Plasma Membrane Redox Enzyme 1S Lnvolved in the Synthesis of 0,- and H,O, by Phytophthora Elicitor- Stimulated Rose Cells'

Plant Physiol. (1995) 107: 1241-1247

Plasma Membrane Redox Enzyme 1s lnvolved in the Synthesis of 0,-and H,O, by Phytophthora Elicitor- Stimulated Rose Cells’

Chung-Kyoon Auh and Terence M. Murphy* Section of Plant Biology, Division of Biological Sciences, University of California, Davis, California 9561 6

suspension-cultured soybean or bean cells treated with An elicitor prepared from the autoclaved cell walls of Phytoph- funga1 elicitor (Bradley et al., 1992). H,O,-mediated protein thora sp. induced O,- generation and H,O, accumulation by cul- cross-linking functions as a rapid defense mechanism to tured cells of Rosa damascena Mill. cv Cloire de Cuilan. N,N- strengthen the cell wall during the hypersensitive re- Diethyldithiocarbamate,a superoxide dismutase inhibitor, blocked sponse, prior to deployment of transcription-dependent H,O, accumulation and caused a dramatic accumulation of 0,-by defenses (Brisson et al., 1994). H,Oz from the oxidative elicitor-treated rose cells. In the absence of N,N-diethyldithiocar- burst was shown to function as a local trigger for the bamate no detectable 0,- was accumulated. Diphenyleneiodo- programmed death of challenged cells and as a diffusible nium, quinacrine, pyridine, and imidazole, inhibitors of the mammalian neutrophil NADPH oxidase responsible for the generation of signal for the induction of cellular protectant genes in O,- during phagocytosis, inhibited O,- generation by elicitor- surrounding cells (Levine et al., 1994). It was suggested treated rose cells. Diphenyleneiodonium also inhibited NADH-de- that 0,- might induce the synthesis of phytoalexin in pendent O,- production by plasma membranes isolated from rose potato tubers infected with fungus (Doke, 1983a). How- cells. None of the four compounds inhibited the peroxidase activity ever, the mechanisms of the active oxygen production in- in the cell-suspension medium. These results demonstrate that elic- duced by elicitors and pathogens are not established. itor-stimulated accumulation of H,OZ comes only from superoxide Although a two-electron transfer to O,, producing H202 dismutase-catalyzed dismutation of 02-.The data are inconsistent directly, is possible (Bienfait and Liittge, 1988), H,O, may with the hypothesis that the synthesis of 0,-is catalyzed by extra- also be formed by the dismutation of O,-. Most investiga- cellular peroxidase and suggest that the enzyme responsible for the tors suggest that O,- dismutation is the source of elicitor- synthesis of O,- by elicitor-treated rose cells might be similar to the mammalian neutrophil NADPH oxidase. or pathogen-induced H202, probably because both O,- and H,02 synthesis have been shown to be induced in various systems, but there is little direct evidence to show the degree to which dismutation of 0,- accounts for the Treatment of suspension-cultured cells of Rosa damascena synthesis of H,O,. Mill. with elicitor prepared from Pkytopktkora sp. rapidly Little is known about the enzymes responsible for the stimulates the synthesis of H,O, (hydrogen peroxide) by reduction of O, to form O,-. Cell wall peroxidase was the cells (Arnott and Murphy, 1991). The rapid and tran- suggested by severa1 groups (Elstner and Heupel, 1976; sient production of active oxygen species by elicited plant Gross et al., 1977; Halliwell, 1978) to produce 0,- by a cells is termed the oxidative burst. Severa1 plant tissues and complex pathway involving a cycling of apoplastic NADH, suspension-cultured cells have been reported to produce NAD, radical, and NADC.More recently it was shown that active oxygen species, including 0,- (superoxide radical) a plasma membrane peroxidase is responsible for the for- and H202, during stimulation by elicitors (Aposto1 et al., mation of O,- at the surface of the plasma membrane 1989; Vera-Estrella et al., 1992; Legendre et al., 1993) or (Chalmers et al., 1986; Askerlund et al., 1987; Vianello and infection by pathogens (Chai and Doke, 1987; Doke and Macri, 1989). It is possible, even likely, that the cell wall Ohashi, 1988; Adam et al., 1989; Devlin and Gustine, 1992). peroxidase and the plasma membrane peroxidase are the These active oxygen species may be involved in plant same enzyme. Peroxidase has been suggested to function in defense responses against pathogens. the oxidative burst (Vera-Estrella et al., 1992). H,O, is required for the peroxidative polymerization of The other hypothesis, which derived from the study of cinnamoyl alcohols leading to lignification of cell walls mammalian neutrophil NADPH oxidase (Cross and Jones, (Harkin and Obst, 1973; Mader and Fiissl, 1982; van Huys- 1991), is that the synthesis of O,- occurs directly at the tee, 1987), which is a common response to pathogen attack extracellular surface of the plasma membrane through a (Kohle et al., 1984). H,O, rapidly mediates oxidative cross- linking of pre-existing structural proteins of the cell wall in Abbreviations: DDC, N,N-diethyldithiocarbamate; DPI, diphenyleneiodonium; LCDC, lucigenin-dependent chemiluminescence; ’ This research was supported by Environmental Protection LMDC, luminol-dependent chemiluminescence; lucigenin, N,N- Agency grant R 814960-01-0. dimethyl-9,9’-biacridium dinitrate; luminol, 5-amino-2,3-dihydro- * Corresponding author; e-mail [email protected]; fax 1,4-phthalazinedione; redox, oxidation/reduction; SOD, superox- 1-916 -752-5410. ide dismutase. 1241 1242 Auh and Murphy Plant Physiol. Vol. 107, 1995 one-electron reduction of molecular oxygen. The trans- search Service, University of California, Davis). The proce- plasma membrane redox components, including a flavin dure for obtaining crude elicitors by autoclavi ng washed and a Cyt, transfer electrons from cytosolic NAD(P)H to funga1 cell walls, based on the work of Ayers et al. (19761, molecular oxygen. Doke (1985) first suggested that plants was described by Arnott and Murphy (1991). The heat- might contain an O,--generating NADPH oxidase in the solubilized, nondialyzable material obtained from mycelial plasma membrane from the demonstration of NADPH- walls of Phytophthoru sp. was used as an elicitor. 4liquots of dependent 02-production in microsomes isolated from elicitor preparation were assayed for total carbohydrate by potato tubers infected by a pathogen. the phenol-sulfuric acid method (Dubois et al., 1956) using There are several redox enzymes in the plasma mem- Glc standards and for protein by the Bradfoi-d method brane that transfer electrons from cytosolic electron donors (Bradford, 1976) with BSA as the standard. The elicitor to electron acceptors in the apoplast. These plasma mem- preparation used in the present experiments had 0.72 brane redox enzyme activities have been proposed to be mg/mL carbohydrate and 0.49 mg/mL pote n. Elicitor involved directly or indirectly in several physiological pro- was added to 10 mL of cell suspension. cesses, including reduction of extracellular iron for uptake by root cells (Bienfait, 1985), formation of an electrochem- lsolation of Plasma Membranes ical membrane potential for the transport of ions (Merller and Crane, 1990), phototropic response to blue light (Kjell- The plasma membranes were isolated using the aqueous bom et al., 19851, and production of 0,- (Vianello and polymer two-phase system from rose cells harvested from Macri, 1989, 1991). 5-d-old cultures as previously described (Mc.rphy and In this study we show that treatment of rose cells with Auh, 1992). Phytophthoru elicitor caused the generation of O,-, which dismutated to H,O, through the action of SOD. In addition, Chemiluminescence Assay for H,O, we show that the oxidative burst in elicitor-treated cells is The concentration of H,O, in the cell suspensions was inhibited by compounds that do not affect the activity of measured by the chemiluminescence of luminol as previ- the rose cell peroxidase but do inhibit the mammalian ously described (Murphy and Huerta, 1990). This chemilu- neutrophil NADPH oxidase complex responsible for the minescence depends on the presence of H,O, and peroxi- respiratory burst during phagocytosis. dase (Seitz, 1978). The assay was conducted in a total volume of 2 mL by placing 0.2 mL of cell suspension, 0.2 MATERIALS AND METHODS mL of 1 mM luminol solution, and 0.02 to 0.1 unit of Chemicals peroxidase in 20 mM potassium phosphate buffer (pH 7.4) in a scintillation vial. The scintillation vial was immediately Luminol, lucigenin, horseradish peroxidase (EC 1.11.1.7), placed in a scintillation spectrometer (model LS8000, Beck- catalase (EC 1.11.1.6), and SOD (EC 1.15.1.1) were pur- man) and LMDC was detected on single-chanxiel mode. chased from Sigma. DDC was obtained from Fisher Chem- Counts were reported every 15 s for 1 min; thcb last two ical (Pittsburgh, PA) and DPI was from Cookson Chemicals values were averaged. Ltd. (Southampton, UK). Chemiluminescence Assay for O,- Cells The accumulated amount of O,- in the cell suspensions Cells of Rosa damascena Mill. cv Gloire de Guilan were was measured by the chemiluminescence of lucigenin, grown in suspension culture as previously described (Mur- which is specific for O,- (Corbisier et al., 1987). The assay phy et al., 1979). Rose cells were grown in 25 or 50 mL of was conducted in a total volume of 2 mL by placing 0.2 mL MXG medium on a rotary shaker (100 rpm) in the dark at of cell suspension and 0.2 mL of 1 mM lucigenin in 0.1 M 26°C. The cell line was maintained by subculturing 0.1 Gly-NaOH buffer (pH 9.0) containing 1 mM EDTA and 1 volume into fresh medium every week. For experiments, mM sodium salicylate. The SOD inhibitor DDC was added cells were subcultured into 50 mL of fresh medium and to the cell suspensions at 1 mM to block the dismutation of grown for 5 d prior to use. Cells from 5-d-old cultures were O,- to H,O, by SOD. The assay of 0,- prodiiction in harvested by filtration and washed three times at 30-min plasma membrane vesicles was conducted in a total vol- intervals by resuspending them in a solution of 1 mM CaC1, ume of 2 mL by combining plasma membrane vejicles (10 and 0.1 mM KC1. Cells were incubated at 26°C with con- pg of protein), 0.1 mM NADH, 0.2 mM lucigenin, and 0.01% stant shaking during the washing procedure and used in (w/v) Triton X-100 in the buffer described above. The experiments 2 h after the last wash. LCDC was also detected on single-channel mode j n a scintillation spectrometer. Counts were reported every 6 s for Elicitor Preparation 30 s; the last two values were averaged.

The elicitor was prepared from a strain of Phytophthora Enzyme Assay sp. isolated from a rose plant showing symptoms of root and crown rot (Arnott and Murphy, 1991). This strain was Peroxidase activity was assayed by measuring the oxi- provided to us by J. Mircetich (Department of Plant Pathol- dation of guaiacol at 470 nm as described by Castjllo et al. ogy and U.S. Department of Agriculture/Agricultural Re- (1984). The assay was run in a total volume of :I mL by Synthesis of 0,-by Elicitor-Stimulated Rose Cells 1243 combining 16 mM guaiacol, 2 mM H,O,, and 20 PL of the 1 O0 cell-free medium in 60 mM potassium phosphate buffer (pH 6.1). h v8 80 RESULTS o 60 Treatment of rose cells with Phytophthora elicitor stimu- 5 lated the accumulation of H,O, in the cell suspension. The .-9 40 elicitor-induced accumulation of H,O, started to increase 5 -a to 10 min after elicitor treatment, reached a maximum at - about 45 min, and decreased thereafter (Fig. 1).Rose cells 2 20 without elicitor treatment accumulated little H,O, (Fig. 1). The accumulation of H,O, depended on the amount of n O 0.2 6 2 6 elicitor (Fig. 2); very small amounts of elicitor (0.2 pL/mL of cell suspension) caused a significant accumulation of Elicitor (pUmL) H,O,. In control tests, elicitor caused no increase in LMDC Figure 2. Dose-dependent accumulation of H20, by rose cells in- in the absence of cells (data not shown). duced by funga1 elicitor. Samples were taken 30 min (black bars) and The effects of catalase and SOD on the elicitor-induced 45 min (gray bars) after elicitor treatment. The light signal for H202 LMDC are shown in Table I. Addition of catalase (100 accumulation obtained 45 min after elicitation from rose cells treated units/mL) to the cell suspension treated with elicitor in- with 6 yL/mL elicitor was set to 100%. The ordinate shows the hibited LMDC very strongly (Table I), confirming that the relative LMDC. Data represent means 2 SD from two independent elicitor-induced increase of LMDC was dependent on the experiments. presence of H,O, in the medium. The addition of catalase also reduced the LMDC of untreated Tose cells (Table I). The above result prompted us to measure the amount of Therefore, under our conditions, H,O, was the species of O,- in the cell suspension directly. The concentration of active oxygen most effective in producing LMDC, although O*- in the medium of suspension-cultured rose cells was since the elicitor-induced LMDC was not completely inhib- measured by LCDC. Addition of DDC (at a final concen- ited by catalase other species of active oxygen might also tration of 1 mM) to the cell suspension caused an accumu- have participated. When rose cells were incubated with lation of O,-. In the presence of DDC, treatment of rose elicitor and SOD (12 units/mL), LMDC was increased (Ta- cells with Phytophthovu elicitor caused a rapid increase in ble I). No detectable H,O, accumulated when rose cells the rate of synthesis of O,- (Fig. 3) in a dose-dependent were incubated with elicitor and the inhibitor of SOD, DDC manner (Fig. 4). In the absence of DDC neither untreated (Table I). These observations indicate that first superoxide nor elicitor-treated rose cells showed an accumulation of radical was formed in response to elicitor treatment and O,- (Fig. 3), demonstrating that in these cells the amount then H,O, was produced by the SOD-catalyzed dismuta- of SOD was sufficient to remove almost a11 the detectable tion of O,- into H,Oz and O,. O,- produced, even when its rate of production was stimulated by elicitor. The inhibitors of mammalian neutrophil NADPH oxi- 100- dase are broadly divided into two groups, those that inhibit

80 - Table 1. Effects of catalase, SOD, and DDC, an inhibitor of SOD, on H,O, accumulation by suspension-cultured rose cells treated 60 - with Phytophthora elicitor Rose cells were treated with elicitor (2 pL/mL) (+ elicitor) or water (- elicitor) and with DDC (1 mM), denatured catalase (heating at 40 - 100°C for 5 min), catalase (100 units/mL), or SOD (1 2 units/mL). All treatments were at zero time. The light signal for H,02 accumulation 20 - obtained from cells treated with elicitor and denatured catalase was set to 100%. Samples were taken 45 min after elicitor treatment and assayed for LMDC. Results are means ? SE from three independent experiments. O 15 30 45 60 75 90 Relative Concentration of H,Oz Time (min) Effector - Elicitnr + Elicitor Appearance of H20, in the medium of suspension-cul- % tured rose cells treated with Phytophthora elicitor, 2 pVmL (O),or DDC 0.03 ? 0.01 0.06 ? 0.03 not treated (O). Elicitor was added to the cell suspension at time zero. Denatured catalase 10.2 ? 2.3 1 0oa The light signal for H,02 accumulation obtained 45 min after elici- Catalase 3.1 ? 0.9 22.8 ? 1.9 tation from elicitor-treated cells was set to 100%. The ordinate shows SOD 2.1 2 0.1 128 2 10 the relative LMDC. Each point is the mean ? SE from five indepen- a The light signal of LMDC for 100% was 7.6 X 10' to 1.2 X 1 dent experiments. Error bars are masked when they are smaller than O* counts min-' (mL suspension)-', depending on the experiment. the symbol size. 1244 Auh and Murphy Plant Physiol. Vol. 107, 1995

1 O0 ,004

20 .I O O 25 50 75 O 15 30 45 60 Diphenyleneiodonium (pM) Time (min) Figure 5. The effect of DPI on O,- production by elicitor-treated Figure 3. Accumulation of O,- formed by suspension-cultured rose rose cells. DPI inhibits neutrophil NADPH oxidase activity by bind- cells treated with elicitor, 2 pL/mL (circles), or not treated (squares). ing to the flavoprotein component (Cross and Jones, 1986). DPI was Curves with filled symbols received 1 mM DDC. Curves with open added 5 min before elicitor treatment; elicitor (2 pL/mL) was added symbols did not receive DDC. Elicitor and DDC were added to the at time O; the light signal for 0,- production was determined 30 min cell suspension at zero time. The light signal for 0,- accumulation after elicitation. The LCDC for O,- accumulation from rose cells obtained 60 min after elicitation from elicitor-treated cells was set to untreated with DPI was set to 100%. The ordinate shows the relative 100%. The ordinate shows the relative LCDC. Each point is the mean LCDC. This experiment was performed three times with similar -C SE from five independent experiments. Error bars are masked when results; the results of a representative experiment are shown. they are smaller than the size of symbols. 0,- production by 84%. Pyridine (20 mM) and imidazole activation of the oxidase and those that exert a direct (10 mM), which bind to the b-type Cyt component (Iizuka et inhibitory effect on the oxidase components (Cross and al., 1985), inhibited 0,- production by about 80%. The Jones, 1991). DPI directly inhibits the NADPH oxidase concentrations of inhibitors used for these experirnents had activity by binding to the flavoprotein component of the no effect on the chemiluminescence reaction by lucigenin oxidase complex (Cross and Jones, 1986,1991). The synthe- and O,- (data not shown). sis of O,- by elicitor-treated rose cells was inhibited by low The effects of these inhibitors on the guaiacol peroxidase concentrations of DPI (Fig. 5). A 50% inhibition of 0,- activity of rose cell medium were tested becawe peroxi- synthesis was observed at about 13 p~ DPI. The effects of dase has been hypothesized to produce O,- in the apoplast other inhibitors of neutrophil NADPH oxidase are shown (Halliwell, 1978) and at the surface of the plasma mem- in Table 11. Quinacrine (0.5 mM), which binds to the fla- brane (Chalmers et al., 1986). As shown in Table 11, none of voprotein component (Cross and Jones, 1991), inhibited Table II. Effects of neutrophil NADPH oxidase inhibitors and peroxidase inhibitors on the production of 0,- by elicitor-treated rose 1O0 cells and on the peroxidase activity of the cell-free medi,m lnhibitors were added to the cell suspensions 5 min before elicitor h 8 80 treatment. Samples were taken 30 min after elicitor treatment and W assayed for LCDC. The light signal for O,- accumulation from o elicitor-treated cells with H,O was set to 100%. Value!, given (in 0 o 6o percent of control) are means 2 SE from three independtmt experi- -I ments. Peroxidase activity was assayed by measuring the o (idation of a, guaiacol at 470 nm. Data shown are means SD from an experiment .I .I 40 t - run in three to six replicates. Two independent preparations of the (d - cell-free medium showed the same results. B 20 Relative lnhibitor Concentration LCDC Peroxidase Activity

0 % of control AA4,* mil- mL- O 0.4 0.8 2 4 H,O (Control) 1 O0 10.8 t 0.6 Elicitor (pUmL) DPI 25 pM nt+b 10.8 2 0.8 Quinacrine 0.5 mM 16.0 ? 1.9 8.3 t 0.2 Figure 4. Dose-dependent accumulation of O,- generated by rose Pyridine 20 mM 21.2 ? 2.6 10.7 t 0.4 cells treated with elicitor. Samples were taken 30 min (black bars) lmidazole 10 mM 22.3 2 5.9 10.1 t 0.5 and 45 min (gray bars) after elicitor treatment. The light signal for SHAM' 5 mM 102 ? 3.8 5.2 :t 0.2 O,- accumulation obtained 45 min after elicitation from rose cells KCN 50 pM nta C' treated with 4 pVmL elicitor was set to 100%. The ordinate shows nt, Not tested. Refer to Figure 5. SHAM, jalicylhy- the relative LCDC. Data represent means -C SD from two independent droxamic acid. experiments. Synthesis of O,- by Elicitor-Stimulated Rose Cells 1245 the four compounds inhibited the peroxidase activity in the verted to H,O, by SOD. DDC at 0.63 mM gives nearly cell-suspension medium except quinacrine, which gave complete inhibition of the activity of purified SOD or of only a 25% inhibition. This activity was inhibited by sali- SOD in cellular supernatant, and it does not interfere with cylhydroxamic acid (5 mM) and KCN (50 WM), as expected. the light signal of LCDC (Corbisier et al., 1987). Phytoph- Salicylhydroxamicacid had no effect on O,- generation by thora elicitor stimulated the generation of O,- by rose cells elicitor-treated rose cells. (Figs. 3 and 4). The rate of O,- accumulation by elicitor- Plasma membrane vesicles isolated from rose cells were treated cells began to increase 5 to 10 min after treatment, able to produce O,- in the presence of NADH (0.1 mM). stayed high between 15 and 45 min, and slowed down after Without plasma membrane vesicles, the light signal of 45 min (Fig. 3). The time course of H,O, accumulation, LCDC was (2.2 ? 0.2) X 106 counts min-' (n = 31, and with measured without DDC (Fig. 11, was coincident with that 10 pg of protein of plasma membrane vesicles the light of O,- production, measured in the presence of DDC signal of LCDC for O,- production was (2.7 ? 0.1) X 107 (Fig. 3). counts min-' (n = 3). This activity was very sensitive to 25 Little is known about the mechanism of generation of PM DPI i(5.4 ? 1.4) X 106 counts min-', about 88% inhi- O,- by pathogen-infected or elicitor-treated plant cells. bitionl but insensitive to 0.1 mM KCN. A second indepen- Doke first demonstrated NADPH-dependent O,- produc- dent preparation of plasma membrane vesicles showed the tion in elicitor-treated protoplasts of potato tubers (1983b) same results (data not shown). and in microsomes isolated from tubers infected by Phyto- phthora infestam (19851, and suggested that plants might DlSCUSSlON contain an O,--generating NADPH oxidase in the plasma membrane. Chai and Doke (1987) also suggested that 0,- Treatment of rose cells with elicitor stimulated the accu- generation in plant leaves infected by P. infestam might be mulation of H,O,. The evidence for this comes from the due to the activation of an O,--generating NADPH oxi- LMDC of elicitor-treated cell suspensions (Figs. 1 and 2) dase. However, the nature of an O,--generating NADPH and from the inhibition of this chemiluminescence by cata- oxidase in plant cells has been confused by the demonstra- lase (Table I). Similar effects of catalase on the elicitor- tion that peroxidase in the presence of salicylhydroxamic induced chemiluminescence were observed in other acid or other phenolic compounds has NADH oxidase- and suspension-cultured cell/pathogen or cell/elicitor interac- O,--generating activity (Askerlund et al., 1987). tions, including white clover lPseudomonas corrugata (Dev- lin and Gustine, 1992), spruce/Heterobasidion annosum In the present study, elicitor-induced O,- generation by rose cells was shown to be sensitive to four inhibitors of the (Schwacke and Hager, 1992), and tomato/CZadosporium ful- num (Vera-Estrella et al., 1992).The fact that catalase inhib- mammalian neutrophil NADPH oxidase (Fig. 5; Table 11) at ited the LMDC of elicitor-treated cells by only 77% sug- concentrations similar to or lower than those effective with gests that a minor fraction of the LMDC could have been mammalian oxidase. Seventy micromolar DPI inhibited stimulated by another species of active oxygen, such as O,- generation by 75%. Addition of 50 WM DPI to phorbol 'OH. myristate acetate-stimulated neutrophils caused 98% inhi- The accumulation of H,O, stimulated by elicitor was bition of O,- production (Cross and Jones, 1986). Quina- almost entirely due to the dismutation of 0,- by SOD crine at 0.5 mM caused 84% inhibition of O,- generation by activity of rose cells. The evidence for this comes from the rose cells. Pyridine and imidazole inhibited O,- generation nearly complete inhibition of the elicitor-induced LMDC by about 80% at 20 mM and 10 mM, respectively. Pyridine by DDC, an inhibitor of SOD, and from the enhancement of inhibited the respiratory burst of phorbol myristate ace- this chemiluminescence by SOD (Table I). It seems unusual tate-stimulated porcine neutrophils by 95% at 150 mM, and that the addition of SOD would increase the rate of accu- similar inhibition was observed with imidazole (Iizuka et mulation of H,O,, given that the cells' own SOD is suffi- al., 1985). None of the four inhibitors, however, inhibited cient to keep the leve1 of O,- very low (Fig. 3). However, a peroxidase activity (Table II), excluding the possibility of a simple mathematical model suggests that these results are role for peroxidase for any more than 15 to 20% of the O,- not contradictory, provided that the formation of 'OH from production by the intact, elicitor-stimulated cells. This does O,- and H,O, through the metal-catalyzed Haber-Weiss not exclude a greater role for peroxidase in the generation reactions is taken into account. Similar effects of SOD were of H,O, in lignifying tissues of developing whole plants, found in white clover/P. corrugata (Devlin and Gustine, however. DPI also strongly inhibited O,- production by 1992) and spruce/H. annosum (Schwacke and Hager, 1992) plasma membrane vesicles isolated from rose cells, interactions, but an opposite effect (inhibition of chemilu- whereas KCN (0.1 mM) did not inhibit it, supporting the minescence by SOD) was observed in the tomato/C. fulvum contention that the elicitor-stimulated synthesis of O,- is interaction (Vera-Estrella et al., 1992). The inhibition of based at the plasma membrane (Doke, 1985). LMDC by DDC also implies that rose cells increased their The NADPH oxidase is thought to consist of at least two rate of synthesis of O,- in response to elicitor treatment. plasma membrane redox components, a flavoprotein and a More direct evidence for the synthesis of O,- was obtained b-type Cyt. DPI and quinacrine inhibit the mammalian by assaying O,- using LCDC, which is specific for O,- neutrophil NADPH oxidase activity by binding to the fla- (Corbisier et al., 1987). Treatment of the cell suspension voprotein, and pyridine and imidazole inhibit it by binding with 1 mM DDC was necessary to measure the accumula- to the b-type Cyt. The mechanisms of O,- generation by tion of O,-, because 0,- was otherwise very rapidly con- neutrophil NADPH oxidase consist of two-electron transfer 1246 Auh and Murphy Plant Physiol. Vol. 107, 1995 from cytosolic NADPH to the flavoprotein component, Cross AR, Jones OTG (1986) The effect of the inhibitor diphe- one-electron transfer to the b-type Cyt component, and nylene iodonium on the superoxide-generating syst'zm of neu- one-electron reduction of O, (Cross and Jones, 1991). The trophils: specific labelling of a component polypeptide of the oxidase. Biochem J 237 111-116 present results suggest that the enzyme responsible for the Cross AR, Jones OTG (1991) Enzymic mechanisms of isuperoxide synthesis of O,- by elicitor-treated plant cells is similar to production. Biochim Biophys Acta 1057: 281-298 the mammalian neutrophil NADPH oxidase (Mehdy, Devlin W, Gustine D (1992) Involvement of the oxidative burst in 1994), and that one function of the flavins and b-type Cyts phytoalexin accumulation and the hypersensitive reaction. Plant in plant plasma membranes (M~llerand Crane, 1990) may Physiol 100 1189-1195 Doke N (1983a) Involvement of superoxide anion generation in to participate in O,- generation. However, this sugges- be the hypersensitive response of potato tuber tissues to infection tion must be qualified by the recognition that the inhibitors with an incompatible race of Phytophtkoru infestuns and to the used, including DPI, also inhibit other enzymes. hyphal wall components. Physiol Plant Pathol 23: 345-357 Doke N (1983b) Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to h yphal wall ACKNOWLEDCMENT components of Phytophtkoru infestuns and specific inhibition of the reaction by suppressors of hypersensitivity. Phfsiol Plant The authors would like to thank Judy Murphy for critica1 read- Pathol 23 359-367 ing of the manuscript and for many helpful comments. Doke N (1985) NADPH-dependent 02-generation in membrane fractions isolated from wounded potato tubers inoculated with Pkytopkthoru infestuns. Physiol Plant Pathol 27: 311-3:!2 Received October 17,1994; accepted December 14, 1994. Doke N, Ohashi Y (1988) Involvement of an O,- ,;enerating Copyright Clearance Center: 0032-0889/95/107/1241/07. system in the induction of necrotic lesions on tobacco leaves infected with tobacco mosaic virus. 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