Role of peroxidases in the Arabidopsis defense response
It is now recognized that various plants exhibit either a plasma membrane-localized NADPH/NADH oxidase-dependent, or a cell wall peroxidase-dependent oxidative burst, or both (for reviews see Apel and Hirt, 2004; Grant and Loake, 2000). It is therefore important to determine in which situations either or both enzymes are involved in the generation of ROS, and the respective roles of the two types of ROS-generation mechanisms in the plant-defense response. In two biochemically well characterized systems, French bean cells and rose cells, different mechanisms are involved in the generation of ROS (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).
Here we determined the extent to which cell wall-localized peroxidases contribute to the generation of ROS in Arabidopsis in response to pathogens and pathogen-derived elicitors. Using an Arabidopsis cell suspension-culture system and an elicitor from the fungal pathogen F. oxysporum, we showed that production of ROS is sensitive to azide and cyanide, inhibitors that in other species have been shown to target primarily cell wall-derived peroxidases. Consistent with these results, the F. oxysporum-elicited production of H2O2 was relatively insensitive to DPI, an inhibitor that is relatively specific for NADPH-oxidases. To confirm that peroxidases play a role in the Arabidopsis oxidative burst, we expressed a cDNA encoding the French bean peroxidase FBP1 in the anti-sense orientation in transgenic Arabidopsis plants. These FBP1 transgenic plants exhibited decreased production of H2O2 in response to the F. oxysporum elicitor and to infection by an avirulent strain of P. syringae; were more susceptible to both virulent and avirulent bacterial and fungal pathogens; had reduced levels of ionically cell wall-bound peroxidase activity; and exhibited lower levels of mRNAs corresponding to two close Arabidopsis homologs of FBP1, At3g49110 and At3g49120.
The reduced levels of the oxidative burst that we observed in transgenic FBP1 lines, as well as their enhanced susceptibility to both virulent and avirulent P. syringae strains, need to be reconciled with previous observations that indicate that NADPH oxidase(s) play a key role in the oxidative burst (Grant et al., 2000b; Torres et al., 2002). In the Grant et al. (2000b) study, H2O2 production following inoculation with Pto DC3000(avrRpm1) was sensitive to 7 μm DPI, and inhibition at this concentration was interpreted as demonstrating that an NADPH oxidase was most likely the origin of ROS detected with cerium chloride staining. Although the concentration of DPI used would favor inhibition of the NADPH oxidase, 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 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 the inhibitor was co-inoculated with either avrB- or avrRpt2-expressing P. syringae strains (Grant et al., 2000a). This was interpreted as indicating that both oxidases and peroxidases could be engaged in generating ROS in Arabidopsis.
By generating a series of Arabidopsis NADPH oxidase mutants, Torres et al. (2002) showed that the AtrbohD and AtrbohF genes were required for production of a full oxidative burst. Because our 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 in our study, in comparison with the Torres et al. (2002) study, is that we observed that the FBP1 anti-sense transgenic plants are highly susceptible to pathogen infection, whereas the atrboh mutants in the Torres et al. (2002) study were not more susceptible. This raises a question as to the respective roles of peroxidases and oxidases in the oxidative burst.
A possible explanation of the role of peroxidases in the oxidative burst comes from recent work of Torres et al. (2005), in which they constructed an lsd1 atrbohD atrbohF triple mutant. The lsd1 (lesions simulating disease) mutant exhibits spreading lesions and, surprisingly, the triple mutant exhibited an enhanced spreading lesion phenotype compared with lsd1, contrary to what was expected if the NADPH oxidase-generated burst was responsible for triggering cell death. Torres et al. (2005) concluded, counter-intuitively, that the role of NADPH oxidases is to limit the spread of a salicylic acid-elicited cell-death program in cells surrounding an infection site. Moreover, they showed that the NADPH oxidases need to be activated by an independent source of ROS to generate their own oxidative burst.
A model, consistent with our observations as well as those of Torres et al. (2005), is that apoplastic peroxidases are an initial rapid source of ROS, and are essential for conferring at least partial resistance independently of any involvement of NADPH oxidases. Subsequently, the peroxidase-generated ROS 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 neighboring cells. If this model is correct, the failure to detect an oxidative burst in Arabidopsis atrbohD/F mutants could be a consequence of the fact that the oxidative burst assays were carried out several hours after elicitation, long after a peroxidase-mediated burst may have occurred (Torres et al., 2002, 2005).
For some species, there is evidence that recognition of virulent and avirulent pathogens produces different oxidative burst profiles, with early ROS production observed in non-host, compatible and incompatible interactions, and a later, more extensive and sustained burst only in R gene-mediated resistance responses (Apel and Hirt, 2004; Grant and Loake, 2000). Data presented here support a role for cell wall-associated peroxidase(s) in both phases, as hydrogen peroxide production is reduced in the FBP1 transgenic line compared with wild-type plants following elicitation with an elicitor from cell walls of F. oxysporum within a 1-h time frame, or elicitation with an avirulent strain of bacteria within a 5-h time frame. One possibility is that peroxidase(s) catalyze ROS production during basal resistance triggered by recognition of pathogen-associated molecular patterns of virulent pathogens, and that this initial oxidative burst is essential for subsequent activation of NADPH oxidase during an R gene-mediated HR, as well as for activation of basal defenses. In this model, peroxidases play an essential role in basal resistance, independently of their role in activating NADPH oxidases, explaining why the FBP1 anti-sense line has a much more severe phenotype than an atrbohD/F mutant. Consistent with this interpretation is our observation that the FBP1 transgenic plants are more susceptible to highly virulent necrotrophic (B. cinerea) and biotrophic (powdery mildew, Golovinomyces orontii) fungal pathogens as well as virulent and avirulent P. syringae strains. Additional support for this model comes from recent work showing differential effects of DPI on H2O2 production following challenge of Arabidopsis leaves with hrpL mutants of P. syringae pv. phaseolicola, or with P. syringae pv. phaseolicola carrying an avirulence gene that triggers an HR. Interestingly, the oxidative burst elicited by the hrpL mutant, which induced papilla formation but not HR, was much less susceptible to DPI inhibition than the burst elicited by the avr gene, suggesting that ROS may be generated by different mechanisms, depending on the challenge (Soylu et al., 2005). Alternatively, overexpressing FBP1 might compromise resistance by preventing oxidative cell-wall cross-linking and the formation of barriers. However, the loss of resistance to avirulent P. syringae argues against an effect solely on physical barriers. Distinguishing between these possibilities requires further biochemical studies, such as those carried out in French bean (Bolwell et al., 2002), and assays of defense gene expression. Insertion mutants that target specifically either At3g49120 or At3g49110 would help clarify the involvement of these two peroxidases during defense. However, lines with insertions in regions of At3g49120 that would be likely to interfere with protein function are not available: although Arabidopsis stock collections list such mutants, it is not possible to obtain them; in at least one case the line could not be maintained, consistent with our own experience. It is likely that tests of the requirement of AtPCb and/or AtPCa for the production of H2O2 in response to potential pathogens will require an inducible system for targeted reduction of At3g49110 and At3g49120 expression.
Although the cationic French bean FBP1 peroxidase shows reasonable similarity to a number of Arabidopsis type III peroxidases, including the anionic AtPA2, which has a putative role in lignification (Ostergaard et al., 2000), expression of anti-sense cDNA coding for FBP1 appears to target specifically At3g49120 and At3g49110 that encode AtPCb and AtPCa, respectively (Valério et al., 2004). Both peroxidases have been grouped by a detailed phylogenetic analysis in a clade of seven out of the 73 type III peroxidases in the Arabidopsis genome that is closest to FBP1 (Duroux and Welinder, 2003). Comparison of FBP1 and AtPCb amino acid sequences indicates that they share 53.1% identity and 65.4% similarity, while FBP1 and AtPCa have 53.3% identity and 65.0% similarity. Furthermore, FBP1, AtPCa and AtPCb are all cationic, have a similar number of potential glycosylation sites, and have an unusual carboxy-terminal extension that is known to be cleaved in the case of FBP1 (Blee et al., 2001). All three have predicted amino-terminal secretion sequences leading to cell-wall localization. These properties of AtPCa and AtPCb are consistent with the data in Figure 5 that show a reduction of a wall-bound cationic peroxidase activity in the FBP1 transgenic plants. AtPCb and AtPCa have been characterized previously (Ostergaard et al., 1998; Tognolli et al., 2002; Welinder et al., 2002). AtPCb is expressed throughout the plant, whereas AtPCa has been detected only in aerial organs (Welinder et al., 2002). Like FBP1, AtPCb is induced in response to a variety of different pathogens and elicitors including virulent and avirulent H. parasitica, avirulent Pto DC3000, B. cinerea, and oligogalacturonides (Maleck et al., 2000; Scheideler et al., 2002; Tao et al., 2003; S. Ferrari, J.P., C.D., J.D. and F.M.A., unpublished data). Less is known about the regulation of AtPCa because it is not represented on the Affymetrix ATH1 GeneChip, although the data in Figure 6 show that it is strongly induced following P. syringae infection. At the protein level, AtPCb is one of only eight extracellular peroxidases detected in the culture medium of liquid-grown 2-week-old seedlings; AtPCa was not detected (Charmont et al., 2005). Data from the FBP1 anti-sense transgenic line do not allow us to determine the relative importance of AtPCb and AtPCa in limiting pathogen growth, as both peroxidases have reduced expression in this line. However, the difference in protein levels prior to infection, and the difficulty of maintaining lines with insertions in At2g49120, suggest that, of the two, AtPCb has the more critical role in defense.
Interestingly, two glutathione peroxidases were also downregulated in uninfected leaves of the FBP1 anti-sense plants. Presumably, these would function in scavenging ROS. It may well be that levels of ROS accumulating over time in these plants under normal light conditions are reduced compared with the wild type, resulting in lower levels of induction of protective peroxidases. It is unlikely that these two glutathione peroxidases are direct targets of the FBP1 anti-sense transgene, as they have both low overall similarity and regions of identity with FBP1 that are no longer than 10 nucleotides.
Susceptibility of transgenic FBP1 plants to pathogens
Despite all efforts, most of the Arabidopsis transgenic FBP1 lines succumbed to opportunistic fungal infections during culturing. However, two T1 lines survived and homozygous lines were derived from one of these. The plants that survived were normal phenotypically, although the homozygous lines formed somewhat larger rosette leaves and inflorescence initiation was delayed compared with Col-0. However, because of their susceptibility to opportunistic infections, even these surviving FBP1 transgenic lines are very difficult to maintain. The FBP1 transgenic lines demonstrated reduced capacity for ROS generation when challenged with the F. oxysporum elicitor in the leaf-disc assay, and exhibited a level of disease susceptibility comparable with previously isolated Arabidopsis enhanced disease susceptibility (eds) mutants including npr1 and pad4 (Glazebrook et al., 1996; Rogers and Ausubel, 1997) when challenged with the virulent bacterial pathogens Psm ES4326 or Pto DC3000.
In contrast to most other eds mutants, however, the transgenic FBP1 plants exhibited enhanced susceptibility to avirulent P. syringae strains. While Psm ES4326(avrRpt2) grew less than 10-fold in wild-type Col-0 plants over 3 days, the same strain multiplied 20 000-fold in line H4 and actually exceeded growth of a near-isogenic virulent strain of Psm by a small but reproducible amount. A number of studies have demonstrated that, in the absence of an R gene-mediated resistance response, bacterial avirulence proteins (including AvrRpt2) have effector or virulence functions (Abramovitch et al., 2003; Chen et al., 2004; Chisholm et al., 2005; Greenberg and Vinatzer, 2003; Hauck et al., 2003; Kim et al., 2005; Metz et al., 2005). Our data suggest that not only basal resistance, but also R gene-mediated resistance, are compromised by a reduction in AtPCb and/or AtPCa activity. Consistent with an increase in bacterial growth, tissue collapse 3 days after inoculation with either virulent or avirulent P. syringae strains was more extensive in the peroxidase anti-sense lines than in the wild type.
The peroxidase anti-sense lines are also unusual in having increased susceptibility to a broad range of virulent pathogens. Many of the mutants isolated on the basis of enhanced susceptibility to P. syringae do not show a similar eds phenotype with G. orontii or B. cinerea (Ferrari et al., 2003; Reuber et al., 1998) and, conversely, a majority of mutants isolated in a screen for enhanced susceptibility to G. orontii are not significantly more susceptible to P. syringae ES4326 (Dewdney et al., 2000). However, FBP1 anti-sense line H4 is strikingly more susceptible than the wild-type to all three of these pathogens, which represent bacterial, biotrophic fungal and necrotrophic fungal phytopathogens.
Although FBP1 anti-sense plants exhibited increased susceptibility to pathogens, they were more resistant to the fungal toxin fumonisin B1. In wild-type plants, fumonisin B1 triggers the generation of reactive oxygen intermediates (Stone et al., 2000), which may be essential for FB1 induction of HR-like cell death (Gilchrist, 1998). A number of studies (Alvarez et al., 1998; Bennett et al., 2005; Delledonne, 2005; Kliebenstein et al., 1999; Mach et al., 2001; Torres et al., 2005) indicate that cell-death programs are impacted by multiple ROS, including superoxide, hydrogen peroxide and nitric oxide, which may activate or repress cellular suicide. Although the precise mechanism of FB1-induced cell death in Arabidopsis is unclear, the attenuation of HR-like cell death in FB1-treated FBP1 anti-sense plants suggests that peroxidase-generated H2O2 is involved. It would be of interest to test whether the atrbohD/F double mutant is also resistant to fumonisin B1.