J Biol Chem, Vol. 274, Issue 37, 26192-26198, September 10, 1999

Identification and Molecular Characterization of Novel Peroxidase with Structural Protein-like Properties^*

Satoshi Morimoto, Norifumi Tateishi, Masako Inuyama, Futoshi Taura, Hiroyuki Tanaka, and Yukihiro Shoyama

From the Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Elicitor treatment or mechanical damage to Scutellaria baicalensis Georgi (skullcap plants) callus causes an immediate insolubilization of a 36-kDa protein into cell walls. The 36-kDa protein was identified as peroxidase 1 by analysis of its internal amino acid sequence and by immunoblotting using affinity-purified anti-peroxidase 1. Insolubilized peroxidase 1 is cross-linked to lignin through covalent bonds, and the cross-linking is catalyzed in the presence of H₂O₂ by peroxidase 1 itself. The properties of insolubilized peroxidase 1 resemble those of defense-related structural proteins (extensins and proline-rich proteins) cross-linked to cell wall. Although the isozymes peroxidases 2 and 3 have enzyme activities similar to peroxidase 1, they are not insolubilized by stress treatment. Molecular characterization established that peroxidase 1 contains regions characteristic of structural proteins, but peroxidases 2 and 3 do not have such regions. These results suggest that among the three isozymes, only peroxidase 1 has a structural protein-like function as well as an enzymatic function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most higher plants can quickly activate various defense systems to protect themselves from pathogen attack, injury, or other forms of stress. Among them, insolubilization of structural proteins has been believed to strengthen the cell wall barrier against pathogen attack (1). Extensins and proline-rich proteins (PRPs)¹ that act as defense-related structural proteins have been identified in both dicotyledons and monocotyledons (2). Extensins and PRPs have been thought to be nonenzymatic and purely structural proteins, and molecular characterization of these proteins has established that they all possess several structural characteristics in common: 1) they are basic; 2) they are abundant in hydroxyproline, proline, lysine, and tyrosine residues; 3) and they have highly repetitive peptide sequence with characteristic motifs (e.g. Ser-Hyp-Hyp-Hyp-Hyp for extensins, and Pro-Pro-Val-Tyr-Lys for PRPs) (2-4). Their insolubilization mechanisms have been also well examined, confirming that insolubilization of both structural proteins, which involves H₂O₂-mediated oxidative cross-linking to cell walls, is catalyzed by coexisting peroxidases (5-7).

Peroxidases are ubiquitous enzymes that catalyze oxidation of cellular components in the presence of H₂O₂. Most higher plants contain a number of peroxidase isozymes, which can be classified into two (anionic and cationic) or three (anionic, neutral, and cationic) subgroups according to their isoelectrophoretic mobilities, and these isozymes exist in cytosol, chroloplast, vacuole, and cell wall (8, 9). Their physiological roles have been extensively investigated, and it has been demonstrated that they catalyze a variety of important reactions, such as indole-3-acetic acid catabolism (10), lignin biosynthesis (11, 12), suberization of cell wall (13), and detoxification of H₂O₂ (9, 14). These peroxidases are soluble in buffers containing detergents or high concentration of salt, whereas peroxidases that cannot be extracted using these buffers are also found in various plants (15-18). Although the latter peroxidases have been shown to be covalently bound to cell walls, almost nothing is known about their physiological importance, their structural characteristics, or the mechanism of covalent bond formation.

Previously, we identified a novel H₂O₂-detoxifying system induced by elicitor treatment in Scutellaria cells and demonstrated that two ionically bound wall peroxidases (peroxidases 1 and 2) effectively metabolize large amounts of H₂O₂ produced during oxidative burst (19). Further studies have revealed that peroxidase 1 possesses quite novel functions; like extensins and PRPs, peroxidase 1 is covalently bound to cell wall in response to elicitor treatment and mechanical damage, and surprisingly, this reaction is catalyzed by the action of peroxidase 1 itself in the presence of H₂O₂. We report here the novel properties and possible physiological roles of peroxidase 1. In addition, we have found that the isozymes peroxidase 2 and the newly identified peroxidase 3 show enzymatic properties similar to peroxidase 1, but they do not become insoluble after stress treatment. Because the divergent and shared properties of these peroxidases are assumed to arise from their structures, we have isolated cDNA clones encoding peroxidases 1-3 and predicted their amino acid sequences. We also describe the primary structures of these Scutellaria peroxidases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stress Treatment of Scutellaria Calluses-- The four-week-old calluses of Scutellaria baicalensis (5 g), which was cultured on Murashige-Skoog medium (21) as described previously (20), were subdivided into 100-ml flasks, and 20 ml of liquid Murashige-Skoog medium was added to each flask. After 200 µl of 10% (v/w) yeast extract dissolved in the liquid Murashige-Skoog medium was added (final concentration of yeast extract, 0.1%), the calluses were incubated at 25 °C for 3 h. Treatment of the callus with H₂O₂ was conducted as described above, except that 100 µl of 1 M H₂O₂ (final H₂O₂ concentration, 5 mM) was used instead of the yeast extract solution. Mechanical damage to callus was done as follows. After an aliquot (0.5 g) of the callus was homogenized with 20 ml of the liquid Murashige-Skoog medium, the intact callus (4.5 g) was added to the homogenate and incubated as above.

Immunological Procedures-- Polyclonal antiserum against peroxidase 1 was generated in white female rabbits using 75 µg of purified peroxidase 1 per injection. The antiserum obtained after the second boost was applied to an affinity column that contains purified peroxidase 1 linked to CNBr-activated Sepharose 4B as described by the manufacturer (Amersham Pharmacia Biotech). Anti-peroxidase 1 was eluted with 2 M MgCl₂. For Western blotting analysis, samples were subjected to SDS-PAGE (12.5% acrylamide gels) and transferred to polyvinylidene difluoride membranes at 1.5 mA/cm² for 1 h with a semidry blotting apparatus. The membranes were blocked overnight with blocking buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% gelatin) and then incubated at room temperature for 1 h with affinity-purified anti-peroxidase 1 diluted in blocking buffer. Antibody binding was visualized using horseradish peroxidase-conjugated secondary antibodies (Wako, Tokyo) and 1-chrolo-4-naphtol (Sigma).

Immunostaining of crude cell wall was conducted as follows. Crude cell wall, which was prepared as described below, was incubated at room temperature for 1 h in blocking buffer containing affinity-purified anti-peroxidase 1 (final dilution, 1:100) and centrifuged at 30,000 × g for 20 min. The cell wall was rinsed in washing buffer (10 mM Tris-HCl, pH 7.5, 0.5% Tween 20) and incubated at room temperature for 2 h with alkaline phosphatase-conjugated secondary antibodies (Wako; final dilution, 1:1000) diluted in the blocking buffer. After the cell wall was rinsed with the washing buffer, antibody binding was detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate according to the method of Mobassaleh et al. (22).

Preparation and Fractionation of Cell Walls from Scutellaria Callus-- To avoid insolubilization of peroxidase 1 during preparation of cell wall, ionically bound wall proteins were removed by washing the 4-week-old callus (100 g) with 1 M NaCl (2,000 ml). The NaCl-washed calluses were homogenized with 50 ml of 50 mM phosphate buffer containing 1% (w/v) Triton X-100. The homogenate was filtered, and the residue was rinsed with 450 ml of the same solvent. The residue was further washed with 99% EtOH (250 ml) and then with hexane (250 ml) and was used as crude cell wall. Fractionation of the crude cell wall was carried out by a modification of the method of Srisuma et al. (23). The crude cell wall (5 g) was heated in water (500 ml) at 85 °C for 1 h and then filtered. Pectin was extracted by incubating the hot water-insoluble residue at 85 °C for 1 h in 0.5% (w/v) ammonium oxalate (500 ml) at 85 °C, dialyzed against water, and lyophilized. The ammonium oxalate-insoluble materials were further incubated at room temperature for 18 h in 1 M NaOH (500 ml). The NaOH-soluble fraction was dialyzed against water, followed by lyophilization to isolate hemicellulose, whereas the NaOH-insoluble fraction was washed with water (500 ml) and dried under vacuum to give lignocellulose.

Purification of Peroxidases-- The fraction containing cationic peroxidases was prepared from 4-week-old callus (300 g) by ammonium sulfate precipitation, followed by chromatography on CM-cellulose (19). Subsequently, this fraction was applied to a hydroxylapatite column (Nacalei Tesque, Kyoto, Japan, 1.0 × 20.0 cm) preequilibrated with 10 mM phosphate buffer (pH 7.0). The column was washed with 160 ml of the same buffer to isolate pure peroxidase 1. Further elution at a flow rate of 1 ml/min with an increasing ionic strength gradient (10-70 mM) of phosphate buffer resulted in separation of two peroxidase activity peaks, eluted at approximately 20 and 30 mM. The fractions (fractions 13-15 and 18-20, each 20 ml) contained peroxidases 2 and 3, respectively. Peroxidase 2 was purified to homogeneity, whereas peroxidase 3 was contaminated with a few minor proteins. After the peroxidase-3-containing fractions were pooled, concentrated as above and dialyzed overnight against 10 mM sodium phosphate buffer (pH 6.0), the dialysate was applied to a CM cellulose column (1.0 × 20.0 cm) preequilibrated with the same buffer. The column was washed with 60 ml of the same buffer, and peroxidase 3 was then eluted at a flow rate of 1.0 ml/min with a 600-ml linear gradient of NaCl (0-0.3 M). The most active fractions (9-11, each 20 ml), which contain peroxidase 3 in a pure form, were used for the determination of enzymatic properties and amino acid sequence.

HPLC Conditions-- An HPLC system (Tosoh, Tokyo, Japan) composed of a CCPM pump and a UV-8000 absorbance detector equipped with a cation exchange column Poros SP/M (PerSeptive Biosystems, 0.46 × 10.0 cm) was used. Peroxidases were eluted at a flow rate of 1 ml/min by a linear gradient of NaCl (0-0.2 M). The effluent was monitored by absorption at 220 nm.

Microsequencing of Peroxidases-- Peroxidases (60-80 µg) were dissolved in 40 µl of digestion buffer (125 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.1% (w/v) SDS, 0.01% (w/v) bromphenol blue, 20% (w/v) glycerol) containing 5% (w/v) mercaptoethanol. Each sample was heated at 95 °C for 3 min, and then 40 µl of the digestion buffer was added. After 20 µl of endoproteinase Glu-C solution (2 unit, Promega) was added to the peroxidase solution, the mixture was incubated at 37 °C for 5 h. For degradation of peroxidases with CNBr, purified peroxidases (each 20 µg) were dissolved in 100 µl of 70% (v/v) formic acid containing 1% (w/v) CNBr and incubated at 30 °C for 20 h under darkness. The samples treated with endoproteinase Glu-C or CNBr were subjected to SDS-PAGE (12.5% acrylamide gels) and transferred to polyvinylidene difluoride membranes as above. The fragments were NH₂-terminally sequenced on an Applied Biosystems 473A protein sequencer.

RNA Extraction and Reverse Transcription-- Four-week-old callus (100 mg) was homogenized with 225 µl of extraction buffer (total RNA extraction kit, Amersham Pharmacia Biotech) containing 4.5 µl of -mercaptoethanol. After 2 M acetate buffer (pH 5.2, 22.5 µl), phenol solution (225 µl), and chloroform-isoamylalcohol (24:1, 45 µl) were added to the homogenate, the samples were well mixed and centrifuged at 9000 rpm for 5 min. Isopropanol (250 µl) was added to the aqueous phase, stored at 20 °C for 30 min and then centrifuged as above. The pellet containing RNA was resuspended again in 70% EtOH (1 ml), centrifuged as above, and dissolved in 20 µl of diethylpyrocarbonate-treated water.

In this study, four cDNA pools were used as templates for PCR. The cDNA pools 1 and 2 were prepared by reverse transcription of the above RNA solution using oligo dT primer (5'-CGA(T)14-3') and oligo dT primer possessing adapter (5'-GACTCGTCTAGAGGATCC-CG(T)17-3'), respectively. Both reactions were carried out with M-MLV reverse transcriptase according to the manufacturer (Toyobo, Osaka, Japan). The cDNA pools 3 and 4 were synthesized by attaching poly(C) and poly(A) tails to cDNA pool 1 using terminal deoxynucleotidyl transferase according to the manufacturer's protocols (Takara, Tokyo, Japan).

Cloning and Sequencing of cDNA encoding Peroxidases-- The following oligonucleotide primers were used in this study: degenerate primers 1 (5'-GGGGTACCTTYCAYGAYTGYTTYGT-3'), 2 (5'-CGGGATCCTTRTTRAAIIINGTRCARTG-3'), 3 (5'-CGGGATCCAARTTICCRCAYTGISWYTG-3'), and 4 (5'-CGGGATCCGCYTGICCDATIGTRTG); gene-specific primers 1 (5'-GGTTTCATTAGCTTCCGAGGCA 3'), 2 (5'-GGGGTA-CCCGAGCATTTGCCAACAAGGG-3'), 3 (5'-GACAACTTCAAAACCTCTGAGGC-3'), 4 (5'-GGGGTACCCGGAGGTGAATCTTTCTCTGC-3'), 5 (5'-GGGGTACCCCTACCTTCTACTCTCACCCA-3'), 6 (5'-ACTTGGAAGCCTCTGACAGA-3'), 7 (5'-GGGGTACCATTGGGGCCCGCAGTCTTTT-3'), 8 (5'-GGGGTACCCCTGGACTCAATCTCAATGC-3'), 9 (5'-ATCGAACCCTCGCAGCGAAT-3'), and 10 (5'-GGGGTACCTTTTCTC-CGGTGAACCCAGT-3'; and adapter primers 1 (5'-GACTCGTCTAGAGGATCCCG-3'), 2 (5'-GACTCGTCTAGAGGATCCCG₁₁-3'), and 3 (5'-GACTCGTCTAGAGGATCCCGT₁₇-3'). All PCR amplifications were carried out in 50-µl reaction mixture consisting of primers (degenerate primer, 2 µM; gene-specific or adapter-specific primer, 0.2 µM), dNTPs (0.2 mM each), and Taq DNA polymerase (1 unit, Roche Molecular Biochemicals).

For cloning of cDNA encoding peroxidase 1, degenerate oligonucleotide primers 1 and 2 were designed to adhere to the internal amino acid sequences. In the first step, a cDNA fragment (450 bp) was amplified using both degenerate primers in the presence of cDNA pool 1 as template (40 cycles of each 1 min at 94, 40 and 72 °C). Furthermore, to amplify 3'-terminal and 5'-terminal regions, RACE was employed. The first PCR was conducted with gene-specific primer 1, adapter primer 1, and template cDNA pool 2 (30 cycles of each 1 min at 94, 55, and 72 °C). A 3'-RACE product (750 bp) was obtained by a second round of PCR with gene-specific primer 2 and adapter primer 1 in the presence of the PCR products from the first reaction as template (30 cycles of each 1 min at 94, 58, and 72 °C). 5'-RACE was created in the same manner. The first PCR was performed with gene-specific primer 3 and adapter primers 1 and 2 using template cDNA pool 3 (five cycles of 1 min at 94, 50, and 72 °C, and then 30 cycles of 1 min at 94, 55, and 72 °C). A second round of PCR (30 cycles of 1 min at 94, 55, and 72 °C) with gene-specific primer 4, adapter primer 1, and the PCR products from the first reaction resulted in a 5'-RACE product (350 bp). PCR products were cloned into phage vectors (M13mp18 and M13mp19), transformed into Escherichia coli competent cells (JM110), and sequenced on an Applied Biosystems 373S DNA sequencer. The obtained sequences were combined using a SeqEd^TM program (Applied Biosystems).

Similar procedures were used for the amplification of cDNAs encoding peroxidase 2. As a first step, degenerate PCR was conducted using degenerate primers 1 and 3 in the presence of template cDNA pool 1 (30 cycles of each 1 min at 94, 42, and 72 °C) and resulted in a 400-bp product. A 3'-RACE product (700 bp) was obtained with gene-specific primer 5, adapter primer 1 and template cDNA pool 2 (30 cycles of each 1 min at 94, 55 and 72 °C). 5'-RACE product was created by first performing PCR with gene-specific primer 1 and adapter primers 1 and 3 using template cDNA pool 4 (5 cycles of 1 min at 94, 50, and 72 °C and then 30 cycles of 1 min at 94, 55, and 72 °C). Then a second round of PCR (30 cycles of 1 min at 94, 55, and 72 °C) with gene-specific primer 7, adapter primer 1, and first PCR products yielded a 5'-RACE product (350 bp). Sequencing of the amplified fragments was carried out as above. Concerning the amplification and sequencing of cDNA encoding peroxidase 3, the same PCR conditions were employed, except that degenerate primer 4 and gene-specific primers 8, 9, and 10 were used instead of degenerate primer 3 and gene-specific primers 5, 6, and 7, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of a Cell Wall Protein Insolubilized in Response to Elicitor Treatment-- Previously, Bradley et al. (1) reported that treatment of French bean (Phaseolus vulgaris L.) or soybean (Glycine max L.) cells with elicitor causes rapid insolubilization of extensins and PRPs in cell walls. Therefore, we investigated whether similar treatment affects extractability of structural proteins in S. baicalensis. After Scutellaria calluses were treated with yeast extract as an elicitor, cell wall proteins were extracted with NaCl and then analyzed by SDS-PAGE. When NaCl-extractable proteins in the elicitor-treated callus were compared with those in the control callus, apparent decreases in a 36-kDa protein were observed in the former callus (Fig. 1A). To obtain structural information on this 36-kDa protein, cell wall proteins resolved by SDS-PAGE were electroblotted onto polyvinylidene difluoride membranes, and the 36-kDa protein was NH₂-terminally sequenced. However, sequencing was unsuccessful because the NH₂ terminus appeared to be chemically blocked. The 36-kDa protein was eluted from the gel and digested using endoproteinase Glu-C. NH₂ terminus sequencing of the hydrolysates (1.2- and 1.6-kDa fragments) surprisingly revealed that both fragments contain motifs very similar to the peroxidase active site (Ala-Ala-Gly-Leu-Ile-Arg-Leu-His-Phe-Asp) and the ligand of heme (Met-Val-Thr-Leu-Ser-Gly-Ala-His-Thr-Leu), respectively (8). However, the motifs characteristic of extensins and PRPs were not found. Previously, we identified two cationic peroxidase isozymes (peroxidases 1 and 2) catalyzing H₂O₂-metabolism in S. baicalensis (19), and the molecular mass of the 36-kDa protein was closer to peroxidase 1 than peroxidase 2. Finally, the 36-kDa protein was identified as peroxidase 1 by Western blotting using affinity-purified anti-peroxidase 1 antibody (Fig. 1B).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Stress-induced changes in the composition of cell wall proteins of Scutellaria calluses. A, SDS-PAGE analysis. After Scutellaria calluses (5 g) were treated under various stress conditions, cell wall proteins were extracted with 20 ml of 2 M NaCl and then with 20 ml of 1 M NaCl. Both extracts were combined, dialyzed against water, and lyophilized. The lyophilized samples (each 70 µg) were subjected to SDS-PAGE (12.5% acrylamide gel), and the gel was stained with Coomassie Brilliant Blue. B, Western blotting analysis. The aliquot (10 µg) of each lyophilized sample was resolved by SDS-PAGE (12.5% acrylamide gel) and processed for immunoblotting with affinity-purified anti-peroxidase 1.

In situ immunostaining of Scutellaria callus was carried out with anti-peroxidase 1 antibody by a method described previously (1), but it did not differentiate between control and elicitor-treated calluses (data not shown). We hypothesized that ionically bound wall peroxidase 1 is not completely removed using this staining method, and therefore, soluble peroxidase-free cell wall fractions, which were prepared by extensive washing the callus with NaCl, were used for immunostaining. Consequently, the immunoreactivity in the isolated cell walls was significantly enhanced by elicitor treatment (Fig. 2), indicating that the decrease in NaCl-extractable peroxidase 1 is due to insolubilization in the cell walls. Although we attempted to extract insolubilized peroxidase 1 from the elicited callus using various methods including SDS extraction (1), acidified cholite treatment (24), and limited proteolysis (25), peroxidase 1 could not be solubilized. This observation, taken together with the results of immunostaining, suggests that peroxidase 1 is rigidly cross-linked to cell wall through covalent bonds.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Immunostaining of cell walls. After cell walls prepared from control and elicitor-treated callus were incubated with affinity-purified anti-peroxidase 1 and then with alkaline phosphatase-conjugated secondary antibodies, antibody binding was visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (samples 2 and 3, respectively). Sample 1 was the control cell wall stained by similar procedures in the absence of anti-peroxidase 1.

Mechanism of Peroxidase Insolubilization-- Fig. 1A shows that mechanical damage also induces similar decrease in NaCl-extractable peroxidase 1. Because elicitor treatment or mechanical damage causes rapid production of H₂O₂ in plant cells, we assumed that H₂O₂ is indispensable for the insolubilization of peroxidase 1. This assumption was strengthen by the fact that treatment of Scutellaria callus with H₂O₂ promotes decreases in NaCl-extractable peroxidase 1 (Fig. 1A). We therefore attempted in vitro insolubilization using H₂O₂, purified peroxidase 1 and isolated cell walls and detected the amount of NaCl-extractable peroxidase 1 by Western blotting. As shown in Fig. 3A, insolubilization of peroxidase 1 was found only in the presence of crude cell wall and H₂O₂. These results indicated that, like extensins and PRPs, insolubilization of peroxidase 1 involves H₂O₂-dependent cross-linking to cell walls. Peroxidase 1 was thought to catalyze the reaction itself because the insolubilization never occurred using inactivated peroxidase 1 (Fig. 3B). Brownleader et al. (7) demonstrated polymerization of extensins by in vitro peroxidase reaction in the absence of cell walls, whereas peroxidase 1 was not polymerized under similar conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   In vitro insolubilization of peroxidase 1. A, conditions required for cross-linking of peroxidase 1. Purified peroxidase 1 (2 µg) dissolved in 200 µl of 10 mM citrate buffer (pH 4.0) was incubated at 25 °C for 2 h in the presence of H2O2 (final concentration, 5 mM) and/or crude cell wall (5 mg). After 1 M NaCl (5 ml) was added to each sample, NaCl extracts were analyzed by Western blotting. B, effect of peroxidase activity on insolubilization. Intact peroxidase 1 or inactivated peroxidase 1 (each 2 µg) was incubated at 25 °C for 2 h in 200 µl of the above citrate buffer containing 5 mM H2O2 and crude cell wall (5 mg). Peroxidase 1 was extracted with 1 M NaCl and analyzed by Western blotting as above. C, binding ability of peroxidase 1 to various cell wall components. Purified peroxidase 1 (2 µg) was dissolved in 200 µl of the above citrate buffer containing 5 mM H2O2. The peroxidase solution was incubated at 25 °C for 2 h in the absence (blank) or presence (each 5 mg) of isolated cell wall component (pectin, hemicellulose, or lignocellulose). NaCl-soluble peroxidase 1 was analyzed by Western blotting as above.

The crude cell walls were further fractionated into pectin, hemicellulose, and lignocellulose (23), and binding of peroxidase 1 to each fraction was tested. Fig. 3C shows that peroxidase 1 bind only to lignocellulose. This lignocellulose fraction from the elicited callus was treated with cellulase and anhydrous hydrofluoric acid, which catalyze cleavage of glycosidic bonds, but treatment using both reagents did not solubilize peroxidase 1, confirming that peroxidase 1 is not bound to the cellulose moiety (data not shown).

Kinetics of Insolubilization-- The kinetics of peroxidase insolubilization were determined by monitoring the band intensity of peroxidase 1 by SDS-PAGE (Fig. 4). NaCl-soluble peroxidase 1 quickly decreased until 30 min after addition of elicitor and thereafter slowly insolubilized. More than 80% of ionically bound wall peroxidase 1 was cross-linked to the cell walls at 60 min after elicitation. Its significant decrease was not detectable after 180 min. The insolubilization of extensins and PRPs, which is initiated within 2-5 min and completed within 20-30 min, is one of the earliest defense responses (1), whereas the insolubilization of peroxidase 1 was somewhat slower. We hypothesized that this lower rate is due to scarcity of H₂O₂, because Scutellaria cells rapidly metabolize large amounts of H₂O₂ produced during oxidative burst (19). Hence, we conducted experiments using an excess of H₂O₂, but only a slight increase in the insolubilization rate occurred (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Kinetics of elicitor-stimulated insolubilization of peroxidase 1. The calluses (1 g) were incubated with 4 ml of 0.1% yeast extract or 5 mM H2O2 at 25 °C for different times (0-180 min). Ionically bound wall proteins were extracted with 1 M NaCl (20 ml), dialyzed against H2O2, and lyophilized. The lyophilized samples were resolved by SDS-PAGE (12.5% acrylamide gels). After the gel was stained with Coomassie Brilliant Blue, the intensity of peroxidase 1 was estimated using an image scanner. The S.D. was always within 5% of the mean of triplicate determinations.

Properties of Other Peroxidase Isozymes-- Our previous studies demonstrated the presence of other isozymes in S. baicalensis during purification of peroxidases 1 and 2 (19), but their properties were not examined. To precisely compare the properties of these isozymes with those of peroxidase 1, we attempted purification of them. A cell wall protein fraction, which was prepared from 4-week-old callus as described previously (19), was applied to a CM-cellulose (CM-52) column and then to a hydroxylapatite column to isolate a new isozyme (peroxidase 3), together with peroxidases 1 and 2. The peroxidase-3-containing fractions were further chromatographed over a CM-52 column, purifying peroxidase 3 to homogeneity (Fig. 5).

View larger version (52K): [in this window] [in a new window]   Fig. 5.   SDS-PAGE of peroxidases. Samples were resolved by electrophoresis on a 12.5% acrylamide gel. Proteins were stained with Coomassie Brilliant Blue.

SDS-PAGE of peroxidase 3 revealed that it has the same molecular mass (34 kDa) as peroxidase 2 (Fig. 5), whereas its pI value (8.5) was cationic, like peroxidases 1 and 2. Peroxidase 3 catalyzed degradation of the flavones baicalein and luteolin, but the other flavones (baicalin, apigenin, oroxylin A, and wogonin) were not substrates of this isozyme. This catalytic ability was identical to that of peroxidases 1 and 2 (19). In the presence of baicalein, its H₂O₂-metabolizing activity (1.29 µkatal/mg of protein) also resembled those of peroxidases 1 (1.38 µkatal/mg of protein) and 2 (1.02 µkatal/mg of protein). Thus, enzymatic properties of these three peroxidases were similar, although the responses of peroxidases 2 and 3 to oxidative stress were apparently different from that of peroxidase 1. The intensity of a 34-kDa band corresponding to peroxidases 2 and 3 was not significantly decreased by elicitor treatment or mechanical damage, contrary to that of peroxidase 1 (Fig. 1A). Furthermore, we evaluated changes in ionically bound wall peroxidases 2 and 3 levels by HPLC. Fig. 6 demonstrates that the levels of NaCl-soluble peroxidases 2 and 3 are not affected by elicitor treatment.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   HPLC analysis of peroxidases 2 and 3 in control callus and elicitor-treated callus. Ionically bound wall peroxidases were extracted with 1 M NaCl (1,000 ml) from control callus (A) or elicited callus (B) (each 100 g). Each extract was concentrated by ultrafiltration and dialyzed against 10 mM phosphate buffer (pH 6.0). The dialyzed sample was loaded directly onto a CM-52 cellulose column (1.5 × 20.0 cm) preequilibrated with the same buffer. After the column was washed with 0.1 M NaCl (100 ml), bound proteins were eluted with 0.4 M NaCl (100 ml) and dialyzed against phosphate buffer (pH 7.0). The dialyzed sample was applied to a hydroxylapatite column (1.0 × 10.0 cm). After the column was rinsed with the same buffer (100 ml), the fractions containing peroxidases 2 and 3 were eluted with 100 mM phosphate buffer (50 ml), dialyzed against water, lyophilized, and analyzed by HPLC.

Molecular Characterization of Peroxidases 1-3-- We next analyzed the amino acid contents of all isozymes. Although extensins and PRPs are characterized by high (hydroxyl)proline and lysine contents (2), peroxidases 1-3 had much lower proline (5.1, 4.3, and 4.0%, respectively) and lysine (2.9, 2.5, and 2.6%, respectively) contents versus 25% hydroxyproline, 15% proline, and 14% lysine for maize extensin (26). None of the peroxidases contained hydroxyproline residues. As amino acid analyses did not provide useful findings to explain difference in properties of these peroxidases, we isolated and sequenced cDNA clones coding for them to reveal their primary structures. A reverse transcription-PCR method was employed to isolate the genes encoding peroxidases. Purified peroxidases were treated with endoproteinase Glu-C and cyanogen bromide, and the resulting fragments were NH₂-terminally sequenced. Internal cDNA fragments were amplified using degenerate oligonucleotide primers, which were designed based on the amino acid sequences. Moreover, cDNA fragments containing 3'-end and 5'-end regions were obtained by 3'- and 5'-RACE. Finally, the cDNA fragments, which were assumed to possess the entire coding region, were amplified using gene-specific primers designed from 3'- and 5'-RACE products. Nucleotide sequences of the amplified fragments were determined from both strands to ensure accuracy.

The cloned genes encoding peroxidases 1-3 consisted of 966-, 975-, and 954-nucleotide open reading frames encoding 322, 325 and 318 amino acids, respectively. The PSORT program predicted that 19, 28, and 24 amino acid residues from each first methionine, which are shaded in yellow in Fig. 7, are signal peptides, thus suggesting that mature peroxidases 1-3 consist of 303, 297, and 294 amino acid residues, respectively. We confirmed that each deduced amino acid sequence contain all fragments obtained by digestion with endoproteinase Glu-C or cyanogen bromide. Mature peroxidase 1 showed 47 and 42% identity with mature peroxidases 2 and 3, respectively, whereas mature peroxidase 2 displayed 59% identity with mature peroxidase 3. 

View larger version (55K): [in this window] [in a new window]   Fig. 7.   Deduced amino acid sequences of peroxidases 1-3. The maximum alignment of the amino acid sequence of peroxidase 1 with those of peroxidases 2 and 3 was carried out using the SIM program. Gaps were introduced to maximize alignment. The bars indicate the same amino acid residues as those in peroxidase 1. The regions shaded in yellow, pink, and light blue are signal peptide, peroxidase active site, and ligand of heme, respectively. The amino acids confirmed by NH2-terminal sequencing of the proteinase or CNBr cleavage products of the purified peroxidases are underlined. Proline-rich regions are boxed. The numbers at the right indicate the positions of amino acids counted from the N terminus of each mature peroxidase.

All peroxidases possess several highly homologous regions including the peroxidase active site (Fig. 7, shaded in pink) and the ligand of heme (shaded in light blue), and all cysteine residues were conserved in mature peroxidases 1-3 (Fig. 7). Concerning tyrosine residue, which is reported to be involved in binding of proteins to lignin and its related compound (25, 27), peroxidase 1 showed a higher tyrosine content (7 residues in deduced mature form) than peroxidase 2 or 3 (5 residues each). Among them, four tyrosine residues were conserved in peroxidases 1-3. Although neither peroxidase had repetitive proline-rich motifs found in extensins and PRPs, we found that only peroxidase 1 possesses three proline-rich regions. The sequences in the first and second proline-rich regions (Ser-Pro-Pro and Pro-Pro-Pro-Ser, respectively) were also found in the amino acid sequence predicted from the nucleotide sequence of tobacco extensin gene (28). Maize extensin gene was shown to encode repeats of a proline-rich motif consisting of 16 amino acids (29), partial sequence of which was identical with that (Pro-Pro-Thr-Pro) in the third proline-rich region. Surprisingly, the amino acid sequence (Val-Val-Pro-Met-Asp-Pro-Pro-Thr-Pro-Ala) of the third region were found to bear 80% identity and 90% homology to a region (Val-Val-Pro-Val-Asp-Ala-Pro-Thr-Pro-Ala) of the structural proteins of the giant cockroach (Blaberus craniifer) (30). In contrast, mature peroxidases 2 and 3 did not possess any proline-rich domains. Thus, molecular characterization established that among the three isozymes, only peroxidase 1 has structural protein-like motifs as well as regions critical for peroxidase activity.

	DISCUSSION

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In present study, we have discovered that peroxidase 1 in S. baicalensis has quite novel properties. Elicitor treatment or mechanical damage of the callus led to a decrease in ionically bound wall peroxidase 1, and this decrease was confirmed by immunostaining to be due to its insolubilization in cell wall. Peroxidase 1 is the first enzyme insolubilized in response to oxidative stress. Chemical and enzymatic treatments could not solubilize peroxidase 1 once it was insolubilized. These properties of peroxidase 1 are identical with those of extensins and PRPs in French bean and soybean cells (1). Because insolubilization of both structural proteins has been believed to strengthen the cell wall barrier against invading pathogen, peroxidase 1 may have a similar function. Plant peroxidases have been shown to have various enzymatic properties, but peroxidases having structural protein-like properties have not been reported.

The mechanism of peroxidase insolubilization is also similar to that of the cross-linking of extensins and PRPs. As soon as Scutellaria callus exhibits oxidative burst in response to elicitor treatment or mechanical damage, peroxidase 1 is oxidatively cross-linked to cell walls in the presence of the resulting H₂O₂. This reaction is catalyzed by peroxidase 1 itself. It is notable that enzyme acts as its substrate. On the other hand, extensins are shown to be polymerized by in vitro peroxidase reaction in the absence of cell wall (7), whereas polymerization of peroxidase 1 has not been observed under similar conditions. For the mechanism of polymerization of extensins, it is proposed that they are polymerized by linking each other through intermolecular tyrosine-tyrosine (dityrosine or isodityrosine) bonds (1, 31). Because the tyrosine content in peroxidase 1 is lower than that in extensins, intermolecular tyrosine-tyrosine linkages may be difficult to be formed in peroxidase 1 as compared with extensins.

We conclude that peroxidase 1 is cross-linked to the lignin moiety in cell wall, based on the facts that this enzyme covalently binds only to lignocellulose and that the cross-linked peroxidase 1 cannot be solubilized by treatment with cellulase or anhydrous hydrofluoric acid. Evans and Himmelsbach (17) reported that during in vitro synthesis of lignin, peroxidase reaction catalyzes formation of tyrosine-lignin bonds between the enzyme and the resulting lignin-like products. Therefore, it is likely that peroxidase 1 is also cross-linked to cell wall through tyrosine-lignin bond. Peroxidase bound to cell walls via tyrosine-lignin bond has been identified in flax plants (Linum usitatissimum) (18) and has been shown to be solubilized by limited proteolysis (25). We treated the cell wall fraction of the elicited Scutellaria callus under similar conditions, but cross-linked peroxidase 1 was not solubilized, suggesting that insolubilized peroxidase 1 may be more rigidly bound to cell wall than flax peroxidase or may resist hydrolysis by proteinase. Constitutive presence of covalently bound wall peroxidases has been confirmed in various plants, including flax plants (15-18), whereas the physiological roles of these enzymes and the mechanisms of covalent bond formation remain unclear.

Insolubilization of peroxidase 1 was initiated within 10 min and almost completed within 60 min. This response is apparently faster than transcription-dependent defense responses, such as phytoalexin accumulation, which are usually observed at more than 2 h after elicitation (32). Accordingly, insolubilization of peroxidase 1 can be regard as one of the earlier defense responses. Previously we demonstrated preexistence of large amounts of peroxidases 1 and 2 in the cell walls of S. baicalensis, and this constitutive expression is considered to enable the rapid response. Interestingly, the activities of ionically bound wall peroxidases were also shown to continue to decrease until 60 min after treatment of Scutellaria cells with elicitor (19). This rapid decrease may be due to cross-linking of NaCl-soluble peroxidase 1. Insolubilization of extensins and PRPs is somewhat more quickly initiated and completed, compared with peroxidase 1. Scutellaria cells immediately metabolize large amounts of H₂O₂ in a response to elicitor treatment, but addition of excess H₂O₂ only slightly enhanced its insolubilization. Therefore, scarcity of H₂O₂ is not the cause of the slower insolubilization rate of peroxidase 1. The lower tyrosine content in peroxidase 1 may account for its slower insolubilization as compared with extensins, because McDougall et al. (27) demonstrated that the presence of tyrosine residues accelerates the cross-linking of synthetic proteins to lignin-like products.

In the present study, we have identified a new isozyme (peroxidase 3), together with peroxidases 1 and 2, in S. baicalensis, whereas peroxidases 2 and 3 are not insolubilized by oxidative stress. Because we assumed that the divergent properties between these peroxidases are due to their structures, we examined their amino acid sequences. Consequently, we confirm that the primary structures of peroxidases 2 and 3 are similar to each other (59% identity) but show lower homology to peroxidase 1 (47 and 42% identity, respectively). Interestingly, tyrosine and proline residues, which are abundant in extensins and PRPs, are conserved in peroxidases 2 and 3, except that peroxidase 2 has one additional proline residue (Pro²⁶¹), whereas peroxidase 1 contains more of both tyrosine and proline residues than peroxidases 2 and 3. A tyrosine residue is assumed to contribute to binding of peroxidase and synthetic proteins to lignin (17, 25, 27), and the locations of the four tyrosine residues (Tyr⁷, Tyr¹⁸⁴, Tyr²³³, and Tyr²³⁴) in peroxidase 1 are identical with those in peroxidases 2 and 3. Because peroxidases 2 and 3 are not insolubilized, the other tyrosine residues (Tyr¹⁰⁴, Tyr¹¹⁷, and Tyr²⁰⁰) seem more significant for insolubilization of peroxidase 1 than the conserved ones. These three residues are located between the first and third proline-rich regions, whereas peroxidases 2 and 3 contain no tyrosine residue except for the conserved residue (Tyr¹⁸⁴) in the corresponding regions. Hence, insolubilization of Scutellaria peroxidase may depend on the number or appropriate location of tyrosine residues in the middle domain containing approximately 100 amino acid residues. Moreover, the novel features are also found in the locations of proline residues in peroxidase 1. Peroxidase 1 possesses three proline-rich regions in its molecule, and these amino acid sequences are also found in those predicted from genes encoding tobacco extensin (28), maize extensin (29), and a giant cockroach structural protein (30), assuming that this isozyme can act as a structural protein. In contrast, peroxidases 2 and 3 have no significant characteristics except for regions with high homology to other plant peroxidases. We examined proline-rich regions in peroxidases from other plants using SwissProt, whereas no proline-rich regions were found in 10 peroxidases with relatively high homology to peroxidase 1, except for rice peroxidase, having only one region (Pro-Pro-Pro).

Thus, we confirm that peroxidase 1 consists of the domains critical for peroxidase activity, the motifs characteristic of structural proteins, and the tyrosine residues presumably involved in cross-linking to cell wall. The properties and molecular characteristics of peroxidase 1 support the notion that like extensins and PRPs, this enzyme also play potentially important roles in stiffening of cell walls in S. baicalensis. Previously we demonstrated that peroxidase 1 effectively metabolizes large amounts of H₂O₂ in the Scutellaria cells. Accordingly, peroxidase 1 is a quite versatile enzyme involved in various defense reactions.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB024437, AB024438, and AB024439 for peroxidases 1-3, respectively.

To whom correspondence should be addressed. Tel.: 81-92-642-6581; Fax: 81-92-642-6545; E-mail: morimoto@shoyaku.phar.kyushu-u.ac.jp.

	ABBREVIATIONS

The abbreviations used are: PRP, proline-rich protein; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; RACE, rapid amplification of the cDNA ends; PCR, polymerase chain reaction; bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES