Introduction
============

Class III peroxidases (EC 1.11.1.7; donor: H~2~O~2~ oxidoreductases; secretory pathway) belong to a large multigenic protein family. In maize, approximately 200 genes correspond with this protein class ([@bib26]). Even more isoenzymes can be generated by post-transcriptional and post-translational modifications ([@bib44]; [@bib47]), and more than 50% of them were estimated to show a stress-induced expression ([@bib34]). Artificial phenolic substrates like guaiacol can be used to detect all class III peroxidases present independent of their different *in vivo* substrate specificities. Probably due to this high number of isoforms, and due to the heterogeneous regulation of their expression, peroxidases are involved in numerous cellular processes during plant development and stress response ([@bib17]; [@bib20]; [@bib33]; Cosi and Dunand, 2009). The majority of class III peroxidases are soluble apoplastic and cell wall-bound isoenzymes. In addition, four membrane-bound peroxidases were identified in plasma membrane (PM) preparations of maize (*Zea mays* L.) roots ([@bib28]; [@bib26]). The biochemical properties and enzyme activities of these peroxidases were characterized in detail ([@bib28]). The full-length amino acid sequences of pmPOX1 (ZmPrx1), pmPOX2b (ZmPrx70), and pmPOX3 (ZmPrx66) were identified. A first *in silico* sequence analysis of the membrane-bound class III peroxidases, suggested a function of these peroxidases in oxidative stress at the apoplastic site of the plant PM ([@bib26]).

Due to two possible catalytic cycles, peroxidative and hydroxylic, peroxidases can detoxify or generate reactive oxygen species like ^•^OH and HOO^•^, polymerize cell wall compounds, and regulate H~2~O~2~ levels ([@bib33]). Large amounts of H~2~O~2~ are produced at the PM due to cellular processes, as a response to stress factors, or to external sources in plant--pathogen interactions ([@bib37]; [@bib6]; [@bib38]; [@bib30]). Oxidative stress causes lipid peroxidation and changes in membrane permeability ([@bib8]; [@bib35]; [@bib36]). Due to these observations it seems likely that PM-bound peroxidases could be substantially involved in the detoxification of the cell and/or membrane repair of the PM, and could probably play an important role to maintain the cell functions under different stress conditions. This hypothesis is further supported by solubilization properties of the tightly membrane-bound haem peroxidases ([@bib28]) suggesting their localization in microdomains (so-called lipid rafts), and the identification of a PM-bound peroxidase in detergent-resistant membrane fractions of *Medicago truncatula* ([@bib22]). A localization in microdomains may allow PM-bound peroxidases to co-localize with ROS producing and detoxifying enzymes in the membrane. Thus PM-bound peroxidases could probably not only detoxify H~2~O~2~ directly at the site of origin to ensure the optimal protection of the membrane, but could also protect specific functional regions of the PM ([@bib24]). However, despite the knowledge about the exact location of a peroxidase, it is often difficult to reveal the function(s) of each single enzyme due to (i) the large number of similar isoenzymes, (ii) their broad substrate specificity, (iii) multiple possible functions, and (iv) the ability of other isoforms to replace the role of a missing peroxidase in knock-out experiments ([@bib17]; [@bib27]).

When plants are attacked by pathogens, they defend themselves with an arsenal of defence mechanisms, that includes *de novo* protein synthesis which is regulated through a complex and interconnected network of signalling pathways. These pathways mainly involve the two signalling molecules methyl jasmonate and salicylic acid (Koornneef and Pieterse, 2008; [@bib48]; [@bib1]). Methyl jasmonate-mediated signalling pathways are implicated in the regulation of antiherbivore defences, wounding, and the induction of systemic acquired resistance ([@bib46]), while the salicylic acid pathway is associated with defence responses against pathogens. However, depending on the pathogen species there are many exceptions and network interactions between both pathways ([@bib39]). In addition, H~2~O~2~ has been demonstrated to be involved in pathogen defence and signalling under oxidative stress ([@bib11]; [@bib27]; [@bib4]; [@bib40]). The gene expression of several soluble class III peroxidases is induced by these factors ([@bib17]). Microbe or elicitor-induced signal transduction pathways lead to (i) the reinforcement of cell walls and lignification, (ii) the production of antimicrobial metabolites (phytoalexins), and (iii) the production of active oxygen species and active nitrogen species ([@bib1]). Elicitors are pathogen-derived messengers that induce plant responses against herbivores or pathogens ([@bib32]). Among the proteins induced during host plant defence, soluble class III plant peroxidases are well known to participate directly or indirectly in a broad range of physiological processes, such as lignin and suberin formation, cross-linking of cell wall components, and synthesis of phytoalexins, or to participate in the metabolism of active oxygen species, switching on the hypersensitive response, a form of programmed host cell death at the infection site associated with limited pathogen development ([@bib1]). Phosphorylation and dephosphorylation events take place during cell signalling in oxidative stress ([@bib13]) and are involved in protein biosynthesis and degradation processes ([@bib41]). A partial gene analysis of *pmPOX2b* revealed several putative *cis*-regulatory elements that suggested a regulation of its gene by wounding, methyl jasmonate, salicylic acid, and elicitors ([@bib26]). In addition, the 5′-untranslated region of *pmPOX2b* contained seven successive elements with sequence similarity to the consensus sequence of antioxidant-responsive elements (ARE), strongly suggesting a regulation of the gene by oxidative stress ([@bib16]).

The aim of the present study was the investigation of the regulation of class III peroxidases under oxidative stress with a special focus on PM-bound isoenzymes. Since gene expression does not always reflect protein abundance at the plant PM, i.e. the average correlation observed in eukaryotes is about 0.5 ([@bib15]), a functional analysis was carried out by biochemical and proteomic approaches. Total guaiacol peroxidase activity was measured in soluble and microsomal fractions of maize roots after wounding or treatment with different signalling compounds (H~2~O~2~, methyl jasmonate, salicylic acid, and elicitors), and compared with changes in PM-bound peroxidase activities. The changes of the protein levels of four plasma membrane-bound class III peroxidases were monitored in response to these multiple stresses.

Materials and methods
=====================

Stress and effector treatments of maize seedlings
-------------------------------------------------

Maize seedlings (*Zea mays* L. cv. *GBL*, Saatenunion, Hannover, Germany) were cultivated in hydroponic cultures in the dark as described earlier ([@bib25]). Effectors were added to the hydroponic culture medium of 5-d-old seedlings under green light, and cautiously but thoroughly mixed. Wounding experiments were performed as cutting of the roots in 2--5 cm long root segments. Cantharidin stock solutions were dissolved in acetone. The final concentration of the solvent during the treatment was ≤0.02%. Control experiments were always performed in parallel using the same procedures and conditions, while effector solutions were replaced by equal amounts of hydroponic medium or solvent. Each sample was analysed using 2--3 independent stress experiments, each experiment with approximately 900 maize seedlings, and 900 control plants that were handled in parallel.

Elicitor preparation
--------------------

Crude preparations of elicitor from *Fusarium graminearum* and *Fusarium culmorum* were prepared by standard procedures ([@bib14]). Conidial suspensions (provided by Frank Maier, Biocenter Klein Flottbek, Hamburg, Germany) were cultivated on agar plates (Corn-meal agar, Difco) at 28 °C until mycelium and new conidia covered the surface of the plate. Fungal mycelium and conidia were collected after the addition of 10 ml sterile water and shaking of the plate (50 U min^−1^) for 2 h at room temperature. The covering solution containing the whole suspension was sonicated for 2 min in a Labsonic 1510 apparatus (B Braun, Melsunger AG, Germany). The protein concentration of the suspension was determined according to [@bib7], then autoclaved at 115 °C for 35 min and used as elicitor extract stock solution.

Chitosan was prepared as described elsewhere ([@bib19]).

Isolation of microsomes, PMs, and soluble proteins
--------------------------------------------------

A soluble protein fraction containing soluble proteins from all the cell compartments, microsomes, and PM was prepared from stressed 5-d-old maize roots by differential centrifugation, and aqueous two-phase partitioning as described earlier ([@bib25]). Briefly, primary roots (80--130 g fresh weight) were harvested, and washed with cold hydroponic medium. The harvest time did not exceed 30 min. Roots were homogenized (three times for 15 s) in a Waring blender in 200 ml of cold HEPES-KOH buffer (250 mM sucrose, 50 mM HEPES, 1 mM EDTA, pH 7.5) supplemented with 1 mM dithiothreitol and 1% polyvinylpyrrolidone. Crude extracts were filtered through a nylon net (125 μm mesh; Hydrobios, Kiel, Germany), supplemented with 1 mM phenylmethylsulphonyl fluoride, and microsomal fractions were isolated by differential centrifugation (10 min at 10 000 *g* and 30 min at 50 000 g). Plasma membranes were purified from microsomal fractions by six steps of phase partitioning \[36 g phase system consisting of 6.5% (w/w) dextran T500 (Pharmacia, Freiburg, Germany), 6.5% (w/w) polyethylene glycol 3350 (Sigma Aldrich, Taufkirchen, Germany), 250 mM sucrose, 5 mM KC1, 5 mM phosphate buffer, pH 7.8\]. Isolated PM were washed in 25 mM sodium acetate-HCl, pH 4.0, 500 mM KCl, 1 mM EDTA, and 0.01% (w/v) Triton X-100 for 30 min at 4 °C under continuous stirring to remove peripheral and adsorbed soluble proteins ([@bib28]). Washed plasma membranes were pelleted at 105 000 *g* for 45 min at 4 °C, and resuspended in acetate buffer (25 mM sodium acetate-HCl, pH 4.0, 1 mM EDTA). The final membrane pellets were stored at −80 °C until use.

The purity of the plasma membrane preparations was estimated by a plant cell compartment antibody marker set (Agrisera, Vännäs, Sweden; see [Supplementary Fig. S1](http://jxb.oxfordjournals.org/cgi/content/full/erp353/DC1) at *JXB* online). Polyclonal antibodies against H^+^-ATPase (plasma membrane), V-ATPase (tonoplast), Sar1 (endoplasmic reticulum, ER), Sec21 (Golgi apparatus), and cytochrome *c* oxidase (COXII, inner mitochondrial membrane) were used for Western-blot analysis. According to the protocol of the distributor, 1 μg protein was loaded on the gel for each sample.

Solubilization of membrane proteins
-----------------------------------

Washed PM (50--200 μg protein) were solubilized with CHAPS at a detergent:protein ratio of 30:1 (w/v) in the presence of 0.5 mM aminocaproic acid in acetate buffer (25 mM sodium acetate-HCl, pH 4.0, and 1 mM EDTA). After incubation for 1 h at 4 °C, solubilised proteins were separated by centrifugation at 13 000 rpm for 1 h at 4 °C (Heraeus Biofuge Type fresco, Kendro, Osterode, Germany).

The PM-bound peroxidase pmPOX2a was further purified from solubilizates of control plants as described earlier ([@bib26]). Briefly, CHAPS/ACA solubilizates were separated by cation exchange chromatography using an HPLC-System (AKTA, GE Healthcare, Freiburg, Germany), peak fractions were concentrated using Centricon YM-10 concentrators (Millipore, Bedford, MA), and purified by additional size exclusion chromatography.

2D-electrophoresis
------------------

Solubilized membrane proteins were separated in the first dimension by isoelectric focusing with mini gels (7.5% (w/v) acrylamide, 0.2% (w/v) *N,N'*-methylene *bis*acrylamide, 3 M urea, 2% (w/v) CHAPS, 0.1% (v/v) TEMED, 1% (w/v) ammonium persulphate) for 3 h at 4 °C with 10 mM phosphoric acid and 20 mM NaOH as anode and cathode buffer, respectively. For a better resolution, different pH gradients \[pH 3--10, pH 8--11; 2% ampholyte (Amersham Pharmacia Biotech, Freiburg, Germany)\] were used for different PM-bound peroxidases. pmPOX3 was visible in both gradients allowing standardization. 24 μl sample were mixed with 8 μl loading buffer (8% ampholyte, 8% (w/v) CHAPS, 40% glycerol). Isoelectric points were calculated in comparison with the pH of gel segments derived from control lanes. Gel lanes of the first dimension were equillibrated in 62.5 mM TRIS-HCl, pH 8.8, 10% glycerol, 0.8% SDS, 0.02% bromophenol blue for 30 min on a shaker (50 min^−1^) and subjected to gradient sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with 4% sample gels and 4--18% gradient polyacrylamide slab gels under non-reducing conditions. Peroxidase protein spots were detected by staining with 1% (v/v) guaiacol and 0.03% (v/v) H~2~O~2~ in 250 mM sodium acetate--HCl, pH 5.0 for 30 min. As described earlier ([@bib26]), under these conditions this detects the haem groups of class III peroxidases. These contain exactly one prosthetic group per monomer and subunit. Thus, the orange colour detected is directly proportional to the protein abundance of the pmPOX. Control and stressed samples were purified, separated, stained, and photographed in parallel and on the same image. Images were taken by a digital camera (Camedia C-4000 ZOOM, Olympus Optical Co. Ltd., Indonesia). For each gel, several pictures were subjected to image analysis (*n*=3), and protein spot intensities were evaluated by Fitswork (Version 3.93, <http://freenet-homepage.de/JDierks/softw.htm>), and ImageJ (Version 1.37; <http://rsbweb.nih.gov> /ij/). pmPOX were identified in comparison with identified isoelectric points and molecular masses of purified proteins from control plants separated under the same conditions.

Enzyme assays
-------------

Peroxidase activities of soluble proteins and membrane fractions were measured as oxidation of guaiacol (8.26 mM, ε=26.6 mm^−1^ cm^−1^) in the presence of 8.8 mM H~2~O~2~ within 1 min in 25 mM sodium acetate-HCl (pH 5.0). The assay (1 ml) contained 10 μl soluble proteins, 25 μl of 1:10 diluted microsomes, 25 μl PM or 25 μl washed PM vesicles, respectively. The reaction was started by the addition of guaiacol and followed spectrophotometrically (DU 7500, Beckmann, Munich, Germany) as the increase of absorbance at 470 nm. All rates were corrected by chemical control experiments, i.e. auto-oxidation rates of guaiacol under these assay conditions in the absence of active samples. Protein concentrations were determined according to [@bib7] in the presence of 0.01% Triton X-100. Data presented were calculated with Microcal Origin (version 5.0, Additive GmbH, Friedrichsdorf/TS, Germany), and significant differences were analysed using one-way ANOVA. When significant differences in data groups were found, single *t* tests were performed between each control and stress treatment data set.

Results
=======

5-d-old maize seedlings were exposed to wound stress, or treated with different signal compounds to investigate different forms of oxidative stress. The effects of a broad range of compounds (H~2~O~2~, methyl jasmonate, salicylic acid, maize pathogen extracts, chitosan, and cantharidin) that are involved in multiple plant signalling cascades were investigated. For each factor, the total protein yield and total class III peroxidase activity was determined for the soluble protein fraction, the microsomal membrane protein fraction, and the non-washed and washed plasma membrane vesicle fraction, respectively.

Total soluble and membrane-bound class III peroxidase activities
----------------------------------------------------------------

[Table 1](#tbl1){ref-type="table"} reveals an overview of total protein yields and total guaiacol peroxidase activities of the four different protein fractions investigated in control plants. [Figures 1A](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"} show the comparison of microsomal and soluble fractions after different stress treatments. Only cantharidin decreased the yield of soluble proteins, whereas the yield of membrane proteins did not change significantly ([Fig. 1A](#fig1){ref-type="fig"}). Guaiacol as artificial phenolic substrate detects mainly class III peroxidases. Relative guaiacol activity is shown on the basis of gram fresh weight of maize roots and on the basis of milligrams of protein. After treatment with H~2~O~2~, total guaiacol peroxidase activity of the membrane protein fraction was reduced by 20%. In contrast, the total activity of the soluble enzymes was not significantly changed ([Fig. 2A, B](#fig2){ref-type="fig"}). Wounding and treatment with methyl-jasmonate slightly reduced the guaiacol rates of the membrane fraction, while the total rate of the soluble proteins was less affected. Treatment with salicylic acid did not exhibit any effect on the total peroxidase activity of membrane proteins ([Fig. 2A, B](#fig2){ref-type="fig"}). The soluble guaiacol activity showed a weak, but significant reduction after treatment with salicylic acid.

###### 

Total soluble, microsomal, PM-bound, and wPM-bound protein yield and class III peroxidase activity of control plants

                                        Yield (μg protein g^−1^ FW)   Guaiacol peroxidase activity   
  ------------------------------------- ----------------------------- ------------------------------ -----------
  Soluble enzymes                       1817±63                       13372±674                      7.58±0.33
  Membrane-bound enzymes (microsomes)   242±18                        517±25                         2.24±0.09
  PM-bound enzymes                      43.1±1.7                      43.9±1.4                       0.89±0.02
  wPM-bound enzymes                     4.8±0.3                       15.4±0.4                       3.40±0.10

Shown are means ±SE (*n*=30). The soluble, membrane-bound (microsomal), plasma membrane-bound, and washed plasma membrane-bound protein fractions of maize seedlings grown in hydroponic cultures were analysed. Plasma membranes were isolated representing peripheral and integral PM-bound proteins. By contrast, thoroughly washed PM vesicles contain mainly integral PM-bound proteins, and, thus, represent the total activity of the four pmPOX of the plant PM (for further details see [Figs 1, 2, and 3](#fig1 fig2 fig3){ref-type="fig"}). FW, fresh weight.

![Effects of stress treatments on total soluble, microsomal, PM-bound, and wPM-bound protein yields from 5-d-old maize roots. Maize seedlings were exposed to different stressors as indicated. Each stress treatment was analysed using 2--3 independent experiments, i.e. each experiment with approximately 900 plant seedlings. Total protein yields were determined in triplicate in each experiment. Relative yields were calculated in comparison with direct controls that were handled in parallel. Shown are means ±SE (*n*=6--9; controls *n*=30). Values that were significantly different from the controls in *t* test analysis were marked at *P* \< 0.001 (\*) and *P* \< 0.05 (\*\*). H~2~O~2~ (2 mM); wounding (cutting); methyl-jasmonate (50 μM); salicylic acid (0.5 mM); *F. graminearum* extract (250 ng protein ml^−1^); *F. culmorum* extract (125 ng protein ml^−1^); chitosan (20 μg ml^−1^); cantharidin (20 μM); FW, fresh weight. For treatment times see [Table 2](#tbl2){ref-type="table"}. Soluble enzymes (grey bars), membrane-bound (microsomes) (dotted bars); PM-bound, checked bars; wPM-bound, solid bars.](jexboterp353f01_ht){#fig1}

![Effects of stress treatments on total microsomal and soluble guaiacol peroxidase activities from 5-d-old maize roots. Maize seedlings were exposed to different stressors as indicated. Each treatment was analysed using 2--3 independent experiments. Total class III peroxidase activities were determined in triplicate in each experiment. Relative peroxidase activities were calculated in comparison with direct controls that were handled in parallel. Shown are means ±SE (*n*=6--9; controls *n*=30). Class III peroxidase activities were measured in the presence of 8.26 mM guaiacol and 8.8 mM H~2~O~2~. Values that were significantly different from the controls in *t* test analysis were marked at *P* \< 0.001 (\*) and *P* \< 0.05 (\*\*). H~2~O~2~ (2 mM); wounding (cutting); methyl-jasmonate (50 μM); salicylic acid (0.5 mM); *F. graminearum* extract (250 ng protein ml^−1^); *F. culmorum* extract (125 ng protein ml^−1^); chitosan (20 μg ml^−1^); cantharidin (20 μM); FW, fresh weight. For treatment times see [Table 2](#tbl2){ref-type="table"}. (Soluble enzymes, grey bars; membrane-bound enzymes (microsomes), dotted bars.](jexboterp353f02_ht){#fig2}

By contrast, the three elicitors tested, i.e. chitosan, and extracts of *Fusarium graminearum* and *F. culmorum*, slightly increased the total peroxidase activities of the membrane fraction, while the total amount of soluble peroxidases did not reveal a significant change ([Fig. 2A, B](#fig2){ref-type="fig"}). Treatment with the phosphatase inhibitor cantharidin reduced both membrane-bound and soluble activities, on the basis of gram fresh weight, whereas the specific activity (μmol min^−1^ mg^−1^ protein) of soluble guaiacol peroxidases appeared not to be affected due to the change in the total protein yield.

Total plasma membrane and washed plasma membrane peroxidase activities
----------------------------------------------------------------------

In order to achieve a more detailed picture of stress and signal compound impact on the membranes and peroxidases of maize roots, plasma membranes (PM) and thoroughly washed plasma membrane (wPM) vesicles were investigated ([Figs 1B](#fig1){ref-type="fig"}, [3](#fig3){ref-type="fig"}).

![Effects of stress treatments on total PM-bound and wPM-bound guaiacol peroxidase activities from 5-d-old maize roots. Maize seedlings were exposed to different stressors as indicated. Each treatment was analysed using 2--3 independent repetitions. Plasma membranes were isolated representing peripheral and integral PM-bound proteins. By contrast, thoroughly washed PM vesicles contain mainly integral PM-bound proteins, and thus represent the total activity of the four pmPOX of the plant PM. Relative peroxidase activities were determined in triplicate for each independent experiment. Shown are means ±SE (*n*=6--9; controls *n*=30; further details see [Fig. 2](#fig2){ref-type="fig"}). Class III peroxidase activities were measured in presence of the peroxidase substrates guaiacol and H~2~O~2~. Values that were significantly different from the controls in *t* test analysis were marked at levels of *P* \< 0.001 (\*) and *P* \< 0.05 (\*\*). PM-bound, checked bars; wPM-bound enzymes, solid bars.](jexboterp353f03_lw){#fig3}

The measurements with PM vesicles represent the total activities of integral and peripheral membrane-bound peroxidases including some activities of soluble peroxidases that bind to the vesicles during the purification procedure ([@bib3]). The latter are not removable without the removal of non-covalent bound peripheral membrane proteins as well. The activities of wPM representing only the integral membrane protein fraction and, thus, the total activities of the four pmPOX, in comparison with the peroxidase activities of the PM and the soluble fraction, at least show the possible tendencies in the regulation of peripheral membrane bound enzymes. After wounding and treatment with salicylic acid, *F. culmorum*, and chitosan the PM protein yields were significantly increased, while decreased values were found in *F. graminearum* and cantharidin-treated samples ([Fig. 1B](#fig1){ref-type="fig"}). The wPM protein yield was enhanced by salicylic acid and *F. graminearum*, and was slightly decreased by H~2~O~2~. By contrast, cantharidin reduced the yield to 50% ([Fig. 1B](#fig1){ref-type="fig"}).

As shown in Fig. 3, the specific guaiacol peroxidase activity of PM vesicles derived from seedlings after wounding or treatment with H~2~O~2~, methyl-jasmonate or salicylic acid, respectively, was reduced by 20--40% ([Fig. 3A, B](#fig3){ref-type="fig"}). The effects on the wPM fractions were similar to the PM fractions with the exception of methyl jasmonate, which revealed a strongly induced guaiacol activity. A stimulation of total guaiacol peroxidase activity was also found for wPM with all the elicitors tested ([Fig. 3B](#fig3){ref-type="fig"}).

2D-PAGE analysis of the four single pmPOX
-----------------------------------------

In addition to the guaiacol peroxidase activities, the stress-dependent protein abundance of the four PM-bound peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) present at the plant wPM was analysed in detail by 2D electrophoresis. For each treatment 6--8 two-dimensional PAGE gels (*n*=18--24 gel pictures) of two to three independent experiments and the same amount of control gels were analysed. A comparison between exemplary gels representing samples treated with salicylic acid and its direct controls is shown in [Fig. 4](#fig4){ref-type="fig"}. The up-regulation of all four pmPOX by salicylic acid is clearly visible.

![2D-PAGE analysis of pmPOX after treatment with salicylic acid. Shown are exemplary gels for a control (A) and salicylic acid treatment (B). Solubilizates of washed PM were separated by isoelectric focusing in the first dimension with two different overlapping ampholyte gradients (A1, B1, pH range 10--8; A2, B2, pH range 8--3) to allow optimal separation of pmPOX, followed by a modified non-reducing gradient SDS-PAGE in the second dimension. Thus, oligomers and haem proteins remain intact. Protein spots were stained with guaiacol and H~2~O~2~. As described earlier ([@bib26]), under these conditions this detects the haem groups of class III peroxidases, i.e. visualizing the protein abundance of the pmPOX (for details see the Materials and methods). pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3 are visible. All four pmPOX are up-regulated in salicylic acid-treated plants.](jexboterp353f04_3c){#fig4}

The PM-bound peroxidases were identified by staining with the peroxidase substrates guaiacol and H~2~O~2~ in comparison with known pI and MW values for pmPOX1, pmPOX2b, and pmPOX3. By contrast, the isoelectric point (9.5) and molecular mass (63 kDa) of pmPOX2a were revealed for the first time, based on a 2D-PAGE analysis of the purified protein after cation exchange chromatography and size exclusion of control wPM solubilizates (data not shown). Thus, the corresponding spot for the monomeric pmPOX2a was easily identified in the 2D-PAGE analysis of stressed wPM solubilizates ([Fig. 4](#fig4){ref-type="fig"}). The non-reducing SDS-PAGE used in the second dimension allows the in-gel detection of haem groups of class III peroxidases (*b*-type cytochromes) by peroxidase substrates as described earlier ([@bib43]; [@bib26]), and the quantification of the spot intensity allows an estimation of the regulation of the different isoenzymes on the protein level due to their constant amount of prosthetic groups.

The total spot intensities as an indicator of the protein levels of the four class III peroxidases were 18.4 for pmPOX1, 84.9 for pmPOX2a, 22.2 for pmPOX2b, and 100.0 for pmPOX3 in control plants (*n*=21 gels/63 gel pictures), when they were normalized to the predominant enzyme. The intensity of the four pmPOX spots is higher in 2D gels using wPM in comparison to PM, since the amount of the pmPOX referred to protein concentration is higher, i.e. the specific activity increased. As shown in [Table 2](#tbl2){ref-type="table"}, the four membrane-bound class III peroxidases were differentially regulated by the treatments tested. H~2~O~2~ had a different effect on the protein level of each single pmPOX: pmPOX1 was not affected, while an increase in spot intensity was observed for pmPOX2b \>pmPOX2a, whereas a strong decrease was observed for pmPOX3. Wounding caused a significant decrease in intensity for pmPOX2b. The methyl jasmonate-treatment increased the intensity of all four pmPOX: pmPOX3 \>pmPOX2b \>pmPOX2a \>pmPOX1. Salicylic acid showed a strong up-regulation for pmPOX2b, whereas pmPOX3 \>pmPOX1 \>pmPOX2a were less affected.

###### 

Effects of different stress factors and signal compounds on the protein level of all four pmPOX from maize seedlings

  Treatment                  Treatment times (h)   Relative protein spot intensity (%)                                                    
  -------------------------- --------------------- ------------------------------------- ----------------------- ------------------------ ----------------------
  Control                                          100.0±17.1 (21/63)                    100.0±15.7 (24/72)      100.0±18.0 (21/63)       100.0±15.3 (24/72)
  H~2~O~2~                   1                     111.0±44.9 (5/15)                     158.9±24.4 (6/18)\*\*   244.8±60.8 (4/12)\*\*    47.7±4.3 (6/18)\*
  Wounding                   1                     100.3±5.1 (6/18)                      87.4±11.2 (8/24)        52.7±4.1 (6/18)\*        79.5±9.3 (8/24)
  Methyl jasmonate           1                     147.9±14.6 (6/18)\*\*                 251.8±25.3 (6/18)\*     267.3±21.2 (4/12)\*      628.0±166.2 (6/18)\*
  Salicylic acid             1                     347.0±81.1 (4/12)\*                   188.8±39.7 (6/18)\*\*   877.4±306.5 (4/12)\*\*   422.3±87.4 (6/18)\*
  *F. graminearum* extract   4                     113.5±18.2 (4/12)                     65.3±6.9(6/18)\*        233.0±42.5 (4/12)\*\*    113.8±23.0 (6/18)
  *F. culmorum* extract      4                     183.0±40.9 (4/12)\*\*                 182.9±23.1 (6/18)\*     165.3±23.8 (4/12)\*      241.0±14.9 (6/18)\*
  Chitosan                   4                     136.7±23.2 (4/12)\*\*                 169.7±20.0 (6/18)\*     256.7±71.5 (4/12)\*\*    981.7±99.0 (6/18)\*
  Cantharidin                1                     72.3±12.3 (4/12)\*\*                  59.4±5.9 (6/18)\*       192.4±25.1 (4/12)\*\*    98.6±15.3 (6/18)

5-d-old maize seedlings were exposed to different stressors as indicated. Each treatment was analysed using 2--3 independent experiments, i.e. each experiment with approximately 900 plant seedlings. Treated and control plants and samples were handled in parallel throughout all subsequent steps. For each experiment solubilizates from isolated wPM were repeatedly separated by 2D PAGE analysis (isoelectric focusing and non-reducing modified gradient SDS-PAGE, [Fig. 4](#fig4){ref-type="fig"}). As described earlier, thus, oligomers and haem proteins remain intact (Mika and Lüthe, 2003). PM-bound peroxidases were identified by staining with guaiacol and H~2~O~2~. Relative spot intensities representing protein abundance were analysed and calculated in comparison with direct controls. Isoelectric focusing was performed in two different overlapping ampholyte gradients to provide optimal separation of the pmPOX. Shown are means ±SE (*n*=analysed 2D gels/gel pictures). SE of controls for single independent treatments were ≤4%. Values that were significantly different from controls in *t* test analysis were marked at *P* \< 0.001 (\*) and *P* \< 0.05 (\*\*). The proteomic analysis revealed strong changes in protein biosynthesis of all four pmPOX proteins after different treatments.

Elicitors, in general, induced the peroxidases, with the exception of the *F. graminearum* extract that decreased the intensity of pmPOX2a significantly and was without an effect on pmPOX1 and pmPOX3. Cantharidin decreased the intensity of pmPOX2a \>pmPOX1, and increased the intensity of pmPOX2b. Again intensity of pmPOX3 was not significantly affected.

Overall the strongest decrease in spot intensity occurred for pmPOX2b by wounding. A maximal up-regulation of 980% was revealed for pmPOX3 after treatment with chitosan. None of the PAGE gels revealed new spots after any of the treatments.

Discussion
==========

After treatment with the different effectors total activities of soluble and microsomal fractions were compared. However, only slight changes were observed for the total activities of microsomal fractions and soluble peroxidases with the majority of stresses ([Figs 1A](#fig1){ref-type="fig"}, [2](#fig2){ref-type="fig"}). Exceptions were H~2~O~2~, chitosan, and cantharidin with effects of ≥20--30% on the peroxidase activity of microsomal fractions. This observation may be explained by the large number of isoenzymes in both fractions that might be differentially regulated. Some of the isoenzymes may be up-regulated whereas others are down-regulated, the overall reaction may cause either no effect or weak changes.

In contrast to microsomal fractions, PM preparations showed multiple strong changes in peroxidase activity by the effectors tested (Fig. 3). The presence of soluble peroxidases, which may be attached to the PM by electrostatic interaction, appears to falsify the results in unwashed PM fractions. The results for PM vesicles in comparison with the total peroxidase activities of the soluble enzymes and wPM vesicles, indicated the presence of possible peripheric membrane-bound peroxidases only in plants treated with cantharidin, while all other apparent contradictory results between PM and wPM fractions (i.e. methyl jasmonate) are easily explained by the attachment of soluble proteins to the PM. [@bib6] showed that soluble apoplastic peroxidases can bind to plasma membranes of French bean (*Phaseolus vulgaris* L.) in the presence of certain stresses.

In control plants, the purified plasma membrane-bound peroxidases showed guaiacol activities of 61.9±24.1 for pmPOX1, 26.5±2.4 for pmPOX2a, 6.9±1.1 for pmPOX2b, and 54.7±1.8 μmol min^−1^ mg^−1^ protein for pmPOX3 after cation exchange chromatography and size exclusion ([@bib26]). Thus, pmPOX1 contributes to the total activity in non-stressed plants with 41.3%, followed by pmPOX3 with 36.5%, pmPOX2a with 17.6%, and pmPOX2b with 4.6%. The expression levels of the four haem proteins in response to stresses and signal compounds were investigated using a proteomic approach with isoelectric focusing and a non-reducing, modified SDS-PAGE as 2D analysis had several advantages. (i) As already stated above, the non-reducing state of the SDS-PAGE allows the in-gel detection of the haem groups of class III peroxidases by peroxidase substrates ([@bib43]; [@bib26]), and the quantification of the spot intensity allows an estimation of the regulation of the different isoenzymes on the protein level due to their constant amount of prosthetic groups. (ii) In contrast, an activity gel staining of native 2D-PAGE gels with guaiacol and hydrogen peroxide would detect their prosthetic groups and enzyme activity, and create a mixture of protein abundance and peroxidase activity data. (iii) In addition, the natural substrates of the four pmPOX might differ. Despite a detailed analysis of the substrate specificity of the pmPOX ([@bib28]), the *in vivo* substrates of these enzymes at the outside of the plant PM remain unclear. Thus, the enzyme activites of the pmPOX that are already constitutively present at the membrane could be additionally regulated, depending on the levels of their natural substrates and regulating factors to provide even faster adaptations to stress responses.

The experimental isoelectric point and relative molecular mass were determined for the pmPOX2a monomer for the first time, while the values for pmPOX1, pmPOX2a, and pmPOX3 were known by earlier 1-dimensional isoelectric focusing, SDS-PAGE analysis, and size exclusion chromatography ([@bib26]). After purification by cation exchange chromatography and size exclusion, pmPOX2a eluted in fractions with average molecular masses of 155 kDa ([@bib28]). However, the present combined 2D-PAGE analysis of both wPM and the purified protein resulted in the detection of the enzyme by guaiacol and hydrogen peroxide. The difference between the native molecular mass and the value derived from 2D gels is often found, and can be explained by post-translational modifications, the formation of protein aggregates during the purification procedure, or the participation of pmPOX2a in a protein complex within the plant PM ([@bib28]). The 63 kDa molecular mass of the pmPOX2a protein spot is typical for monomeric glycosylated class III peroxidases, as already described for pmPOX2b, and pmPOX3 and other soluble plant peroxidases ([@bib26]). Isoelectric points of class III peroxidases show the complete range of the spectrum, while molecular masses are often within a range of 28--60 kDa ([@bib17]). The 2D-PAGE analysis revealed additional high molecular mass spots ([Fig. 4](#fig4){ref-type="fig"}) that either comprise residual amounts of pmPOX3 due to aggregate formation during the purification procedure, or might be haem proteins. It was shown for in-gel staining with other peroxidase substrates, i.e. tetramethylbenzidine and hydrogen peroxide, that, in addition to class III peroxidases, haem and copper-containing proteins were non-specifically stained ([@bib29]). The native molecular mass of pmPOX3 (40 kDa) in size exclusion chromatography fractions did not suggest the existence of a pmPOX3-containing complex.

Oxidative stress
----------------

H~2~O~2~ plays a crucial role in plant development and cell signalling ([@bib11]). Total activity of wPM decreased after H~2~O~2~ treatment in order to induce oxidative stress directly ([Fig. 3](#fig3){ref-type="fig"}). The increase in intensity of pmPOX2a and pmPOX2b in comparison to the controls ([Table 2](#tbl2){ref-type="table"}) suggests an up-regulation of these isoenzymes by H~2~O~2~. The discrepancy between total activity and protein up-regulation of pmPOX2a and pmPOX2b can be explained by the simultaneous down-regulation of pmPOX3 ([Table 2](#tbl2){ref-type="table"}), which is usually the dominant peroxidase at the protein level and reveals a high activity level. In contrast to pmPOX2a and pmPOX2b, the down-regulation of pmPOX3 suggests that this enzyme might not be involved in membrane protection. Due to these results and the indications that pmPOX could be part of lipid rafts ([@bib24]) it can be hypothesized that the overall peroxidase activity at the PM might be reduced to allow H~2~O~2~ signalling, while certain microdomains of the membrane and, thus, specific functions might be protected. A thylakoid-bound peroxidase in chloroplasts contains an additional hydrophobic domain at the C-terminus that is missing in soluble cytosolic and stroma ascorbate peroxidases, and it seems to enable the protein to bind next to photosystem I to the thylakoid membrane ([@bib2]). This microcompartmentation of the membrane-bound peroxidase is an elegant solution to protect the thylakoid membrane against destruction and to detoxify H~2~O~2~ directly at the site of origin.

Mechanical stress
-----------------

Wounding, applied as cutting, caused a strong decrease in the guaiacol activity of unwashed PM preparations, whereas no effect was observed with washed PM (Fig. 3). The stronger effect of wounding on the activity of unwashed PM may be explained partially by the contribution of soluble proteins attached to the PM during preparation ([@bib3]). The decrease in total guaiacol activity of washed PM suggested that PM-bound peroxidases may not be involved in the wound response. This hypothesis was further supported by the decrease in abundance of pmPOX2b (Table 2). The decreases of the protein levels of pmPOX2a and pmPOX3 were not significant and pmPOX1 was not affected. In contrast to PM-bound peroxidases from maize roots, extracellular soluble peroxidases isolated from wheat roots were demonstrated to be involved in the wound response ([@bib31]).

The total guaiacol activity of wPM revealed a strong increase after treatment with the plant hormone methyl jasmonate ([Fig. 3](#fig3){ref-type="fig"}). Data shown in [Table 2](#tbl2){ref-type="table"} demonstrated the up-regulation of all PM isoenzymes by methyl jasmonate, and pmPOX2a showed its highest response to this signal compound. This observation suggests a function of PM-bound peroxidases in systemic acquired resistance and/or herbivore stress. The low responses of the pmPOX after cutting do not exclude reactions of the pmPOX to herbivores. The strong up-regulation of all pmPOX at the protein level in the presence of methyl jasmonate could indicate their involvement in herbivore stress as well. [@bib18] showed that different types of wounding triggered the expression of different class III peroxidases from rice (*Oryza sativa* L.), thus, representing an even more complicated network of responses. Soluble peroxidases are well known as pathogen-related proteins involved in systemic acquired resistance and plant defence reactions ([@bib1]).

Both methyl jasmonate and salicylic acid (0.1--0.5 mM) increase plant tolerance against oxidative stress ([@bib48]). As shown in Fig. 3, treatment with salicylic acid caused weak effects on total activities of PM preparations. In contrast to this observation, the spot intensity of all four PM-bound peroxidases increased and suggested an up-regulation of the enzymes. However, data observed for PM preparations appear contradictory to the increases in intensity. As shown in [Fig. 3](#fig3){ref-type="fig"}, the effects on total activity depend on the basis used for calculation. The specific activity in μmol min^−1^ mg^−1^ protein decreased, whereas total activity in nmol min^−1^ g^−1^ FW increased. It may be speculated, that besides peroxidases, several other proteins were up- or down-regulated by the treatment. A significant up-regulation of other proteins changes the specific activity calculated for the peroxidases.

Pathogen defence
----------------

Up-regulation of PM-bound peroxidases by methyl jasmonate and salicylic acid suggests not only a function in mechanical stress but also in pathogen response. The classical answer of animal and plant cells to pathogen attack is an oxidative burst, a sudden production of active oxygen species appearing briefly and/or hours after certain stresses ([@bib20]; [@bib27]; [@bib45]; Sumimoto *et al.*, 2008). The production of reactive oxygen species can be observed after the application of fungal elicitors as well ([@bib32]). In the present study, chitosan and extracts of *F. graminearum* and *F. culmorum* were used for the induction of an oxidative burst. Both fungi are major pathogens of cereals. As shown in Fig. 3, treatment with all three elicitors increased guaiacol peroxidase activity of wPM fractions. This observation was confirmed by the increase in protein abundance for PM-bound peroxidases (Table 2). However, the *F. graminearum* extract caused a decrease in intensity of pmPOX2a, whereas the other two elicitors increased the intensity. This suggested a differential regulation of pmPOX2a in the responses of maize plants against the different microbes. Recent data suggest a fine-tuning of the pathogen defence and the corresponding signal cascades to single species and their properties (Koornneef and Pieterse, 2008). Pathogen defence responses include cell wall strengthening by lignin and suberin formation, cross-linking of cell wall components, biosynthesis of defence molecules, i.e. phytoalexins, and the metabolism of active oxygen species ([@bib27]; [@bib1]). Peroxidases can be involved in all of these processes ([@bib1]). The high affinity of several pmPOX for hydroxycinnamyl alcohols ([@bib28]) that are used by apoplastic peroxidases for lignin biosynthesis ([@bib17]) in combination with their very strong increases in abundance after contact of the plants to pathogen elicitors suggest a function in cell wall strengthening besides membrane protection.

The contribution of protein phosphorylation and dephosphorylation in signal transduction during plant--pathogen interaction has been demonstrated frequently ([@bib41]). Treatment of plants with the phosphatase inhibitor cantharidin results in a decrease of total guaiacol peroxidase activity in PM preparations (Fig. 3). The effect on washed PM, however, indicated a weak decrease in peroxidase activity. As shown in Table 2, the protein level of pmPOX2b was shown to be up-regulated, whereas pmPOX2a and pmPOX1 were significantly down-regulated after treatment with cantharidin. These data suggest a differential regulation of PM-bound peroxidases. Elicitor treatment of cell cultures induced a rapid change in the phosphorylation status of extracellular peroxidases ([@bib9]). Sequence analysis of the pmPOX revealed 15--21 putative phosphorylation sites in each of the proteins (NetPhos2.0 prediction tool; [@bib5]). Thus, results observed after treatment with cantharidin could be based on direct regulation by phosphorylation/dephosphorylation of the proteins or indirect regulation due to effects on *de novo* biosynthesis or the degradation of peroxidases.

Conclusions
===========

The present study demonstrates the involvement of membrane-bound class III peroxidases from maize seedlings in stress responses for the first time. The regulation of pmPOX2b confirmed the predictions made due to the occurrence of *cis*-regulatory elements found by gene analysis ([@bib26]). The total peroxidase activity of the maize root wPM and the protein abundance of all four pmPOX were changed after five or more treatment types, indicating a tight control and adjustment of the enzymes to multiple stress functions. As postulated before, the tremendous amount of peroxidase isoenzymes and their tight, differential, and overlapping regulation seem to have evolved to deal with every possible stress situation ([@bib33]). Using the strongest effects on the pmPOX as guidelines, the results of the present study indicate a regulation of all four pmPOX by methyl-jasmonate and salicylic acid signalling. The data at hand showed that all four membrane-bound peroxidases are involved in pathogen defence with different responses to each elicitor, and probably different roles. pmPOX2b showed the broadest response of all four enzymes, reacted to every stress and effector used, and revealed the highest up-regulation by H~2~O~2~ treatment. Thus, the present study suggests a broad function of PM-bound peroxidases in oxidative stress. In the future, more detailed analysis approaches might shed light on the complex functional network of plasma membrane-bound, apoplastic, and cell wall peroxidases.

Supplementary data
==================

[Supplementary data](http://jxb.oxfordjournals.org/cgi/content/full/erp353/DC1) can be found at *JXB* online.

**[Supplementary Fig. S1](http://jxb.oxfordjournals.org/cgi/content/full/erp353/DC1).** Marker analysis of plasma membrane-enriched fractions.

Supplementary Material
======================

###### \[Supplementary Data\]

The authors wish to thank Aslihan Ayar, Janis Hantke, and Natalie Cyranck, for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Lu-668/4-2) and by the University of Hamburg (PhD student\'s grant no. HmbNFG to DH).

[^1]: Present address: Queensland Institute of Medical Research, Infectious Diseases and Immunology Division, 300 Herston Road, Herston 4029, Queensland, Australia.
