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Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells
Plant Science 161 (2001) 273– 278 www.elsevier.com/locate/plantsci
Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells Fumiya Kurosaki *, Aoi Yamashita, Munehisa Arisawa Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical Uni6ersity, Sugitani, Toyama 930 -0194, Japan Received 10 November 2000; received in revised form 22 February 2001; accepted 13 March 2001
Abstract Biosynthetic activity of carrot phytoalexin 6-methoxymellen was induced in cell suspension culture by the treatment with oligogalacturonide elicitor; however, the elicitor-induced activity appreciably reduced in the presence of suramin, a potent inhibitor of GTP-binding proteins. In contrast, addition of G-protein activators, such as mastoparan or GTP-g-S, to carrot cell culture triggered 6-methoxymellein production even in the absence of uronide elicitor. An appreciable GTPase activity was found in purified plasma membrane of cultured carrot cells, and the hydrolytic activity was significantly increased by the addition of elicitor. Carrot plasma membrane was capable of associating with GTP-g-S, and the binding ability was markedly increased in the presence of elicitor. However, the binding activity markedly decreased when the membrane preparation was pre-incubated with GTP but not with ATP. These observations strongly suggest that a certain GTP-binding protein located at plasma membrane of cultured carrot cells plays an important role in the oligogalacturonide elicitor-induced 6-methoxymellein production. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Daucus carota; Phytoalexin biosynthesis; Signal transduction; GTP-binding protein; Plasma membrane; Oligogalacturonide elicitor
1. Introduction Higher plants defend themselves through a variety of mechanisms when they are challenged by pathogenic microorganisms [1–3]. However, several defense reactions preventing the microbial invasion take place concomitantly in infected plant cells, and this sometimes makes the physiological analyses of the individual defense responses difficult. Production of carrot phytoalexin, 6methoxymellein (6MM), is triggered by the contact of the cells with oligogalacturonide elicitor that is liberated by partial hydrolysis of carrot cell walls by the action of extracellular pectinase or proteases secreted by invading fungi [4]. We have demonstrated [4 – 7] that oligogalacturonide elicitor exhibits highly specific activity of inducing * Corresponding author. Tel.: + 81-76-4342281/2637; fax: + 8176-4345052. E-mail address: [email protected] (F. Kurosaki).
phytoalexin production in cultured carrot cells, and other defensive responses, such as chitinase induction, lignification and hypersensitive cell death, are essentially not observed by the stimulation with this class of elicitor. 6MM production in carrot cells is controlled primarily by the rate of transcription of genes encoding the biosynthetic enzyme proteins, and the increase in Ca2 + level in the cytoplasm of the cells is an essential early event in eliciting 6MM production [8]. We have demonstrated [9–12] that the elevation of cytoplasmic Ca2 + concentration in cultured carrot cells is triggered by at least two independent mechanisms. First, a rapid breakdown of phosphatidylinositol in plasma membrane of carrot cells takes place upon the contact with elicitor molecules, and this results in the liberation of a second messenger, inositoltrisphosphate, that derives Ca2 + from the internal store into cytoplasmic space [9]. As the second mechanism, we have shown [10 –12] that, in response to the stimulation
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by oligogalacturonide elicitor, cyclic AMP (cAMP) concentration in cultured carrot cells increases, and the nucleotide messenger in the elevated levels evokes gating of Ca2 + channels at the plasma membrane to initiate Ca2 + influx into the cytoplasm of the cells. We have proposed, therefore, that the dual mechanisms, activation of phosphatidylinositol cycle [9] and gating of cAMP-dependent cation channel(s) [10–12] are responsible for the increase in cytoplasmic Ca2 + triggered by the reception of elicitor molecules. However, biochemical events that evoke the activation of phospholipase C and cAMP generating system have not yet been elucidated. In our primary study, we reported [8] that the direct addition of cholera toxin to carrot cell culture is capable of triggering 6MM production in the cells, suggesting the possibility that GTP-binding protein is involved in the early events of the transmembrane signaling pathway to liberate the second messengers in carrot cytosol. In the present study, we attempted to elucidate whether GTPbinding protein(s) functions in the downstream stage of oligogalacturonide elicitor reception in the signal transduction cascade for the induction of 6MM production in cultured carrot cells.
2. Materials and methods
dichlorophenoxyacetic acid. The cell suspensions were maintained on an Innova 2300 rotary shaker (150 r.p.m.; New Brunswick Scientific Co.) at 26°C under darkness, and 15 ml aliquots of the culture were transferred to the fresh medium at a regular interval (14 days).
2.3. Induction of 6MM biosynthesis in cultured carrot cells Elicitor-active oligogalacturonides were prepared by partial hydrolysis of pectin fraction of cultured carrot cells as described previously in detail [9,14]. Aliquots of 14-day-old culture (early stationary phase, 5 ml each) were transferred to six-well flat bottom plates, and 10 mM K-acetate buffer (pH 5.5) containing 5 mg oligogalacturonide elicitor (50 ml) was added to the culture. If necessary, GTP analogs or G-protein inhibitors were added alone or 15 min prior to the exposure of carrot cells to elicitor, and four replicate cultures were prepared for each treatment. After incubation at 26°C for 24 h, the replicates were combined, and the cultured cells were harvested by vacuum filtration. The amounts of 6MM accumulated in the carrot cells were determined by a densitometric scan after the separation of the cell extracts by thin-layer chromatography according to the method described previously in detail [14].
2.1. Chemicals 6MM was isolated from fungi-infected carrot roots according to the method described previously in detail [4]. ATP, ATP-g-S, GTP, GDP-b-S and GTP-g-S were purchased from Boehringer Mannheim, while mastoparan and polyethylene glycol 3350 were from Sigma. Dextran T-500 and suramin were obtained from Pharmacia and Wako, respectively. [33P]GTP (specific activity, 111 TBq/mmol) and [35S]GTP-g-S (specific activity, 46.2 TBq/mmol) were obtained from New England Nuclear. All other chemicals were of reagent grade.
2.2. Carrot cell culture Cultured carrot cells (ft-2525 strain) derived from the root tissues were grown in 85 ml Murashige and Skoog’s liquid medium [13] in the presence of 3% (w/v) sucrose and 4.5 mM 2,4-
2.4. GTPase acti6ity of plasma membrane of cultured carrot cells Plasma membrane of cultured carrot cells was prepared by aqueous two-phase partitioning method as described previously in detail [11,15] with several modifications. In brief, the microsome fraction of cultured carrot cells (14 days old) was subjected to partitioning with 10 mM Hepes– KOH buffer (pH 7.2) containing 250 mM sucrose, 30 mM KCl, 5.6% (w/w) polyethylene glycol 3350 and 5.6% (w/w) Dextran T-500. The resultant upper phase was removed and mixed again with the freshly prepared lower phase, and plasma membranes in the upper phase were recovered by centrifugation (156 000×g, 30 min). Protein concentration was determined by the method of Bradford [16]. Purity of the membrane was assessed by measuring several marker enzymes, and the results had been reported previously [11,12].
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GTPase activity in carrot plasma membrane was determined by measuring the radioactivities of [33P]orthophosphate released by hydrolysis of [g33 P]GTP. The assay mixture consisted of 50 mM Tris– HCl (pH 7.6), 10 mM MgSO4, 25 mM KCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 5 mM dithiothreitol, purified plasma membrane of cultured carrot cells (50 mg protein) and 1 mM [g-33P]GTP (3.7 kBq) in a total volume of 1 ml. In a parallel experiment, 5 mg oligogalacturonide elicitor was also added to the assay mixture, and a set of the assay was also carried out in the presence of 100 mM NaF. The reaction was run at 0°C, and, at regular intervals, 100 ml aliquots of the reaction mixtures were removed. To the samples were added 100 ml of 10% (v/v) perchloric acid, 100 ml molybdate reagent and 300 ml isobutanol according to the method of Sacchi et al. [17], and [33P]phosphate released from radiolabeled GTP was recovered in the isobutanolic phase by blending. One hundred microliter aliquots were removed from the alcohol phase, and the radioactivities were determined after mixing with 3.5 ml commercial scintillation cocktail (ACS II; Amersham).
2.5. GTP-binding acti6ity of plasma membrane of cultured carrot cells GTP-binding activity of plasma membrane of cultured carrot cells was determined by the incubation of the highly purified membrane with [35S]GTP-g-S. The assay mixture consisted of, in a total volume of 200 ml, 20 mM Tris–HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, 25 mM MgSO4, 25 mM KCl, 50 mg plasma membrane, and, if necessary, 5 mg oligogalacturonide elicitor. The binding reaction was initiated by the addition of 50 nM [35S]GTP-g-S (7.4 kBq). In some experiments, 1 mM GTP or ATP was added to the assay mixture 10 min prior to the start of the reaction. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 1 ml ice-cold binding buffer free from GTP-g-S, and the mixtures were transferred to a microfiltration apparatus (Bio-Dot; Bio-Rad Laboratories), respectively. The samples were filtered through a nitrocellulose membrane (0.22 mm) by rapid suction, and the membrane filter was successively washed with the GTP-g-S-omitted binding buffer (300 ml each, three times). The appropriate areas of the filter
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adsorbing the membrane proteins were punched out with a cork bore, and the radioactivities associated with the proteins were determined.
3. Results
3.1. Effect of GTP analogs on 6MM production in cultured carrot cells In order to elucidate the possible participation of GTP-binding proteins in the induction of 6MM biosynthesis in cultured carrot, we first tested the effect of GTP analogs on the phytoalexin production in the cells. Several GTP-related reagents were added to cultured carrot in the absence or presence of oligogalacturonide elicitor, and their effects on 6MM production in the cells were examined (Table 1). Stimulation of 6MM production in cultured carrot cells was observed by the addition of mastoparan, a well-known oligopeptide activator of GTP-binding proteins, even in the absence of elicitor. Similarly, GTP-g-S, a non-hydrolyzable GTP analog that locks G-proteins in the active state, exhibited a significant activity of inducing Table 1 Effect of GTP-binding protein-related reagents on elicitor-induced 6MM biosynthesis in cultured carrot cellsa Treatment
Relative activity of 6MM biosynthesis (%) Experiment 1
Oligogalacturonide 100 elicitor Control (not treated) Not detectable Mastoparan (10 mM) 59 ATP-g-S (100 mM) 7 GDP-b-S (100 mM) 2 GTP-g-S (100 mM) 39 Elicitor+ATP-g-S (100 94 mM) Elicitor+GDP-b-S (100 89 mM) Elicitor+GTP-g-S (100 141 mM)
Experiment 2 100 Not detectable 68 3 5 51 110 82 159
a Cultured carrot cells were treated with various GTP-binding protein-related reagents in the presence or absence of oligogalacturonide elicitor, and 6MM accumulated in the cells was determined after 24 h incubation. The results are presented as percentages to the amounts of 6MM in positive controls as 100% that were treated with oligogalacturonide elicitor (0.55 and 0.40 mg 6MM/ml culture, respectively).
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3.2. Change in GTP hydrolytic acti6ity of the plasma membrane of cultured carrot cells
Fig. 1. Effect of suramin on oligogalacturonide elicitor-induced 6MM production in cultured carrot cells. Carrot cell culture was treated with various concentrations of suramin 15 min prior to the addition of oligogalacturonide elicitor and, after incubation for 24 h, 6MM accumulated in the cells were determined. The results were expressed as percentages to the amounts of 6MM in controls as 100% that did not receive suramin (0.47 and 0.38 mg 6MM/ml culture, respectively).
6MM biosynthesis without the presence of elicitor. In contrast, either ATP-g-S (non-GTP analog) or GDP-b-S GTP (GTP analog locking G-proteins in the inactive state) did not trigger the biosynthesis of the phytoalexin in carrot cell culture. Oligogalacturonide elicitor-induced 6MM production was markedly potentiated when GTP-g-S was added prior to the treatment of carrot culture with elicitor, and an approximately 1.4- to 1.6-fold increase in the phytoalexin accumulation was observed in repeated experiments. In contrast, it appeared that pretreatment of carrot cells with ATP-g-S did not affect elicitor-induced 6MM production. Although GDP-b-S is a well-known Gprotein inhibitor, cultured carrot cells previously treated with this reagent showed only very low reduction in the biosynthetic activity of 6MM after being challenged by elicitor A similar apparent discrepancy in the effect of GTP analogs has been also reported in soybean cells infected by Pseudomonas syringae [18], and these phenomena would be probably due to uptake problems for the GTP analog in the present experimental conditions. Suramin, a reagent that interferes with the GTP-binding site of G-proteins, showed an appreciable inhibitory activity against oligogalacturonide elicitor-induced 6MM production in cultured carrot cells when added 10 min prior to challenge with elicitor (Fig. 1). The elicitor-induced 6MM production decreased with the increase in suramin concentration, and it reduced to approximately 40% the level of control in the presence of 50 mM suramin.
Involvement of GTP-binding protein in the induction of phytoalexin production in cultured carrot cells was further confirmed by examining change in GTP hydrolytic activity of the carrot plasma membrane on the contact with oligogalacturonide elicitor. As shown in Fig. 2, an appreciable activity of GTP hydrolysis was observed in the plasma membrane of carrot cells even if the membrane preparation was not treated with elicitor. The activity of GTP hydrolysis was almost completely inhibited in the presence of 100 mM NaF [17], suggesting that the apparent liberation of orthophosphate was due to the specific hydrolysis by GTPase. The hydrolytic activity of carrot plasma membrane was significantly enhanced when oligogalacturonide elicitor was involved in the assay mixture, and, in repeated experiments, the activities were found to be 1.4- to 1.6-fold higher than those of non-treated controls after incubation for 20 min. As was in control treatment, GTPase activity of the elicitor-treated membrane was almost completely inhibited if NaF was involved in the assay mixture.
Fig. 2. Change in GTP hydrolytic activity of plasma membrane of cultured carrot cells by the treatment with oligogalacturonide elicitor. Plasma membrane prepared from cultured carrot cells was incubated with [33P]GTP in the absence ( ) or presence ( ) of oligogalacturonide elicitor, and the radioactivities of [33P]orthophosphate liberated from the nucleotide were determined. Four replicate experiments were performed at the time point of 20 min, and the results are presented as the means and standard deviations. In parallel experiments, the assay was also carried out in the presence of 100 mM NaF in the absence () or presence ( ) of elicitor.
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4. Discussion
Fig. 3. Change in GTP-binding activity of the plasma membrane of cultured carrot cells by the treatment with oligogalacturonide elicitor. Plasma membrane prepared from cultured carrot cells was incubated with [35S]GTP-g-S in the absence (1) or presence (2) of oligogalacturonide elicitor, and the radioactivities associated with the membrane were determined. In some experiments, plasma membrane was incubated with 1 mM GTP (3) or 1 mM ATP (4) prior to the addition of [35S]GTP-g-S. Data are presented as the means and standard deviations obtained from four independent experiments.
3.3. Change in GTP-binding acti6ity of the plasma membrane of cultured carrot cells Plasma membrane preparation of cultured carrot cells was incubated with [35S]GTP-g-S and the binding ability of the membrane toward the radiolabeled nucleotide was determined in the presence or absence of oligogalacturonide elicitor (Fig. 3). It was shown that an appreciable GTP-g-S-binding activity was found to associate with carrot plasma membrane even in the absence of elicitor. However, the nucleotide-binding activity located at the membrane increased approximately 1.7-fold the non-treated control if oligogalacturonide elicitor was present in the assay mixture. When the membrane was previously incubated with 1 mM GTP, GTP-g-S-binding activity in elicitor-treated carrot plasma membrane markedly decreased. The level of the binding in the GTP-pretreated membrane was much lower than that of the constitutive activity found in the control treatment. It appeared that this reduction of the binding ability by pre-incubation with nucleotides was GTP specific, and the decrease in the activity was not observed when GTP was replaced by ATP.
As well as in animal cells and eukaryotic microorganisms [19,20], it has been demonstrated that GTP-binding proteins are involved in various transmembrane signaling processes in higher plant cells [21,22]. Occurrence of several G-proteins, both heterotrimer complexes consisting of a-, b-, and g-subunits and monomeric proteins of low molecular weight, has been demonstrated in a variety of plant sources [21,22], and several genes encoding these proteins have been cloned [21]. In the present study, employing the combination of carrot oligogalacturonides, a highly ‘specific’ experimental system, it has been demonstrated that: (1) 6MM production in cultured carrot cells was induced by different class of G-protein activators, GTP-g-S and mastoparan, even in the absence of oligogalacturonide elicitor; (2) elicitor-induced 6MM production was appreciably inhibited by the treatment with suramin in a dose-dependent manner; (3) GTP hydrolytic activity observed in the plasma membrane of cultured carrot cells was appreciably enhanced in the presence of uronide elicitor; and (4) GTP-binding activity of the plasma membrane of carrot cells was significantly increased in the presence of uronide elicitor, and the activity was specifically inhibited by the pretreatment of the membrane with GTP. These observations strongly suggest that a certain GTP-binding protein(s) plays an important role in the early stage of signal transduction mechanisms of phytoalexin 6MM production in cultured carrot cells induced by stimulation with oligogalacturonide elicitor. It has been recently shown [23,24] that GTPbinding proteins participating in the hypersensitive response and oxidative burst in infected plant cells are small molecular weight monomers but not heterotrimers. In contrast, in the present study, it has been shown that mastoparan, an activator of trimer-type G-proteins, stimulates 6MM production even in the absence of elicitor. These results apparently suggest that phytoalexin production and other defense responses in higher plants might be mediated by different classes of GTP-binding proteins. However, mastoparan activates calmodulin, a key modulator protein of Ca2 + cascade, as well as trimer G-proteins. As reported previously [8,10 –12], 6MM production in cultured carrot cells is mediated by the activation of calmodulin,
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and therefore further study is necessary to determine the subclass of G-proteins involved in 6MM production in carrot cells. In our previous work, it has been already demonstrated that concomitant activation of phosphatidylinositol cycle [9] and gating of cAMP-dependent cation channel(s) [10–12] results in the elevation of the cytoplasmic Ca2 + level. This fact would provide primary information about the target effecters of elicitor-activated Gprotein. Phospholipase C and/or adenylate cyclase or their related proteins located at the plasma membrane would be important candidates of the effecter components of the G-protein specifically activated by oligogalacturonide elicitor. Therefore, the combination of uronide elicitor and cultured carrot cells appears to provide a convenient experimental system to investigate the functional relationship between GTP-binding protein and its effecter(s). Identification and characterization of G-protein activated by uronide elicitor and elucidation of its effecter components are in progress in our laboratory.
Acknowledgements
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
This work was supported in part by a Grant-inAid from the Ministry of Education, Science and Culture, Japan.
[17]
References
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[1] J. Friend, Plant phenolics, lignification, and plant disease, Prog. Phytochem. 14 (1980) 197 – 261. [2] R.A. Dixon, M.J. Harrison, C.J. Lamb, Early events in the activation of plant defense responses, Annu. Rev. Phytopathol. 32 (1994) 479 –501. [3] J. Ebel, A. Mithoefer, Early events in the elicitation of plant defense, Planta 206 (1998) 335 –348. [4] F. Kurosaki, Y. Tsurusawa, A. Nishi, Partial purification and characterization of elicitors for 6-methoxymellein production in cultured carrot cells, Physiol. Plant Pathol. 27 (1985) 209 –217. [5] F. Kurosaki, N. Tashiro, A. Nishi, Induction of chitinase and phenylalanine ammonia-lyase in cultured carrot cells treated with fungal walls, Plant Cell Physiol. 27 (1986) 1587–1591. [6] F. Kurosaki, N. Tashiro, A. Nishi, Induction, purification and possible function of chitinase in cultured carrot cells treated with fungal mycelial walls, Physiol. Mol. Plant Pathol. 31 (1987) 201 –210. [7] F. Kurosaki, N. Tashiro, A. Nishi, Role of chitinase and
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chitin oligosaccharides in lignification response of cultured carrot cells treated with mycelial walls, Plant Cell Physiol. 29 (1988) 527 – 531. F. Kurosaki, Y. Tsurusawa, A. Nishi, The elicitation of phytoalexins by Ca2 + and cyclic AMP in carrot cells, Phytochemistry 26 (1987) 1919 –1928. F. Kurosaki, Y. Tsurusawa, A. Nishi, Breakdown of phosphatidylinositol during the elicitation of phytoalexin production in cultured carrot cells, Plant Physiol. 85 (1987) 601 –604. F. Kurosaki, A. Nishi, Stimulation of calcium influx and calcium cascade by cyclic AMP in cultured carrot cells, Arch. Biochem. Biophys. 302 (1993) 144 –151. F. Kurosaki, H. Kaburaki, A. Nishi, Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells, FEBS Lett. 340 (1994) 193 –196. F. Kurosaki, Role of inward K+-channel located at carrot plasma membrane in signal cross-talking of cyclic AMP with Ca2 + -cascade, FEBS Lett. 408 (1997) 115 –119. T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant. 15 (1962) 473 –479. F. Kurosaki, Y. Kizawa, A. Nishi, Derailment product in NADPH-dependent synthesis of dihydroisocoumarin 6hydroxymellein by elicitor-treated carrot cell extracts, Eur. J. Biochem. 185 (1989) 85 –89. P. Graef, E.W. Weiler, ATP-driven Ca2 + -transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelia communis, Physiol. Plant. 75 (1989) 469 –478. M.M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein –dye binding, Anal. Biochem. 72 (1976) 248 –254. G.A. Sacchi, L. Pirovano, G. Lucchini, S. Cocucci, A low-molecular-mass GTP-binding protein in the cytosol of germinated wheat embryos, Eur. J. Biochem. 241 (1996) 286–290. V.K. Rajasekhar, C. Lamb, R.A. Dixon, Early events in the signal pathway for the oxidative burst in soybean cells exposed to avirulent Pseudomonas syringae pv glycinea, Plant Physiol. 120 (1999) 1137 –1146. A.G. Gilman, G-proteins: transducers of receptor-generated signals, Annu. Rev. Biochem. 56 (1987) 615 –649. H.R. Bourne, D.A. Sanders, F. McCormick, The GTPase superfamily: conserved structure and molecular mechanism, Nature 349 (1991) 117 –127. N. Terryn, M. Van Montagu, D. Inze, GTP-binding proteins in plants, Plant Mol. Biol. 22 (1993) 143 – 152. R.M. Mulligan, J. Chory, J.R. Ecker, Signaling in plants, Proc. Natl. Acad. Sci. USA 94 (1997) 2793 –2795. T. Kawasaki, K. Henmi, E. Ono, S. Hatakeyama, M. Iwano, H. Satoh, K. Shinamoto, The small GTP-binding protein Rac is a regulator of cell death in plants, Proc. Natl. Acad. Sci. USA 96 (1999) 10922 –10926. F. Kieffer, F. Simon-Plas, B.F. Maume, J.P. Blein, Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst, FEBS Lett. 403 (1997) 149–153.
Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells Fumiya Kurosaki *, Aoi Yamashita, Munehisa Arisawa Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical Uni6ersity, Sugitani, Toyama 930 -0194, Japan Received 10 November 2000; received in revised form 22 February 2001; accepted 13 March 2001
Abstract Biosynthetic activity of carrot phytoalexin 6-methoxymellen was induced in cell suspension culture by the treatment with oligogalacturonide elicitor; however, the elicitor-induced activity appreciably reduced in the presence of suramin, a potent inhibitor of GTP-binding proteins. In contrast, addition of G-protein activators, such as mastoparan or GTP-g-S, to carrot cell culture triggered 6-methoxymellein production even in the absence of uronide elicitor. An appreciable GTPase activity was found in purified plasma membrane of cultured carrot cells, and the hydrolytic activity was significantly increased by the addition of elicitor. Carrot plasma membrane was capable of associating with GTP-g-S, and the binding ability was markedly increased in the presence of elicitor. However, the binding activity markedly decreased when the membrane preparation was pre-incubated with GTP but not with ATP. These observations strongly suggest that a certain GTP-binding protein located at plasma membrane of cultured carrot cells plays an important role in the oligogalacturonide elicitor-induced 6-methoxymellein production. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Daucus carota; Phytoalexin biosynthesis; Signal transduction; GTP-binding protein; Plasma membrane; Oligogalacturonide elicitor
1. Introduction Higher plants defend themselves through a variety of mechanisms when they are challenged by pathogenic microorganisms [1–3]. However, several defense reactions preventing the microbial invasion take place concomitantly in infected plant cells, and this sometimes makes the physiological analyses of the individual defense responses difficult. Production of carrot phytoalexin, 6methoxymellein (6MM), is triggered by the contact of the cells with oligogalacturonide elicitor that is liberated by partial hydrolysis of carrot cell walls by the action of extracellular pectinase or proteases secreted by invading fungi [4]. We have demonstrated [4 – 7] that oligogalacturonide elicitor exhibits highly specific activity of inducing * Corresponding author. Tel.: + 81-76-4342281/2637; fax: + 8176-4345052. E-mail address: [email protected] (F. Kurosaki).
phytoalexin production in cultured carrot cells, and other defensive responses, such as chitinase induction, lignification and hypersensitive cell death, are essentially not observed by the stimulation with this class of elicitor. 6MM production in carrot cells is controlled primarily by the rate of transcription of genes encoding the biosynthetic enzyme proteins, and the increase in Ca2 + level in the cytoplasm of the cells is an essential early event in eliciting 6MM production [8]. We have demonstrated [9–12] that the elevation of cytoplasmic Ca2 + concentration in cultured carrot cells is triggered by at least two independent mechanisms. First, a rapid breakdown of phosphatidylinositol in plasma membrane of carrot cells takes place upon the contact with elicitor molecules, and this results in the liberation of a second messenger, inositoltrisphosphate, that derives Ca2 + from the internal store into cytoplasmic space [9]. As the second mechanism, we have shown [10 –12] that, in response to the stimulation
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by oligogalacturonide elicitor, cyclic AMP (cAMP) concentration in cultured carrot cells increases, and the nucleotide messenger in the elevated levels evokes gating of Ca2 + channels at the plasma membrane to initiate Ca2 + influx into the cytoplasm of the cells. We have proposed, therefore, that the dual mechanisms, activation of phosphatidylinositol cycle [9] and gating of cAMP-dependent cation channel(s) [10–12] are responsible for the increase in cytoplasmic Ca2 + triggered by the reception of elicitor molecules. However, biochemical events that evoke the activation of phospholipase C and cAMP generating system have not yet been elucidated. In our primary study, we reported [8] that the direct addition of cholera toxin to carrot cell culture is capable of triggering 6MM production in the cells, suggesting the possibility that GTP-binding protein is involved in the early events of the transmembrane signaling pathway to liberate the second messengers in carrot cytosol. In the present study, we attempted to elucidate whether GTPbinding protein(s) functions in the downstream stage of oligogalacturonide elicitor reception in the signal transduction cascade for the induction of 6MM production in cultured carrot cells.
2. Materials and methods
dichlorophenoxyacetic acid. The cell suspensions were maintained on an Innova 2300 rotary shaker (150 r.p.m.; New Brunswick Scientific Co.) at 26°C under darkness, and 15 ml aliquots of the culture were transferred to the fresh medium at a regular interval (14 days).
2.3. Induction of 6MM biosynthesis in cultured carrot cells Elicitor-active oligogalacturonides were prepared by partial hydrolysis of pectin fraction of cultured carrot cells as described previously in detail [9,14]. Aliquots of 14-day-old culture (early stationary phase, 5 ml each) were transferred to six-well flat bottom plates, and 10 mM K-acetate buffer (pH 5.5) containing 5 mg oligogalacturonide elicitor (50 ml) was added to the culture. If necessary, GTP analogs or G-protein inhibitors were added alone or 15 min prior to the exposure of carrot cells to elicitor, and four replicate cultures were prepared for each treatment. After incubation at 26°C for 24 h, the replicates were combined, and the cultured cells were harvested by vacuum filtration. The amounts of 6MM accumulated in the carrot cells were determined by a densitometric scan after the separation of the cell extracts by thin-layer chromatography according to the method described previously in detail [14].
2.1. Chemicals 6MM was isolated from fungi-infected carrot roots according to the method described previously in detail [4]. ATP, ATP-g-S, GTP, GDP-b-S and GTP-g-S were purchased from Boehringer Mannheim, while mastoparan and polyethylene glycol 3350 were from Sigma. Dextran T-500 and suramin were obtained from Pharmacia and Wako, respectively. [33P]GTP (specific activity, 111 TBq/mmol) and [35S]GTP-g-S (specific activity, 46.2 TBq/mmol) were obtained from New England Nuclear. All other chemicals were of reagent grade.
2.2. Carrot cell culture Cultured carrot cells (ft-2525 strain) derived from the root tissues were grown in 85 ml Murashige and Skoog’s liquid medium [13] in the presence of 3% (w/v) sucrose and 4.5 mM 2,4-
2.4. GTPase acti6ity of plasma membrane of cultured carrot cells Plasma membrane of cultured carrot cells was prepared by aqueous two-phase partitioning method as described previously in detail [11,15] with several modifications. In brief, the microsome fraction of cultured carrot cells (14 days old) was subjected to partitioning with 10 mM Hepes– KOH buffer (pH 7.2) containing 250 mM sucrose, 30 mM KCl, 5.6% (w/w) polyethylene glycol 3350 and 5.6% (w/w) Dextran T-500. The resultant upper phase was removed and mixed again with the freshly prepared lower phase, and plasma membranes in the upper phase were recovered by centrifugation (156 000×g, 30 min). Protein concentration was determined by the method of Bradford [16]. Purity of the membrane was assessed by measuring several marker enzymes, and the results had been reported previously [11,12].
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GTPase activity in carrot plasma membrane was determined by measuring the radioactivities of [33P]orthophosphate released by hydrolysis of [g33 P]GTP. The assay mixture consisted of 50 mM Tris– HCl (pH 7.6), 10 mM MgSO4, 25 mM KCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 5 mM dithiothreitol, purified plasma membrane of cultured carrot cells (50 mg protein) and 1 mM [g-33P]GTP (3.7 kBq) in a total volume of 1 ml. In a parallel experiment, 5 mg oligogalacturonide elicitor was also added to the assay mixture, and a set of the assay was also carried out in the presence of 100 mM NaF. The reaction was run at 0°C, and, at regular intervals, 100 ml aliquots of the reaction mixtures were removed. To the samples were added 100 ml of 10% (v/v) perchloric acid, 100 ml molybdate reagent and 300 ml isobutanol according to the method of Sacchi et al. [17], and [33P]phosphate released from radiolabeled GTP was recovered in the isobutanolic phase by blending. One hundred microliter aliquots were removed from the alcohol phase, and the radioactivities were determined after mixing with 3.5 ml commercial scintillation cocktail (ACS II; Amersham).
2.5. GTP-binding acti6ity of plasma membrane of cultured carrot cells GTP-binding activity of plasma membrane of cultured carrot cells was determined by the incubation of the highly purified membrane with [35S]GTP-g-S. The assay mixture consisted of, in a total volume of 200 ml, 20 mM Tris–HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, 25 mM MgSO4, 25 mM KCl, 50 mg plasma membrane, and, if necessary, 5 mg oligogalacturonide elicitor. The binding reaction was initiated by the addition of 50 nM [35S]GTP-g-S (7.4 kBq). In some experiments, 1 mM GTP or ATP was added to the assay mixture 10 min prior to the start of the reaction. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 1 ml ice-cold binding buffer free from GTP-g-S, and the mixtures were transferred to a microfiltration apparatus (Bio-Dot; Bio-Rad Laboratories), respectively. The samples were filtered through a nitrocellulose membrane (0.22 mm) by rapid suction, and the membrane filter was successively washed with the GTP-g-S-omitted binding buffer (300 ml each, three times). The appropriate areas of the filter
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adsorbing the membrane proteins were punched out with a cork bore, and the radioactivities associated with the proteins were determined.
3. Results
3.1. Effect of GTP analogs on 6MM production in cultured carrot cells In order to elucidate the possible participation of GTP-binding proteins in the induction of 6MM biosynthesis in cultured carrot, we first tested the effect of GTP analogs on the phytoalexin production in the cells. Several GTP-related reagents were added to cultured carrot in the absence or presence of oligogalacturonide elicitor, and their effects on 6MM production in the cells were examined (Table 1). Stimulation of 6MM production in cultured carrot cells was observed by the addition of mastoparan, a well-known oligopeptide activator of GTP-binding proteins, even in the absence of elicitor. Similarly, GTP-g-S, a non-hydrolyzable GTP analog that locks G-proteins in the active state, exhibited a significant activity of inducing Table 1 Effect of GTP-binding protein-related reagents on elicitor-induced 6MM biosynthesis in cultured carrot cellsa Treatment
Relative activity of 6MM biosynthesis (%) Experiment 1
Oligogalacturonide 100 elicitor Control (not treated) Not detectable Mastoparan (10 mM) 59 ATP-g-S (100 mM) 7 GDP-b-S (100 mM) 2 GTP-g-S (100 mM) 39 Elicitor+ATP-g-S (100 94 mM) Elicitor+GDP-b-S (100 89 mM) Elicitor+GTP-g-S (100 141 mM)
Experiment 2 100 Not detectable 68 3 5 51 110 82 159
a Cultured carrot cells were treated with various GTP-binding protein-related reagents in the presence or absence of oligogalacturonide elicitor, and 6MM accumulated in the cells was determined after 24 h incubation. The results are presented as percentages to the amounts of 6MM in positive controls as 100% that were treated with oligogalacturonide elicitor (0.55 and 0.40 mg 6MM/ml culture, respectively).
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3.2. Change in GTP hydrolytic acti6ity of the plasma membrane of cultured carrot cells
Fig. 1. Effect of suramin on oligogalacturonide elicitor-induced 6MM production in cultured carrot cells. Carrot cell culture was treated with various concentrations of suramin 15 min prior to the addition of oligogalacturonide elicitor and, after incubation for 24 h, 6MM accumulated in the cells were determined. The results were expressed as percentages to the amounts of 6MM in controls as 100% that did not receive suramin (0.47 and 0.38 mg 6MM/ml culture, respectively).
6MM biosynthesis without the presence of elicitor. In contrast, either ATP-g-S (non-GTP analog) or GDP-b-S GTP (GTP analog locking G-proteins in the inactive state) did not trigger the biosynthesis of the phytoalexin in carrot cell culture. Oligogalacturonide elicitor-induced 6MM production was markedly potentiated when GTP-g-S was added prior to the treatment of carrot culture with elicitor, and an approximately 1.4- to 1.6-fold increase in the phytoalexin accumulation was observed in repeated experiments. In contrast, it appeared that pretreatment of carrot cells with ATP-g-S did not affect elicitor-induced 6MM production. Although GDP-b-S is a well-known Gprotein inhibitor, cultured carrot cells previously treated with this reagent showed only very low reduction in the biosynthetic activity of 6MM after being challenged by elicitor A similar apparent discrepancy in the effect of GTP analogs has been also reported in soybean cells infected by Pseudomonas syringae [18], and these phenomena would be probably due to uptake problems for the GTP analog in the present experimental conditions. Suramin, a reagent that interferes with the GTP-binding site of G-proteins, showed an appreciable inhibitory activity against oligogalacturonide elicitor-induced 6MM production in cultured carrot cells when added 10 min prior to challenge with elicitor (Fig. 1). The elicitor-induced 6MM production decreased with the increase in suramin concentration, and it reduced to approximately 40% the level of control in the presence of 50 mM suramin.
Involvement of GTP-binding protein in the induction of phytoalexin production in cultured carrot cells was further confirmed by examining change in GTP hydrolytic activity of the carrot plasma membrane on the contact with oligogalacturonide elicitor. As shown in Fig. 2, an appreciable activity of GTP hydrolysis was observed in the plasma membrane of carrot cells even if the membrane preparation was not treated with elicitor. The activity of GTP hydrolysis was almost completely inhibited in the presence of 100 mM NaF [17], suggesting that the apparent liberation of orthophosphate was due to the specific hydrolysis by GTPase. The hydrolytic activity of carrot plasma membrane was significantly enhanced when oligogalacturonide elicitor was involved in the assay mixture, and, in repeated experiments, the activities were found to be 1.4- to 1.6-fold higher than those of non-treated controls after incubation for 20 min. As was in control treatment, GTPase activity of the elicitor-treated membrane was almost completely inhibited if NaF was involved in the assay mixture.
Fig. 2. Change in GTP hydrolytic activity of plasma membrane of cultured carrot cells by the treatment with oligogalacturonide elicitor. Plasma membrane prepared from cultured carrot cells was incubated with [33P]GTP in the absence ( ) or presence ( ) of oligogalacturonide elicitor, and the radioactivities of [33P]orthophosphate liberated from the nucleotide were determined. Four replicate experiments were performed at the time point of 20 min, and the results are presented as the means and standard deviations. In parallel experiments, the assay was also carried out in the presence of 100 mM NaF in the absence () or presence ( ) of elicitor.
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4. Discussion
Fig. 3. Change in GTP-binding activity of the plasma membrane of cultured carrot cells by the treatment with oligogalacturonide elicitor. Plasma membrane prepared from cultured carrot cells was incubated with [35S]GTP-g-S in the absence (1) or presence (2) of oligogalacturonide elicitor, and the radioactivities associated with the membrane were determined. In some experiments, plasma membrane was incubated with 1 mM GTP (3) or 1 mM ATP (4) prior to the addition of [35S]GTP-g-S. Data are presented as the means and standard deviations obtained from four independent experiments.
3.3. Change in GTP-binding acti6ity of the plasma membrane of cultured carrot cells Plasma membrane preparation of cultured carrot cells was incubated with [35S]GTP-g-S and the binding ability of the membrane toward the radiolabeled nucleotide was determined in the presence or absence of oligogalacturonide elicitor (Fig. 3). It was shown that an appreciable GTP-g-S-binding activity was found to associate with carrot plasma membrane even in the absence of elicitor. However, the nucleotide-binding activity located at the membrane increased approximately 1.7-fold the non-treated control if oligogalacturonide elicitor was present in the assay mixture. When the membrane was previously incubated with 1 mM GTP, GTP-g-S-binding activity in elicitor-treated carrot plasma membrane markedly decreased. The level of the binding in the GTP-pretreated membrane was much lower than that of the constitutive activity found in the control treatment. It appeared that this reduction of the binding ability by pre-incubation with nucleotides was GTP specific, and the decrease in the activity was not observed when GTP was replaced by ATP.
As well as in animal cells and eukaryotic microorganisms [19,20], it has been demonstrated that GTP-binding proteins are involved in various transmembrane signaling processes in higher plant cells [21,22]. Occurrence of several G-proteins, both heterotrimer complexes consisting of a-, b-, and g-subunits and monomeric proteins of low molecular weight, has been demonstrated in a variety of plant sources [21,22], and several genes encoding these proteins have been cloned [21]. In the present study, employing the combination of carrot oligogalacturonides, a highly ‘specific’ experimental system, it has been demonstrated that: (1) 6MM production in cultured carrot cells was induced by different class of G-protein activators, GTP-g-S and mastoparan, even in the absence of oligogalacturonide elicitor; (2) elicitor-induced 6MM production was appreciably inhibited by the treatment with suramin in a dose-dependent manner; (3) GTP hydrolytic activity observed in the plasma membrane of cultured carrot cells was appreciably enhanced in the presence of uronide elicitor; and (4) GTP-binding activity of the plasma membrane of carrot cells was significantly increased in the presence of uronide elicitor, and the activity was specifically inhibited by the pretreatment of the membrane with GTP. These observations strongly suggest that a certain GTP-binding protein(s) plays an important role in the early stage of signal transduction mechanisms of phytoalexin 6MM production in cultured carrot cells induced by stimulation with oligogalacturonide elicitor. It has been recently shown [23,24] that GTPbinding proteins participating in the hypersensitive response and oxidative burst in infected plant cells are small molecular weight monomers but not heterotrimers. In contrast, in the present study, it has been shown that mastoparan, an activator of trimer-type G-proteins, stimulates 6MM production even in the absence of elicitor. These results apparently suggest that phytoalexin production and other defense responses in higher plants might be mediated by different classes of GTP-binding proteins. However, mastoparan activates calmodulin, a key modulator protein of Ca2 + cascade, as well as trimer G-proteins. As reported previously [8,10 –12], 6MM production in cultured carrot cells is mediated by the activation of calmodulin,
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and therefore further study is necessary to determine the subclass of G-proteins involved in 6MM production in carrot cells. In our previous work, it has been already demonstrated that concomitant activation of phosphatidylinositol cycle [9] and gating of cAMP-dependent cation channel(s) [10–12] results in the elevation of the cytoplasmic Ca2 + level. This fact would provide primary information about the target effecters of elicitor-activated Gprotein. Phospholipase C and/or adenylate cyclase or their related proteins located at the plasma membrane would be important candidates of the effecter components of the G-protein specifically activated by oligogalacturonide elicitor. Therefore, the combination of uronide elicitor and cultured carrot cells appears to provide a convenient experimental system to investigate the functional relationship between GTP-binding protein and its effecter(s). Identification and characterization of G-protein activated by uronide elicitor and elucidation of its effecter components are in progress in our laboratory.
Acknowledgements
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
This work was supported in part by a Grant-inAid from the Ministry of Education, Science and Culture, Japan.
[17]
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