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Accumulation of a Basic Peroxidase Isoenzyme in Leaves of Arabidopsis thaliana Following Inoculation with Pseudomonas syringae pv. syringae or Wounding.


Pathogens inflict billions of dollars of damage on crop plants every year. However, despite the importance of plant diseases, the mechanisms by which plants suppress the growth of potential pathogens are not well understood. One way plants may defend themselves from disease is by accumulating peroxidase. We found that inoculating leaves of Arabidopsis thaliana with Pseudomonas syringae pv. syringae resulted in an increase in soluble peroxidase activity. This increase in activity was localized to the site of inoculation and was associated with the accumulation of a basic peroxidase isoenzyme (p19.7). This peroxidase isoenzyme also accumulated in leaves following wounding and was observed in extracts prepared from stems, flowers, siliques, and roots. These results suggest that the basic Arabidopsis peroxidase isoenzyme may play a role in disease resistance and in growth and development.


Peroxidases (EC catalyze the oxidation of organic compounds in the presence of hydrogen peroxide. Although many studies have been conducted on plant peroxidases, the precise functions of the enzyme remain uncertain. In plants, peroxidases are believed to help strengthen the cell wall by producing lignin (Grisebach 1981) and by cross-linking phenolic residues of cell wall polysaccharides and glycoproteins (Fry 1986). Peroxidases are also thought to be involved in the metabolism of auxin (Ludwig-Muller and Hilgenberg 1992). Furthermore, peroxidases are believed to play a role in disease resistance by strengthening the cell wall (Bradley et al. 1992) and by generating antimicrobial radicals (Peng and Kuc 1992). Studies of the functions of peroxidase have been complicated by the occurrence of multiple peroxidase isoenzymes (Hoyle 1977). These isoenzymes can be distinguished by their isoelectric points (pI) and are thought to perform different functions in different tissues at different times in plant d evelopment.

Because of the wealth of molecular information known about Arabidopsis thaliana and because Arabidopsis produces fewer peroxidase isoenzymes than other crucifers (Hoyle 1977), several molecular studies of peroxidase have been performed with this plant. In Arabidopsis, several full-length peroxidase clones (prxCa, prxCb, prxEa, atp A2, atp la, atp 2a) have been isolated and characterized (Intapruk et al. 1991; Intapruk et al. 1993; Intapruk et al. 1994a; Intapruk et al. 1994b; Kjaersgard et al. 1997; [phi]stergaard et al. 1996). All of these clones share a high degree of sequence identity with other plant peroxidase clones. In addition to these full-length Arabidopsis peroxidase cDNA and genomic clones, numerous partial cDNA clones have been isolated (Newman et al. 1994).

A number of agents have been reported to increase peroxidase activity or to induce the accumulation of peroxidase mRNA in Arabidopsis. Davis and Ausubel(1989) observed that suspension-cultured cells of Arabidopsis treated with alpha-1 ,4-endopolygalacturonic acid lyase accumulated mRNA of a number of enzymes involved in phenylpropanoid biosynthesis and also peroxidase. Similarly, Richards et al. (1998) and Sharma and Davis (1994) reported that exposure of Arabidopsis to high amounts of aluminum and ozone can induce the expression of a number of stress-related genes including peroxidase. In addition, Lummerzheim et al. (1993,1995) reported that foliar applications of lead nitrate and inoculations with Xanthomonas campestris pv. campestris can stimulate peroxidase activity and elicit the accumulation of peroxidase mRNA. Furthermore, while studying systemic acquired resistance in Arabidopsis, Summermater et al. (1995) observed that peroxidase activity increased in leaves following inoculation with Pseudnmonas s yringae pv. syringae. In this investigation, we further characterized the peroxidase induced by inoculation with P. s. syringae.


Plant and Bacterial Growth Conditions

Arabidopsis were grown in 12.7 cm plastic pots in a soil mixture consisting of equal parts of peat and perlite covered with 1-2 cm of fine vermiculite. Seeds of Arabidopsis ecotype Columbia, obtained from the Arabidopsis Biological Resource Center at the Ohio State University, were scattered on top of the fine vermiculate. The pots were covered with plastic wrap and placed into 55.8 x 27.9 x 7.0 cm plastic trays containing tap water. The plastic wrap was removed following germination approximately one week after planting. Plants were subirrigated continuously and were grown under continuous illumination (3500 lux) using four cool-white fluorescent bulbs at 23-27[degrees]C.

Pseudomonas syringae pv. syringae PSSD20 van Hall (Smith et al. 1991) was obtained from Dr. R. Hammerschmidt at Michigan State University. Bacteria were cultured at 25[degrees]C on King's B agar (Schaad 1988) or in King's B broth on a rotary shaker (90 revolutions [min.sup.-1]).

Inoculations and Wounding

Inoculum was prepared by diluting overnight liquid cultures of P. s. syringae in sterile 10mM Mg[Cl.sub.2] (Whalen et al. 1991). The inoculum was adjusted to the desired concentration by measuring the absorbance of the bacterial suspension at 600 nm. Leaves of approximately 5-week-old Arabidopsis plants were infiltrated with a 1 x [10.sup.8] cfu [ml.sup.-1] suspension of P. s. syringae using a needleless syringe (Tsuji et al. 1992). For wounding experiments, leaves were crushed between the serrated ends of a 25.4 cm long metal forceps. Leaves were then harvested at daily intervals and stored at --20[degrees]C.

Peroxidase Assays

Extracts were prepared by homogenizing tissues in 4[degrees]C 100 mM sodium phosphate buffer, pH 6.5 using a pre-cooled mortar and pestle. The homogenate was then centrifuged for 5 mm at 12,000 x g, and the supernatant was used as a crude enzyme preparation. Peroxidase activity was determined colorimetrically using guaiacol as a substrate (Hammerschmidt et al. 1982). Protein concentrations were determined by the method of Bradford (1976) using chicken egg albumin as a standard.

Localization of Peroxidase

Two and four days postinoculation of Arabidopsis leaves with a 1 x [10.sup.8] cfu [ml.sup.-1] suspension of P.s. syringae, inoculated areas undergoing the hypersensitive response were separated from healthy uninoculated areas of the same leaf, using a razor blade. Extracts were separately prepared from the inoculated and uninoculated tissues of the same leaf and assayed for peroxidase activity as described above. Leaves of uninoculated plants were used as controls.

Inoculated leaves of Arabidopsis were also detached at the base of the petiole and placed in glass petri dishes containing 0.48 mM 4-chloro-1-napthol, 50 mM Tris, 0.2 M NaCl, 17% MeOH, and 0.01% [H.sub.2][O.sub.2]. The leaves were incubated at 25[degrees]C on a rotary shaker. An insoluble purple product indicative of peroxidase activity was visible within an hour. The leaves were maintained in the 4-chioro-1-napthol solution overnight to allow for the extraction of chiorophylls from the leaves.


Peroxidase isoenzymes were separated by agarose gel electrophoresis according to the manufacturer's instructions (Modern Biology, Inc.). Equal amounts of protein (10 [micro]g) were loaded in each lane. Cytochrome C (pI 10.2) and a mixture of horseradish peroxidase isoenzymes (pI 6.4, 7.1, and 9.0) served as standards. Following electrophoresis, peroxidase activity was detected in the gels using 4-chloro-1-napthol as a substrate (Modern Biology, Inc.).


Appearance of Inoculated and Wounded Leaves

Immediately after inoculation of leaves of Arabidopsis ecotype Columbia with P. s. syringae, the leaves appeared water-soaked. This water-soaked appearance disappeared several hours after inoculation. Approximately 24 hours after inoculation, macroscopic tissue collapse characteristic of the hypersensitive response was visible. The necrotic area did not spread beyond the initial site of inoculation and little or no yellowing was observed.

Inoculation of leaves of Arabidopsis with a 1 x [10.sup.8] cfu [ml.sup.-1] suspension of P. s. syringae resulted in confluent necrosis after 24 hours. Less extensive and more sporatic tissue collapse was observed using a 1 x [10.sup.7] cfu [ml.sup.-1] suspension of P. s. syringae. Very little or no macroscopic hypersensitive response was observed using concentrations of 1 x [10.sup.6] cfu [ml.sup.-1] or less. No visible necrosis was observed after infiltrating leaves with 10 mM [MgCl.sub.2] in the absence of any bacteria.

Gently crushing leaves of Arabidopsis between the serrated ends of a large metal forceps resulted in numerous, small, water-soaked areas. These areas of tissue maceration gradually dried and appeared a faint gray three days after wounding. Tissues surrounding the wound sites remained green.

Peroxidase Activity of Inoculated and Wounded Leaves

Inoculated and wounded leaves were analyzed for changes in soluble peroxidase activity (Figure 1). Leaves harvested immediately after inoculation with P. s. syringae or wounding possessed very little peroxidase activity. However, peroxidase activity increased thereafter with the greatest peroxidase activity observed on the fifth day (P. s. syringae) or the second day (wounding) of the experiment. Peroxidase activity remained low through the duration of the experiments in untreated control leaves (Figure 1).

Localization of Peroxidase Activity

To determine the location of the peroxidase activity of Arabidopsis leaves inoculated with P. s. syringae, extracts were prepared from inoculated and uninoculated regions of the same leaf and then the extracts were assayed for peroxidase activity (Figure 2). At two and four days postinoculation with P. s. syringae, inoculated regions of Arabidopsis leaves possessed higher levels of peroxidase activity than uninoculated regions of the same leaf. The mean peroxidase activity of the uninoculated regions of inoculated leaves was similar to the mean peroxidase activity of the control leaves at day two, but higher at day four (Figure 2).

The location of the peroxidase activity in inoculated Arabidopsis leaves was also examined by incubating detached inoculated leaves in a 4-chloro-1 naptholsolution. Purple stain indicative of peroxidase activity was observed in the necrotic regions undergoing the hypersensitive response and in the healthy tissues immediately surrounding the necrotic cells.

Peroxidase Isoenzymes

Electrophoresis of extracts prepared from leaves inoculated with P. s. syringae revealed the induction of a prominent basic peroxidase isoenzyme (Figure 3). The Arabidopsis peroxidase isoenzyme migrated in the gel at a distance between those of the basic horseradish peroxidase isoenzyme (pI 9.0) and cytochrome C (pI 10.2). We have estimated that the basic Arabidopsis leaf peroxidase isoenzyme has an isoelectric point of approximately 9.7.

Electrophoresis of extracts prepared from wounded Arabidopsis leaves also revealed the presence of a basic peroxidase isoenzyme (Figure 4). This isoenzyme migrated in the gel at the same distance as the peroxidase isoenzyme induced by P. s. syringae. The induction of the same peroxidase isoenzyme in the absence of P. s. syringae strongly suggests that the basic peroxidase isoenzyme originated from Arabidopsis and not from P. s. syringae.

Electrophoresis was also performed on extracts prepared from Arabidopsis stems, flowers, immature siliques, and roots (Figure 5). The stem extract was observed to possess at least two basic peroxidase isoenzymes. The extracts prepared from flowers and siliques each contained at least one basic peroxidase isoenzyme. The root extract was observed to possess at least four basic and one acidic peroxidase isoenzymes. The stem, flower, silique, and root extracts each possessed the same basic peroxidase isoenzyme as the one initially identified from Arabidopsis leaf extracts (Figure 5).


The pattern of increase in peroxidase activity was different between bacterial inoculation and wounding. Similar to the observations of Summermatter et al. (1995), we found that soluble leaf peroxidase activity increased nearly linearly after inoculation with P. s. syringae. In contrast, the increase in soluble peroxidase activity was more delayed after wounding and peaked earlier than compared to bacterial inoculation. We observed that wounding leaves using a serrated metal forceps elicited a pattern of increase in peroxidase activity similar to that reported by spraying leaves with lead nitrate (Lummerzheim et al. 1995). Since P. s. syringae and wounding elicited different patterns of peroxidase accumulation, Arabidopsis may have a mechanism to distinguish between biotic and abiotic agents.

The peroxidase localized to the inoculated regions may be involved in strengthening the cell wall. Peroxidases are believed to strengthen the cell wall by cross-linking cell wall components (Fry 1986) and by catalyzing the synthesis of lignin (Grisebach 1981) and other polyphenolic compounds (Lummerzheim et al. 1995). Alternatively, the increase of peroxidase activity may be related to increased tryptophan oxidase activity (Ludwig-Muller and Hilgenberg 1992). The product of tryptophan oxidase, indole-3-acetaldoxime, can be metabolized to indole-3-acetonitrile (Helmlinger et al. 1985). We have found that indole-3-acetonitrile can reduce the growth of P. s. syringae in culture (Tsuji, unpublished data). Peroxidase may play a role in disease resistance by contributing to the synthesis of antimicrobial metabolites.

Peroxidase activity also increased in uninoculated regions of inoculated leaves. These results suggest that P. s. syringae-inoculated areas may be producing a signal that is stimulating peroxidase activity in healthy, uninoculated regions of the same leaf. Although peroxidase is not induced systemically in Arabidopsis (Summermatter et al. 1995), this is the first report of a bacterium inducing local changes in leaf peroxidase activity.

Increases in soluble leaf peroxidase activity in Arabidopsis were associated with the accumulation of a basic peroxidase isoenzyme. We have estimated that the basic Arabidopsis leaf peroxidase isoenzyme has an isoelectric point of approximately 9.7. This estimate is in agreement with the isoelectric point estimate for the most basic peroxidase isoenzyme observed by Intapruk et al. (1991) and Ludwig-Muller and Hilgenberg (1992). Although the basic peroxidase isoenzyme has been previously reported in Arabidopsis leaves, this paper is the first report of such an isoenzyme in roots, flowers, stems, and immature siliques. The presence of the same basic peroxidase isoenzyme (pI 9.7) in all these tissues suggests that this peroxidase isoenzyme may play a role in growth and development.


The authors thank Siena Heights University for financial support and Dr. Ray Hammerschmidt for providing the culture of P. s. syringae.


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