localization of hydrogen peroxide accumulation during the ... · ing cross-linking reactions...

The Plant Cell, Vol. 9, 209-221, February 1997 O 1997 American Society of Plant Physiologists

Localization of Hydrogen Peroxide Accumulation during the Hypersensitive Reaction of Lettuce Cells to Pseudomonas syringae pv phaseolicola

Charles S. Bestwick,' Ian R. Brown, Mark H. R. Bennett, and John W. Mansfield Department of Biological Sciences, Wye College, University of London, Wye, Near Ashford, Kent TN25 5AH, United Kingdom

The active oxygen species hydrogen peroxide (H202) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H202 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H202 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H202 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H202, unless the responding cell was undergoing the HR. Cata- lase treatment to decompose H,O, almost entirely eliminated staining, but 3-amino-l,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H202 detected. H202 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCI, reacts with excess H202 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H202 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H,Oz is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment.

INTRODUCTION

The active resistance of plants to colonization by bacteria and fungi is often expressed by the hypersensitive reaction (HR) of challenged plant cells (Ingram, 1978; Klement, 1982; Mansfield, 1990; Tenhaken et al., 1995). The HR can be rec- ognized as the rapid and localized death of cells in response to an avirulent pathogen; it has been observed during most interactions involving race-specific resistance and also in many examples of non-host resistance (Heath, 1989; Mansfield, 1990; Mansfield et al., 1997). A second form of resistance, more commonly found in non-host reactions, is the highly localized alteration of the cell wall at sites attacked by fungi or bacteria. Modification of the cell wall per se is often associated with the formation of a papilla or apposition at reaction sites (Ride, 1986; Nicholson and Hammerschmidt, 1992; Bestwick et al., 1995). In phytopathogenic bacteria, hypersensitive response and pathogenicity (hrp) genes determine the ability to multiply within susceptible plants and to cause

Current address: Division of Biochemical Sciences, Rowett Re- search Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK. To whom correspondence should be addressed.

a macroscopic HR in either resistant varieties of their host or in non-host plants (Bonas, 1994). Hrp mutants, although failing to cause the HR, do cause defense responses in plants, including the alteration of adjacent cell walls and papilla formation (Jakobek and Lindgren, 1993; Bestwick et al., 1995; Brown et al., 1995; Fett and Jones, 1995).

The term active oxygen species (AOS; also referred to as reactive oxygen species or reactive oxygen intermediates) describes radicals and other nonradical but reactive species derived from oxygen. The rapid production and accumulation of AOS, primarily the superoxide anion (O,. -) and hydrogen peroxide (H202), have been shown to occur in several plant-microbe interactions (for recent reviews, see Mehdy, 1994; Baker and Orlandi, 1995; Low and Merida, 1996). In aqueous solutions, O,' ~ exists in acid base equilib- rium with the protonated form, the hydroperoxyl radical HO,. (Sutherland, 1991). Both 0,'- and HO,. species undergo dismutation to yield H,O2. Increased AOS production may exert a toxic effect within cells via lipid peroxidation, protein degradationhodification, and DNA damage (re- viewed in Fridovich, 1986; Wolff et al., 1986; lmlay and Linn, 1988). In a number of studies, researchers have detected increased lipid peroxidation during the initial stages of the HR

21 O The Plant Cell

(Adám et al., 1989, 1995; Croft et al., 1990; May et al., 1996). Despite the temporal association of 0,. - production with lipid peroxidation, the superoxide anion, unlike Hop., is not capable by itself of performing H abstraction, and because it is charged, it is also unable to penetrate the hydrophobic in- terior of membranes (Halliwell and Gutteridge, 1989). H202 has a similarly low reactivity toward biological molecules. But in the presence of trace quantities of metal ions, H202 may participate in both the Fenton and Haber-Weiss reactions to form the highly reactive hydroxyl radical (HO’). For- mation of HO’ probably accounts for much of the in vivo toxicity associated with excessive H202 production. H,O, is relatively stable, can diffuse freely throughout the cell, and hence may be able to participate in HO’ formation at sites distant from its production (Baker and Orlandi, 1995).

Various enzyme systems have been proposed to generate AOS in plants. These include a neutrophil-analogous, membrane-bound NADPH oxidase (Doke, 1983; Levine et al., 1994; Auh and Murphy, 1995; Doke and Miura, 1995; Dwyer et al., 1996), lipoxygenase (Croft et al., 1990), and apoplastic peroxidases (Vera-Estrela et al., 1992; Bolwell et al., 1995). Adam et al. (1 995) have suggested that H202 accumulation may occur by a reduction in H202 scavenging and an increase in H,OZ production involving coordinated changes in peroxidase, superoxide dismutase, and catalase activities.

In many plants, treatment with inhibitors of protein synthesis has been found to prevent or delay the HR, implying a requirement for active metabolism within the plant cell leading to cell collapse (Woods et al., 1989; He et al., 1993). The occurrence of lesion mimics (notably in Arabidopsis), which rapidly form HR-like lesions in the absence of an avirulent pathogen, suggests that the HR is under genetic control. The plant’s response may therefore represent a form of programmed cell death (pcd; Dietrich et al., 1994; Greenberg et al., 1994). Levine et al. (1994) have suggested that H202 may initiate pcd events leading to the HR.

The most common form of pcd in animal cells is mani- fested in apoptosis. This descriptive term is used to define cells that demonstrate particular morphological and biochemical features associated with their death. Typical ultrastructural features of apoptosis include chromatin condensation, cell surface blebbing, chromatin migration to the nuclear mar- gins, nuclear fragmentation, and formation of several membrane-bound apoptotic bodies (Collins et al., 1992; Buja et al., 1993; Kerr et al., 1994). Biochemically, increased pro- tease and endonuclease activities and internucleosomal DNA cleavage have been reported (Arends and Wyllie, 1991; Martin et al., 1994). Some of these features have been associated with developmental and pathological cell death in plants (Mittler and Lam, 1995; Levine et al., 1996; Ryerson and Heath, 1996; Wang et al., 1996), but the HR often lacks the classic ultrastructural features associated with apoptosis (Bestwick et al., 1995; Mittler and Lam, 1996). AOS have been shown to induce apoptosis in mammalian cells, but the involvement of AOS in the execution of apoptotic cell death is controversial (Buttke and Sandstrom, 1995; Jacobson, 1996).

In plants, H,Op production is also involved in the defen- sive modifications that occur within the walls of challenged cells (Bolwell, 1993; Wojtaszek et al., 1995). In some cases, alteration to cell wall structure may be an extremely rapid response. Bradley et al. (1992) identified a rapid H,O,-dependent insolubilization of a proline-rich protein and putative hydroxyproline-rich glycoprotein (HRGP) within 2 min of application of an elicitor to soybean cell suspensions. HRGPs have been shown to accumulate at sites of bacterial attachment to plant cell walls and also within the papillae that form adjacent to bacteria or sites of attempted fungal penetration (O’Connell et al., 1990; Bestwick et al., 1995; Brown et al., 1995). The accumulation of lignin or ligninlike materials has been observed in numerous incompatible interactions (Ride, 1986; Nicholson and Hammerschmidt, 1992). Polymeriza- tion of hydroxycinnamyl alcohols in the final stages of lignin biosynthesis and the intramolecular and intermolecular cross-linking of HRGPs are both catalyzed by isoperoxidase activities. In the presence of H202, peroxidase is also thought to mediate cross-linking between numerous other cell wall components (liyama et al., 1994). The detection of cross-linking within tissues is complicated by the highly localized nature of reactions that occur at the subcellular level in plant cells (Bestwick et al., 1995; Brown et al., 1995). Al- terations to wall structure involving H20p may restrict diffu- sion of host-derived nutrients to the pathogen and pathogen-derived toxins to the host and alter the availability of wall linkages to cell wall-degrading enzymes produced by some potential pathogens (Friend, 1981; Brisson et al., 1994). The production of H202 may also lead to the development of an antimicrobial environment within the apoplasm (Peng and Kuc, 1992).

In addition to its proposed roles as (1) a direct cause of membrane damage, (2) a component of structural defense, (3) a signal molecule operating within the challenged cell, and (4) an antimicrobial compound, H202 has also been hy- pothesized to act as a signal molecule in the induction of systemic acquired resistance (Chen et al., 1993). There is, however, very little information on the precise localization of H202 production or accumulation during plant-pathogen interactions. Direct attempts to localize AOS by light microscopy have included the use of nitro blue tetrazolium salt to detect 0,. ~ (Adám et al., 1989), potassium iodidektarch to detect H202 (Olson and Varner, 1993), and compounds that either fluoresce or have their fluorescence quenched when oxidized by H,Op (Yahraus et al., 1995). Detection of H,02 as a product of enzyme catalysis has formed the basis of the localization at the subcellular level of enzyme activities that generate the AOS in animals and plants, for example, NADH oxidase and glycolate oxidase (Briggs et al., 1975; Kausch et al., 1983; Kausch, 1987). The histochemical as- says used are based on the reaction of HP02 with CeCI, to produce electron-dense insoluble precipitates of cerium perhydroxides, Ce[OH],OOH and Ce[OH],OOH. This ultrastructural technique allows the precise localization of sites of H202 accumulation or production and has been used by

H,Op Localization and the HR 21 1

Czaninski et al. (1993) in a study of endogenous H,Op production in lignifying tissues.

Here, we report use of the CeCI, procedure to localize H,Op within lettuce cells challenged by wild-type and hrpD mutant strains of Pseudomonas syringae pv phaseolicola. Because both strains cause localized cell wall alterations, including HRGP and phenolic deposition, but only the wild type causes the HR (Bestwick et al., 1995), responses specifically associated with the HR were determined. Our experiments reveal the highly localized accumulation of H202 occurring within regions of the cell wall adjacent to wild-type bacteria during the early stages of the HR. Results obtained support the proposals that H202 may have an antibacterial role and also may be a primary cause of membrane damage in plant cells undergoing the HR during non-host resistance.

RESULTS

Localization of H202 in Uninoculated Tissue

In noninoculated leaves or tissue away from bacterial colo- nies, precipitates of electron-dense cerium perhydroxides, indicating the presence H202, were located predominantly within the cell walls of xylem vessels with secondary thick-

ening and occasionally within the walls of surrounding cells (Figure 1A). Junctions between mesophyll cells, typically the sites of bacterial attachment to the plant cell wall, were usu- ally free of staining (Figure 1 B).

Localized Accumulation of H202 Is Detected at Reaction Sites

After injection, as inoculum droplets evaporated and were absorbed, bacterial cells were drawn into contact with plant cell walls. Wild-type and hrp mutant bacteria caused similar responses within the first 5 hr of inoculation. The plasma membrane adjacent to bacteria became convoluted, and amorphous material, initiating papilla formation, was depos- ited between the plant plasma membrane and cell wall. After 5 hr, deposition of HRGPs and phenolics was already observed in the altered cell walls adjacent to attached bacteria. Papillae continued to expand at sites inoculated with the hrp mutant, but vesiculation of the cytoplasm and cytoplasmic disorganization, indicative of the HR, occurred extensively only in plant cells next to wild-type bacteria, as reported in detail by Bestwick et al. (1995).

Preliminary observations, after CeCI, staining of samples 5 and 8 hr after inoculation, revealed H202 accumulation at reaction sites. Formation of the cerium perhydroxides was

Figure 1. Detection of H,Op in Uninoculated Tissue.

(A) Staining with CeCI, to form electron-dense deposits of cerium perhydroxides localized to spiral thickening of the xylem vessels and parts of the associated cell wall (arrows). Bar = 5 pm. (B) Absence of staining from a typical junction between two spongy mesophyll cells. Bar = 1 pm. c, chloroplast: CV, central vacuole; IS, intercellular space; XV, xylem vessel.

Figure 2. Localization of H2O2 Accumulation in Cells Adjacent to Wild-Type f . s. phaseokola.

(A) Electron-dense deposits of cerium perhydroxides extending from the site of attachment of wild-type bacteria (large arrow) into the surrounding plant cell wall (small arrows). The small papillae formed were also intensely stained 8 hr after inoculation. Note that the cytoplasm of responding cells is already vesiculated near the bacteria but that the nucleus (N), close to the reaction site, shows no signs of chromatin condensation or nuclear fragmentation (i.e., apoptotic features). Bar = 2 pm. (B) Restriction of H202 to one of a pair of adjacent mesophyll cells 5 hr after inoculation. Note that the one shared wall of two cells is intensely stained but that staining only extends into the individual wall of one cell (arrow). Bar = 1 pm. B, bacterium; c, chloroplast; CV, central vacuole; IS, intercellular space; m, mitochondrion; N, nucleus; V, vesiculation.

H,Op Localization and the HR 213

particularly striking in the plant cell walls adjacent to wild- type bacteria and was remarkably localized. The most dense deposits were found next to bacteria, and they extended with reducing intensity into the surrounding walls (Figure 2A). Staining was also frequently observed in material en- capsulating the bacteria. At some sites, although bacterial cells were lodged between two mesophyll cells, staining with CeCI, was detected predominantly in one cell, indicating differential activation of the oxidative burst (Figure 2B).

Quantitative Analysis Reveals a Prolonged Burst of H202 during the HR

To assess H202 accumulation objectively, we developed a four-point scale to assess the appearance of deposits of cerium perhydroxides within the plant cell wall as follows: O, none; 1, faint and patchy; 2, dense but patchy; 3, dense and confluent deposits. The minimum degree of deposit assigned to the categories is illustrated in Figures 3A to 3D. Figure 3 also provides additional data on the localization of deposits within responding cells.

The accumulation and persistence of H202 were then quantified at different times after inoculation, following as- signment of a total of at least 30 reaction sites to categories. The results presented in Table 1 clearly show that H202 accumulation was an early response observed in the interaction with wild-type bacteria that caused the HR. A prolonged burst of H202 accumulation was apparent between 5 and 8 hr after inoculation with the wild-type strain. Analysis of data for the hrpD mutant and wild-type bacteria 5 and 8 hr after inoculation, using xz analysis of contingency tables prepared by grouping staining into low (categories O or 1) and high (categories 2 and 3) classes, revealed statistically significant differences between the strains at P < 0.001. The strong staining of a low percentage of cells challenged by the hrp mutant (after 8 and 16 hr) corresponded to the low numbers of cells found to undergo cytoplasmic collapse in this interaction (Bestwick et al., 1995).

The possibility that wild-type bacteria might produce H202 in the plant was discounted by experiments on the induction time for the HR. lnduction time, which was first demon- strated by Klement and Goodman (1967), defines the time during which protein synthesis must be performed by bacteria in the plant to induce a macroscopic HR. In the lettuce- P.s. phaseolicola interaction, the induction time is 2 to 2.5 hr, after which the prevention of bacterial protein synthesis does not stop the HR. Results presented in Table 2 show that when bacteria were inactivated by streptomycin injection, H,Op continued to accumulate only if the induction time had passed, that is, if the HR continued to develop. The production of H202 was therefore shown to be a plant response.

In an attempt to determine the concentrations of H202 required to caúse the staining observed in the cell wall, we injected noninoculated leaves with solutions of H202 ranging

from 1 FM to 3 M and immediately processed samples with CeCI,. Faint staining (equivalent to category 1) was observed after injection with 100 pM H,O,, and category 2 staining was frequently observed with concentrations in excess of 100 mM. However, dense, category 3 staining was observed only with concentrations of or in excess of 1 M H202. It was necessary to process tissues within 5 min after the application of exogenous H202, because the ability to detect cerium perhydroxide formation in the cell wall rapidly declined after this time. Tissues injected with H202 were also examined by electron microscopy after 12 hr, and the number of collapsed cells was estimated. Treatment with 0.1 M H202 had caused cytoplasmic collapse in -0 to 10% of the mesophyll cells; 1 and 3 M H202 caused 25 to 30% and 80 to 90% cell collapse, respectively.

Papilla Formation 1s Not Associated with Major Accumulation of H20,

As previously reported (Bestwick et al., 1995), papilla development occurred in response to both wild-type and hrp mutant strains. Initially, papilla development was slightly more rapid in response to the wild-type bacterium; however, by 16 hr after inoculation, large papillae were more frequently observed in cells next to the hrp mutant. High levels of H2O2, indicated by either dense but patchy or confluent cerium perhydroxide formation, were detected within small papillae produced in response to the wild-type strain during the early stages of the interaction (Figure 4A). In more developed papillae (>0.3 pm thick), observed in the first 8 hr after inoculation, staining was often most intense in the outer layers of paramural deposits (Figure 4B). By 12 hr after inoculation, 50% of the lettuce cells next to wild-type bacteria had collapsed, and after 24 hr, the figure had increased to 85% (Bestwick et al., 1995). During the later stages of cell collapse (after 16 and 24 hr), staining was either absent from or located at the edge of papillae (Figure 4C). Although papillae adjacent to the hrpD mutant appeared morphologically identical to those formed after challenge with wild-type bacteria throughout the time course, as shown in Figure 4D, they typically lacked dense deposits of cerium perhydroxides after staining with CeCI,.

Effects of lnhibitors on H202 Accumulation

Detecting deposits of cerium perhydroxides at reaction sites raised severa1 questions. For example, how specific was CeCI, staining for H,02, and was the accumulation of H,02 generated by peroxidase or NADPH oxidase activities? In attempts to answer these questions, CeCI, staining was examined in the presence of catalase (to decompose H202), aminotriazole (ATZ; to inhibit catalase in the plant), potassium cyanide and sodium azide to inhibit peroxidases, and diphenylene iodonium chloride (DPI) to inhibit neutrophil-like

21 4 The Plant Cell

Figure 3. Minimum Intensity of Staining Used to Quantify H202 Accumulation in the Cell Wall as Detected by the Formation of Cerium Perhy- droxides at Reaction Sites.

(A) No staining. (B) Category 1 : faint and patchy staining. (C) Category 2: dense but patchy staining. . . (D) Category 3: dense and confluent staining. (A) and (B) show tissue 16 and 8 hr after inoculation with the hrpD mutant, respectively; (C) and (D) show tissue 8 and 5 hr after inoculation with wild-type P. s. phaseolicola, respectively. B, bacterium; CV, central vacuole; IS, intercellular space. Bars = 0.5 pm.

HzOp Localization and the HR 215

Table 1. Quantitative Assessment of Staining with CeCI, to Reveal H,Oz Accumulation within the Cell Wall Adjacent to Bacteria

Strain and Time after Inoculation

Percentage of Sites in Each Category of Staininga

O 1 2 3 0 1 2 3

(hr) (Without Aminotriazole) (With Aminotriazole)

Wild type 3 70 20 10 O 70 30 O O 5 10 10 2 78 7 12 12 69 8 40 10 5 45 30 3 14 53

1 6b 66 18 16 O 19 50 31 O

hrpD mutant 3 80 20 O O 83 14 3 O 5 52 24 8 8 63 19 3 16 8 58 14 14 14 64 6 19 11

16 77 3 17 3 80 9 O 11

=At least 30 sites were examined at each time from three replicate leaf samples. The minimum degrees of deposit assigned to each category of staining are as follows: O, none; 1, faint and patchy; 2, dense but patchy; and 3, confluent and dense staining. For illustra- tions, see Figure 3.

By 16 hr, most cells had collapsed.

NADPH oxidase (Levine et al., 1994; Bolwell et al., 1995; Murphy and Auh, 1996). Sites were assessed for CeCI, staining, and deposits were quantified. These data are sum- marized in Tables 1 and 2.

The specificity of CeCI, staining for H202 was demon- strated by the striking reduction observed after treatment with catalase. By contrast, ATZ did not cause major changes; however, at the later time points during the HR, increased staining in categories 1 and 2 was detected. Treat- ment with cyanide and azide effectively prevented intense staining, whereas DPI caused a less dramatic but also statistically significant reduction (Table 2).

DISCUSSION

Increases in cerium perhydroxide production were clearly localized to sites of bacterial attachment to lettuce cell walls. The inhibition of perhydroxide formation by exogenous catalase confirms that H202 was the species being detected. In plant cells, catalase is principally located in peroxisomes, although cytoplasmic forms have been reported. The catalase inhibitor ATZ rapidly diffuses into cells, and its failure to cause increased H202 accumulation in the cytoplasm or peroxisomes demonstrates that reduced catalase activity does not necessarily lead to a corresponding increase in HzO, concentration. A similar conclusion has been reached in a number of studies (Ferguson and Dunning, 1986; Volk and

Feierabend, 1989; Amory et al., 1992). Amory et al. (1992), examining the failure of ATZ to induce H202 accumulation in pea seedlings, considered that any potential increase in detectable H202 must be prevented by its use in oxidative- reductive processes or scavenging by alternative antioxi- dant systems. The minor increase in H202 detection within lettuce tissues treated with ATZ 16 hr after inoculation, by which time cells had collapsed during the HR, suggests that H202 may be scavenged finally by catalase released from degrading peroxisomes.

H202 accumulation was localized to sites of wall alterations and papilla formation adjacent to attached bacteria. As such, it may be argued that H202 is produced in response to a requirement for peroxidase-mediated cross-linking reactions in the cell wall and papilla, both of which accumulate peroxidase substrates, such as HRGPs and phenolic compounds (Bestwick et al., 1995). However, by 16 hr after inoculation, although papilla formation was far more prevalent in the interaction with the hrp mutant, H202 accumulation was much less than observed in response to the wild-type strain. Is it possible, therefore, that papillae formed in response to the hrp mutant are less cross-linked? Given the identical appearance of papillae developing in response to Hrp- or Hrp+ bacteria and their similar impregnation with HRGPs and phenolic compounds (Bestwick et al., 1995), it appears that the same potential for cross-linking exists in both interactions.

The H,O,-dependent insolubilization of preexisting cell wall proteins has been reported in bean and soybean cells challenged with fungal elicitors and bacteria (Bradley et al., 1992; Brisson et al., 1994; Wojtaszek et al., 1995). In response to elicitors, such insolubilization is extremely rapid (Bradley et al., 1992). However, in response to avirulent bacteria, cross- linking is apparently much slower, occurring during the initial stages of visible hypersensitive cell collapse in soybean, some 24 hr after inoculation (Brisson et al., 1994). In soybean, as with other plant-pathogen interactions, the production of H202 in response to both elicitors and bacteria is a relatively rapid response (Levine et al., 1994; Baker and Orlandi, 1995). Increased AOS production is therefore not necessarily correlated with immediately detectable oxidative cross- linking. In the context of wall alterations, it will be interesting to localize sites of peroxidase activity relative to H,O, production and/or accumulation during these interactions.

We suggest that CeCI, is principally able to react with excess H,O, that is not being rapidly metabolized during cross-linking reactions and that this excess H202 is a primary factor in the development of membrane damage. Pre- viously, we were unable to follow the sequence of collapse observed in a few cells challenged with the hrp mutant, but now it appears that there is a correlation between the number of cells collapsing (-10%; Bestwick et al., 1995) and the number of sites associated with high levels of H202.

Classically, two bursts of AOS production can be observed during incompatible plant-pathogen interactions. The first burst appears to be nonspecific, being produced in response to numerous elicitors, wounding, avirulent and

Figure 4. Variable Staining within Papillae and Collapsing Cells.

(A) Dense cerium perhydroxide deposits obscure papillae (asterisks) produced 8 hr after inoculation with the wild-type strain. Note that both the cell wall and paramural deposit are stained. Bar = 0.5 pm. (B) Staining of a large papilla produced 8 hr after inoculation with wild-type bacteria, showing the presence of H202 (arrow) at the papilla-plant cell membrane interface. Bar = 0.5 pm. (C) Sites of positive staining for H202 (arrows) dispersed within a cell undergoing cytoplasmic collapse during the late stages of the HR in response to wild-type bacteria, 16 hr after inoculation. Note that the cytoplasmic faces of the large papillae are associated with cerium perhydroxides. Bar = 1 pm. (D) Lack of staining in papillae observed 16 hr after inoculation with the hrpD mutant. Note that neither the cell wall nor paramural deposits are stained. Bar = 1 pm. 6, bacterium; CV, central vacuole; IS, intercellular space; m, mitochondrion; asterisk, papilla deposit.

H202 Localization and the HR 217

virulent pathogens, and hrp mutants (Doke, 1983; Aposto1 et al., 1989; Baker and Orlandi, 1995). The second burst was thought to be HR specific and a requirement for HR development; however, Glazener et al. (1996) have reported that hrmA mutants o f f . s. syringae, while failing to cause the HR in tobacco, do cause a biphasic oxidative burst. Further- more, Baker and Orlandi (1995) have pointed out that the levels of exogenous HzOz required to initiate plant cell death are far in excess of those observed in the HR. The conflict- ing evidence for a causal role for HzO2 in the HR may be partly reconciled in view of the highly localized accumulation of HzO2 detected in our work.

In noninoculated leaves, high concentrations of H202 are required to induce levels of staining similar to those observed during the HR. It is, however, difficult to define the in situ concentration of HZOz at sites of bacterial attachment. The detection of H202 will be influenced by various antioxi- dant mechanisms, and it is unlikely that the activity of such systems will remain unchanged throughout the development of the HR (Croft et al., 1990; Adam et al., 1995). Neverthe- less, the concentrations of H,Op required to induce cell collapse are comparable to those required to mimic the degree of cerium perhydroxide deposition observed during the HR. At reaction sites, concentrations of H2O2 may be sufficient to initiate toxic effects on the plasma membrane, leading to the distinctive vesiculation observed in ultrastructural studies (Bestwick et al., 1995). The availability and release of “cata- lytic” Fez+ from storage or transport proteins or iron-containing enzymes may be important in regulating HzO2 toxicity to plant cells. A decrease in intracellular pH, providing conditions that are favorable for Fez+ release, has been reported during the HR (Baker and Orlandi, 1995) and may therefore enhance conversion of H202 to the active hydroxyl radical.

In lettuce cells, locally high levels of H,0, are maintained during the first 8 hr after inoculation in tissues undergoing the HR. During this period, cell viability was reduced to 50%, electrolyte leakage increased, and the first signs of cell decompartmentalization were detected (Bestwick et al., 1995). Oxidative stress is therefore probably maintained during the critical phases of membrane damage in lettuce. The maintenance of AOS production may be a more important factor in cell collapse than is a transient biphasic burst.

Variation in the level of H202 within individual cells pro- foundly affects the mechanism and type of cell death observed in mammalian cell types. Severe oxidative stress has been shown to induce necrosis directly, whereas lower levels of AOS induce apoptosis/pcd or even cell proliferation (Dypbukt et al., 1994). We suggest that during the HR, a specifically increased production of HzO2 may be viewed as part of a suicidal response, regardless of whether ultimate cell death is via a “toxic shock,” that is, caused directly by excessive production of H2O2 or indirectly through activation of pcd. It is also possible that HzO, released into the apoplasm is directly toxic to the pathogen.

The source of H2O2 production is unclear, but its direc- tional accumulation in the cell wall and general absence

Table 2. Effects of Inhibitory Treatments on the Accumulation of H202 in the Cell Wall Adjacent to Wild-Type Bacteria 5 Hr after Inoculation

Percentage of Sites in Each Category of Staininga

Treatment o 1 2 3

None

Affecting bacteriab Streptomycin before induction

Streptomycin after induction

Heat-killed bacteria

timeC

timeC

Affecting the plantb Catalase KCN 1 mM KCN 3 mM Azide 1 mM Diphenylene iodonium chloride

8 PM

10 10 8 72

67 33 O O

8 O 8 8 4

82 18 O O

75 25 O O 41 16 8 5 67 33 O O 9 2 8 0 0 40 20 40 O

aCategories are as described in Table 1 and as illustrated in Figure 3. Statistical analysis using x2 contingency tests showed that all treatments, except for streptomycin after the induction time, significantly reduced the frequency of sites in categories 2 and 3 com- pared with O and 1 combined (P < 0.001). Major effects that were expected on either the bacteria or plant. Injection was with streptomycin before or after completion of the

induction time at 0.5 and 3 hr after inoculation, respectively.

from the cytoplasm suggest that the principal site is on the extracellular surface of the plasma membrane or within the apoplasm. The lack of staining in the cytoplasm does not re- flect an inability of CeCI, to enter plant cells. In both inoculated and noninoculated lettuce leaves, staining of the tonoplast was occasionally encountered (data not shown), demonstrating the potential penetration of CeCI, throughout the cytoplasm. Under similar incubation conditions, Kausch et al. (1 983) have reported deposition of cerium perhydroxides in peroxisomes of root parenchyma cells.

Studies of the HR in soybean and potato have suggested that the biphasic oxidative burst is the result of a membrane-bound neutrophil-like NADPH oxidase generating Oz. -, which rapidly dismutes to HzO2 (Doke, 1983; Levine et al., 1994; Doke and Miura, 1995; Dwyer et al., 1996). An elicitor-induced oxidative burst in rose cells has also been correlated with NADPH and NADH oxidase activities (Auh and Murphy, 1995; Murphy and Auh, 1996). In all of these studies, the production of AOS was sensitive to inhibition by DPI, a potent inhibitor of neutrophil NADPH oxidase.

Although DPI inhibited H,02 production in lettuce, the peroxidase inhibitors cyanide and azide were much more effective. Because neither azide nor cyanide inhibits neutrophil-like NADPH oxidase activity (Bolwell et al., 1995;

218 The Plant Cell

Murphy and Auh, 1996), there appears to be a fundamen- tally different mechanism of H202 generation in lettuce from that suggested to operate in potato, rose, and soybean cells (Levine et al., 1994; Auh and Murphy, 1995; Doke and Miura, 1995; Murphy and Auh, 1996). It should be noted that DPI is also known to inhibit the activities of heme proteins, such as peroxidases (Deme et al., 1994; Dwyer et al., 1996). Peroxi- dases can generate H202 via H,O,-independent oxidation of a number of substrates, for example, NADH, NADPH, thiols, and certain phenols (Halliwell, 1978; Mader and Amberg- Fisher, 1982; Vianello and Macri, 1991; Pichorner et al., 1992; Bolwell et al., 1995). In bean cells, Bolwell et al. (1995) have presented evidence for the involvement of an apoplastic peroxidase activity in elicitor-induced H202 production that was insensitive to DPI but inhibited by cyanide. The involvement of peroxidase and components of the cell wall in the generation of AOS during the HR in lettuce merits further investigation. Perhaps significantly, protoplasts (from carrot) have been shown to be unable to generate an elicitor-induced oxidative burst (Bach et al., 1993).

The localized accumulation of H202 is consistent with the existence of a physically close link between the putative receptor controlling recognition of the challenging bacterium and activation of AOS production. Confirmation of the molecular organization of the signaling cascade leading to AOS production will require the identification of both the bacterial ligand and the plant’s receptor, which are both effective in generating the non-host HR in lettuce.

50 mM 3-(N-morpholino)propanesulfonic acid (Mops) at pH 7.2 for 1 hr. Tissues were then fixed in 1.25% (v/v) glutaraldehyde/l.25% (v/v) paraformaldehyde in 50 mM sodium cacodylate (CAB) buffer, pH 7.2, for 1 hr. After fixation, tissues were washed twice for 10 min in CAB buffer and postfixed for 45 min in 1 % (v/v) osmium tetroxide in CAB. Tissues were then washed again in CAB buffer (twice for 10 min) and dehydrated in a graded ethanol series (30, 50, 70, 80, and 90% [v/v] ethanol; 15 min each change and then three changes of 100% ethanol of 20 min each in duration). Samples were transferred to two changes of propylene oxide of 20 min each in duration and progressively embedded in Eponaraldite (Agar Aids, Bishop’s Stortford, UK). Alternatively, tissues were dehydrated in a graded acetone series, transferred directly into acetone-resin mixtures, and progressively embedded. Tissues were then subjected to 12 hr in pure resin, fol- lowed by a change of fresh resin for 4 hr, and placed in blocks and polymerized at 60°C for 48 hr. Blocks were sectioned (70 to 90 nm) on a Reichert-Ultracut E microtome (Milton Keynes, UK), using a di- amond knife (Diatome, Bienne, Switzerland), and mounted on un- coated copper grids (300 mesh). Sections were examined using a transmission electron microscope (model 7000; Hitachi, Tokyo, Ja- pan) at an accelerating voltage of 75 kV.

Treatment with Inhibitors

Inoculated and noninoculated tissue segments were excised and incubated for 30 min in 50 mM Mops, pH 7.2, containing either 1 or 3 mM potassium cyanide, 1 mM sodium azide, 8 pM diphenylene iodonium chloride (DPI), 25 pg mL-l bovine liver catalase, or 20 pM 3-amino-I ,2,4-triazole (ATZ). Control tissues were incubated in Mops devoid of further additions. Tissues were then transferred to CeCI, solutions supplemented with inhibitors at the stated concentrations, incubated for 1 hr, and processed for transmission electron microscopy as described above.

METHODS

Effect of Bacterial Viability on H202 Production Reagents

Unless stated otherwise, all reagents used were analytical or equivalent grade and supplied by BDH (Poole, Dorset, UK) or Sigma (Poole, Dorset, UK).

Maintenance of Plants and Bacteria

Lettuce plants (Lactuca sativa cv Diana) were grown from seed and inoculated by injection with suspensions of bacteria (1 O8 colony- forming units mL-l), as described by Bestwick et al. (1995). The strains of Pseudomonas syringae pv phaseolicola used were the wild-type race 6 strain S21-WT and the hrpD mutant S21-533 (Nollenburg et al., 1990).

Transmission Electron Microscopy

Samples were fixed, embedded, sectioned, and stained for conven- tional electron microscopy, as described by Bestwick et al. (1995). The histochemical method developed for localization of hydrogen peroxide (H202), based on the generation of cerium perhydroxides, was as follows. Tissue pieces (4 to 2 mm2) were excised from inoculated leaf panels and incubated in freshly prepared 5 mM CeCI, in

The induction time for confluent expression of the hypersensitive reaction (HR) was determined by injecting aqueous solutions of the an- tibiotic streptomycin sulfate (200 pg mL-’) at 30-min intervals into leaf panels previously inoculated with bacteria, and the effect on confluent expression of the HR and H202 production was observed.

ACKNOWLEDGMENTS

This work was supported by a grant from the United Kingdom Agri- cultural and Food Research Council.

Received September 16, 1996; accepted December 13, 1996.

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DOI 10.1105/tpc.9.2.209 1997;9;209-221Plant Cell

C S Bestwick, I R Brown, M H Bennett and J W Mansfieldcells to Pseudomonas syringae pv phaseolicola.

Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce

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