JXB Advance Access originally published online on May 23, 2006
Journal of Experimental Botany 2006 57(8):1817-1827; doi:10.1093/jxb/erj216
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RESEARCH PAPER |
Production of reactive oxygen species in Arabidopsis thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum: implications for basal resistance
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK
To whom correspondence should be addressed. E-mail: p.bolwell{at}rhul.ac.uk
Received 2 March 2006; Accepted 24 March 2006
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Abstract |
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The present understanding of ROS generation in the defence response of Arabidopsis thaliana is reviewed. Evidence suggests that the apoplastic oxidative burst generated during basal resistance is peroxidase-dependent. The ROS generated during this basal resistance may serve to activate NADPH oxidase during the R-gene-mediated hypersensitive response. The processes involved in the production of reactive oxygen species in A. thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum are investigated in the present work. This system appears analogous to the production of ROS during the basal resistance response in French bean, which is peroxidase-dependent. A panel of modulators effective in other pathogen elicitor and plant cell systems has been used to investigate the Arabidopsis signalling pathways and the plant cell responses involved. Thus as in other systems, an early calcium influx into the cytosolic compartment, a rapid efflux of K+ and Cl–, and extracellular alkalinization of elicited cell cultures has been found. However the alkalinization is not sufficient to stimulate the apoplastic oxidative burst by itself, unlike in French bean, although vectorial ion fluxes are needed. A secretory component which is sensitive to monensin and N-ethylmaleimide and insensitive to brefeldin A may also be necessary for the release and provision of substrates for peroxidase-dependent generation of H2O2.
Key words: Arabidopsis thaliana, calcium, elicitation, hydrogen peroxide, oxidative burst, secretion
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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One of the earliest events in the plant defence response against pathogen attack is the production of reactive oxygen species (ROS) including hydrogen peroxide and superoxide (Lamb and Dixon, 1997; Wojtaszek, 1997; Bolwell and Wojtaszek, 1997). It is now recognized that various plants, including the model plant Arabidopsis, exhibit different oxidative burst phases with an early production of ROS in both compatible and incompatible interactions and a later burst shown only in R-gene-mediated resistance responses (Grant and Loake, 2000; Apel and Hirt, 2004). It is also now established that the major sources of ROS can be plasma membrane-localized NADPH/NADH oxidase-dependent which can generate superoxide or cell wall-localized peroxidase-dependent which can generate hydrogen peroxide directly or both sources (Bolwell, 1999). Depending upon the host and pathogen species involved, it is important to dissect the relative contribution of plasma membrane NADPH oxidases or cell wall peroxidases (Bolwell et al., 1998; Martinez et al., 1998; Grant J et al., 2000; Grant M et al., 2000; Torres et al., 2002) in relation to the production and role of ROS in basal resistance and in the hypersensitive response (HR).
NADPH oxidases, also referred to as respiratory burst oxidases, have been implicated in biotic interactions, abiotic stress responses, and development in different plant species and have received considerable attention in Arabidopsis thaliana (Torres and Dangl, 2005). Of the ten-gene family of Atrboh genes encoding homologues of the mammalian NADPH oxidase gp91phox (Keller et al., 1998; Torres et al., 1998), AtrbohD and AtrbohF genes were identified as required for the production of a full oxidative burst in response to avirulent strains of the bacterial and oomycete pathogens Pseudomonas syringae and Hyaloperonospora parasitica, respectively (Torres et al., 2002). However, neither the atrbohD or atbrohF mutants, either singly or doubly, were more susceptible to either P. syringae or H. parasitica. More recently, a lsd1-atrbohD-atrbohF triple mutant has been constructed in which the lsd1 (lesions simulating disease) mutation exhibits spreading lesions. Since this phenotype was found to be enhanced in the triple mutant, it was concluded that the role of NADPH oxidases is to limit the spread of a salicylic acid-elicited cell death programme in cells surrounding an infection site (Torres et al., 2005). However, in the context of the present study, it was also shown that the NADPH oxidases need to be activated by an independent source of ROS to generate their own oxidative burst. The source of that ROS is likely to be generated during the basal response and these data place the NADPH oxidases as having a function in the HR only.
In comparison, there has not been as much investigation of peroxidase-dependent ROS production in Arabidopsis. However, use of inhibitors has indicated its existence (Grant J et al., 2000; Soylu et al., 2005) and this needs to be reconciled with previous observations that indicate that NADPH oxidase(s) play a key role in the oxidative burst (Grant M et al., 2000; Torres et al., 2002). In the earlier study (Grant M et al., 2000), H2O2 production detected with cerium chloride staining following inoculation with DC3000 (avrRpm1) was sensitive to 7 µM diphenylene iodonium (DPI) and inhibition at this concentration was interpreted as demonstrating that an NADPH oxidase was most likely the origin of ROS. Although DPI has been claimed to be a specific inhibitor of NADPH oxidases and the concentration of DPI used would favour its inhibition the peroxidase system is also sensitive to DPI, albeit with lower specificity, and 7 µM DPI would also inhibit peroxidase-generated ROS to some extent (Frahry and Schopfer, 1998). In order to distinguish peroxidase-dependent processes the cytochrome inhibitors potassium cyanide and sodium azide have been used since NADPH oxidase, being a flavoprotein, is insensitive to these. Thus in another study utilizing a ROS-responsive GST-luciferase gene expression system, the oxidative burst proved to be sensitive to both 3 µM DPI and 1 µM azide when either inhibitor was co-inoculated with either avrB- or avrRpt2-expresssing P. syringae strains (Grant J et al., 2000). This was interpreted as indicating that both oxidases and peroxidases could be engaged in generating reactive oxygen species in Arabidopsis. Subsequently, a structural study has investigated the response of Arabidopsis to either mutant or avirulent P. syringae phaseolicola using cerium chloride to study the accumulation of H2O2 at reaction sites (Soylu et al., 2005). The strongest H2O2 response was found during the HR activated by avirulent strains and other mutant strains. Accumulation of H2O2 during the HR, but not during wall alterations, was strongly suppressed by inhibition of NADPH oxidase with DPI, but at 8 µM, and therefore under the reservations with respect to specificity cited above. However, the differential effect of DPI suggests an alternative source of H2O2 to modify the plant cell wall, such modifications being a major feature of basal resistance (Schulze-Lefert, 2004).
Therefore an alternative source of ROS to that from NAPH oxidase is involved in basal (non-host) resistance as suggested from knock out and inhibitor data in Arabidopsis. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a French bean type III peroxidase (FBP1; Blee et al., 2001) exhibited an impaired oxidative burst in response to avirulent strains of P. syringae as shown by a lack of diaminobenzidine detectable ROS at the cellular level in the leaf and cerium hydroperoxide staining in cell wall appositions at the subcellular level. Moreover FBP1 antisense plants were more susceptible than wild-type plants to both fungal and bacterial pathogens (Bolwell et al., 2002; LV Bindschedler, J Dewdney, FM Ausubel, GP Bolwell, unpublished data). Transcriptional profiling and RT-PCR analysis showed that the antisense FBP1 transgenic plants had reduced levels of specific peroxidase-encoding mRNAs that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H2O2 during the Arabidopsis defence response and in conferring resistance to a wide range of pathogens (LV Bindschedler, J Dewdney, FM Ausubel, GP Bolwell, unpublished data). Because these results show that peroxidases are also required for H2O2 production in response to the same avirulent P. syringae strains, it seems likely that both membrane-associated NADPH oxidases and cell-wall bound peroxidases are required to generate H2O2. A major difference between peroxidase knock-downs and the atrobh mutants in the Torres et al. (2002) study, however, is that the FBP1 anti-sense transgenic plants were highly susceptible to pathogen infection and this was correlated with a decrease in ROS production whereas the atrboh mutants were not impaired in resistance despite a reduction in ROS production. One possibility of reconciling the role of peroxidases and their consequent effect on the NADPH oxidases encoded by members of the atrboh gene family is that apoplastic peroxidases are an initial rapid source of ROS and are essential for conferring at least partial resistance. The peroxidase-generated ROS could be the source predicted to activate NADPH oxidases, which in turn generate a plasma membrane associated oxidative burst, the primary role of which is to limit the extent of cell death in neighbouring cells.
In addition to such whole plant studies, cell suspension cultures have contributed to an understanding of the biochemistry and the signalling events involved in the generation of ROS. They probably model the basal resistance triggered by recognition of PAMPs (pathogen associated molecular patterns) of virulent pathogens (Navarro et al., 2004). For example, in the two biochemically well-characterized systems, French bean cells and rose cells, different mechanisms are involved in the generation of ROS in response to specific fungal cell-wall derived PAMPs which are ineffective in the other system (Bolwell et al., 1998). In French bean, the peroxidase appears to be the dominant mechanism; in rose cells, the oxidase with additional specificity towards NADH as well as NADPH is dominant (Bolwell et al., 1998). On the other hand, other plants including cotton exhibit both mechanisms of ROS generation, albeit temporally separated (Martinez et al., 1998). In bean cell cultures an apoplastic peroxidase has been shown to be responsible for ROS production after elicitation with a cell-wall crude extract of C. lindemuthianum. The oxidative burst is dependent upon three components in bean, a cell wall peroxidase, the release of a reductant and/or a substrate, and pH change which gives rise to extracellular alkalinization (Bolwell et al., 2002). Initial studies on Arabidopsis thaliana suspension-cultured cells revealed an azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H2O2 in response to a Fusarium oxysporum cell wall preparation (Bolwell et al., 2002; LV Bindschedler, J Dewdney, FM Ausubel, GP Bolwell, unpublished data). This system has been subjected to further characterization in the present study and early signalling events compared with the well-characterized peroxidase-dependent burst in French bean.
Amongst the earliest signalling events involved in the oxidative burst, an increase in cytosolic Ca2+, which in some tissues occurs within s of elicitation, is thought to be a primary signal essential for the subsequent down-stream events, which include the production of ROS (Chandra and Low, 1997; Rajasekhar et al., 1999; Blume et al., 2000; Grant M et al., 2000). This leads to the induction of defence-related genes such as those encoding PR proteins and those involved in phytoalexin production (Blume et al., 2000) underlined by important changes in the level of a number of proteins including antioxidant enzymes and molecular chaperones (Ndimba et al., 2003; Chivasa et al., 2006). Beside the early calcium influx into the cytosolic compartment, a rapid efflux of K+ and Cl– and extracellular alkalinization of elicited cell cultures has also been observed (Scheel, 1998; Fellbrich et al., 2000). Extracellular alkalinization appears to be essential for the apoplastic oxidative burst in French bean (Bolwell et al., 1995). Using modulators of cAMP, calcium and G proteins to treat French bean (Phaseolus vulgaris) cells before the addition of an elicitor from Colletotrichum lindemuthianum, evidence was obtained for the involvement of these signalling components in the early steps of signal transduction leading to the production of the apoplastic oxidative burst (Bindschedler et al., 2001). A similar range of modulators have been tested on Arabidopsis cells treated with an elicitor from Fusarium oxysporum in the present work.
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Chemicals
All chemicals were purchased from Sigma (Poole, Dorset, UK).
Cell cultures and elicitation
Arabidopsis suspension-cultured cell lines were a kind gift of Professor AR Slabas, Durham University, UK, and were maintained in MSMO medium (Sigma) containing sucrose (30 g l–1), naphthalene acetic acid (0.5 mg l–1), and kinetin (0.05 mg 1–1) under white light (fluorescent cool white) at a fluence rate of 30±10 µmol m–2 s–1 in a 16/8 h light/dark regime. Cell cultures were subcultured every week by transferring 2 ml of 1-week-old culture to 100 ml of fresh medium in a sterile 250 ml flask.
F. oxysporum f.s. matthioli race 1 (Kistler et al., 1991) was used for the derivation of the elicitor. The elicitor preparation was prepared by culturing F. oxysporum as a yeast form in half-strength potato dextrose broth (Sigma P-6685), collecting yeast cells by centrifugation, resuspending in 100 ml of 500 mM KH2PO4, and centrifuging at 5000 g in an SS-34 rotor (Sorvall) for 30 min. The pellet was resuspended in 200 ml 50 mM KH2PO4 and centrifuged as above. The pellet was then sequentially washed and centrifuged in chloroform/methanol (1:1 v/v) and acetone. The pellet was resuspended in ddH2O (10 g pellet l–1) and autoclaved for 30 min at 121 °C. The elicitor preparation was stored at –20 °C until used. The F. oxysporum elicitor at 100 µg ml–1 glucose equivalents, the optimal concentration, induced a robust and highly reproducible oxidative burst.
For elicitation, 20 ml of 5-d-old Arabidopsis cell cultures were transferred to a 100 ml beaker and agitated on a rotating platform at 180 rpm. The oxidative burst modulators were added and cells incubated for an optimized period of time prior to addition of the elicitor, unless stated differently. The optimum preincubation periods were determined in French bean to be 30 min for monensin, nigericin, valinomycin, verapamil, and dibutyryl cAMP and 5 min for N-ethylmaleimide (NEM), forskolin, W7, and A23187 [GenBank] (Bindschedler et al., 2001). NEM, verapamil, and dibutyryl cAMP were dissolved in water, Brefeldin A, valinomycin, W7, and A23187 [GenBank] in DMSO, and forskolin, monensin, and nigericin in ethanol. As a control, cells were incubated with the corresponding solvent or buffer as used for the modulator tested.
To examine the effects of EGTA and Ca2+ on the elcitation response, cells were harvested by centrifugation at 50 g for 1 min, washed with sucrose (3% w/v) and resuspended in sucrose (3% w/v) containing 1 mM EGTA, pH 5.7. The resuspended cells were preincubated for 15 min before addition of elicitor.
Detection of hydrogen peroxide and other ROS
H2O2 production by elicited Arabidopsis cells was specifically monitored with xylenol orange, where hydroperoxides are reduced by ferrous ions in acid solution forming a ferric product–xylenol orange complex, detected spectrophotometrically at 560 nm (Gray et al., 1999) as described previously (Bindschedler et al., 2001). ROS production was also monitored using a luminol-based assay described by Bolwell et al. (1998).
Cell death
Cell death was assayed 24 h and 44 h after elicitation by incubating 1 ml of cells with 0.05% (w/v) Evans blue. Unbound dye was removed by extensive washing with ddH2O. Dye bound to dead cells was solubilized by grinding in 50% methanol containing 1% (w/v) SDS and incubating at 50 °C (Levine et al., 1994). The A600 was used to monitor cell death. Cell samples were also checked after the washing stage and observed by light and fluorescence microscopy.
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The oxidative burst in Arabidopsis suspension-cultured cells
The oxidative burst in Arabidopsis cells was measured either using the luminol method or colorimetrically using xylenol orange. Since the cells are green the luminol assay cannot be used directly because the chemiluminescence is quenched. Therefore the methods were compared using medium following removal of cells by centrifugation or filtration. The medium was used directly for the xylenol orange method (Bindschedler et al., 2001) while horseradish peroxidase was added to the supernatant in borate buffer before determination using the luminol method (Bolwell et al., 1995). Although similar levels of ROS were determined in both assays there was much greater variation in level of detectable ROS and in timing of the burst when the luminol assay was used (data not shown). Although the reason for this is unknown, since the ROS detected by xylenol orange was elicitor specific and shown to be due to H2O2 by its abolition in the presence of catalase (Table 1; Bindschedler et al., 2006), this assay was routinely used for the rest of the study.
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The kinetics of the oxidative burst in response to an elicitor derived from cell walls of Fusarium oxysporum have been examined and it was found that the production of ROS peaks at about 60–80 min, therefore the response appears to be slower than that in the French bean cultures. Maximal levels of H2O2 in the medium, as measured by the xylenol orange assay, reach about 25 µM and the high level of ROS generated persisted for up to 120 min (Fig. 1A). Inhibitor studies indicate the operation of an apoplastic peroxidase-dependent oxidative burst (Table 1). DPI hardly inhibits the oxidative burst in elicited cells even at high concentration, while ROS were not produced with KCN or NaN3 inhibitors of peroxidase at 1 or 2 mM which nearly completely blocked the oxidative burst.
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Cell death
Using the Evans blue assay and quantitative determination at 600 nm in a series of five determinations, elicited cells showed a 99% increase in cell death after 24 h (A600 0.533:0.267) and a 77% increase after 44 h (A600 0.758: 0.428) over control cells. This cannot be accurately computed in terms of cell numbers due to non-random clustering of dead cells and the size of the cell-suspension clumps. Attempts to disperse clumps would lead to leakage of Evans blue dye. However, an estimate can be given for elicited cells which ranged between 5–50% cell death in different clumps and an average of around 31% at 44 h after elicitation (data not shown).
pH-dependence of the burst
Extracellular alkalinization has been reported for a number of elicitation systems. An increase in pH from 5.1 to 6.0 has been reported in these same Arabidopsis suspension-cultured cells in response to an elicitor preparation (Toppan and Esquerre-Tugaye, 1984) from the maize pathogen F. moniliforme (Chivasa et al., 2006). Furthermore, it increased from 5.7–6.0 in Arabidopsis cells treated with harpin (Clarke et al., 2005). An elicitor from F. oxysporum f.s. matthioli race 1 found growing on Col-0 has been used, to which Col-0 is resistant. The pH also rapidly increased during the oxidative burst from 5.7 to a maximum of 6.45 (Fig. 1A). This was less than for bean which increased to around pH 7.2.
The pH dependence of the burst was also tested by buffering the cell culture medium (Fig. 1B). French bean cells which generate ROS as a response to an elicitor from Colletotrichum lindemuthianum (Bolwell et al., 1995) can also produce a small burst simply when cell cultures are exposed to a high pH (7.5), while ROS production was inhibited by maintaining a low pH. On the contrary, Arabidopsis cell response to F. oxysporum elicitor was inhibited by buffering the medium at high pH. The pH shift due to alkalinization of the medium of Arabidopsis cell cultures after elicitation is less important than was observed for bean. This might be due to a lower buffering capacity of Arabidopsis cell walls in the cultures, as the cell mass is smaller than for the bean cell cultures. In comparison with French bean the extracellular alkalinization per se does not seems to be essential in Arabidopsis for peroxidase activity to generate hydrogen peroxide although the ion fluxes associated with H+ influx are required for ROS generation, as shown by the next series of experiments.
Effect of K+ ionophores
Potassium ionophores were previously shown to be effective inhibitors of the oxidative burst in French bean cells (Bolwell et al., 1995). When administered to French bean cells, 30 min before the addition of elicitor, both nigericin and valinomycin at 2 µM caused ion leakage which led to an equlibration in extracellular pH of 5.7 in the former and 7.5 in the latter. This prevented the elicitor-dependent pH change and resulted in total inhibition of the oxidative burst. 2 µM nigericin under the same conditions of 30 min preincubation also reduced H2O2 production in Arabidopsis cells over the whole time-course, showing that the inhibition was not due to a delayed burst (Fig. 2). However, both nigericin and valinomycin were rather less effective in inhibiting the burst in Arabidopsis cells compared with French bean cells (Table 1).
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Effect of modulators of calcium on the oxidative burst
Figure 3A shows that chelation of calcium with EGTA inhibited the burst, while added Ca2+ in the form of CaCl2 in excess of the EGTA restored and amplified the burst. The ionophore A23187 [GenBank] has frequently been used to examine the role of intracellular Ca2+ in signal transduction. Addition of concentrations of ionophore to French bean cells preincubated for 5 min before the addition of elicitor led to up to 5-fold increase of the oxidative burst at 50 µM A23187 [GenBank] (Bindschedler et al., 2001). Similar experiments on Arabidopsis (Fig. 3B), further confirms that a nearly 2-fold increase in the oxidative burst can be modulated by A23187 [GenBank] with a maximum enhancement at 10 µM without further increase at higher concentrations (not shown). In both cases the ionophore alone, in the absence of elicitor, did not cause a significant production of hydrogen peroxide, suggesting that regulatory events other than an influx of Ca2+ are required for the oxidative burst.
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In French bean, the Ca2+ channel blocker, verapamil, inhibited the apoplastic oxidative burst when the cells were preincubated with the blocker for 30 min before addition of the elicitor in French bean which was maximum at 100 µM and gave a 75% inhibition (Bindschedler et al., 2001). In comparison, H2O2 production in elicited Arabidopsis cell cultures under the same conditions was only inhibited by 34% when tested up to 200 µM (Table 1; Fig. 3C).
W7 is known to be an inhibitor of calmodulin-dependent proteins and calmodulin-dependent regulatory events including the activation of calmodulin-dependent kinases and plant CDPKs. Therefore its mode of action should be to block calcium-dependent effects as CDPKs are activated when calmodulin is binding Ca2+. In French bean the effect of W7 was found to be concentration-dependent, with an 80% inhibition at 100 µM when the cells were preincubated with the inhibitor for 5 min before the addition of elicitor (Bindschedler et al., 2001). W7 produced a similar level of inhibition of 65% in Arabidopsis (Table 1).
Effect of modulators of cAMP
Forskolin, a cell-permeable derivative and a potent activator of adenylyl cyclase has been used extensively in mammalian systems to increase intracellular cAMP and to study cAMP-dependent physiological effects. Using the xylenol orange assay an oxidative burst was observed with the addition of 1, 10, or 100 µM forskolin in French bean cells and at 100 µM also induced alkalinization of the apoplasm (Bindschedler et al., 2001). Forskolin was similarly active in Arabidopsis cells and induced a 2-fold increase in H2O2, while dibutyryl cyclic AMP was not (Table 1). This may indicate compartmentation differences in accessibility to their respective site of action. However, since forskolin can have additional effects, the evidence for cAMP participation in the oxidative burst in Arabidopsis is much less than for French bean cells.
Effect of inhibitors of exocytosis
Although primarily a Na+ ionophore, monensin is also an effective inhibitor of secretion in plant cells (Zhang et al., 1993) and this property has been linked to its effect in elicited cells (Bolwell, 1999). Monensin is a particularly potent inhibitor in Arabidopsis (Table 1; Fig. 4A, B) and at 10 µM showed 75% inhibition of the oxidative burst. NEM (N-ethylmaleimide) has been reported as an inhibitor of exocytosis in animal cells where the target is NEM sensitive factor (NSF). This is involved in binary SNARE complexes that consist of a syntaxin and a SNAP25 protein and usually bind a third partner, a membrane-anchored v-SNARE that resides in vesicles (Jahn et al., 2003). It has also been shown to have specific effects on membrane transport in plant cells (Brandizzi et al., 2002). An NSF and number of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors exist in the Arabidopsis genome and include syntaxins (Sanderfoot et al., 2000; Schulze-Lefert, 2005). NEM proved to be an effective inhibitor of the oxidative burst in Arabidopsis producing inhibition up to 65% (Fig. 4C, D).
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Brefeldin A specifically and reversibly blocks translocation of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus without affecting endocytosis or lysosome function and causes disassembly of the Golgi complex and ER swelling in a variety of mammalian cell lines at <40 ng ml–1. Brefeldin A has been used frequently as an inhibitor of exocytosis in plant cells at higher concentrations (Brandizzi et al., 2002; Chatre et al., 2005). However, it had no effect on the oxidative burst in Arabidopsis when used at these concentrations effective in blocking protein transport between ER and Golgi (Fig. 4E).
Although they may have other effects, the differential potency of the three inhibitors of secretion can be interpreted that, although exocytosis is a significant component of the apoplastic oxidative burst, it is limited to the unloading of post-Golgi vesicles. In French bean leaf cells the site of hydrogen peroxide production coincides with the papillae, which are the product of such directed secretion (Brown et al., 1998) and this is further borne out in Arabidopsis (Soylu et al., 2005).
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Discussion |
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Several lines of evidence from various plant species suggest that the sources of reactive oxygen species are different during non-host (basal) resistance and during the hypersensitive response (HR), but also that these sources may interact with each other. The apoplastic oxidative burst during basal resistance is peroxidase-dependent (Solyu et al., 2005; LV Bindschedler, J Dewdney, FM Ausubel, GP Bolwell, unpublished data). It now appears that an earlier source of ROS may serve to activate NADPH oxidase during the HR (Torres et al., 2005). By deduction, this source could be from the basal resistance response. Genetic evidence and use of inhibitors in Arabidopsis thaliana plants have provided data that would comply with such a model but there is no direct evidence as yet, since compromising the peroxidase-dependent apoplastic oxidative burst also affects the HR. Some of these aspects are also difficult to determine in the plant and can be investigated in detail in model systems. Both transcriptional analysis of flagellin- and chitin-treated suspension-cultured cells (Ramonell et al., 2002; Navarro et al., 2004) and proteomic analysis of cells treated with an elicitor preparation (Toppan and Esquerre-Tugaye, 1984) from the maize pathogen F. moniliforme (Chivasa et al., 2006) have shown the applicability of modelling responses in this way in Arabidopsis.
The processes involved in the production of reactive oxygen species in Arabidopsis thaliana cell-suspension cultures in response to an elicitor from Fusarium oxysporum are investigated in the present work. This system appears analogous to the basal resistance response since inhibitor studies indicate that the bulk of the H2O2 produced is peroxidase-dependent (LV Bindschedler, J Dewdney, FM Ausubel, GP Bolwell, unpublished data). Use of modulators shows that the associated signalling appears to be partly the same as in other systems. An early calcium influx into the cytosolic compartment, a rapid efflux of K+ and Cl– and extracellular alkalinization of elicited cell cultures probably occurs. However, the alkalinization does not appear to be the trigger per se in this system but, nevertheless, the ion fluxes are still required to activate the burst.
The selection of the modulators has come primarily from their previous use in studying the three-component oxidative burst in French bean cells where the signalling involved in the apoplastic generation of hydrogen peroxide is well studied (Bolwell et al., 1995, 1999, 2002). The requirement for K+ movement was demonstrated by the inhibition of the burst as a result of treatment with the ionphores, valinomycin and nigericin, which would act to equilibrate K+ ions and prevent the vectorial transport in response to elicitor action. This, in turn, would alter the pH change, although the action of buffers indicate that alkalinization itself is not necessary for the oxidative burst in Arabidopsis. Alkalinization, however, does mediate salicylic acid transport in Arabidopsis (Clarke et al., 2005).
The Ca2+ dependence of the burst has also been confirmed in Arabidopsis. Thus, the Ca2+ channel blocker, verapamil, which modulates movement of Ca2+ into cells through voltage-sensitive calcium channels in mammalian cells, is also known to exert inhibitory effects on Ca2+ influx in plant cells (Volotovski et al., 1998). In the present study, verapamil inhibited the elicitor-induced production of ROS, suggesting that at least some of the events triggered by the elicitor were mediated via an extracellular Ca2+ flux into the cytosol. The involvement of calcium in events upstream of the oxidative burst was confirmed by the increase in ROS production in the presence of the ionophore A23187. [GenBank] It has also been shown that an increase in cytoplasmic calcium is required for the production of ROS in Arabidopsis leaf cells (Grant M et al., 2000). Although Ca2+ may directly activate the NADPH oxidase without a direct requirement for a calcium-activated protein kinase (Romeis et al., 2000), its role in a peroxidase-dependent apoplastic oxidative burst is obscure. A target for Ca2+ influx would be CDPKs, as it has been observed that a specific form appears to be involved in the phosphorylation of PAL following elicitation (Allwood et al., 2002). The effect of W7 would indicate a possible role of CDPKs in the apoplastic oxidative burst, but it is difficult to reconcile its role with an action on the ion influxes or the extracellular peroxidase. If there is an involvement of these kinases, it is likely to be on the provision of the substrate, which would constitute the third element of the three-component system required for the apoplastic production of hydrogen peroxide (Bolwell, 1999; Bolwell et al., 2002). Another mode of transduction in Arabidopsis could be through the action of calmodulin on cyclic nucleotide-gated ion channels (Kohler et al., 1999) and, as in animals, Ca2+/calmodulin may attenuate the activity of these CNGCs by increasing their apparent affinity for cyclic nucleotides.
Of the cyclic nucleotides, cAMP is now considered to be a potent secondary messenger in plants (Assmann, 1995; Bolwell, 1995; Newton and Smith, 2004). Its involvement in the defence response of French bean cells was indicated by the enhancement of the burst when cells were treated with forskolin, the exogenous application of the cAMP homologue, or application of the G-protein activator, cholera toxin (Bindschedler et al., 2001), and reinforced by the observed transient increase in cAMP observed in elicited bean cells within 15 min (Bolwell, 1992). Such data indicate that an early event such as the oxidative burst and a later event such as the induction of PAL in French bean defence responses probably both require common steps in the early events of the signalling pathway involving a G protein and cAMP. With respect to the Arabidopsis system, only forskolin produced significant enhancement of the burst, so it is less conclusive that this signalling is involved although there is growing evidence of a link between cyclic nucleotides and ion channels which would provide indirect activation of ROS production. In mammals, it has been shown that both cAMP and cGMP can stimulate cyclic nucleotide-gated cation channels leading to an influx of calcium (Qiu et al., 2000). A similar system probably exists in plants, since both cyclic nucleotides induced an increase of Ca2+ in tobacco (Volotovski et al., 1998). Moreover, cyclic nucleotide-gated cation channels have been cloned in Arabidopsis (Leng et al., 1999; Clough et al., 2000) and tobacco (Arazi et al., 2000). In elicited Arabidopsis cells, NO has been shown to be produced much earlier than H2O2 and cGMP appears to be required for the induction of NO-dependent PCD (Clarke et al., 2000). cAMP may directly act on cyclic-nucleotide gated channels and induce an increase in [Ca2+]cyt and alkalinization of the medium. A G protein 
-subunit may also activate plasma membrane Ca2+ channels (Aharon et al., 1998).
A secretory component may also be necessary for the release and provision of substrates for peroxidase-dependent generation of H2O2 (Bolwell, 1999). Use of inhibitors of the secretory system such as monensin, brefeldin A, and NEM shows differential inhibition of ROS production. The potent inhibitory effect of monensin and NEM, together with the lack of effect of brefeldin A, can be interpreted as an involvement of post-Golgi vesicles only in the apoplastic oxidative burst. Notwithstanding, the secretory system has been shown to be responsible for many of the aspects of overall resistance (Schulze-Lefert, 2004). In the context of the oxidative burst this could involve unloading of substrates emanating from vesicles or larger organelles such as peroxisomes. There is both structural and genetic evidence for such involvement. Rearrangement and directed secretion of vesicles and organelles to sites of pathogen adhesion and/or attempted penetration has been observed in several systems leading to papilla formation (Freytag et al., 1994; Kobayashi et al., 1997; Bestwick et al., 1998; Brown et al., 1998; McLusky et al., 1999; Schmelzer, 2002) including Arabidopsis (Solyu et al., 2005). Hydrogen peroxide is localized to these sites (Bestwick et al., 1998; Brown et al., 1998). Elicitor systems mimic these wall changes, but without the localized deposition. Callose, for example is deposited evenly around the wall (Robertson et al., 1999). These mechanisms leading to apoplastic defence are beginning to be dissected genetically. For example, characterization of a series of penetration mutants that allow inappropriate ingress by barley powdery mildew in Arabidopsis include a plasma membrane-resident syntaxin which represents one of 24 sequence-related family members in the Arabidopsis genome which codes for soluble N-ethylmaleimide-sensitive factor adaptor protein receptors (Sanderfoot et al., 2000; Collins et al., 2003) and explains how NEM may be effective in blocking the oxidative burst in this study. There is strong evidence for the existence of active SNARE protein-dependent and vesicle-associated resistance responses at the cell periphery. Interestingly, a second penetration mutant encodes a glycosyl hydrolase, localizes to peroxisomes, and acts as a component of an inducible preinvasion resistance mechanism and could be involved in provision of metabolites (Lipka et al., 2005). Appearance of metabolites in the wall following elicitation include fatty acids, malate, citrate, and succinate in French bean cells (Bolwell et al., 1999, 2002). These are also found in Arabidopsis together with malonate and fumarate (VS Butt, P Finch, DR Davies, GP Bolwell, unpublished data). Since these are indicators of peroxisomal metabolism it may be that peroxisomes are unloaded into the wall as well as secretory vesicles. These compounds may also indicate metabolism related to the provision of substrates for H2O2 generation although the precise identity of these is still unknown.
In summary, the response of Fusarium elicitor-treated Arabidopsis suspension-cultured cells to various modulators is similar to that shown by French bean (Table 1) and complies with the three-component model for the apoplastic generation of hydrogen peroxide (Bolwell, 1999; Bolwell et al., 2002).
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Footnotes |
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* Present address: The BioCentre, University of Reading, Reading RG6 6AS, UK.
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Abbreviations |
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HR, hypersensitive response; NEM, N-ethylmaleimide; ROS, reactive oxygen species.
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