Peroxidative Degradation of Sphagnum Acid (P-hydroxy-/3-[carboxymethyl]-cinnamic Acid)

1) Botanisches Institut der Universitat Kiel, Olshausenstr. 40, D-2300 Kiel, FRG 2) Institut fur Allgemeine Mikrobiologie der Universitat Kiel, Olshausenstr. 40, D-2300 Kiel,

FRG

Received October 23,1985' Accepted July 18,1986

Summary

P-hydroxy-/3-[carboxymethyl]-cinnamic acid (sphagnum acid) is degraded in vitro by a per­oxidase isolated from Sphagnum magellanicum. 10.3/Lmol CO2 are evolved during the degrada­tion of 7.0 /Lmol sphagnum acid. By high-resolution mass spectrometry the formula of the main degradation product was determined with C IOH s0 4• A peroxidase was detected ultracytochem­ically in the cell walls of completely differentiated Sphagnum magellanicum chlorocytes. The reaction product is limited strictly to the innermost layer of the cell wall, adjacent to the cyto­plasm. Thus, peroxidase activity was found at the same site in the cell where the proposed sub­strate, sphagnum acid, is localized. The data document for the first time the occurrence of per­oxidase in the cell walls of bryophytes.

Key words: Sphagnum magellanicum Brid., cell wall, oxidative decarboxylation, peroxidase, sphagnum acid, ultracytochemicallocalization.

Introduction

Peat mosses do not contain lignin. The signals for the ,6-0-4 and phenylcoumaran structures are lacking in the aliphatic range of the l3C NMR spectra. However, the typical signals of p-hydroxyphenyl residues are obtained (Nimz and Tutschek, 1977). Cell walls of Sphagnum species show two extraordinary characteristics: they exhibit an intense red stain after treatment with Millon's reagent; the cellulose is masked, so that its histochemical detection is impossible. These phenomena are caused by phenolic substances incorporated into the cell walls at late stages of differentiation. The main component masking cellulose has been identified as p-hydroxy-,6-[ carboxy­methylJ-cinnamic acid, known as sphagnum acid (Tutschek et aI., 1973; Tutschek, 1975). All the thirty Sphagnum species analysed contain this substance in variable amounts. It is established by use of glyphosate that sphagnum acid is synthesized via the shikimate pathway. The content varies with seasons and within different parts of the plants. Outside the Sphagnales detection of sphagnum acid has thus far failed (Tutschek, 1979 a; Rudolph and Samland, 1985). It is possible to degrade synthetic sphagnum acid in vitro by a peroxidase isolated from Sphagnum magellanicum (Tutschek, 1979 b; Wachter and Rudolph, 1984).

Abbreviations: DAB = 3,3'-diaminobenzidinetetrahydrochloride, POD = peroxidase, HPLC = high performance liquid chromatography, RPC = reversed phase column.

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450 DORIS WACHTER, HANS]ORG RUDOLPH and HORST VOLKER

In the present paper we report on a first characterization of the main degradation product of the peroxidative degradation of sphagnum acid and the detection of a per­oxidase at the same site in the cell where the proposed substrate, sphagnum acid, is lo­cated.

Materials and Methods

Degradation of sphagnum acid in vitro. Sphagnum acid was synthesized according to Wachter and Rudolph (1984). For enzyme

preparations fresh material was used. The cell walls were homogenized in phosphate buffer in a cell-homogenizer MSK (Braun Melsungen) by means of glass-beads (Rudolph and Samland, 1985). The assay for determination of peroxidase activity was based on Bergmeyer (1974).

For degradation of sphagnum acid we used the following incubation mixture: 7.0/tmol sphagnum acid in 4 ml 0.1 M acetate buffer, pH 8.8; 2 ml POD, 39 nkat ml- I ; 2 ml 1.19 mmol ml- I H 20 2; final pH-value 5.1.

For CO2 determination the infrared gas analyser UNOR 4N (Maihak) was used. The reac­tion ceased after about 30 min (20°C) as indicated by no further CO2 evolution. For isolation of the main degradation product «x" the reaction mixture was extracted three times with methylene chloride. After evaporation of the solvent the residue was dissolved in EtOH. For purification the solution was applied to cellulose layers and chromatographed in n-butanol-ace­tic acid-water (3 : 2 : 95). The band, detected by fluorescence (UV 360 nm) was scraped off and suspended in EtOH. The suspension was filtered and the solvent evaporated. The isolated pro­duct is stable when stored under nitrogen at + 4°C in the dark. The reaction mixture was an­alysed qualitatively and quantitatively by HPLC/RPC (Kontron Instruments) before starting and after finishing of the degradation process.

Ultracytochemical assay

Sphagnum magellanicum was cultivated according to Rudolph and Voigt (1986). Only the apical parts of green gametophytes were analysed cytochemically. If not stated otherwise, the following procedures were carried out at room temperature.

Leaflets of 1.5 - 2.0 mm in length were excised from the buds. The prefixation was carried out for 1 h in 0.5 % glutaraldehyde (in 0.05 M cacodylate buffer, pH 7.2, + 0.3 % CaCb), the main fixation for 3 h in 5.0 % glutaraldehyde. The fixative was removed by several changes of cacodylate buffer (3 h).

The cytochemical assay was a modification of the method of Graham and Karnovsky (1966). Five mg DAB were dissolved in 10 ml acetate buffer (0.2 M, pH 5.0) to which H 20 2 (final concentration 0.005 %) was added. The leaflets were incubated for 10 min, rinsed thoroughly in cacodylate buffer (0.05 M, pH 7.2), and postfixed in 1 % OS04 (in 0.05 M cacodylate buffer, pH 7.2, 4°C). After 6 h the leaflets were washed in cacodylate buffer and distilled water, de­hydrated in a graded series of ethanol followed by propylene oxide, and embedded in Spurr's resin (1969). The controls included: 1. Inactivation of the enzyme by heat denaturation (5 min during glutaraldehyde fixation at 100°C), then incubation of leaves in complete medium. 2. In­cubation in a medium lacking H 20 2. 3. Addition of inhibitors; preincubation for 30 min in KCN (1 mM) or NaN3 (0.1 M) in cacodylate buffer (0.05M, pH 7.2), followed by incubation in the complete medium containing KCN (1 mM) or NaN3 (0.1 M). Sections of leaflets were cut with a Reichert Om U2-microtome and poststained with uranyl acetate and lead citrate (Rey­nolds, 1963). Micrographs were taken either with a Zeiss-Photomicroscope II on Kodak Plus-X­Pan or with a Philips EM 300 on Kodak electron microscope film 4489.

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Fig. 1: Light micrograph of a cross section of a completely differentiated Sphagnum magel/ani· cum leaflet, consisting of chlorocytes (C) and hydrocytes (H) with wall thickenings; • pore; Bar: 10Jlm.

Fig. 2: Chlorocyte treated with DAB and H 20 2 showing precipitate (= site of peroxidase) at the innermost stratum of the cell wall (arrows); Bar 1 Jlm.

Fig. 3: Peroxidase activity at the innermost stratum of the cell wall (CW) and the plasmalemma (PL) in a chlorocyte poststained with uranyl acetate and lead citrate; chloroplast (P); Bar: O.5Jlm.

Fig. 4: Site of precipitate in a chlorocyte not poststained; Chloroplast (P); Cell wall (CW); Bar: O.5Jlm.

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452 DORIS WACHTER, HANS]ORG RUDOLPH and HORST VOLKER

Results

Degradation 0/ sphagnum acid by peroxidase in vitro

Under the conditions employed 99.5 % of sphagnum acid was degraded by the ac­tion of peroxidase within 30 minutes. The reaction is characterized by the evolution of 10.3 p.moles CO2 per 7.0 p.moles substrate. Several reaction products could be de­tected by HPLC. The main degradation product called «x» was isolated. It is charac­terized by a bright blue fluorescence in UV360nm light. The Rf-value on cellulose layers in n-butanol-acetic acid-water is 0.6. The substance is neither stained with Pau­ly's nor with Millon's reagent. The UV spectrum, measured in Methanol (Uvasol)l HCI shows a maximum at 312 nm. The molecular formula C lOH g0 4 and the molecu­lar weight 192 were determined on the basis of its high-resolution mass spectrum. A scheme of the reaction is proposed in Fig. 7.

Ultracytochemicallocalization 0/ peroxidase

Fig. 1 shows a survey picture of a cross-section of a completely differentiated Sphag­num magellanicum leaflet: living chlorocytes alternate with dead hydrocytes. For the cytochemical assays only such completely differentiated leaflets with a length of 1.5-2.0 mm were used because at this stage of development the incorporation of phenolic substances into the cell walls is complete.

After incubation in a medium containing DAB and H 20 2 the innermost stratum of the cell wall, adjacent to the cytoplasm, was intensely stained with fine-grained reac­tion product (Figs. 2+3). Staining only occurs in cell walls of chlorocytes, never in cell walls of completely differentiated hydrocytes.

Precipitate is strictly limited to ca. 0.1 p.m on the innermost stratum of the wall. In most cells the plasmalemma also exhibits staining.

To establish that the contrast is due to enzyme activity, a series of controls was used. From a section not poststained (Fig. 4) it is evident that the reaction product oc­curring in the cell wall cannot be attributed to a contamination with lead. Material in­cubated in the absence of H 20 2 shows an almost complete lack of reaction product (Fig. 5). In contrast to the leaflets that were incubated in media containing H 20 2 , the structure of cell organelles, e.g. nucleus, chloroplasts, mitochondria, is well pre­served. In the cell walls of heat-treated samples no enzymatic activity occurred, as evi­dent by lack of staining (Fig. 6A). In the presence of the inhibitor KCN there is a complete lack of reaction product (Fig. 6 C). NaN3-treated material exhibited only very low levels of activity (Fig. 6 B).

Discussion

In comparison to higher plants peroxidase has rarely been studied in bryophytes (e.g. Georgiev and Bakardjieva, 1973; Hebant, 1977; Hilgenberg et aI., 1978; Tutschek, 1979 b; Jaeger-Wunderer, 1980; Krzakowa, 1981; Szweykowski and Zie­linski, 1983; Zielinski et aI., 1985).

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Degradation of Sphagnum acid 453

5

Fig. 5: Chlorocyte after treatment with DAB but without H 20 2; Bar: 1 p.m.

Fig. 6: Control reactions: after heat denaturation 6A; in the presence of NaN3 6B or KCN 6C. Bar: 0.5 p.m.

The data presented in the present work document for the first time the occurrence of peroxidase in the cell walls of Sphagnum. The peroxidase of Sphagnum magellani­cum consists of five acidic and five basic isoenzymes and degrades in vitro sphagnum

J. Plant Physiol. Vol. 126. pp. 449-456 (1987)

454 DORIS WACHTER, HANS]ORG RUDOLPH and HORST VOLKER

HO

/ C02 H

CH2

& C02 H

~ I H

Sphagnum Acid

C11 H10 Os

Fig. 7: Formation of «x» from sphagnum acid by peroxidative decarboxylation.

acid, the main phenolic compound of cell walls of peat mosses (Tutschek, 1979 b). Such degradation reactions of e.g. phenolic compounds by peroxidases have been re­peatedly described in higher plants (Berlin and Barz, 1975; Barz, 1977; Barz and Hosel, 1979; Krisnangkura and Gold, 1979).

The peroxidative degradation of sphagnum acid is characterized by the evolution of 1.47/Lmoles CO2 per 1 /Lmol substrate degraded.

The formula of the unknown degradation product «x» was determined by high-res­olution mass spectrometry with C IOH g0 4• The substance is neither stained with Pau­ly's nor with Millon's reagent. This product of peroxidative degradation in vitro also occurs in all Sphagnum species analysed; furthermore there is also a correlation be­tween the sphagnum acid content and peroxidase activity (Rudolph and Samland, 1985).

The occurrence of peroxidase in cell walls of phanerogams is well documented par­ticularly in the context of lignification (e.g. van Huystee and Cairns, 1982; Gaspar et aI., 1982; Goldberg, 1985). The method of Graham and Karnovsky (1966) has been also successfully used for cytochemical localization of peroxidases in cell walls not in­volved in lignification. In onion root tips the reaction product is located predomi­nantly in the area of the middle lamella (Goff, 1975). In the root hairs of cress, perox­idase activity has been found in the cell wall as well as in the dictyosomes, the associated vesicles and in the ribosomes of ER-cristernae (Zaar, 1979).

In the cell walls of Sphagnum magellanicum chlorocytes the reaction product occurs in a clearly defined region of the cell wall adjacent to the cytoplasm. This means that the cytochemical assay illustrates that in these cell walls peroxidase activity can be de­tected at the same site where sphagnum acid is located. Results of control incuba­tions indicate that the reaction is catalyzed by peroxidase. Thus, the data presented, support the interpretation that peroxidase degrades sphagnum acid not only in vitro but also in vivo.

Up to now the significance of sphagnum acid is not clear. Does the sphagnum acid represent an end product of aberrant biosynthetic pathways or is this cinnamic acid derivative together with other phenolic compounds partially responsible for the ap­parent resistance to decomposition shown by Sphagnum tissues (e.g. Dickinson, 1983; Berch and Fortin, 1983)? The antimicrobial nature of phenolics is well known (e.g.

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Degradation of Sphagnum acid 455

Swain, 1977; Friend, 1979; Venere, 1980; Alfenas et aI., 1982}. It is also often noted that positive correlations between disease resistance of plants and peroxidase activity suggest that peroxidase-rich cell walls may act as a barrier by their oxidizing capacity (e.g. Kosuge, 1969; Benedict, 1971; Urs and Dunleavy, 1975; Venere, 1980; Alfenas et aI., 1982). The low level of microbial activity in Sphagnum capitula is well known. First unpublished results indicate that sphagnum acid inhibits to a great extent the germination rate of Phycomyces blakesleeanus spores, the fermentation rate of Sac­charomyces cerevisiae as well as the development of ChIorella pyrenoidosa. Further in­vestigations have to be carried out to clear up finally the significance of sphagnum acid and its peroxidative degradation products.

Acknowledgements

We thank Professor Dr. Janice Glime for critically reading the English text, and Jiirgen Wilschke for the HPLC-data.

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