B reached '50% of the elicitor-stimulated levels, though at buffer. Chemiluminescence was recorded for 10 sec after the considerably lower initial rates, and were of similar duration. last injection. The signal was calibrated using 50 mM xanthine In contrast, nystatin induced external Cl2 and K1 levels almost and 0.025–25 milliunits xanthine oxidase or KO2. identical