Journal of Experimental Botany, Vol. 54, No. 383, pp. 647-656,
February 1, 2003
© 2003 Oxford University Press
Multiple signalling pathways mediate fungal elicitor-induced ß-thujaplicin biosynthesis in Cupressus lusitanica cell cultures
Received 4 April 2002; Accepted 7 October 2002
1 Laboratory of Forest Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
2 Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
3 Present address and to whom correspondence should be sent: Department of Biochemistry, 104 Willard Hall, Kansas State University, Manhattan, KS 66506 USA. Fax: +1 785 532 7572. E-mail: jzhao{at}ksu.edu
Abbreviations: EGTA, ethylene glycol-bis-ß-aminoethylether-N,N,N’,N'-tetraacetic acid; G-proteins, GTP-binding proteins; DPI, diphenylene iodonium; MeJA, methyl jasmonate.
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Abstract |
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The biosynthesis of a phytoalexin, ß-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H2O2, or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of ß-thujaplicin. The elicitor signalling pathway involved in ß-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of ß-thujaplicin. A23187 also enhanced the elicitor-induced production of ß-thujaplicin. EGTA, LaCl3, and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of ß-thujaplicin. These results suggest that Ca2+ influx is required for elicitor-induced production of ß-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of ß-thujaplicin or enhanced the elicitor-induced production of ß-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of ß-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of ß-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca2+ flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca2+ signalling pathway increased lipoxygenase activity. These observations suggest that Ca2+ and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of ß-thujaplicin.
Key words: Calcium influx, Cupressus lusitanica, G-proteins, jasmonate signalling, signal transduction, ß-thujaplicin.
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Introduction |
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Plant–microbe interactions and plant defence responses, as well as the signal transduction pathways involved, have been studied extensively and continue to be topics of active research and discussion (Scheel, 1998). It is generally accepted that pathogen or elicitor recognition on the cell surface or in the cell initiates many cellular signalling processes that further activate multiple defence responses. The earliest event in plant cells exposed to a fungal elicitor is the perception of elicitor signals by receptors on the plasma membrane (Legendre et al., 1992; Zimmermann et al., 1997; Roos et al., 1999). Perception of elicitor signals activates receptor-coupled effectors, such as GTP-binding proteins (G-proteins) or protein kinases, which further activate ion fluxes (Legendre et al., 1992; Ligterink et al., 1997; Aharon et al., 1998). These ion fluxes seem to be necessary and sufficient for the induction of oxidative burst, defence gene activation and phytoalexin accumulation (Jabs et al., 1997; Trewavas and Malhó, 1998). Among these ion fluxes, Ca2+ influx and the transient calcium pulse in the cytosol are the most important events in plant defence responses because calcium plays a central role in various signal transduction systems (Bush, 1995; Trewavas and Malhó, 1998; Keller et al., 1998). In addition, It has been demonstrated that some downstream reactions, including H2O2 and jasmonate signalling pathways, and protein phosphorylation and dephosphorylation, are also involved in elicitor-induced defence responses (Mahady et al., 1998; Rajasekhar et al., 1999; Menke et al., 1999). Despite these considerable achievements, many aspects of plant defence responses and their signal transduction pathways remain to be determined. For example, G-proteins and their involvement in signal transduction and their relationships to plant secondary metabolism, as well as cross-talk among different signalling pathways are largely unknown (Scheel, 1998; Millner, 2001).
ß-Thujaplicin is a phytoalexin with strong antimicrobial activity and wide utilization in cosmetics, clinical products and other areas (Zhao and Sakai, 2001). However, ß-thujaplicin is mainly contained in the heartwood oleoresin in some Cupressaceae trees at low levels. Cupressus lusitanica cell culture was shown to produce a high level of ß-thujaplicin when treated with a yeast elicitor or methyl jasmonate (Zhao et al., 2001a). After elicitation, a transient increase in NADPH oxidase activity was observed, followed by a transient production of H2O2 (Zhao and Sakai, 2001). These observations suggest that elicitor-induced de novo biosynthesis of ß-thujaplicin may involve signalling events, since it becomes apparent that a number of signalling pathways mediate the fungal elicitor reorganization and the subsequent transduction to downstream responses. Therefore, there was interest in the signalling pathways that may be involved in yeast elicitor-induced accumulation of ß-thujaplicin. The information obtained from the study will certainly facilitate the improvement of the production of ß-thujaplicin by plant cell culture. Because pharmacological methods, together with other biochemical and molecular approaches, have been successfully used to identify various second messengers in signal transduction systems (Mahady et al., 1998; Rajasekhar et al., 1999; Kurosaki et al., 2001), these methods were employed to reveal the possible signalling pathways that mediate elicitor-induced biosynthesis of ß-thujaplicin.
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Materials and methods |
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Plant cell culture and elicitor treatment
The Cupressus lusitanica suspension cultures from callus were established as previously described (Zhao et al., 2001a; Zhao and Sakai, 2001). About 2.5 g of fresh cells were inoculated into 20 ml production medium in a 100 ml flask and incubated on a rotary shaker (120 rpm) at 23±2 °C in the dark. For the time-course study, an autoclave-sterilized yeast elicitor (YE, 1 mg ml–1), or water (control) was added to the cell cultures after filter sterilization, and the cell cultures were collected at specified intervals for the analysis of ß-thujaplicin and enzyme activity. The YE was a 70–80% (v/v) ethanol-insoluble oligosaccharide fraction prepared from yeast extract. Five-day-old suspension cultures were treated with YE, methyl jasmonate (MeJA), and H2O2 for 24 h, and then were collected for analysis.
Chemical reagents
Calcium ionophore A23187, melittin, cholera toxin, mastoparan, and suramin were obtained from Sigma, and these reagents were prepared in 0.5% DMSO solution or sterile distilled water. Lanthanum chloride and verapamil hydrochloride were obtained from Wako Pure Chemicals (Osaka, Japan), and these reagents were dissolved in water or 0.5% DMSO solution. Diphenylene iodonium (DPI) and ethylene glycol-bis-(ß-aminoethyl)ether-N,N,N’,N'-tetraacetic acid (EGTA) were obtained from ICN (Tokyo, Japan) and these reagents were dissolved in 0.5% DMSO solution. [
-32P] GTP and [
-32P] GTP were obtained from ARC (St Louis USA).
Treatment of C. lusitanica suspension cultures with pharmacological reagents
Five-day-old C. lusitanica suspension cells were used in all pharmacological experiments. In single treatment experiments, YE, A23187, melittin, cholera toxin, and mastoparan were filter-sterilized and added to 5-d-old cell cultures, respectively. For combination treatments with YE, A23187, melittin, cholera toxin, mastoparan, suramin, EGTA, DPI, LaCl3, and verapamil were filter-sterilized and added to cell cultures 20–40 min prior to YE addition. The control cells received the same volume of 0.5% DMSO solution. For combination treatments with A23187, EGTA or calcium channel inhibitors were added to the cell cultures 20 min before treatment with A23187, and the control received only the same volume of solvent. After incubation, the cell cultures were harvested for ß-thujaplicin and enzyme assays.
Lipoxygenase activity assay
Cells were collected by filtration under vacuum, and then frozen immediately at –80 °C. For lipoxygenase activity assay, the cells were homogenized in an ice bath with 0.1 M TRIS-HCl buffer (pH 8.5) containing 1% PVP (w/v), 1 mM CaCl2, 5 mM DTT, and 10% (v/v) glycerol. The homogenate was centrifuged at 11 000 g for 20 min at 4 °C, and the supernatant was used as the enzyme extract. Lipoxygenase was assayed according to Axelrod et al. (1981). 50 mg of linoleic acid was added to 50 mg Tween 20 and mixed with 10 ml of Na2HPO4 buffer (0.1 M, pH 8.7) by stirring and ultrasonic dispersion. The solution was cleared by addition of 250 µl of 1 M NaOH and diluted to 25 ml with the buffer. One ml of the enzyme reaction mixture contained 50 µl enzyme extract, 0.95 ml Na2HPO4 buffer and 5 µl of substrate solution. The increase in absorbance was monitored at 234 nm.
Plasma membrane isolation
The plasma membrane was isolated and purified according to the method of Wheeler and Boss (1987) with slight modifications. Plant cells were homogenized into powder in liquid nitrogen, and suspended in a cold extraction buffer containing 0.1 mM TRIS-HCl (pH 7.4), 1 mM EGTA, 0.25 M sucrose, 1% (w/v) cross-linked polypyrrolidone, 5 mM each of 2-mercaptoethanol and DTT, and protein inhibitor cocktail. The homogenate was mixed and centrifuged at 10 000 g for 15 min. The supernatant was centrifuged at 100 000 g at 4 °C for 1 h to pellet the microsomes. To separate the plasma membrane, the microsomal pellet was resuspended in 1.5 ml of the extraction buffer and layered on a 6.3% (w/v) PEG 3350/Dextran T500 polymer two-phase gradient in K2HPO4 buffer (pH 7.2) containing 0.25 M sucrose and 30 mM KCl. The two-phase system was inverted 70 times at 4 °C and then centrifuged in a swinging bucket rotor (600 g) for 10 min at 4 °C. The upper phase was removed and diluted to 30 ml with the grinding buffer, and centrifuged at 100 000 g for 1 h at 4 °C. The pellet was washed in 30 mM TRIS-HCl (pH 7.4) containing 15 mM MgCl2 and centrifuged at 100 000 g for 1.5 h and washed with the same buffer. The plasma membrane was resuspended in 30 mM TRIS-HCl (pH 7.4), 15 mM MgCl2, frozen in liquid nitrogen, and stored under –80 °C until use.
GTPase activity assay
GTPase activity in the plasma membrane was determined by measuring the amount of 32P released by hydrolysis of [-32P] GTP. The assay mixture consisted of 50 mM TRISHCl (pH 7.6), 10 mM MgSO4, 25 mM KCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 5 mM DTT, plasma membrane of the cultured cells, and 1 µM [-32P] GTP (37 MBq) in a total volume of 1 ml. Two sets of experiments were conducted. For the time-course of GTPase activity, the plasma membrane fractions from cells treated with YE for different times were used. For the effects of G-protein modulators on GTPase activity, the plasma membrane from non-elicited cells was used in parallel experiments by adding YE, cholera toxin, elicitor plus suramin, NaF or water, respectively. The reaction was run at 0 °C, and, at regular intervals, 100 µl aliquots of the reaction mixtures were removed. To the samples were added 100 µl of 10% (v/v) perchloric acid, 100 µl molybdate reagent, and 300 µl isobutanol according to the method of Sacchi et al. (1996). 32PO4 released from radiolabelled GTP was recovered in the isobutanolic phase by vertexing. One-hundred microlitre aliquots were removed from the alcohol phase, and the radioactivity was determined.
GTP-binding activity assay
GTP-binding activity of the plasma membrane from the cultured cells was determined by the incubation of the purified membrane with [-32P] GTP. The assay mixture consisted of 20 mM TRISHCl (pH 7.6), 1 mM EDTA, 1 mM DTT, 25 mM MgSO4, 25 mM KCl, 100 mM NaF, and the plasma membrane in a total volume of 200 µl. The binding reaction was initiated by the addition of 2 µM [-32P] GTP (9.25 MBq). In the time-course experiment, the plasma membrane from cells elicited for different times was tested. For the effects of G-protein modulators using the plasma membrane from non-elicited cultured cells, YE, cholera toxin, and suramin were added as indicated. In the experiments for testing binding specificity, 200 µM GTP, CTP, TTP or ATP was added to the assay mixture 10 min prior to the start of the reaction or [-32P] GTP at various concentrations was added. After incubation at 0 °C for 30 min, the reactions were stopped by the addition of 1 ml ice-cold binding buffer free of GTP, and the mixtures were transferred to a filtration apparatus. The samples were filtered through a nitrocellulose membrane (0.22 µm) by rapid suction, and the membrane filter was successively washed three times with 300 µl of the GTP-free binding buffer. The radioactivity of the filter adsorbing the membrane proteins was counted after mixing filter membrane with scintillation cocktail in vials. Protein content was determined by the Bradford method (Bradford, 1976) using a Bio-Rad kit with BSA as a standard.
Extraction and determination of ß-thujaplicin
Fresh cells were collected by vacuum filtration through a Miracloth, then weighed and quickly frozen. After homogenization, the homogenate was extracted twice with the same volume of ethyl acetate. To extract ß-thujaplicin, the medium was similarly extracted twice with ethyl acetate. The ethyl acetate extracts from the cell homogenates and the medium were combined and dried under vacuum and the residues were then treated with boron trifluoride methanol solution to form a ß-thujaplicin-BF2 complex. The samples were analysed by HPLC as described by Endo et al. (1988) with minor modifications. A Waters 996 system with a PDA detector monitoring at 254 nm; an Inertsil ODS-3 column (4.6x 150 mm, 5 µm) and a mobile phase composed of water/methanol (55/45, v/v) at a flow rate of 1 ml min–1 were utilized. Vanillin was used as an internal standard. Identification of ß-thujaplicin by GC-MS and 13C-NMR was reported elsewhere (Zhao et al., 2001a). Biomass was expressed by fresh weight.
All data were generated from triplicate independent experiments. Statistical analysis was carried out using Student’s t-test.
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ß-Thujaplicin and lipoxygenase were stimulated by YE, MeJA and H2O2
As shown in Fig. 1A, both YE and methyl jasmonate significantly stimulated biosynthesis of ß-thujaplicin in C. lusitanica cell cultures. The addition of exogenous H2O2 alone also stimulated the production of ß-thujaplicin. A comparison of the effects of YE, MeJA and H2O2 on the production of ß-thujaplicin shows that YE gave the greatest productivity whereas MeJA and H2O2 were less effective, producing only about a half of the ß-thujaplicin stimulated by YE. Previous studies have already shown that H2O2 or the oxidative burst mediates the induction of ß-thujaplicin by YE (Zhao and Sakai, 2001). To determine how H2O2 stimulates ß-thujaplicin accumulation and if the jasmonate signalling pathway is also an integral part of the elicitor signalling pathway leading to the accumulation of ß-thujaplicin, the activity of lipoxygenase, an important enzyme in the octadecanoid pathway to the biosynthesis of jasmonate (Bell et al., 1995; Schaller, 2001) was assayed.
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Results in Fig. 2 show that lipoxygenase was rapidly activated by YE, reaching an extremely high level after 12–24 h of treatment, and then decreasing to the basal level. This suggests that YE treatment stimulates the biosynthesis of jasmonate. MeJA and H2O2 treatments also stimulated lipoxygenase activity (Fig. 1B). Treatment of the cell cultures with H2O2 (10–24 mM) caused a significant increase in lipoxygenase activity, indicating that H2O2 promoted enzymatic lipid peroxidation leading to the accumulation of jasmonate. The results may suggest that the oxidative burst that is induced by YE may mediate YE-induced accumulation of ß-thujaplicin via the octadecanoid pathway. Since exogenous MeJA could up-regulate lipoxygenase activity and production of ß-thujaplicin, MeJA may stimulate an endogenous jasmonate signalling pathway to activate the biosynthesis of ß-thujaplicin in C. lusitanica cell cultures. All these data suggest that the jasmonate signalling pathway is an integral part of elicitor signal transduction leading to the production of ß-thujaplicin. The pathway could be a downstream part of the elicitor signal transduction in C. lusitanica cells just as in other plants (Gundlach et al., 1992; Mueller et al., 1993; Menke et al., 1999).
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Effects of calcium signalling inhibitors on the YE-induced accumulation of ß-thujaplicin
An increasing body of evidence suggests that Ca2+ influx is required for the oxidative burst, activation of defence genes, and the biosynthesis of phytoalexin or other secondary metabolites (Mahady and Beecher, 1994; Zimmermann et al., 1997; Trewavas and Malhó, 1998). A previous study showed that pretreatment of C. lusitanica cell cultures with 10 mM EDTA significantly inhibited elicitor-induced ß-thujaplicin accumulation, suggesting some metal ions may play important roles in the elicitor-induced accumulation of ß-thujaplicin (Zhao et al., 2001a). Inhibitors that affect the Ca2+ signalling pathway were used here to assay the effects of calcium flux on elicitor induction of ß-thujaplicin. EGTA is supposed to chelate apoplastic Ca2+ specifically and then limit Ca2+ influx; LaCl3 is often used as a Ca2+ channel inhibitor; verapamil is a phenylalkylamide and specially inhibits voltage-gated Ca2+-channels (Bush, 1995). As shown in Fig. 3, pretreatment of C. lusitanica cell cultures with 3 mM and 5 mM EGTA, or verapamil at different concentrations, inhibited the accumulation of ß-thujaplicin by about 40–60% over the control. On the other hand, the calcium channel blocker LaCl3 (0.3–1 mM) inhibited the accumulation of ß-thujaplicin induced by YE. These results indicate that extracellular calcium availability and Ca2+ influx may be required for the elicitor-induced production of ß-thujaplicin. These results are in agreement with other reports on calcium-mediated accumulation of phytoalexin or other secondary metabolites upon elicitor treatments (Mahady and Beecher, 1994; Jabs et al., 1997; Zhao et al., 2001b).
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Effects of A23187 on production of ß-thujaplicin
To confirm that extracellular Ca2+ influx is required for ß-thujaplicin biosynthesis, A23187, a well-known calcium ionophore, was applied to C. lusitanica cell cultures. After 12 h of treatment with A23187 at various concentrations, an increase in accumulation of ß-thujaplicin was observed (Fig. 4A). That effect was dose-dependent since A23187 at high concentrations (50 µM) suppressed ß-thujaplicin accumulation. However, pretreatment of the cell cultures with LaCl3 and EGTA inhibited the A23187-induced production of ß-thujaplicin, suggesting that Ca2+ influx is required for the elicitor-induced ß-thujaplicin biosynthesis. In addition, a combination of YE and A23187 significantly improved the production of ß-thujaplicin by 47% relative to YE treatment alone or by 42% relative to A23187 treatment alone (Fig. 4B). However, the promoting effects of YE and A23187 were partially suppressed by pretreatments of the cell with 10 mM EGTA or 0.5 mM LaCl3 (Fig. 4B). These results further strengthen the hypothesis that the YE-induced biosynthesis of ß-thujaplicin most probably involves Ca2+ influx. However, since all these calcium chelator or channel inhibitors only partly inhibited elicitor-induced ß-thujaplicin production, other signalling pathways that might participate in the elicitor-induced production of ß-thujaplicin could not be excluded.
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G-proteins are involved in the production of ß-thujaplicin in C. lusitanica cell cultures
The occurrence of G-proteins, including heterotrimeric complexes consisting of
-, ß-, and -subunits, and monomeric small proteins, has been demonstrated in a large number of plants; and these G-proteins are involved in various transmembrane signalling processes in higher plant cells (Sacchi et al., 1996; Saalbach et al., 1999; Millner, 2001). Some studies have shown that G-protein may also participate in plant defence responses, most probably by coupling with receptors to mediate the elicitor signal to other second messengers (Legendre et al., 1992; Aharon et al., 1998; Park et al., 2000). G-protein activators were first used to assay the involvement of G-proteins in the biosynthesis of ß-thujaplicin. Cholera toxin is a potent agonist of G-protein by preventing GTP hydrolysis. Mastoparan is a 14 amino residue oligopeptide from wasp venom, which can activate G-proteins by promoting the nucleotide exchange of G-protein -subunits in a manner similar to native receptors; Melittin is an amphiphilic oligopeptide with 25 amino resides that also activated G-proteins with potent efficiency (Mahady et al., 1998). In the absence of YE, these G-protein activators significantly improved the production of ß-thujaplicin (Fig. 5A). Among them, 5 µg ml–1 mellitin and 10 µg ml–1 melittin gave more than a 2-fold accumulation of ß-thujaplicin than did the control. Mastoparan also promoted the production of ß-thujaplicin, though its effect was weaker than the other two reagents. Cholera toxin at 2 µg ml–1 and 4 µg ml–1 promoted ß-thujaplicin production by about 2- or 3-fold over the control. In the presence of YE, these G-protein activators further increased the production of ß-thujaplicin (Fig. 5B). Suramin, a reagent that can uncouple G-proteins and receptor, is often used as an inhibitor of G-protein action in plants (Rajasekhar et al., 1999; Kurosaki et al., 2001). When suramin (50–100 µM) was added to the cell cultures prior to YE treatment, a significant decrease in accumulation of ß-thujaplicin was observed. These observations suggest that G-proteins may be involved in the biosynthesis of ß-thujaplicin and the YE-induced biosynthesis of ß-thujaplicin. It is likely that the YE signals are perceived by G-protein-coupled receptors on the surface of C. lusitanica cells.
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Elicitor treatment activated both GTP-binding activity and GTPase activity
To obtain further evidence for the involvement of G-proteins in YE-induced production of ß-thujaplicin, G-protein activity was assayed in the cell cultures treated or non-treated with YE. Because GTP-binding activity and GTPase activity are the two most important characteristics of G-proteins and are closely related to their structure and function, two activities were assayed in the plasma membrane purified from the cultured cells.
GTP-binding activity in the plasma membrane showed a gradual increase after YE treatment for 20 min and then slowly decreased (Fig. 6A). specific binding activity of the plasma membrane to GTP was also observed when unlabelled ATP, CTP, GTP, TTP at 100-fold excess compared with [-32P] GTP concentration were used during the assay. Except for GTP, all other nucleotides in excess did not obviously affect the GTP-binding activity of the plasma membrane (less than 8%), whereas excess unlabelled-GTP significantly (85%) decreased the amounts of [-32P] GTP bound to the plasma membrane. When [-32P] GTP was used at different concentrations to assay the GTP-binding activity of the plasma membrane, an enzymatic kinetics-like curve for GTP binding activity was obtained (Fig. 6A). These data all suggest that the binding activity of the plasma membrane to GTP is specific in these assays. Under identical conditions, suramin pretreatment significantly inhibited the elicitor-induced increase in GTP-binding activity, whereas cholera toxin treatment alone stimulated GTP-binding activity to a significant degree compared with the control (Fig. 6B). The results suggest that the GTP-binding activity of the plasma membrane could be affected by signal–receptor interactions, or could be receptor-coupled. These observations support the specificity of modulating effects of suramin and cholera toxin on G-protein functions observed in these pharmacological studies.
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Another important feature of G-proteins is GTPase activity that hydrolyses GTP bound to G-proteins and thus inactivate G-proteins. This activity is necessary for the regulation and recycling of G-proteins. The experiments show that GTPase activity increased over 15 min in the plasma membrane from YE-treated cells. Fifteen minutes after elicitor treatment, GTPase activity of the plasma membrane decreased (Fig. 7A). In addition, incubation of the plasma membrane in vitro with [
-32P] GTP increased the GTPase activity over 20 min (Fig. 7B). Therefore, the elicitor stimulated GTPase activity of G-proteins in the plasma membrane both in vivo and in vitro. The GTPase activity assay also showed that cholera toxin decreased GTPase activity, whereas suramin partially blocked GTPase activity induced by YE (Fig.7B). GTPase activity of the plasma membrane was suppressed almost completely in the presence of 100 mM NaF in the reaction mixtures, which is in agreement with the intrinsic GTPase property (Sacchi et al., 1996). Those results are consistent with the properties and the specificity of two reagents: cholera toxin suppresses GTP hydrolysis and suramin uncouples G-proteins to the receptor and thereafter inactivates GTPase activity of G-proteins. This behaviour of G-proteins in biochemical studies support the conclusions drawn from pharmacological evidence on ß-thujaplicin accumulation.
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Ca2+ and G-protein signalling pathways affected lipoxygenase activity
Since G-protein activation, Ca2+ release, and the jasmonate signalling pathway may mediate the elicitor signal to ß-thujaplicin accumulation, it was tested whether G-proteins and Ca2+ signals are connected to the jasmonate signalling pathway. To elucidate the effects of the Ca2+ signal and G-proteins on the jasmonate signalling pathway, lipoxygenase activity was assayed in the cell cultures treated with activators or inhibitors that can modulate function and signalling of G-proteins or Ca2+. Figure 8 shows that all functional inhibitors of G-proteins and Ca2+ suppressed the YE-induced increase in lipoxygenase activity. On the other hand, the activators of G-proteins and the Ca2+ signalling pathway stimulated lipoxygenase activity, alone or together with YE. Inhibition of the oxidative burst by DPI also partially suppressed the elicitor–induced increase in lipoxygenase activity, which is consistent with H2O2 stimulation of lipoxygenase activity. These observations suggest that G-proteins, Ca2+, and the oxidative burst may mediate the elicitor signals to the jasmonate signalling pathway, which finally leads to the biosynthesis of phytoalexin.
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Jasmonate production ubiquitously takes place in plants as a signal to alter gene expression in response to biotic and abiotic stresses. In many fungal elicited plant cells such as rice, tobacco and yew, a transient increase of endogenous jasmonate is followed by defence gene expression and the accumulation of secondary metabolites (Gundlach et al., 1992; Mueller et al., 1993; Menke et al., 1999). The jasmonate signalling pathway is also involved in the biosynthesis of protease inhibitors, PR-proteins, and phytoalexins in wounding-, elicitor-, UV-, or MeJA- induced plant defence responses (Bell and Mullet, 1993; Mueller et al., 1993; Schaller, 2001). The present results strongly support that the jasmonate signalling pathway is involved in the MeJA- and YE-induced production of ß-thujaplicin. A recent report has shown a mechanism by which jasmonate induced-gene expression is involved in the biosynthesis of plant secondary metabolites at a molecular level. Jasmonate may activate target genes through an ORCA3 transcription factor with a conserved jasmonate-response domain (van der Fits and Memelink, 2000). These observations suggest that the jasmonate signalling pathway is a sophisticated part of plant defence signal transduction and the accumulation of defence compounds.
The current studies also suggest that exogenous H2O2 could induce the biosynthesis of endogenous jasmonate biosynthesis and initiate the jasmonate pathway to activate the biosynthesis of ß-thujaplicin. Except for activating the octadecanoid pathway, H2O2 treatment also causes lipid peroxidation in plants. The fatty acid peroxides from lipoxygenase-catalysed or non-enzymatic lipid peroxidation can differentially activate plant defence response and cell death (Lam et al., 2001). The results in this paper suggest that H2O2- or the oxidative burst-induced lipoxygenase may play a critical role in the mediation of elicitor-induced production of ß-thujaplicin. However, how YE signals are transferred to the production of H2O2 and the jasmonate pathway, as well as to the biosynthesis of ß-thujaplicin is not clear. This study shows that receptor-coupled G-proteins and Ca2+ most probably are involved in the signalling processes. In other words, C. lusitanica cell cultures employ conserved signalling components such as the calcium ion and G-proteins in the transduction of the elicitor signal to downstream defence responses, such as the jasmonate signalling pathway.
It is clear that extracellular signals could be first transferred to downstream reactions by the receptor-coupled components, such as G-proteins and protein kinases. The existence and involvement of G-proteins in plants and plant signalling processes were once doubted, but the continuously increasing experimental data from molecular cloning of G-protein genes and the physiological characterization of G-proteins in various plant resources have greatly changed the situation (Saalbach et al., 1999; Mason and Botella, 2001; Millner, 2001). pharmacological and biochemical evidence has been provided here for the involvement of G-proteins in elicitor-induced biosynthesis of a phytoalexin. The results in this study from the use of melittin, mastoparan, and cholera toxin demonstrate that G-proteins may be involved in the production of ß-thujaplicin induced by YE. Suramin treatment suggests that YE signal transduction in C. lusitanica cells may be mediated by receptor-coupled G-proteins. Biochemical analysis further confirmed that YE treatment indeed activates G-protein activity, GTP-binding and GTPase activities, and that the effects of these activators or inhibitors of G-protein function used in the study were specific. Therefore, receptor-coupled G-proteins were suggested to be involved in elicitor signal transduction leading to ß-thujaplicin production.
As a universal second messenger, calcium has been proved to be involved in various physiological processes, including biotic and abiotic stress processes (Mahady and Beecher, 1994; Trewavas and Malhó, 1998; Zhao et al., 2001b). Like the defence responses of other plant cells exposed to fungal elicitors, Ca2+ influx may be one of the earlier committed events in C. lusitanica cell cultures after challenge by YE. Using A23187 and various Ca2+ signalling inhibitors, it is proposed that Ca2+ influx is required for ß-thujaplicin accumulation. Moreover, these results show that lipoxygenase, a key enzyme for jasmonate biosynthesis, may be regulated by G-protein and Ca2+ signals. It extends the roles of G-proteins and Ca2+ influx to the jasmonate signalling pathway, about which little is known. Only some events, such as Ca2+ influx, activation of phospholipase C, and H2O2 production, have been reported as G-protein effectors in plants (Aharon et al., 1998; Saalbach et al., 1999; Park et al., 2000).
Taken together, a signal transduction network may be proposed that incorporates the current experimental results and progress made in other plants (Fig. 9). Most probably, the first perception of YE signals (oligosaccharide molecules) by C. lusitanica cells activates a receptor-coupled G-protein, and then the active G-proteins further switch on ion channels. Ca2+ influx and the subsequent Ca2+ wave may initiate many other signalling pathways, such as a calmodulin-dependent protein kinase cascade, the oxidative burst and, eventually, the jasmonate biosynthesis and signalling pathway. These signals finally activate the expression of ß-thujaplicin biosynthesis. Until recently, there was little evidence for Ca2+-activation of jasmonate and G-protein-mediating secondary metabolite accumulation (Mahady et al., 1998; Kurosaki et al., 2001). This study provides evidence for these events.
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On the other hand, care should be taken in the interpretation of results from pharmacological methods because of the specificity of the reagents. The activators or an inhibitor of G-protein functions have mainly been based on animal studies and their specificity in plants is not clear. For example, mastoparan, a most commonly used G-protein activator, can also activate NDPK, which is believed to interact with the G-protein
-subunit. Similarly, melittin and suramin may also affect other signalling enzymes, besides G-proteins. However, these reagents have been successfully used in many previous plant studies (Legendre et al., 1992; Mahady et al., 1998; Rajasekhar et al., 1999). While other possible interpretations of these results could not be excluded, the biochemical analysis of G-proteins provides solid evidence for the specificity of some reagents, and is in agreement with the results obtained from the pharmacological study. Therefore, the present experiments and results could be used to derive some meaningful and trustworthy conclusions. In conclusion, G-proteins and Ca2+ are suggested to mediate the elicitor signal transduction to the jasmonate signalling pathway, which is further involved in elicitor-induced production of ß-thujaplicin. These results will lead to a more profound understanding of biosynthesis and regulation of ß-thujaplicin in the cell cultures, and direct further elucidation of the elicitor signalling network in C. lusitanica cell cultures. |
Acknowledgements |
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This work was supported by grant No. 12099345 from the Japan Society for the Promotion of Science (JSPS), which is gratefully acknowledged. This work was also partially supported by the scientific research fund (No.11876040 and No.13306013) from the Japanese Ministry of Education, Science and Culture. We are grateful to Dr Xuemin Wang and Dr Ruth Welti for reading the manuscript.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aharon GS, Gelli A, Snedden WA, Blumwald E. 1998. Activation of a plant plasma membrane Ca2+ channel by TGalpha1, a heterotrimeric G protein alpha-subunit homologue. FEBS Letters 424, 17–21.[CrossRef][Web of Science][Medline]
Axelrod B, Cheesbrough TM, Laakso S. 1981. Lipoxygenase from soybeans. Methods in Enzymology 17, 441–451.
Bell E, Creelman RA, Mullet JE. 1995. A chloroplast lipoxygenase is required for wound-induced jasmonate acid accumulation in Arabidopsis. Proceedings of the National Academy of Sciences, USA 92, 8675–8679.
Bell E, Mullet JE. 1993. Characterization of an Arabidopsis-lipoxygenase gene response to methyl jasmonate and wounding. Plant Physiology 103, 1133–1137.[Abstract]
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[CrossRef][Web of Science][Medline]
Bush DS. 1995. Calcium regulation in plant cells and its role in signaling. Annual Review of Plant Physiology and Plant Molecular Biology 46, 95–122.[CrossRef][Web of Science]
Endo M, Mizutani T, Matsumura M, Moriyasu M, Ichimaru M, Kato A, Hashimoto Y. 1988. High-performance liquid chromatographic determination of hinokitiol in cosmetics by the formation of difluoroborane compounds. Journal of Chromatography 455, 430–433.[CrossRef][Web of Science][Medline]
Gundlach H, Muller MJ, Kutchan TM, Zenk MH. 1992. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proceedings of the National Academy of Sciences, USA 89, 2389–2393.
Jabs T, Tschöpe M, Colling C, Hahlbrock K, Scheel D. 1997. Elicitor-stimulated ion fluxes and O2– from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proceedings of the National Academy of Sciences, USA 94, 4800–4805.
Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C. 1998. A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs. The Plant Cell 10, 255–266.
Kurosaki F, Yamashita A, Arisawa M. 2001. Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells. Plant Science 161, 273–278.
Lam E, Kato N, Lawton M. 2001. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411, 848–853.[CrossRef][Medline]
Legendre L, Heinstein PF, Low PS. 1992. Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells. Journal of Biological Chemistry 267, 20140–20147.
Ligterink W, Kroj T, Zur Nieden U, Hirt H, Scheel D. 1997. Receptor-mediated activation of a MAP kinase in pathogen defence of plants. Science 276, 2054–2057.
Mahady GB, Beecher CWW. 1994. Elicitor-stimulated benzophenanthridine alkaloid biosynthesis in bloodroot suspension cultures is mediated by calcium. Phytochemistry 37, 415–419.[CrossRef]
Mahady GB, Liu C, Beecher CWW. 1998. Involvement of protein kinase and G proteins in the signal transduction of benzo phenanthridine alkaloid biosynthesis. Phytochemistry 48, 93–102.[CrossRef][Web of Science][Medline]
Mason MG, Botella JR. 2001. Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with G beta. Biochimica et biophysica Acta 1520, 147–153.
Menke FL, Parchmann S, Mueller MJ, Kijne JW, Memelink J. 1999. Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in Catharanthus roseus. Plant Physiology 119, 1289–1296.
Millner PA. 2001. Heterotrimeric G-proteins in plant cell signalling. New Phytologist 151, 165–174.[CrossRef]
Mueller MJ, Brodschelm W, Spannagl E, Zenk MH. 1993. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proceedings of the National Academy of Sciences, USA 90, 7490–7494.
Park J, Choi H-J, Lee S, Lee T, Yang Z, Lee Y. 2000. Rac-related GTP-binding protein in elicitor-induced reactive oxygen generation by suspension-cultured soybean cells. Plant Physiology 124, 725–732.
Rajasekhar VK, Lamb C, Dixon RA. 1999. Early events in the signal pathway for the oxidative burst in soybean cells exposed to avirulent Pseudomonas syringae pv. glycinea. Plant Physiology 120, 1137–1146.
Roos W, Dordschbal B, Strighardt J, Hieke M, Weiss D. 1999. A redox-dependent G-protein-coupled phospholipase A of the plasma membrane is involved in the elicitation of alkaloid biosynthesis in Eschscholtzia californica. Biochimica et Biophysica Acta 1448, 390–402.[Medline]
Scheel D. 1998. Resistance response physiology and signal transduction. Current Opinion in Plant Biology 1, 305–310.[CrossRef][Web of Science][Medline]
Saalbach G, Natura G, Lein W, Buschmann P, Dahse I, Rohrbeck M, Nagy F. 1999. The alpha-subunit of a heterotrimeric G-protein from tobacco, NtGP alpha 1, functions in K+ channel regulation in mesophyll cells. Journal of Experimental Botany 50, 53–61.
Sacchi GA, Pirovano L, lucchini G, Cocucci S. 1996. A low-molecular-mass GTP-binding protein in the cytosol of geminated wheat embryos. Europe Journal of Biochemistry 241, 286–290.
Schaller F. 2001. Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. Journal of Experimental Botany 52, 11–23.
Trewavas AJ, Malhó R. 1998. Ca2+ signalling in plant cells: the big network! Current Opinion in Plant Biology 1, 428–433.[CrossRef][Web of Science][Medline]
van der Fits L, Memelink J. 2000. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289, 295–297.
Wheeler JJ, Boss WF. 1987. Polyphosphoinositides are present in plasma membranes isolated from Fusogenic carrot cells. Plant Physiology 85, 389–392.
Zhao J, Fujita K, Yamada J, Sakai K. 2001a. Improved ß-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate. Applied Microbiology and Biotechnology 55, 301–305.[CrossRef][Web of Science][Medline]
Zhao J, Hu Q, Guo YQ, Zhu WH. 2001b. Elicitor-induced indole alkaloid biosynthesis in Catharanthus roseus cell cultures is related to Ca2+-influx and the oxidative burst. Plant Science 161, 423–431.[CrossRef]
Zhao J, Sakai K. 2001. Involvement of peroxidases and hydrogen peroxide in the metabolism of ß-thujaplicin in fungal elicitor-treated Cupressus lusitanica suspension cultures. In: Morohoshi N, Komamine A, eds. Molecular breeding of woody plants. Progress in Biotechnology, Vol. 18. Amsterdam: Elsevier Science, 263–272.
Zimmermann S, Nurnberger T, Frachisse JM, Wirtz W, Guern J, Hedrich R, Scheel D. 1997. Receptor-mediated activation of a plant Ca2+-peameable ion channel involved in pathogen defence. Proceedings of the National Academy of Sciences, USA 94, 2751–2755.
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