Some Properties of Syringaldazine Oxidase, a Peroxidase Specifically Involved in the Lignification Processes

RENEE GOLDBERG, ANNE-MARIE CATESSON and YVETTE CZANINSKI

Ecole Normale Superieure et Universite P. et M. Curie, Laboratoire de Botanique, 24 rue Lhomond, F-75231 Paris Cedex OS, France

Received December 3, 1982 . Accepted February 22, 1983

Summary

Cell wall peroxidases from poplar stem were investigated through parallel biochemical and histochemical techniques. Oxidation of syringaldazine was obtained only in lignifying cells. That it was a true peroxidasic activity was demonstrated by its absolute requirement for exoge­neous HzOz for in vitro assays. However syringaldazine oxidation could be obtained in situ in the absence of exogeneous hydrogen peroxide probably because of the production of HzOz by the lignifying cell walls themselves. Syringaldazine oxidase activity was strongly bound to the cell walls and fairly resistant to heat inactivation. It exhibited a very high affinity towards its substrate. The KM value was 100 to 1000 times higher with syringaldazine than with guaiacol.

Key words: Populus X euramericana, lignification, peroxidase, syringaldazine.

Introduction

Cell wall peroxidases have widely been suggested to be involved in the final step of lignification processes (Higuchi, 1957; Mader, 1976; Mader et a1., 1977). Peroxidases are thought to mediate the biogenesis of hydrogen peroxide (Elstner and Heupel, 1976; Gross, 1977) as well as the oxidative polymerisation of hydroxylated cinnamyl alcohols. Mader et a1. (1980) have reported that different isoperoxidases were involved in these two mechanisms. In 1978 Catesson et al. demonstrated the special affinity of peroxidases from lignifying cell walls for syringaldazine, a substrate introduced for histochemical tests by Harkin and Obst (1973). The specificity towards syringaldazine of at least one of the peroxidases involved in lignification was recently underlined by Fleuriet and Deloire (1982) in the course of their investiga­tions on wounded tomato fruits. These authors noticed that the lignification of the cells bordering the wounded zone was closely correlated with the apparition of a syringaldazine oxidase activity corresponding to a single isoenzyme.

In order to determine the specific properties of syringaldazine oxidase activity we undertook a comparative study of the peroxidases from young poplar stems in the differentiating xylem of which a syringaldazine-oxidase was previously localized. Pre-

Abbreviations list: DAB = 3,3'-diaminobenzidine, DDC = sodium dietyldithiocarbamate, SYR = syringaldazine.

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268 RENEE GOLDBERG, ANNE-MARIE CATESSON and YVETIE CZANlNSKI

liminary results were reported at the Second Cell Wall Meeting (Goldberg et ai., 1981). Further investigations on this material through parallel biochemical and his­tological approaches enable us to present here a more comprehensive picture of the characters of the syringaldazine oxidase activity.

Material and Methods Poplars (Populus x euramericana var. I. 214) were grown in a greenhouse from cuttings on

perlite and mineral solution. Samples were taken from the four intenl0des immediately below the first fully expanded leaf.

Histochemical techniques

Transverse sections were hand-cut from fresh material. Alternatively small segments of stem were fixed in buffered glutaraldehyde for 1 hand sectionned on a pre-cooled microtome. Sec­tions were incubated at room temperature in either the syringaldazine (SYR), diaminoben­zidine (DAB) or guaiacol medium. These respectively consisted of: 0.1 per cent alcoholic syrin­galdazine (pH 5) and 0.03 % H202 for 2 min (Catesson, 1980); 0.5 per cent 3,3'-diaminoben­zidine buffered at pH 5.0 or pH 9.0 and 0.03 % H202 for 10 min (Catesson, 1980); 48 nM guaiacol, 50 roM sodium cacodylate at pH 7.0 and 0.02 % H202 for 5 or 10 min (Fielding and Hall, 1978 a). Control experiments were performed by incubating sections in media lacking substrate or in complete media with either 0.01 M KCN or 0.02 M sodium diethyldithio­carbamate (DDC) added. In some instances sections were washed for severals hours in 1 M NaCI before incubation.

Progressive lignification was followed by staining some sections with HCI-phloroglucinol. Sections were directly examined under a light microscope. The amount of reaction product was visually estimated for each cell type and photographs taken. However good contrast between stained and unstained areas was difficult to obtain on a black and white film, especially with the pink-stained material incubated in the syringaldazine medium. After guaiacol incubation, the reaction was difficult to estimate because either the staining intensity was too weak or there were diffusion artefacts.

Preparation of enzyme fractions

The isolated stem segments were cleft longitudinally; medullar and cortical (epidermis, bark and most of the cortical parenchyma) regions were then discarded. Outer (fiber, phloem and cambium) and inner (xylem) remaining tissues were separated, ground in liquid nitrogen and lyophylized. Enzymatic extractions were performed according to Ridge and Osborne (1970) and Parish (1975) with an additional step: all enzymic extracts were reduced to 10 ml under nitrogen in an Amicon ultrafiltration cell (membrane PM 10), a process allowing also the desalt­ing of the extracts. Three fractions were solubilized: «cytoplasmic enzymes», hightly- or strongly-bound wall enzymes (the so called «ionically-» and «covalently-bound enzymes»). The absence of peroxidasic activity in the commercial (Sigma) pectinase and cellulase used in the experiments was checked.

Peroxidasic assays

Syringaldazine-H20 2 solutions were made up fresh for each experiments: 1.8 mg syringal­dazine was first dissolved in 500 1'1 hot methanol, the solution adjusted to 50 ml with H20 and 501'1 H 20 2 added. Thus the standard conditions for syringaldazine spectrophotometric assays were for a total volume of 4.1 ml: 0.2JLmol syringaldazine, 6.5 JLmol H 20 2, 200JLmol phosphate buffer pH 7.5 and 100 I' of the enzyme preparation. The oxidation of syringaldazine was

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Properties of syringaldazine oxidase 269

estimated by monitoring the absorbance at 530 nm. For guaiacol oxidation, the assay mixture consisted of 20/Lmol of guaiacol, 20/Lmol of H202, 200/Lmol of phosphate buffer pH 6.0 and 50/Ll of the enzyme preparation. The formation of tetraguaiacol was followed at 470 nm.

Heat stability

Heat inactivation was studied by pre-incubating sections in cacodylate buffer (pH 7.4) at 60, 70, 80, 90 or 100°C. The heat treatment was stopped after 2, 4, 6, 8 or 10 min by an immediate transfer of the sections from the hot bath to an iced one. For in vitro assays, heat treatments were performed on buffer solutions heated to a specified temperature before injections of diluted enzyme. When the defined incubation periods were attained, the solutions were rapidly cooled by immersing the test tubes in an ice water bath. Samples were then assayed as described above.

Results and Discussion

L Estimation o/syringaldazine oxidase activity

Lightly-bound wall enzymes were solubilized by NaCl and tested with syringal­dazine as substrate. A bright pink coloration developed only when hydrogen perox-

Fig. 1: Absorption spectrum of ox­idized syringaldazine. A: wavelength mnm.

0.60

0.40

0.20

00

Vi

0.0 B

600

Ill.

Fig. 2: Oxidation of syringaldazine by cell wall extracts. A: time course of the reaction estimated by absorbance at 530nm. B: initial velocity (L O. D. min-1 at 530nm) as a function of the enzyme volume (in /Ll) added to the assay mixture.

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270 RENEE GoLDBERG, ANNE-MARIE CATESSON and YVETTE CZANINSKI

ide was present in the assay mixture. Without enzyme extract or with a boiled extract, the solution containing syringaldazine and H202 remained colorless. The absorbance of oxidized syringaldazine was maximum at 530 nm as illustrated in Fig. 1. The coloration was unstable and decreased after nearly 30 minutes. Fur­thermore because of the low solubility of syringaldazine in aqueous solution irregu­larities appeared on the plot; nevertheless it was still possible to estimate graphically the initial velocity of the reaction (Fig. 2 A) and to check that this velocity was pro­portional to the amount of enzyme extract present in the assay (Fig. 2 B).

Fig. 3: Histochemical localization of peroxidasic activities on trans-sections of poplar stems with an actively dividing cambium. Incubations were carried out at room temperature in three different reaction media. A: syringaldazine + H202; B: DAB pH 5.0 + H20 2; C: DAB pH 5.0 without H202. Note that in A and C, only the lignifying xylem and fibers are stained. There is no reaction in young vessel walls during the enlargement stage (arrowheads). In B, the phloem and cortex are also stained with DAB. CZ: cambial zone; F: fibers, Ph: phloem; X: xylem. x 60.

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Properties of syringaldazine oxidase 271

II Localization of syringaldazine oxidase activity

1. Histochemical localization (Fig. 3).

Syringaldazine-oxidase activity was always exclusively observed in differentiating xylem cells or phloem fibers (Fig. 3 A). No reaction occured in cambial derivatives enlarging into xylem cells. Positive syringaldazine staining of the primary wall began only with the onset of secondary wall deposition. In older cells, both primary and secondary walls stained a bright pink gradually fading as xylem maturation was achieved. No staining was observed in fully mature xylem cells or phloem fibers. On the other hand, with DAB (Fig. 3 B) or even guaiacol as substrate, cell wall staining was observed in most stem tissues excepted the cambial initials and mature xylem or fibers. However DAB staining was weaker in lignifying secondary walls than in primary ones while reaction intensity with syringaldazine was similar for both. These results confirm the exclusive association of syringaldazine-oxidase with lig­nifying walls. It was demonstrated for the first time in Dianthus (Catesson et al., 1978)

and found afterwards in other materials (Perez-Rodriguez, 1979; Goldberg et al., 1981; Fleuriet and Deloire, 1982; Le, 1982). Furthermore at least two different perox­idasic activities followed one another in differentiating xylem cell walls. During the cell enlargement stage, the walls could be stained with DAB but did not react with syringaldazine. The following stage was characterized by the switching on of both lignification processes and syringaldazine oxidase activity.

Coloration with syringaldazine was so strong that no decrease in intensity could be seen after a several hour wash in NaCl, contrary to what was observed for sections incubated in DAB. However the washing medium reacted with syringaldazine in presence of H 202 showing that some extraction had actually taken place.

Table 1: KM values for syringaldazine and guaiacol oxidase activities. The constants were estimated from double reciprocal plots. The experiments were repeated three times; the differ­ences between the successive measures are given in the table.

Syringaldazine oxidase Guaiacol oxidase

Cell wall enzymes

1.25 ± 0.25· 10-5 M 3.4 ± 1.1 .10-3 M

«Cytoplasmic» enzymes

1.25 ± 0.15· 10-5 M 16.3 ±3.2 .10-3 M

When H 202 was omitted from the incubation medium a positive staining was still observed with syringaldazine. With DAB as substrate, a positive reaction also occured but, in this instance, it was localized only in lignifying cell walls (Fig. 3 C). There was no staining with guaiacol in these conditions. However with DAB as well as with syringaldazine, strong reactions were only obtained with protracted incuba­tion periods. For instance with syringaldazine as substrate a 10 min incubation fol­lowed by a 15 min wash in buffer was needed. Staining remained positive when incubations were run in the dark, showing that substrate photooxidation could be excluded.

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272 RENEE GOLDBERG, ANNE-MARIE CATESSON and YVETTE CZANINSKI

In view of the negative results obtained with substrates for polyphenoloxidases (Goldberg et aL, 1981) as well as the necessary presence of H202 to oxidize in vitro syringaldazine and DAB, it appears that in the in situ oxidation of these substrates could be due to the production of H202 by lignifying walls according to the scheme proposed by Gross et aL (1977). The amount of endogeneous H 202 was probably enough 0 allow reaction both with DAB and syringaldazine for which wall perox­idases have a high affinity. On the other hand, this amount might be insufficient to oxidize guaiacol for which enzyme affinity is lower (Table 1).

2. Biochemical data (Fig. 4)

Only solubilized cell wall activities were estimated. After NaCI and subsequent pectinase-cellulase treatments the cell walls still exhibited significant peroxidase activ­ities. The values reported in Figure 4 for cell wall activities are then underestimated since they represent only a part of the enzymes located in the cell walL Even when only solubilized activities were taken into account, about 80 % of the syringaldazine

s G

c

Fig. 4: Distribution of syringaldazine (S) and guaiacol oxidase (G) activities. Cytoplasmic and cell wall activities from outer (1) and inner (II) tissues were added and normalized to 100. Each kind of activity is expressed as a percent of the total activity solubilized from the tissues of the four investigated internodes. C: cytoplasmic activity; W: activity solubilized from the cell walls with a saline treatment (~) and a subsequent pectinase - cellulase treatment (Ii).

oxidase activity was present in the cell walls. The soluble extracts (= «cytoplasmic» extracts according to Ridge and Osborne, 1970) obtained from the outer tissues exhibited also a slight activity. They represent the enzyme activities non associated with cell walls (vacuolar, membrane-bound or other activities). On the other hand guaiacol oxidase activities were distributed quite differently between the cell wall and the cytoplasm.

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Properties of syringaldazine oxidase 273

Ill. Properties of syringaldazine oxidase activity

1. Kinetic measurements

Lineweaver Burk plots were constructed from initial velocity data and KM values determinated after linear regression analysis (Table 1). The affinity for syringaldazine was 102 to 103 times higher than the affinity for guaiacol and appeared very high com­pared with values reported for other phenolic compounds (Kim et al., 1980) or lignin precursors (Gibson and Liu, 1981) used as hydrogen donors. Cytoplasmic and cell wall extracts exhibited identical KM for syringaldazine. It may then be suggested that both activities result from the same molecule. On the contrary the affinities for guaiacol of cytoplasmic and cell wall extracts were different. We have previously reported (Goldberg et al., 1981) that both extracts contained several isoperoxidases able to react with guaiacol and benzidine. Therefore any changes in the relative amounts of isoenzymes in the two extracts may induce differences of the apparent KMvalues.

2. Inhibitory treatments

Two different chemical inhibitors were tested. KCN is known to act on the activ­ity of cell wall peroxidases. DOC is generally considered as a phenoloxidase inhibitor (Okun et al., 1970) but it inhibits also completely phloem peroxidases (Catesson, 1980).

a) In vivo sensitivity to KCN and DOC

In the cell walls of lignifying tissues, SYR- and DAB-oxidases were completely inhibited in the presence either of KCN or DOC. On the other hand, in the primary wall of very young vessels, during the enlargement stage, a noticeable peroxidasic activity was present even when DOC was added to the DAB medium. It may be due either to a DOC-resistant isoenzyme or to an incomplete inactivation (see also Gold­berg et aI., 1981).

b) In vitro sensitivity to KCN and DOC

Syringaldazine and guaiacol oxidase activities were completely suppressed by 10-4 M KCN. The kinetic pattern depicted in Fig. 5 via a Dixon plot (Dixon, 1953) indicates non-competitive inhibition of syringaldazine oxidase by cyanide. In our extracts guaiacol oxidase was also inhibited non competitively by cyanide whereas competitive inhibition was reported by Sessa and Anderson (1981) for soybean peroxidase. With both hydrogen donors the inhibition constant K; was about 3 /LM. This value is identical to the K; reported by Fridovich (1963) for horseradish peroxidase with guaiacol as hydrogen donor but it is higher than the inhibition constant obtained by Sessa and Anderson (1981) for soybean peroxidase with guaiacol. Populus peroxidases are then apparently less sensitive than soybean peroxidases to inhibition with cyanide.

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274 RENEE GOLDBERG, ANNE-MARIE CATESSON and YVETTE CZANINSKI

A

6

8 16 o 8 16 (KCN)10~

Fig. 5: Effect of cyanide on oxidation of syringaldazine (A) and guaiacol (B). A and B1: oxida­tion bl a cell wall extract. B2 oxidation by a cytoplasmic extract. Substrate concentration: 4· 10- M Ci:r --*) and 1· 10-5 M (_-II) for syringaldazine; 5· 10-3 M (0--0) and l'10-3 M (e--e) for guaiacol.

B

0.10 0.0

1 20 40 5 min. 10 min.

~ig. 6: Tin:e course. of the inhibiting effect c:f DDC on cell wall feroxidase activities. A: oxida­tIOn of syrmgaldazme, 0: no DDC, 1: l' 10 5 M DDC, 2: 2· 10- M DDC 3: 4· 10-5 M DDC B: oxidation of guaiacol, 0: no DDC, 1: 2' 10-5 M DDC, 2: 1· 10-4 M DDC, 3: 2· 10-4 M DDC

DDC could also suppress both activities but its inhibiting effect showed some spe­cial features (Fig. 6). When DDC concentration in the assay mixture exceded 10-5 M (with syringaldazine) or 10-4 M (with guaiacol), a lag time occured, the duration of which increased with inhibitor concentration. This delay may represent the time necessary for a partial destruction of an inactive DDC-substrate complex (it can indeed be suppressed when the substrate and the inhibitor are incubated together 20 min before the enzymatic extract is added). In addition to this lag time, DDC induced also a decrease of the oxidation rate. Castillo et al. (1981) have recently reported similar observations with Pelargonium extracts which promoted both a lag time and an inhibition of the oxidation of guaiacol by horseradish peroxidase.

3. Heat inactivation

Plant peroxidases are in general very stable to thermal inactivation (Reed, 1975). The destruction time for peroxidases varies according to the substrate used (Nebesky et al., 1950), ionic strength of the donor (Wilder, 1962) and molecular species (Sriva­stava and Van Huystee, 1977; Nessel and Mader, 1977). Heat inactivation assays were

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Properties of syringaldazine oxidase 275

performed either on sections (<<in vivo» experiments) or on extracted enzymes (<<in vitro» experiments).

a) In vivo inactivation

Experiments performed on freshly-cut sections showed a marked resistance of wall peroxidases towards heat inactivation. Results obtained after a 10 min incubation at different temperatures are recorded on Table 2. Secondary wall activities appeared to be more sensitive than activities from primary walls. While the first ones generally disappeared following a 10 min incubation at 80°C, the second ones needed to be heated at 90 °C to be destroyed. Syringaldazine oxidase activity from vessel primary walls was the most resistant. Staining of these walls was still obtained after 4 min at 100°C. However the strongest resistance to heat inactivation was observed for DAB oxidase in xylem primary walls (Table 2) and sieve tube walls (Catesson, 1980).

Table 2: Heat inactivation of peroxidasic activities in differentiating cell walls as observed on sections of fresh material.

Substrate 20/60° 70° 80° 90°C

enlarging vessels primary walls Syringaldazine lignifying vessels [ primary walls + +

secondary walls + +

enlarging vessels primary walls + + DABpH9 lignifying vessels [ primary walls + +

secondary walls +/-

enlarging vessels primary walls + + + DAB pH 5 lignifying vessels [ primary walls + + +

secondary walls +

In all instances, heat inactivation was easier to obtain with glutaraldehyde fixed material in which all xylem peroxidases were inactivated by a short incubation (6 min) at 80°C.

The differences observed between primary and secondary walls and between cells may be due either to local variations of the level of peroxidase activities or to the pres­ence of isoenzymes with different heat resistances.

b) In vitro inactivation

A thermal activation of cell wall syringaldazine oxidase activity occured during the first two minutes of a thermal treatment at 50°C. Then thermal inactivation occured and it followed the typical kinetic pattern of a single first-order reaction (Fig. 7 A). With guaiacol as substrate (Fig. 7B), at least two first order reactions could be observ­ed as previously reported for peroxidase in sweet corn (Yamamoto et al., 1961). Therefore two groups of isoperoxidases with different heat stabilities were probably

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276 RENEE GOLDBERG, ANNE-MARIE CATESSON and YVETTE CZANINSKI

7fP

... u cl

2 .

... 1.0 c: Q)

~ Q) 0.

'" 3

10 minutes

B

10

Fig. 7: Effects of heat on the stability of syringaldazine (A) and guaiacol (B) oxidase activities as a function of time. Vertical axis: percent of the in­itial activity expressed in a loga­rithmic scale.

present in the extracts, each being inactivated according to a first order reaction. Each straight line of the inactivation curve was then considered separately in studying the effect of temperature. The D values (decimal reduction time for inactivation of perox­idase activity) were calculated for each first order reaction and the thermal destruc­tion coefficients (z) were obtained graphically by plotting the D values (on a loga­rithmic scale) against the temperatures; the results are reported in Table 3. Cyto­plasmic and cell wall guaiacol oxidase activities from inner and outer tissues presented different thermal destruction coefficients probably due to changes in the relative amounts of isoenzymes existing in the different fractions.

Table 3: Thermal denaturation coefficient (z) of peroxidase activities from outer (I) and inner (II) tissues.

Cell wall activities

Syringaldazine oxidase 29°C Guaiacol oxidase

- thermolabile - thermoresistant

23°C 18 °C

II

41°C 21°C

Cytoplasmic activities

II

28°C 15 °C

It appears then noteworthy that enzymes linked to wall constituants present a very high resistance to heat denaturation. Similar observations were made on Nico­tiana tabacum sections (ie, 1982). Furthermore, when the wall enzymes are solu­bilized they become more sensitive to thermal inactivation.

Conclusion

The results presented here confirm the specificity of syringaldazine as a substrate for peroxidases involved in lignification processes since syringaldazine oxidase activ­ity was closely restricted to lignifying cells. This may be due to the close relationship

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Properties of syringaldazine oxidase 277

existing between this compound and lignin precursors. That syringaldazine oxidation was also obtained with horseradish peroxidase (Kaplan, 1979) may be explained by the heterogeneity of the commercial product, a mixture of enzymes from different tissues, including differentiating xylem. This apparent discrepancy could be resolved by a parallel biochemical and histochemical study such as the work done by Fielding and Hall (1978 a and b) or the present one. Such approaches are very scarce and their advantages must be underlined.

One of the most striking properties of syringaldazine oxidase activity is its strong affinity towards the substrate. This activity is also fairly resistant to heat inactivation. The enzyme appears to be strongly bound to the cell walls. A similar result was obtained by Fleuriet and Deloire (1982) on wounded tomato fruits. In this instance, the authors were able to demonstrate that a single isoenzyme with syringaldazine oxidase activity appeared during the lignification characterizing the wound-healing processes. The induction after wounding of a specific isoenzyme associated with lig­nification was postulated earlier by Rhodes and Wooltorton (1978).

These results as well as our own observations pose again the question of peroxidase regulation (Van Huystee and Cairns, 1980). The onset of a syringaldazine activity concommitant to lignification could be accounted for either by de novo synthesis or post-synthesis alterations of enzyme molecules or the unmasking of a pre-existing iso­enzyme (Legrand et aI., 1976; Castillo et aI., 1981).

Acknowledgements

This work was supported by CNRS (LA 311) and DGRST grants. We are very grateful to Dr. Bernard Monties for valuable suggestions and helpful discussions.

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