Journal of Experimental Botany, Vol. 51, No. 345, pp. 685-693,
April 2000
© 2000 Oxford University Press
Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture
1 Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464–8601 Japan
2 Nagoya University Bioscience Center, Nagoya University, Chikusa-ku, Nagoya, 464–8601 Japan
Received 29 October 1999; Accepted 16 November 1999
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2, cell line BY-2) catalyse the salicylic acid (SA)-dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration. Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA-induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS. The mechanism of peroxidase-catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction. The results showed that SA induced the peroxidase inhibitor-sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected. Similar results were obtained using HRP. It was also observed that SA suppressed the H2O2-dependent formation of hydroxy radicals in vitro. The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase-catalysed reaction, as the intermediate signals. The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2. In addition, it was observed that superoxide dismutase-insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase-catalysed reactions. The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide.
Key words: Active oxygen species, calcium, Nicotiana tabacum, peroxidase, salicylic acid, signal transduction.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Salicylic acid (SA) has been claimed to be essentially involved in systemic acquired resistance (SAR) against viral, fungal and bacterial pathogens (Gaffney et al., 1993
; Delaney et al., 1994). It was proposed that SA signalling leading to SAR is mediated by active oxygen species (AOS) derived from H2O2, since SA specifically binds to catalase and inhibits it (Chen et al., 1993). Thus SA inhibits the decomposition of H2O2 produced in plants. However, it was not studied whether SA directly induces generation of AOS or not.
Calcium is required as a secondary messenger for certain processes in plant defence mechanisms (Knight et al., 1991; Messiaen et al., 1993; Levine et al., 1996; Sanders et al., 1999). Ca2+ is essential for SA-induced chitinase accumulation in tobacco leaves (Raz and Fluhr, 1992) and carrot suspension culture (Schneider-Müller et al., 1994).
The first evidence for direct interaction between the SA-induced AOS and cytosolic free calcium concentration ([Ca2+]c) in tobacco suspension culture has already been shown (Kawano et al., 1998). Extracellularly localized tobacco peroxidases catalyse the SA-dependent formation of superoxide (
) which, in turn, induces an increase in [Ca2+]c. However, generation of other AOS such as H2O2 and hydroxy radicals (HO·) in response to SA addition has not been studied. The present study was carried out in an attrempt to understand the mechanism of SA-dependent production of AOS catalysed by tobacco peroxidases, and horseradish peroxidase used as a model system of peroxidase reaction, through AOS-sensitive chemiluminescence (CL), and electron spin resonance (ESR) spectroscopy. How peroxidases relay the SA signal to the changes in [Ca2+]c was also discussed.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Plant material
Tobacco suspension culture cells (Nicotiana tabacum L. cv. Bright Yellow-2, cell line BY-2), which express apoaequorin specifically in the cytosol (Takahashi et al., 1997
), were grown in the dark and used 3 d after subculturing.
Chemicals
Chemically synthesized coelenterazine and recombinant monodehydroascorbate reductase were generous gifts from Professor M Isobe, Nagoya University and from Dr Miyake, Nara Institute of Science and Technology, respectively. A Cripridina luciferin-derived chemiluminescent reagent (CLA, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) and Tiron were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). SA and luminol were from Wako Pure Chemical Industries (Osaka, Japan), 3-amino-1,2,4-triazole, N,N'-dimethylthiourea (DMTU) from Nacalai Tesque Inc. (Kyoto, Japan), and 1,10-phenanthroline and 2,2'-bipyridyl from Dojindo Laboratories, Inc. (Kumamoto, Japan). CuZn-superoxide dismutase (SOD) and 5,5'-di-methyl-1-pyrroline-N-oxide (DMPO) were from Sigma (St Louis, MO, USA) and horseradish peroxidase (HRP) was from Worthington Biochemical Co. (Freehold, NJ, USA).
SA was dissolved in ethanol and diluted with water to the appropriate concentration keeping the ethanol concentration at 2% (v/v). To 200 µl of the tobacco cell suspension, 10 µl of chemical solutions were added (final ethanol concentration, 0.1%).
[Ca2+]c and AOS monitoring
The generation of and changes in [Ca2+]c were monitored by the -specific CL of CLA and the Ca2+-dependent luminescence of aequorin, respectively, as described previously (Kawano et al., 1998). The calcium-sensitive luminescent protein aequorin was reconstituted by addition of 1 µM coelenterazine to the suspension culture of apoaequorin-expressing tobacco cells, 8 h prior to the measurements of [Ca2+]c. The aequorin-luminescence was measured with a CHEM-GLOW Photometer (American Instrument Co., Md., USA) equipped with a pen recorder (Rikadenki Co., Tokyo, Japan), and expressed as relative luminescence unit (rlu). After each experiment all remaining aequorin was discharged with 1 M CaCl2 and 10% ethanol, and the resultant luminescence was measured to estimate the amount of remaining aequorin. Cytosolic Ca2+ concentration was calculated using the calibration equation: pCa=0.332558(-logk)+5.5593 where k is a rate constant equal to luminescence counts per second divided by total counts (Knight et al., 1996; Takahashi et al., 1997).
By measuring CLA- and luminol-CL as described in the measurement of [Ca2+]c, generation of and H2O2 were monitored, respectively. CLA and luminol were added to the cell suspension at final concentrations of 1 µM and 10 µM, respectively. Productions of and H2O2 were estimated using calibration curves for KO2-dependent CLA-CL and H2O2- dependent luminol-CL, respectively.
Detection of HO· by electron spin resonance (ESR) spectroscopy
Tobacco cell culture was pre-incubated with 500 µM DMPO, used as spin trapper for HO·, for 5 min prior to the addition of SA to the culture. Immediately after SA addition, the culture was filtered through a nylon mesh (40 µm pore size). The resultant cell-free culture medium (175 µl) was placed in a flat-shaped quartz ESR cell. ESR spectra of DMPO-OH signal were collected on a JEOL-TE200 ESR spectrometer (X-band) with a sweep width of 5 mT, a 100 kHz modulation frequency, 60 s sweep time, a time constant of 0.1 s and a microwave power of 10 mW, at room temperature of 20–25 °C. The reaction for Cu-catalysed HO· formation was initiated by the addition of 1 mM CuSO4 to the reaction mixtures containing 100 mM K-phosphate (pH 7.0), 10 mM H2O2, 500 µM DMPO, and various concentrations of SA. Immediately after the addition of CuSO4, 175 µl of the reaction mixture was placed in the flat-shaped quartz ESR cell and ESR spectra were recorded as described above. For the test of UV-driven H2O2 decomposition, the reaction mixtures containing 100 mM K-phosphate (pH 7.0), 10 mM H2O2, 500 µM DMPO, and various concentrations of SA were placed in a flat-shaped quartz ESR cell and irradiated with UV using an UV-illuminator (FI-3 S type, Toshiba) for 1 min, with 1 cm of distance from the light source, at room temperature. Then ESR spectra of DMPO-OH signal were collected as described above.
Monodehydroascorbate (MDA) determination
For measurements of SA-induced production of MDA in tobacco cell suspension culture and HRP reaction mixture, the ESR method described by Pietri et al. (Pietri et al., 1990) was used. The ESR measurements were conditioned as described for detection of DMPO-OH.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Effect of pH on measurements of luminol- and CLA-CL and aequorin luminescence
The effect of pH on measurements of luminol- and CLA-CL and aequorin luminescence was tested at pH ranging from 5.1–9.3. None or only very low intensity luminol-CL induced by 0.5 mM SA was detected at acidic pH (Table 1
). At alkaline conditions the yield of luminol-CL is greater but it is still detectable at pH 7.0. CLA-CL detected at pH 5.1–7.0 was not much different (Table 1). The SA-induced increase in aequorin luminescence was highest at pH 5.1, and it gradually decreased as pH was elevated to 7.5. The basal level of aequorin luminescence was almost same between pH 5.8 and 7.5, but it sharply increased at alkaline conditions and interfered with the measurements of SA-induced increase in [Ca2+]c (data not shown). The sharp drop in aequorin luminescence at pH 8.0 was due to an elevated level of basal luminescence. Therefore, the profile of time course and magnitude of SA-induced increase in [Ca2+]c were affected at alkaline conditions.
|
Profiles of SA-induced production of AOS and increase in [Ca2+]c
The addition of 0.5 mM SA induced an immediate and transient generation of measured with CLA-CL at pH 5.8, reaching the maximum value within 0.1 s (Fig. 1, trace A). Then the production of H2O2 measured with luminol-CL at pH 7.0, was also induced by 0.5 mM SA, reaching the maximum value c. 2.0 s later (trace B). Lastly, the level of [Ca2+]c measured with aequorin luminescence at pH 5.8 showed a gradual increase, reaching the maximum value c. 2 min after SA addition (Fig. 1, trace C). The addition of 0.1% ethanol as a solvent control to the cell suspension showed no detectable effect on AOS and [Ca2+]c. When the productions of and H2O2, and [Ca2+]c were measured at pH 7.0, the profiles of the time-course and the magnitude of CLA-CL, and the aequorin luminescence induced by SA was similar to the traces A and C shown in Fig. 1. The form but not the magnitude of luminol-CL induced by SA was also similar to the trace B in Fig. 1. These observations suggest that SA treatment induced the production of and H2O2, and an increase in [Ca2+]c, in this order.
|
The production of
and H2O2 induced by 0.5 mM SA at pH 7.0 were estimated to be 2.52±0.45 nmol (n=8) and 10.40±1.35 nmol (n=8) per ml culture, respectively. The maximal [Ca2+]c induced by 0.5 mM SA at pH 7.0 was estimated to be 614.0±2.5 nM (n=6) using the calibration equation described in Materials and methods.
Effects of SA on the production of hydroxy radicals (HO·)
The addition of 5 mM H2O2 to the tobacco cell suspension culture incubated with 500 µM DMPO induced the 1 : 2 : 2 : 1 quartet ESR signal (Fig. 2). This ESR quartet represents the formation of a DMPO-OH spin adduct as a consequence of HO· formation (Togashi et al., 1994). A scavenger of HO·, DMTU (1 mM), completely suppressed the formation of the DMPO-OH adduct, confirming that the observed ESR signal represents the formation of HO·. Chelators of iron and copper, 2,2'-bipyridyl (1 mM) and 1,10-phenanthroline (1 mM), inhibited the H2O2-dependent HO· formation. This suggests that conversion of H2O2 to HO· in the cell culture is a metal-catalysed Fenton type reaction. The addition of SA (0.1–2.0 mM tested) to the cell suspension culture did not result in the formation of DMPO-OH (the results of 1 mM SA are shown in Fig. 2). The effect of 5 mM H2O2 on formation of DMPO-OH was completely inhibited by 1 mM SA (Fig. 2). This suggests that SA is a strong scavenger of HO·.
|
Scavenging of HO· by SA was tested in an in vitro HO·-generating system containing 10 mM H2O2 and 1 mM CuSO4 (Fig. 3
). The H2O2-dependent increase in DMPO-OH signal in CuSO4 solution was inhibited almost completely by 1 mM SA, and by half by 10 µM SA. The effect of SA on the formation of HO· was also tested in UV-dependent HO·-generating system. The increase in DMPO-OH signal induced by irradiation of 10 mM H2O2 with UV light was inhibited almost 70% by 1 mM SA, and slightly by 10 µM SA.
|
Detection of monodehydroascorbate (MDA) in tobacco suspension culture
The addition of 10 mM SA to the tobacco cell suspension culture induced an ESR spectrum displaying a characteristic doublet signal of MDA (hyperfine splitting constant=0.188 mT, g value=2.005; cf. Pietri et al., 1990; Togashi et al., 1994), but no signal of MDA was detected in the cell suspension treated with various concentration of H2O2 ranging from 0.1 to 10 mM. A typical result with 10 mM H2O2 is shown in Fig. 4. The signal disappeared in the presence of 3 µM MDA reductase and 0.2 mM NADPH (data not shown) confirming that the doublet signal represents MDA. The MDA signal was detectable only when the concentration of SA added alone was higher than 5 mM (data not shown).
|
Effects of ascorbate, CuZn-SOD and SHAM on measurements of , H2O2, MDA, and [Ca2+]c in the tobacco suspension culture
The addition of 1 mM ascorbate to the tobacco suspension culture enhanced the production of MDA induced by 0.5 mM SA by 3-fold (data not shown), but it effectively inhibited the SA-dependent production of and H2O2, and an increase in [Ca2+]c (Fig. 5). Addition of CuZn-SOD also inhibited the SA-dependent production of and the increase in [Ca2+]c, but not the increase in MDA (Fig. 5). Prior treatment with 5 mM SHAM strongly inhibited all four biochemical changes induced by SA.
|
HRP-catalysed production of AOS
The addition of 1 mM SA to HRP (150 units ml-1) at pH 7.5 resulted in minute increases in CLA-CL and luminol-CL (Fig. 6). The SA-induced increases in CLA-CL and luminol-CL were markedly enhanced by addition of 0.1 µM H2O2 to HRP prior to SA addition. This suggests that only a trace of H2O2 is required to initiate the SA-induced production of both and H2O2. The HRP-catalysed production of both and H2O2 was sensitive to a 5 mM SHAM.
|
HRP-catalysed production of MDA
The SA-dependent production of MDA was also examined in an in vitro reaction system consisted of 150 units ml-1 HRP, 1 mM H2O2 and 1 mM ascorbate (Fig. 7). An intensive signal of MDA induced by 1 mM SA was observed with ESR spectrometry (Fig. 7A). The MDA signal disappeared in the presence of 3 µM MDA reductase and 0.2 mM NADPH (data not shown) confirming that the doublet signal represents MDA. The yield of MDA signal was dependent upon both the concentration of SA (Fig. 7B) and pH (Fig. 7C). Reaction mixture lacking either of HRP, ascorbate, H2O2, or SA produced none or very little MDA (Fig. 7A), suggesting that the maximal production of MDA essentially requires HRP, ascorbate, H2O2, and SA.
|
Effects of HRP on the SA-induced increase in [Ca2+]c in the tobacco suspension culture
The addition of HRP (150 units ml-1) to tobacco cell suspension culture significantly enhanced an increase in [Ca2+]c induced by 0.5 mM SA and also shortened the time reaching the maximum level of [Ca2+]c (Fig. 8).
|
) HRP (150 units ml-1) for 2 min prior to addition of SA (0.5 mM). Vertical bars represent SE (n=4). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Determination of SA-induced AOS
The addition of SA induced an immediate increase in CLA-CL and luminol-CL in tobacco cell suspension culture (Fig. 1
). CLA-CL specifically indicates the generation of (and 1O2 to a lesser extent), but not that of H2O2 or HO· (Nakano et al., 1986). Measurement of luminol-CL had been applied for the detection of an oxidative burst in elicitor-treated tobacco plants, potato tubers and cell suspension cultures of spruce, tomato and sweet pepper (Park et al., 1998; Sanchez et al., 1993; Schwacke and Harger, 1992). The luminol-CL is inhibited in the presence of catalase, but not SOD, suggesting that most of the CL are due to H2O2 (Park et al., 1998; Sanchez et al., 1993).
The addition of H2O2 to tobacco suspension culture induces an increase in [Ca2+]c (Price et al., 1994). The increase in [Ca2+]c in tobacco cell suspension culture, induced by various concentration of H2O2 has previously been reported (Takahashi et al., 1998). The lowest concentration of H2O2 required for a detectable increase in [Ca2+]c was c. 200 µM. In the present work, SA (0.5 mM, pH 7.0) induced the production of c. 10 µM H2O2 and a marked increase in [Ca2+]c in the cell suspension culture. Exogenous application of the same concentration of H2O2 failed to induce any detectable levels of [Ca2+]c increase (data not shown). In vivo production of H2O2 might efficiently access the site of effect, in combination with , acting as an inducer of an increase in [Ca2+]c in the SA signal transduction.
Inhibition of HO· production by SA
The addition of exogenous H2O2 to the cell suspension culture resulted in the generation of HO· by a transition metal-catalysed Fenton-type reaction (Fig. 2). The generation of HO· was lowered by SA which could chelate the transition metals, thus reducing the formation of HO· and/or could act directly as a HO· scavenger. Exogenously administered SA acts as an efficient scavenger of HO· in animals (Sagone and Husney, 1987; Aubin et al., 1998). SA reacts with HO· and yields hydroxylated and decarboxylated products, such as catechol, 2,3- and 2,5-dihydroxybenzoic acids (Sagone and Husney, 1987; Das et al., 1989). Therefore, these products have been shown to be reliable indicators for the production of HO·. Recently, the role of SA as a HO· scavenger in plants has been suggested (Fry, 1998).
Production of MDA prior to generation of
The addition of ascorbate to the tobacco culture inhibited the SA-dependent production of and H2O2, subsequently, the increase in [Ca2+]c was also inhibited (Fig. 5). These results are consistent with the observation that a decrease in AOS lowered the increase in [Ca2+]c in the SA-treated tobacco cell suspension culture (Kawano et al., 1998).
The most characterized enzymes yielding MDA in plants are ascorbate oxidase and ascorbate peroxidase. No phenolics are involved in the oxidation reaction of ascorbate to MDA catalysed by ascorbate oxidase and ascorbate peroxidase (Heber et al., 1996; Miyake et al., 1996). Thus, the SA-dependent production of MDA is not due to these enzymes. In addition, MDA may be formed from ascorbate during photosynthesis and also by the interaction with HO· and (Heber et al., 1996). Since the present study was carried out using dark-grown non-pigmented tobacco cells, involvement of the photosynthetic process is excluded. As SA inhibits production of HO· (Figs 2, 3), involvement of HO· is also excluded. A role for in the SA-dependent production of MDA could, therefore, be excluded for two reasons. First, the interaction of ascorbate with produces MDA and H2O2 (Heber et al., 1996), while the addition of ascorbate to the cell culture resulted in the complete inhibition of the SA-dependent production of H2O2 in the present study (Fig. 5). Second, CuZn-SOD did not inhibit the SA-dependent production of MDA in the tobacco cell culture (Fig. 5). An involvement of peroxidase was indicated since a peroxidase inhibitor, SHAM, strongly inhibited the SA-induced production of MDA in tobacco cell culture. Taken together, it is concluded that the SA-dependent production of MDA is a consequence of the peroxidase-catalysed oxidation of SA, which occurs prior to the generation of .
MDA as an indicator for generation of SA·
The addition of SA to the cell suspension culture induced an ESR spectrum displaying a characteristic doublet signal of MDA, but no MDA signal was induced by H2O2 (Fig. 4), indicating that the production of MDA by SA is not due to SA-induced H2O2.
A mechanism of SA-dependent extracellular production of in tobacco suspension culture has previously been proposed (Kawano et al., 1998) as follows:
 | (1) |
 | (2) |
 | (3) |
 | (4) |
), compound I of GPX, but not that of ascorbate peroxidase, can be reduced by SA to compound II. During the GPX-catalysed reaction, SA and H2O2 may act as the donor and acceptor of electron exchange, respectively, finally yielding SA·. It has been reported that oxidation of SA by compounds I and II of ascorbate peroxidase is very slow (Kvaratskhelia et al., 1997). Thus ascorbate peroxidase may not be involved in the rapid formation of SA·.
Plant peroxidases in the apoplastic space are considered to catalyse the generation of phenoxyl radicals from several phenolic compounds, although only indirect evidence has been obtained (Takahama and Yoshitama, 1998). The life-time of phenoxyl radicals produced during the peroxidase reaction was too short to detect their signals by conventional ESR measurements. However, several reports have indicated that ESR detection of MDA is a reliable measurement for peroxidase-catalysed phenoxyl radical formation, since ascorbate is highly reactive towards phenoxyl radicals (Packer et al., 1979; Sipe et al., 1997; Tyurin et al., 1997; Ritov et al., 1996; Ramakrishna Rao et al., 1990). Ascorbate-mediated ESR method was employed in the present study for the detection of SA· according to the following reaction:
 | (5) |
HRP-catalysed production of AOS
SA addition to HRP induced the production of both and H2O2 (Fig. 6). By analogy it is suggested that the SA-induced production of H2O2 in tobacco cell suspension culture, which follows that of , is not merely due to inhibition of catalase by SA, although SA effectively inhibits catalase activity (Chen et al., 1993; Kawano et al., 1998). Furthermore, the addition of a catalase inhibitor, 3-amino-1,2,4-triazole did not induce a detectable increase in H2O2 in tobacco suspension culture (data not shown).
The SA-induced production of both and H2O2 was markedly enhanced by the addition of 0.1 µM H2O2 to HRP prior to SA addition (Fig. 6). Thus, H2O2 might initiate the peroxidase-catalysed oxidation of SA, subsequently producing and H2O2. This is consistent with previous observations that the SA-dependent generation of requires both H2O2 and extracellularly secreted GPXs in tobacco cell suspension culture (Kawano et al., 1998). It remains unclear at present how the conversion of to H2O2 proceeds in the HRP reaction. In plants, the conversion of apoplastic into H2O2 could be carried out by cell wall-localized CuZn-SOD (as proposed by Ogawa et al., 1997).
HRP-catalysed production of MDA
The SA-dependent production of MDA in the HRP system required HRP, ascorbate, H2O2, and SA. Therefore, MDA production may reflect the generation of SA· by HRP. This suggests that in the tobacco suspension culture, endogenous ascorbate, H2O2, and peroxidase activity are required for SA-induced production of MDA which indicates the peroxidase-catalysed production of SA·.
Enhancement of SA-induced increase in [Ca2+]c by addition of HRP in tobacco suspension culture
Since HRP catalysed the SA-dependent generation of and H2O2 that may induce an increase in [Ca2+]c, the addition of HRP may result in an enhanced increase in [Ca2+]c in response to SA in the tobacco suspension culture. As expected, the SA-induced increase in [Ca2+]c was significantly enhanced in the HRP-treated cell suspension (Fig. 8). The significant role of peroxidase in the transduction of SA signal was further confirmed.
|
Conclusion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
In conclusion, a model is proposed for the mechanism of SA action in the generation of AOS, which induces the expression of the defence-related genes via an increase in [Ca2+]c (Fig. 9
). SA and a trace of H2O2 are used for the SA·-generating peroxidase reaction. Then the resultant SA· reacts with O2 to produce that triggers an increase in [Ca2+]c. The increased [Ca2+]c may induce further physiological responses including the induction of PR genes. Ascorbate re-converts SA· to SA yielding MDA. An excess of ascorbate may inhibit the SA·-dependent generation of . While the generation of is going on, SA inhibits the decomposition of H2O2 by catalase and a Fenton-type reaction. SA also lowers the HO· level by blocking the Fenton reaction and by directly trapping HO·. Thus SA protects the cells from highly reactive HO·, while producing the less reactive and H2O2 through a peroxidase-catalysed reaction, as the intermediate signals.
|
|
Acknowledgments |
|---|
The authors are grateful to Professor K Asada of Fukuyama University and Dr C Miyake of Nara Institute of Science and Technology for valuable suggestions. The authors thank Professor M Cormier for permission to use the apoaequorin cDNA, Professor AJ Trewavas and Dr MR Knight for the generous gift of pMAQ2, Professor M Isobe for the generous gift of chemically synthesized coelenterazine, and Drs K Uchida, Y Morimitsu and Y Makino for valuable technical advice in ESR spectrometry.
|
Notes |
|---|
3 To whom correspondence should be addressed. Fax: + 81 52 789 5206. E-mail: h44787a{at}nucc.cc.nagoya\|[hyphen]\|u.ac.jp
4 Present address: Ohio State University Neurobiotechnology Center, 202 Rightmire hall, 1060 Carmack road, Columbus, OH 43210, USA.
|
Abbreviations |
|---|
AOS, active oxygen species; [Ca2+]c, cytosolic free Ca2+ concentration; CL, chemiluminescence, CLA, Cripridina luciferin-derived chemiluminescent agent; DMPO, 5,5'-di-methyl-1-pyrroline-N-oxide; DMTU, N,N'-dimethylthiourea; ESR, electron spin resonance; GPX, guaiacol peroxidase; HO·, hydroxyl radical; HRP, horseradish peroxidase; MDA, monodehydroascorbate;
, superoxide anion,; SA, salicylic acid; SA·, SA free radical; SAR, systemic acquired resistance; SOD, superoxide dismutase.. |
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Aubin N, Curet O, Deffois A, Carter C.1998. Aspirin and salicylate protect against MPTP-induced dopamine depletion in mice. Journal of Neurochemistry 71, 1635–1642.[ISI][Medline]
Chen Z, Silva H, Klessig DF.1993. Active oxygen species in the induction of plant systemic acquired resistance induced by salicylic acid. Science 262, 1883–1886.
Das DK, George A, Liu X, Rao PS.1989. Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate. Biochemical and Biophysical Research Communications 165, 1004–1009.[ISI][Medline]
Delaney TP, Uknes S, Vernooij B, Friedrich L, Weymann K, Negrotto D, Gaffney T, Gul-Rella M, Kessmann H, Ward E, Ryals J.1994. Central role of salicylic acid in plant defense resistance. Science 266, 1247–1250.
Durner J, Klessig DF.1995. Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses. Proceedings of the National Academy of Sciences, USA 92, 11312–11316.
Fry SC.1998. Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals. Biochemical Journal 32, 507–515.
Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals, J.1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261, 754–756.
Heber U, Miyake C, Mano J, Ohno C, Asada K.1996. Monodehydroascorbate radical detected by electron paramagnetic resonance spectrometry is a sensitive probe of oxidative stress in intact leaves. Plant and Cell Physiology 37, 1066–1072.
Kawano T, Sahashi N, Takahashi K, Uozumi N, Muto S.1998. Salicylic acid induces extracellular generation of superoxide followed by an increase in cytosolic calcium ion in tobacco suspension culture: the earliest events in salicylic acid signal transduction. Plant and Cell Physiology 39, 721–730.
Knight H, Trewavas AJ, Knight MR.1996. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. The Plant Cell 8, 489–503.[Abstract]
Knight MR, Cambell AK, Smith SM, Trewavas AJ.1991. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytosolic calcium. Nature 352, 524–526.[Medline]
Kvaratskhelia M, George SJ, Thorneley RNF.1997. Salicylic acid is a reducing substrate and not an effective inhibitor of ascorbate peroxidase. Journal of Biological Chemistry 272, 20998–21001.
Levine A, Pennel RI, Alvarez ME, Palmer R, Lamb C.1996. Calcium-mediated apoptosis in a plant hypersensitive disease resistance response. Current Biology 6, 427–437.[ISI][Medline]
Messiaen J, Read ND, Van Cutsem P, Trewavas AJ.1993. Cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts. Journal of Cell Science 104, 365–371.[Abstract]
Miyake C, Sano S, Asada K.1996. A new assay of ascorbate peroxidase using the coupled system with monodehydroascorbate radical reductase. In: Obinger C, Burner U, Ebermann R, Penel C, Greppin H, ed. Plant peroxidases: biochemistry and physiology. Vienna: University of Geneva, 386–389.
Nakano M, Sugioka K, Ushijima Y, Goto T.1986. Chemiluminescence probe with Cripridina lucigenin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate . Analytical Biochemistry 159, 363–369.[ISI][Medline]
Ogawa K, Kanematsu S, Asada K.1997. Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification. Plant and Cell Physiology 38, 1118–1126.
Packer JE, Slater TF, Wilson RL.1979. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278, 737–738.[Medline]
Park HJ, Doke N, Miura Y, Kawakita K, Noritake T, Komatsubara H.1998. Induction of sub-systemic oxidative burst by elicitor-stimulated local oxidative burst in potato plant tissue: a possible systemic signaling in systemic acquired resistance. Plant Science 138, 197–208.
Pietri S, Culcasi M, Stell, L, Cozzone PJ.1990. Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study. European Journal of Biochemistry 193, 845–854.[ISI][Medline]
Price AH, Taylor A, Ripley SJ, Griffiths A, Trewavas AJ, Knight MR.1994. Oxidative signals in tobacco increase cytosolic calcium. The Plant Cell 6, 1301–1310.[Abstract]
Ramakrishna Rao DN, Fischer V, Mason RP.1990. Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. Detection of the glutathione disulfide radical anion and the ascorbyl radical. Journal of Biological Chemistry 265, 844–847.
Raz V, Fluhr R.1992. Calcium requirement for ethylene-dependent responses. The Plant Cell 4, 1123–1130.
Ritov VB, Menshikava EV, Goldman R, Kagan VE.1996. Direct oxidation of polyunsaturated cis-parinaric fatty acid by phenoxyl radicals generated by peroxidase/H2O2 in model systems and in HL-60 cells. Toxicological Letters 87, 121–129.[ISI][Medline]
Sagone AL, Husney RM.1987. Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems. Journal of Immunology 138, 2127–2183.
Sanchez LM, Doke N, Kawakita K.1993. Elicitor-induced chemiluminescence in cell suspension cultures of tomato, sweet pepper and tobacco plants and its inhibition by suppressors from Phytophthora spp. Plant Science 88, 141–148.
Sanders D, Brownlee C, Harper JF.1999. Communicating with calcium. The Plant Cell 11, 691–706.
Schneider-Müller S, Kurosaki F, Nishi A.1994. Role of salicylic acid and intracellular Ca2+ in the induction of chitinase activity in carrot suspension culture. Physiological and Molecular Plant Pathology 45, 101–109.
Schwacke R, Hager H.1992. Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+ and protein kinase activity. Planta 187, 136–141.
Sipe HJ, Corbett JT, Mason RP.1997. In vitro free radical metabolism of phenolphthalein by peroxidases. Drug Metabolism and Disposition 25, 468–480.
Takahama U, Yoshitama K.1998. Hydoxycinnamic acid esters enhance peroxidase-dependent oxidation of 3,4-dihydroxyphenylamine. Differences in the enhancement among the esters. Journal of Plant Research 111, 97–100.
Takahashi K, Isobe M, Knight MR, Trewavas AJ, Muto S.1997. Hypoosmotic shock induces increase in cytosolic Ca2+ in tobacco suspension-culture cells. Plant Physiology 113, 587–594.[Abstract]
Takahashi K, Isobe M, Muto S.1998. Mastoparan induces an increase in cytosolic calcium ion concentration and subsequent activation of protein kinases in tobacco suspension culture cells. Biochimica et Biophysica Acta 1401, 339–346.[Medline]
Togashi H, Shinzawa H, Yong H, Takahashi T, Noda H, Oikawa K, Kamada H.1994. Ascorbic acid radical, superoxide, and hydroxyl radical are detected in reperfusion injury of rat liver using electron spin resonance spectroscopy. Archives of Biochemistry and Biophysics 308, 1–7.[ISI][Medline]
Tyurin VA, Carta G, Tyurina YY, Banni S, Day BW, Corobgiu FP, Kagan VE.1997. Peroxidase-catalysed oxidation of beta-carotene in HL-60 cells and in model systems: involvement of phenoxyl radicals. Lipids 32, 131–142.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Kadota, S. Fujii, Y. Ogasawara, Y. Maeda, K. Higashi, and K. Kuchitsu Continuous Recognition of the Elicitor Signal for Several Hours is Prerequisite for Induction of Cell Death and Prolonged Activation of Signaling Events in Tobacco BY-2 Cells Plant Cell Physiol., September 1, 2006; 47(9): 1337 - 1342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Liszkay, E. van der Zalm, and P. Schopfer Production of Reactive Oxygen Intermediates (O2{middle dot}-, H2O2, and {middle dot}OH) by Maize Roots and Their Role in Wall Loosening and Elongation Growth Plant Physiology, October 1, 2004; 136(2): 3114 - 3123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Avsian-Kretchmer, Y. Gueta-Dahan, S. Lev-Yadun, R. Gollop, and G. Ben-Hayyim The Salt-Stress Signal Transduction Pathway That Activates the gpx1 Promoter Is Mediated by Intracellular H2O2, Different from the Pathway Induced by Extracellular H2O2 Plant Physiology, July 1, 2004; 135(3): 1685 - 1696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kadota, T. Goh, H. Tomatsu, R. Tamauchi, K. Higashi, S. Muto, and K. Kuchitsu Cryptogein-Induced Initial Events in Tobacco BY-2 Cells: Pharmacological Characterization of Molecular Relationship among Cytosolic Ca2+ Transients, Anion Efflux and Production of Reactive Oxygen Species Plant Cell Physiol., February 15, 2004; 45(2): 160 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ranieri, A. Castagna, J. Pacini, B. Baldan, A. Mensuali Sodi, and G. F. Soldatini Early production and scavenging of hydrogen peroxide in the apoplast of sunflower plants exposed to ozone J. Exp. Bot., November 1, 2003; 54(392): 2529 - 2540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. WHITE and M. R. BROADLEY Calcium in Plants Ann. Bot., October 1, 2003; 92(4): 487 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fujisawa, T. Atsumi, K. Satoh, and H. Sakagami Interaction between 2-Ethoxybenzoic Acid (EBA) and Eugenol, and Related Changes in Cytotoxicity J. Dent. Res., January 1, 2003; 82(1): 43 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Klusener, J. J. Young, Y. Murata, G. J. Allen, I. C. Mori, V. Hugouvieux, and J. I. Schroeder Convergence of Calcium Signaling Pathways of Pathogenic Elicitors and Abscisic Acid in Arabidopsis Guard Cells Plant Physiology, December 1, 2002; 130(4): 2152 - 2163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, and A. Pugin Analysis and Effects of Cytosolic Free Calcium Increases in Response to Elicitors in Nicotiana plumbaginifolia Cells PLANT CELL, October 1, 2002; 14(10): 2627 - 2641. [Abstract] [Full Text] [PDF]
|
|