Journal of Experimental Botany, Vol. 54, No. 384, pp. 935-941,
March 1, 2003
© 2003 Oxford University Press
Reactive oxygen species production in association with suberization: evidence for an NADPH-dependent oxidase
Received 29 August 2002; Accepted 6 November 2002
Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada
1 To whom correspondence should be addressed. Fax: +1 519 661 3935. E-mail: bernards{at}uwo.ca
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Abstract |
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In response to wounding, potato tubers generate reactive oxygen species (ROS) in association with suberization. Immediately following wounding, an initial burst of ROS occurs, reaching a maximum within 30 to 60 min. In addition to this initial oxidative burst, at least three other massive bursts occur at 42, 63 and 100 h post-wounding. These latter bursts are associated with wound healing and are probably involved in the oxidative cross-linking of suberin poly(phenolics). The source of ROS is likely to be a plasma membrane NADPH-dependent oxidase immunorelated to the human phagocyte plasma membrane oxidase. The initial oxidative burst does not appear to be dependent on new protein synthesis, but the subsequent bursts are associated with an increase in oxidase protein components. Oxidase activity is enhanced in vitro by hydroxycinnamic acids and conjugates associated with the wound healing response in potato.
Key words: NADPH-oxidase, potato, reactive oxygen species, Solanum tuberosum, suberization, wound healing.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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When plants are wounded, they form a protective layer next to the exposed surface to prevent dehydration and potential penetration by opportunistic pathogens. This physical barrier termed suberin comprises a specific cell wall modification characterized by both a poly(phenolic) domain and wax-embedded poly(aliphatic) domain (reviewed in Bernards and Lewis, 1998; Bernards, 2002). Suberin is also formed developmentally and is found in the dermal cells of underground tissues, the Casparian band and in the cork cells of bark tissue (Esau, 1977). The oxidative coupling of the poly(phenolic) component of suberin is thought to follow a peroxidase/H2O2 free radical process analogous to that of lignification (Kolattukudy, 1980). It was recently demonstrated in this laboratory that H2O2 is essential for suberization in potatoes (Razem and Bernards, 2002), but the origin of the H2O2 has remained an open question.
The generation of reactive oxygen species (ROS), which includes superoxide (O.2–), hydrogen peroxide (H2O2), and the hydroxyl radical (OH.), is ubiquitous in biological systems, and occurs either through signal-regulated processes or as an unavoidable by-product of metabolic reactions under both stress and normal conditions (Bolwell, 1996). In plants, ROS are produced during the cross-linking of cell wall components, after exposure to high and low temperatures and light intensities, air pollutants such as ozone, ultraviolet light, herbicides and/or during pathogen attack and mechanical injury (reviewed in Kuzniak and Urbanek, 2000). The rapidly induced production of ROS under stress conditions (i.e. the oxidative burst) initially results in the production of O.2–, which then disproportionates to H2O2 either spontaneously or via superoxide dismutase. Both O.2– and H2O2 have been shown to act directly or indirectly in plant defence and signal transduction (Bolwell, 1996; Kuzniak and Urbanek, 2000; Yoshioka et al., 2001; Vranová et al., 2002). The conversion of O.2– and H2O2 to OH., catalysed by transition metals (Haber–Weiss reaction), accounts for the severe toxicity of ROS in plants (Wojtaszek, 1997). Plants usually keep the levels of ROS under tight control by the production of scavenging enzymes and non-enzymatic antioxidants (Wojtaszek, 1997; Kuzniak and Urbanek, 2000; Møller, 2001; Vranová et al., 2002).
During the hypersensitive response (HR) to pathogen attack and mechanical stress, ROS play a role in killing pathogens and in eliminating the damaged cells (necrosis) in a process widely considered to be analogous to the defensive mechanism of mammalian phagocytes (Lamb and Dixon, 1997). The key enzyme that contributes to ROS formation in phagocytes is a plasma membrane-bound NADPH-dependent oxidase (Segal and Abo, 1993), the activation of which involves the assembly of at least three cytosolic (p67phox, p47phox, p40phox) and two plasma membrane-associated (gp91phox, p22phox) components (Segal and Abo, 1993; Jones, 1994). Upon activation, p67phox, p47phox, and p40phox translocate and dock with gp91phox and p22phox at the plasma membrane. This process involves the phosphorylation of at least one cytosolic component (p47phox) by protein kinase C (PKC) (Jones, 1994).
A role for NADPH-dependent oxidases in the generation of the H2O2 in plants is beginning to emerge. For example, a plasma membrane NADPH-dependent oxidase homologous to the phagocyte oxidase has been demonstrated during pathogen attack (Doke, 1985; Mehdy, 1994; Lamb and Dixon, 1997; Xing et al., 1997; Yoshioka et al., 2001) and lignification (Ogawa et al., 1997; Ros Barceló, 1998). In this study the observations regarding the involvement of an NADPH-dependent oxidase in the polymerization of the poly(phenolic) domain of suberized potato tubers (Bernards and Razem, 2001; Razem and Bernards, 2002) have been extended to characterize the source and the induction of the H2O2 generating system in wound-healing potato tubers.
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Materials and methods |
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Tissue wounding and plasma membrane isolation
Potato (Solanum tuberosum L. cv. Russet Burbank) tubers were cut (i.e. wounded) transversely under sterile conditions and incubated at 25 °C for up to 7 d as described earlier (Bernards and Lewis, 1992). Suberizing layers were collected at various times post-wounding by gently separating them from the underlying, unsuberized tissue using a razor blade (Bernards and Lewis, 1992). Suberized layers were collected at the time of wounding (i.e. 0 h post-wounding (hpw)) followed by 1 h intervals up to 6 hpw and at 6 h intervals thereafter. These time points were chosen to ensure a complete picture of the timing of ROS production post-wounding. After collection, suberized layers were frozen in liquid N2, ground in a pre-chilled mortar and stored at –20 °C until used. Microsomal fractions were obtained by homogenizing ground tissue in extraction buffer (25 mM Tris-HCl buffer, pH 7.5 (3 ml g–1) containing 250 mM sucrose, 1.0 mM EDTA, 10 mM KCl, 1 mM MgCl2, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), and 0.1 mM freshly prepared DTT). The homogenate was filtered through four layers of cheesecloth and centrifuged for 10 min (15000 g) at 4 °C. After buffer-exchange using Econo-Pac 10 DG chromatography columns (BioRad) pre-equilibrated with extraction buffer (minus PMSF), the eluent was centrifuged at 105 000 g for 60 min (4 °C) and the pellet used to isolate plasma membranes by aqueous two phase partitioning (three cycles) as described by Sandelius and Morre (1990) with minor modifications by Xing et al. (1997). Membranes were reconstituted in reconstitution buffer (10 mM potassium phosphate buffer, pH 7.8, 250 mM sucrose, and 1 mM DTT) at 10 µl g–1 initial tissue, and either used immediately or frozen in liquid nitrogen and stored at –20 °C until used. Cytosolic proteins were obtained from the 105 000 g supernatant using a concentration cell with a 10 kDa molecular mass cut-off membrane, giving a final protein concentration of approximately 2 mg ml–1 as measured by the Bradford protein assay (Bradford, 1976).
SDS-PAGE and Western blotting
Membrane and cytosolic proteins (approximately 10 µg) were loaded on a discontinuous SDS-PAGE (10% separation gel) minigel system (BioRad) and separated according to manufacturer’s instructions. Proteins were transferred to nitrocellulose using a tank-blotting chamber (BioRad), according to the manufacturer’s instructions. Blots were blocked for 60 min at room temperature or overnight at 4 °C in blocking buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05 Tween 20, and 5% milk powder). After washing with washing buffer (TBS, 0.05% Tween 20), blots were incubated with primary antibodies raised against gp91phox (1:1000), p67phox (1:1000), p47phox (1:1000), p40phox (1:1000), (the kind gift of Dr A Segal, University College London, UK and Dr Terence Murphy, University of California, Davis, USA) for 60 min at room temperature. Blots were washed four times (5 min each wash) in washing buffer and subsequently incubated with secondary antibodies (goat:anti-rabbit conjugated with alkaline phosphatase) for 60 min. Blots were washed as above and finally with ddH2O (10 min). A chemiluminescent detection system (ECLTM Western Blotting Detection Reagents) prepared shortly before film development was used according to the manufacturer’s instructions. Film was exposed for 30 s.
Enzyme assays
NADPH-dependent oxidase activity was measured indirectly using the superoxide dismutase (SOD)-inhibited reduction of nitroblue tetrazolium (NBT) as described by Van Gestelen et al. (1997). The reaction was started by the addition of 0.1 mM NADPH and the reduction of NBT (0.1 mM) was monitored at 530 nm at 30 °C. Oxidase activity was calculated by taking the difference between apparent reaction rates with and without SOD (50 units ml–1) in the reaction mixture and using an extinction coefficient of 12.8 mM–1 cm–1. Other additions to the reaction medium are discussed in the text.
Peroxidase activity was measured spectrophotometrically in reaction buffer (50 mM Tris-acetate buffer, pH 5.0, 100 mM KCl, 50 mM CaCl2) containing 30 mM H2O2, 0.1 mg tetramethylbenzidine-HCl (TMB), and 50 µl soluble protein in a 1 ml final volume. The oxidation of TMB was monitored at 450 nm.
Treatment with chloramphenicol
Thin tuber sections (0.5 cm) thick were soaked for 1 min in 1 mM chloramphenicol before incubation at 25 °C. The browning surface was carefully collected at the time points shown and processed as above.
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Results |
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Time-course generation of O.2– post-wounding
The production of ROS was measured in wound-healing potato tubers from 0–168 h post-wounding (hpw). The wounding was carried out under sterile conditions to avoid any possibility of pathogen interference and there was no evidence of microbial contamination at any time during the experiments (data not shown). The generation of O.2– sharply increased within 1 hpw (Fig. 1) and then gradually declined. This initial oxidative burst appeared to be independent of new protein synthesis (Fig. 1) and could be significantly inhibited by washing of the tuber tissue. The initial oxidative burst was followed by three other, larger bursts of O.2– generation at approximately 42, 63, and 100 hpw (Fig. 2). The maximum activity of O.2– generation appeared to be at 42 hpw. By contrast, peroxidase activity showed a continuous, gradual increase until it reached a maximum at 100–120 hpw (Fig. 2).
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Effect of DPI and NaN3 on in vitro generation of O.2– in wounded potato tubers
The generation of O.2– could be reconstituted in vitro by the addition of purified plasma membranes to cytosolic protein extracts prepared from suberizing tissues 42 hpw (Figs 3, 4). The complete system yielding maximum activity (77.9±2.9 µmol g–1 tissue h–1) included plasma membrane (PM) and cytosolic protein extracts, as well as all buffer components. The production of O.2– was evident with PM alone, but at much lower rate (approximately 30%) than with the complete system. It was not possible to measure O.2– production when PMs were excluded from the reaction mixture (Fig. 3). The exclusion of NADPH from the reaction medium decreased O.2– generation in vitro by more than 87% (Fig. 3). The addition of DPI, an NADPH-dependent oxidase inhibitor (Segal and Abo, 1993) decreased oxidase activity by over 80% whereas NaN3 (a haem protein inhibitor) had no significant effect on O.2– generation (Fig. 3). The inhibitory effect of NaN3 on peroxidase was verified in in vitro assays (data not shown). The cytosolic fraction prepared from tissues at 1 hpw could not be substituted for that prepared from tissues at 42 hpw (Fig. 4). Interestingly, O.2– generation by PM prepared from tissues at 1 hpw was not significantly inhibited by DPI (Fig. 4). The complete system yielding maximum activity from tissues at 1 hpw (17.7±1.5 µmol g–1 tissue h–1) included PM and cytosolic protein extracts, as well as all buffer components.
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Effect of phenolics on in vitro generation of O.2– in 42 hpw suberized tissues
The addition of certain phenolics (0.1 mM) to the assay mixture stimulated the generation of O.2– in vitro. Of the compounds tested, only chlorogenic acid, p-coumaroyltyramine, and ferulic acid significantly increased the generation of O.2– (Fig. 5). Pretreatment of tuber slices with 1 mM phenolics (ferulic acid, feyloylputrescine, p-coumaroyltyramine, or a mixture of the three), right after wounding had little or no effect on the timing of the secondary oxidative bursts (data not shown). Other compounds tested (e.g. coniferin, 2,3-dichloro-1,4-naphthoquinone, SDS, Triton X-100) had no effect on O.2– generation or gave inconsistent results (data not shown).
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Western blotting analysis
Plasma membrane and cytosolic protein extracts from suberizing tubers were probed with antibodies raised against human p40phox, p47phox, p67phox, and gp91phox. The anti- p40phox, p47phox, and p67phox antibodies (used as a mixture) detected proteins with appropriate Mr in the cytosolic fraction extracted at 42 hpw (Fig. 6). No proteins in the PM fraction were recognized by these antibodies. By contrast, anti-gp91phox antibodies recognized an appropriate Mr protein in PM preparations of 42 hpw potato tubers, but none in the cytosolic protein fraction.
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All oxidase components tested (i.e. p40phox, p47phox, p67phox, and gp91phox), were induced by wounding (Fig. 7), albeit with different patterns. Whereas levels of p40phox and p47phox increased steadily throughout the time-course, p67phox appeared to reach a maximum at 24 hpw. By contrast, the accumulation of gp91phox was slower than the cytosolic components and appeared to peak at approximately 48 hpw, before declining slightly.
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Discussion |
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Wound-induced suberization: pattern of ROS production
In potato tubers, wounding triggers the formation of a periderm (i.e. suberized layer) in the tissue immediately below the site of damage. The initial deposition of wound suberin in potato requires approximately 18 h (Lulai and Corsini, 1998) and reaches a state in which the suberized layer has sufficient structural integrity to be peeled off intact by 3 d post-wounding (Razem and Bernards, 2002). Within 30 min of wounding, however, there is a burst in the generation of ROS that reaches a peak in 1 h (Fig. 1). This initial burst of ROS production probably results from plant-derived signals, since wounding was carried out under sterile conditions and was greatly reduced when cut tubers were washed prior to incubation (Fig. 1). The initial burst was not inhibited by DPI indicating that it may not derive from an NADPH-dependent oxidase. This observation is supported by the fact that cytosolic proteins extracted from tissue collected at 1 hpw cannot substitute for those extracted from tissue collected at 42 hpw (Fig. 4) and vice versa, even when added in excess (data not shown). This is in apparent contrast to earlier reports (Doke, 1985), but since the timing of the initial burst (i.e. maximum at 1 hpw) is not directly relevant to suberin macromolecular assembly, it was not studied further. Nevertheless, it is possible that the H2O2 resulting from the initial burst may act as a messenger molecule. The insensitivity of the initial burst to protein synthesis inhibitors (e.g. chloramphenicol, Fig. 1) suggests that it is not dependent on any new protein synthesis, but rather on pre-existing components.
After the initial oxidative burst, three subsequent, larger bursts in ROS generation occurred at 42, 63, and 100 hpw (Fig. 2). By contrast to the initial ROS burst, these subsequent bursts were coincident with increases in oxidase protein components, particularly gp91phox (Fig. 7). Oxidase activity may be regulated by various mechanisms including fluctuations in the levels of endogenous activators and inhibitors, phosphorylation of the cytosolic components and the subsequent assembly of the oxidase components into a functional complex at the plasma membrane. These regulatory mechanisms may result in the oscillations in oxidase activity apparent in Fig. 2, without an associated change in the amounts of protein. The timing of the secondary ROS generation coincides with phenolic polymerization and the establishment of structural integrity in the suberized layer. A similar regulated generation of H2O2 has been reported for lignifying Pinus taede cell cultures (Nose et al., 1995) and suggests that the synthesis of H2O2 for the polymerization of cell wall phenolics is under tight biosynthetic control (Segal and Abo, 1993; Jones, 1994).
The origin of H2O2 in suberized potato tubers
The origin of ROS has been a source of debate for many years. Several mechanisms for ROS generation in plants have been presented in the literature (see Wojtaszek, 1997, for a review), including the NADPH-dependent oxidase (Doke, 1985; Desikan et al., 1996; Groom et al., 1996; Ogawa et al., 1997; Bolwell et al., 1998; Ros Barceló, 1998; Tenhaken and Rübel, 1998; Amacucci et al., 1999; Yoshioka et al., 2001; Simon-Plas et al., 2002), peroxidases (Bestwick et al., 1998; Bolwell et al., 1998), and polyamine oxidase (Angelini and Federico, 1989). With respect to H2O2 required for wound-induced suberization in potato tubers, evidence for an NADPH-dependent oxidase was recently provided based on the use of inhibitors, particularly DPI (Bernards and Razem, 2001; Razem and Bernards, 2002). Briefly it was demonstrated that the production of H2O2 during wound-healing is inhibited by DPI, a suicide inhibitor of the phagocyte plasma membrane NADPH-dependent oxidase (Segal and Abo, 1993; Jones, 1994) at low µM levels (Bernards and Razem, 2001). Although, DPI has also been shown to inhibit the H2O2 generating activity of peroxidases, it is only able to do so at higher concentrations (e.g. >1 mM) (Bolwell et al., 1998).
The present results more clearly suggest the involvement of an NADPH-dependent oxidase in the generation of ROS in suberizing potato tubers, and provides at least three new lines of evidence. First, peroxidases and oxidases follow a different time-course of induction during wound healing (Fig. 2). Whereas peroxidase showed a relatively linear and gradual increase in activity over 5 d post-wounding, the oxidase showed three transient bursts during the same time frame (Fig. 2). Furthermore, NaN3, a peroxidase inhibitor, failed to block O.2– generation by purified plasma membranes in vitro, even in the presence of cytosolic proteins (Fig. 3). Second, antibodies raised against phagocyte plasma membrane NADPH-dependent oxidase proteins cross-reacted with plasma membrane and cytosolic proteins of appropriate Mr, isolated from suberizing potato tubers (Fig. 6). An oxidase showing high homology with human gp91phox has recently been cloned from tomato (Amacucci et al., 1999) and its activity induced by fungal cell walls, arachidonic acid, and salicylic acid in potatoes (Yoshioka et al., 2001). Furthermore, an oxidase was shown to be the source of ROS generation in tobacco (Simon-Plas et al., 2002), an indication that the NADPH-dependent oxidase-mediated generation of ROS is prevalent in the Solanaceae. Third, the oxidase activity measured herein was found exclusively in plasma membrane preparations and maximum activity was only achieved with complete systems containing PM and cytosolic protein fractions. Washing the PM with high salt concentrations (e.g. 0.7 M NaCl) failed to remove the activity (data not shown). Furthermore, the exclusion of NADPH from the reaction medium significantly decreased ROS generation (Fig. 3).
Role for hydroxycinnamic acids and associated conjugates in ROS generation
In addition to a small amount of monolignols, the poly(phenolic) domain of suberin contains a significant amount of hydroxycinnamic acids (Bernards et al., 1995; Negrel et al., 1996). Preliminary metabolite profiling of potatoes during suberization (Razem and Bernards, 2002), indicates that p-coumaroyltyramine, may play a significant role in potato suberization since it accumulates in DPI-treated tissues. Of the phenolic components tested in this study, p-coumaroyltyramine had the greatest stimulatory effect on the potato oxidase (Fig. 5). By contrast, p-coumaroylputrescine, which accumulates in wound-induced tubers whether treated with DPI or not, has no effect on the activity of the oxidase (Fig. 5).
The activation of NADPH-dependent oxidases by phenolic compounds is well established. For example, ferulic acid, and caffeic acids showed a strong stimulatory activity toward cauliflower inflorescence oxidases (Askerlund et al., 1987), while genistein, an isoflavone that accumulates in elicited soybean hypocotyls, is the most potent activator of NoxII, a plasma membrane-associated oxidase of soybean (Graham and Graham, 1999). Thus a trend toward species relevant phenolics as regulators of NADPH-dependent oxidases is emerging and, in the case of wound-healing potato tubers, p-coumaroyltyramine is a potential candidate regulatory molecule. While ferulic and chlorogenic acids also stimulated potato oxidase activity, the levels were much lower (Fig. 5), even though they accumulated to the same extent as p-coumaroyltyramine during wound healing (Razem and Bernards, 2002).
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Concluding remarks |
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Wounding of potato tubers triggers the massive production of ROS that could serve different purposes. The initial burst that follows mechanical injury is probably a spontaneous and less regulated production of ROS aimed at providing a first line of defence. It could also serve as a second messenger for the synthesis of phenolics and proteins. The subsequent, larger ROS bursts are probably involved in the suberization process and occur several hours apart from each other. It was possible to reconstitute the ROS generating system in vitro and to stimulate its activity by wound-induced hydroxycinnamic acids. Based on these data, and earlier observations (Bernards and Razem, 2001; Razem and Bernards, 2002), it is believed that the source of ROS during wound-healing in potato tubers is probably a plasma membrane-associated NADPH-dependent oxidase that is immunorelated to human plasma membrane oxidase. The failure of cytosolic proteins isolated from tissue collected at 1 hpw to compensate for those isolated from tissue collected at 42 hpw (Fig. 3), coupled with the apparent accumulation of p40phox, p47phox and p67phox over the same time frame strongly supports the conclusion that the generation of H2O2 during wound-induced suberization is under strict regulatory control.
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