Peroxidases in Acetabularia: their possible role in development

Differentiation

Differentiation (1984) 27: 175-181

$;

Springer-Verlag 1984

Peroxidases in Acetabularia :their possible role in development Th6rhe Vanden Driessche', Claire Kevers', Thomas Gaspar and Roland Caubergs

' Dkpartement de Biologie molkulaire, Universitt Libre de Bruxelles, rue des Chevaux 67, B-1640 Rhode-St-Genese, Belgium Institut de Botanique, B22, Universite de LiBge-Sart Tilman, B-4000 Liege, Belgium Rijksuniversitair Centrum, Groenenborgerlaan, 171, B-2020 Antwerpen, Belgium

Abstract. Crude enzymatic extracts from Acetabularia exhibit very low peroxidase activity after a lag period. Starch gel electrophoresis of extracts from growing algae shows a single, extremely anodic band. Extracts of small, slowgrowing or cap-bearing algae, which do not grow any more, do not exhibit any peroxidase band. Cytochemical staining with benzidine reveals changes in both the quantity and distribution of peroxidase along the polarized Acetabularia cell. The homogenous staining of small algae becomes distributed along a negative apico-basal gradient when the algae initiate their rapid growth phase. This polarized pattern is repeated on the hair whorls. A similar developmental sequence directs cap growth, with an initial intense staining reaction of the primordium, which later leaves only the corona inferior stained blue. Finally, the Acetabularia cell remains slightly blue at the edges of the rhizoidal outgrowths and cap rays. Crude extracts of Acetabularia induce a lag in standard horseradish peroxidase (HRP) activity. The inhibitor is always present in small and growing algae; it is sometimes absent or less active in cap-bearing algae. In no case does it change the kinetics of the HRP reaction with guaiacol. The lag is completely suppressed by pretreatment with either H,O, or ascorbate oxidase. The changes in peroxidase activity, correlated with developmental stage and according to a polarized gradient, suggest that the enzyme could be involved in some way in the control of morphogenesis in Acetabularia. An inhibitor of peroxidase activity, which disappears as the cap matures, might, in turn, exert a regulatory function.

Introduction Growth and cap morphogenesis are two major developmental processes in the unicellular giant alga Acetabularia. Both processes are under the direct control of blue light [8], the former requiring less energy than the latter [9]. A blue-light receptor evidenced by a blue-light-induced absorption change (LIAC) has recently been shown in a plasmalemmaenriched fraction from Acetabularia [6]. Since this receptor activity is based upon oxidoreduction [28], and indoleacetic acid (IAA) is likely to take part in the regulation of Acetabularia development [30], peroxidases might be candidates as mediators of some step between signal reception and transduced effects. The present study describes experiments in which peroxidase activity is demonstrated in Acetabularia, discusses its

possible localization, demonstrates the existence of an inhibitor of peroxidase activity and presents information about its nature. Changes in peroxidase activity were related with the developmental stage. Methods Culture of the algae Acetabularia mediterranea was cultivated under standard laboratory conditions [18]. Since the cultures were not axenic, the algae in some experiments were treated overnight once or twice (at 1-weekinterval) with a mixture of antibiotics. The last treatment took place 2 days before the experiment. The antibiotics mixture (modified after [14]) contained 100 mg penicillin, 100 mg streptomycin, 20 mg neomycin and 20,000 units of nystatin. The numbers of algae used (for samples of approximately equal fresh weight) for the study of peroxidase activity at different developmental stages were very different, since the average weight of 100 algae is 27 mg for 5- to 10-mmlong algae, 370mg for 20- to 25-mm-long algae (before cap formation), and 700 mg for cap-bearing algae. Preparation and assay of peroxidases A . Spectrophotometric determination and starch gel electrophoresis. Crude extracts were prepared from 600 mg to 1 g fresh Acetabularia ground in 0.5-1.5 ml phosphate buffer (0.66 M, pH 6.1) with a Mikro-Dismembrator (Braun; 15 and 25 s) and centrifuged at 1,000 g for 5 min. Guaiacol peroxidase activity was assayed spectrophotometrically (Pye Unicam SP8-100) by measuring the increase in absorbancy at 420 nm (incubation mixture, 7.8 ml phosphate buffer, pH 6.1, 1 ml H,O,, 0.2 volume and 1 ml guaiacol solution, 1% and 0.2 ml extract). The peroxidase isozyme pattern was determined by vertical starch gel electrophoresis and staining with benzidine, according to Darimont and Gaspar [lo]. In some preliminary assays and in Experiments 1 and 2 (Table l), Polyclar (PVP) was added to the grinding buffer. B. Cytochemical method. The reaction of Madelung was carried out in the presence of H,O,, as has been described by Langeron [17] and Pearse [26], with benzidine (Fluka; 40 mg dissolved in 100 ml acetic acid acetate buffer, 0.2 M, pH 5.0) in the presence of 5% H,O,.

176 Table 1. Peroxidase band developed by extracts of Acetubuluriu of different dcvelopmental stages Expt.

Treatment with antibiotics

Pretreatment with

Length of morphological stage

"202

5-10 mm

Rapid growth phase'

k20 mm

f12mm

+ + +

1 2 3

-

4 a

+ + +

No band

1 anodic

-

Cap-bearing

I anodic 1 anodic 1 anodic No band

1 anodic

No band

In our culture conditions, thc rapid growth phase begins when the algae about 1 2 m m in length (expt. 1 ) and finishes when they arc about 2&25 mm in length (expt. 4). Thc algae were at the intermediary stage in the other experiments

Testing a peroxiduse inhibitor in Acetabularia Since enzymatic extracts (prepared as described) exhibited, after a lag period, very low activity (see Results and [5]), the possibility of the presence of an inhibitor was raised. Variable amounts of extracts were added to reference horseradish peroxidase (HRP; Boehringer) in the presence of guaiacol. The algae or their extracts were pretreated (or not) with H,O, or with ascorbic oxidase (10 units for 0.5 ml extract; Boehringer). Plasmalemma-enrichedfraction This fraction was obtained from 8-12 g fresh weight by the two aqueous phase system separation technique which has been described by Widell and Larsson [31].

Essentially the same result was obtained on starch gel electrophoresis: a single band of anodic peroxidase was present (Table 1, expt. 3). Comparison between algae at different developmental stages In two experiments, algae of different developmental stages were studied. The stages considered were: in Experiment 4, small algae of 5-10 mm in length, algae in their rapid growth phase and cap-bearing algae; in Experiment 1, the latter two stages. In all experiments, extracts from the 20-mm-long algae had a band corresponding to acid peroxidases. No band was found when the extracts were prepared from either small or cap-bearing algae. Plasmalemma enriched fractions

Activity

of crude extracts

Crude extracts exhibit very low peroxidase activity after a lag period of about 1 min. The use of PVP in the preparation of the extract reduces the lag period and enhances enzyme activity [5]. Starch gel electrophoresis: effect of H 2 0 , pretreatment One band is detectable using benzidine as reagent: it appeared at the extreme anodic side and did not correspond to any of the numerous bands shown by HRP. The gels did not develop a peroxidase band after staining with guaiacol. In the first experiments, the algae were not pretreated with H,O,, and, the benzidine-revealed band was apparent in the presence of PVP in some experiments (Table 1, expts. 1, 2) but not in all (not shown). In contrast, when the Acetabuluria had been pretreated with H,O,, the peroxidase band was always seen on the gels (Table 1 expts. 3 4 ) . Since the cultures were not axenic and bacteria may contain peroxidases, the first experiments were carried out on algae that had been submitted to two treatment with antibiotics at 1-week interval (as in the case for Table 1, expts. 1, 2). It was then decided to compare the results obtained with algae treated and untreated with antibiotics. A large sample of algae at the end of their growing phase was separated in two: half of them were treated with the antibiotics, whereas the others were not (Table 1 expt. 3).

In two experiments, one with untreated algae and one with algae treated with H,O,, plasmalemma fractions were assayed for their peroxidase content, as evaluated by starch gel electrophoresis. In no case could a band which reacted with benzidine be detected. Cytochernical study and polar distribution of peroxidases

Acetabularia cells stained very differently with the cytochemical peroxidase reaction according to their developmental stage. Small algae (5-10 mm long) stained uniformly blue with the peroxidase-benzidine reagent. No gradient was readily apparent on the stalk. The stain corresponded to coloured spots of the cell wall. However, the hair whorls (typically one corona at this stage) had only their basal article deeply coloured ; the second article was slightly coloured, and the apical article was unstained (Fig. 1). In 12-mm-long Acetabularia, the stalk was entirely stained with the reagent, but a negative apico basal gradient was now apparent (Fig. 1). At this stage, the algae initiate their rapid growth phase. The whole rhizoid was as deeply stained as the basal part of the stalk. Similarly, apical hair whorls displayed differences in staining intensity according to the article number. Remnants of older hairs were very lightly stained. At this stage, the algae are more vacuolated than previously, and H 2 0 , treatment resulted in a contraction of the cytoplasm, leaving some empty portions in the stalk. This made the stained spots of the cell wall more

117

1

2

3

4

Fig. 1. Schematic drawing. Left: a small, non-rapidly growing Acetabularia; the stalk is entirely stained (only the last article of the hair whorl is unstained.) Right: an alga in its growth phase; it repeatedly displays an alternate pattern of stained non-growing and unstained growing regions along the stalk and with the article number of the hairs. The colour is due to the blue product of the benzidine-peroxidase reaction Fig. 2. Stained portion of the rhizoid ( R ) and basal part of an alga in its growing phase. It shows the retraction of the cytoplasm (arrow) which leaves the coloured cell wall. The stain is concentrated in spots Fig. 3. Apical part of an alga in which the cap has been recently initiated. It stains deep blue

Fig. 4. Cap with slightly stained edges

178 Table 2. Lag (in seconds) before HRP reacts with guaiacol in the presence of variable amounts (in milliliters) of Acetabularia extracts'

of different developmental stages (Acetabularia pexoxidase does not react with guaiacol) Small algae

Extract

Algae in their growing phase

lag

0.1 0.2

Expt. 1

Expt. 2 Expt. 3 b

Expt. 4'

Treated with antibiotics Extract lag 21 .o 57.5

Cap-bearing algae Untreated with antibiotics Extract lag 0.2 0.4

17.5 38.5

19.5 65

0.2 0.4

1-2 16

0.2 0.4

0 0

0.1 0.2 0.3

19.0 39.5 54

0.1 0.2 0.4

55 87.5 230

0.1 0.2 0.4

45 65

0.2

45 105

0.2

90

0.4 0.6

190 305

0.2

50 130 190

0.4 0.6

0.2 0.4 0.6

0.1 0.2 0.4

a

lag

0.4

0.2

0.4 0.6

Exp. 5

Extract

3 6 11.5

0.1 0.2 0.4

220

140

55 110 I50

3 6 10.5

0.1 0.2 0.4

0 4.5 9

Although several assays were carried out, only the first result is given, since inhibition decreases with time (see text) Extracts of algae still in their growing phase, but reaching the end of this phase. See also Fig. 5 The growing algae were also at the end of the growing phase

Fig. 5. Kinetics of the HRP-catalyzed oxidation of guaiacol recorded at 470 nm in the presence of increasing volumes of Acetabularia extract. From left to right: three sets of curves corresponding to extracts prepared respectively from small, rapidly growing and cap-bearing

AcetabuIaria. The slope of the curve is unaffected, but there is an increasing lag before the start of the reaction. The beginning of the recording on the abscissa corresponds to the moment at which HRP is added to the reaction mixture, which is then rapidly vortexed and injected into the spectrophotometer cuvette. The zero time of the three assays in each experiment are at approximately the same point. The horizontal bar represents 5 sec. C,control; 0.1, 0.2 and 0.4, the extract volume (in milliliters) added to comparable reaction mixtures. The ordinate is the guaiacol oxidation due to the HRP activity

apparent (Fig. 2). An accurate estimation of the width and intensity of the stained spots has not been attempted; it is possible that both vary with the developmental stage of the algae. When a primordium was formed, it stained deeply blue with the benzidine reagent (Fig. 3), indicating that a high peroxidase concentration is locally reached in the apical part. However, the general apicobasal gradient of the stalk remained evident. In the developing young cap, the rays were densely stained. Stalks of Acetabularia bearing a welldeveloped cap were very lightly stained; the apicobasal gradient remained visible. The rhizoid stained blue more strongly than the stalk, but the density of the staining decreased and may be localized only at the extreme end of

the rhizoidal outgrowths. The caps (of almost full size) were slightly stained on the Corona inferior and more clearly stained at the edge of the cap rays (Fig. 4). The initial dense benzidine coloration of the rays had faded away by this stage. Inhibitory effect of Acetabularia crude extract on horseradish peroxidase acrivity

Five series of exeriments, four of them comprising three different developmental stages, were carried out (Table 2). The addition of an extract prepared from small or growing algae always induced a lag in the reaction of HRP with guaiacol (which does not immediately react with Acetabu-

179

laria peroxidase; see Methods). When the extract was prepared from capbearing algae, it induced a lag in two out of four experiments. (The two others displayed no inhibitory effect.) Above a minimum volume (50 pl to 0.2 ml, depending on the experiment), a fraction of extract prepared from 1 g fresh weight in 1 ml buffer induced a lag approximately proportional to the volume added to the reaction mixture for standard HRP activity measurment (Fig. 5). The quantitative value of the lag seems to be typical of the sample (Table 2, expt. 4). The inhibitory effect, whatever the developmental stage, is only a lag which, after a certain time, allows the reaction to proceed with the same kinetics as in the control (Fig. 5). In experiment 4 (Table 2), however, with the relatively hight inhibitory concentration brought about in the reaction mixture by 0.4 ml Acetabularia extract, a change in the slope of the curve was observed. Pretreatment with antibiotics did not change the kinetics of the reaction. The quantitative differences between “ sterile” and “non-sterile” Acetabularia were not further investigated. Towarh the characterization of the inhibitor

The inhibitor is labile, and its activity decreases after 0.5 h and disappears when it is left at room temperature for 3 h. It is thermolabile (85’ C for 5 min). Algae pretreated or not with H,O, were compared. The untreated algae were those of Experiment 1 (Table 2; both were either treated with antibiotics or untreated) and Experiment 4 (Table 2). After pretreatment with H,O,, the extracts no longer displayed an inhibitory effect. In a parallel study, it has been found that ascorbic acid is present in Acetabularia [S]. Ascorbic acid alone is capable of inducing the lag in HRP activity in the standard reaction mixture, since treatment of the extract with ascorbic acid oxidase suppresses the lag (see Fig. 2 of [4]). Aliquots of algae from the three developmental stages of Experiment 5 (Table 2) were similarly submitted to treatment with ascorbic acid oxidase. This resulted in the complete disappearance of the lag.

Discussion

The existence of peroxidase activity in Acetabularia, suggested by spectrophotometry of crude extracts, was ascertained both by gel electrophoresis (at least during the rapid growth phase) and cytochemistry. The obviously higher activity of peroxidase during the growing phase suggests that it might be involved in Acetabularia developmental control. This would nicely tit the data of Kof and Kefeli [21] who have noticed a larger amount of IAA in capbearing algae compared with growing ones. Implicit in the reasoning, however, is the involvement of peroxidase activity in IAA catabolism, a point which will be discussed together with the electrophoretic quality of the enzyme. Cytochemical examination always gave positive results, thereby demonstrating peroxidase activity in all cases, although it was very different in intensity and distribution according to the developmental stages. The fresh weights used for the electrophoretic determination were always approximately the same, but the ratio between cytoplasm, vacuole and membranes may have been different, and this may explain why no electrophoretic band could be seen

when extracts of small algae were used. Inhibitors may also mask peroxidase activity [12]. Clearly, peroxidase activity is developmentallyregulated in Acetabularia and changes both in quantity (the content being higher in growing algae than in smaller or mature algae) and distribution. Our cytochemical study of peroxidase localization suggests that the enzyme is located in the cell wall. When plasmalemma-enriched fractions were assayed in gels, no peroxidase band could be detected, regardless of whether or not the Acetabularia were submitted to pretreatment with H,O,. Special care was always taken in order to avoid loss of enzymes, and Ca2+ was included in the extraction medium. However, low-speed centrifigation of Acetabularia treated with KCl under vacuum (in order to detach the enzymes located in the periplasm or loosely bound to the external surface of the plasmalemma) gives a solution devoid of peroxidase activity, whereas the algae themselves retain such activity [5]. This is in contradiction with the preceeding conclusion. It should also be recalled that the staining of material including surfaces can produce artefacts, with some molecules becoming bound [l]. Thus it cannot be decided, as yet, whether or not peroxidase is associated with the external face of the plasmalemma. Concerning the electrophoretic characterization of peroxidase activity, it is often assumed [12] that, in higher plants, cathodic peroxidases are IAA oxidases and, consequently, are probably involved in in vivo IAA metabolism. However, anodic enzymes have been shown to have only a negligible IAA-oxidase activity in vitro, when purified, in the absence of H,O,, in a single species (Lycopersicm [22]). Moreover, evidence for two IAA degradation pathways has recently been presented [15]. Finally, compound I (an enzyme intermediate with a higher oxidation number than peroxidase) may be involved in IAA oxidation [13]. It should be borne in mind, however, that some basic peroxidase activity might have escaped detection if it were complexed with an inhibitor. An IAA-like substance has been demonstrated in Acetabularia [30], but its exact nature is not yet known. Its very low content in the alga, together with the difficulty of producing large quantities of algae of a comparable physiological stage, makes it very difficult to purify the growth regulator and study its evolution in the course of development. A higher content of IAA-like substance in capdeveloping algae, as compared with growing ones, has been reported [21]. Indirect arguments [29] suggest that the content favourable to growth and the favourable conformational state are associated with the alga apex during the rapid growth phase.’ It is of special interest in this context to follow the evolution of the peroxidase activity at the cellular level. In the course of development, it is subjected to important changes. Small non-growing algae uniformly stain blue with the peroxidase-benzidine reagent. A negative apico-basal gradient appears with cytochemical staining when the algae are actively growing, leaving the growing apex unstained. Similarly, the distal and still-growing articles of the hairs are unstained in contrast to the proximal ones. The young primordium is deeply coloured, but the cap rays are not. Clearly, both the developmental sequence of events and the polarity in Acetabularia suggest that a high peroxidase activity corresponds to non-growing periods or regions. Moreover, at the end of the developmental cycle, only the tip of the cap

180

pea shoots, spinach and wheat leaves, and in the unicellular rays and the edges of the rhizoidal outgrowths are lightly alga Eremosphaera viridus [20]. The oxidation of ascorbate stained, as if peroxidase activity reflected a “stop growing” to dehydroascorbate uses H,O,, which is converted to H,O signal. and gives the homogenate a catalase-like activity. InterestThe lignification of the higher-plant cell wall depends ingly, when H,O, is used as a co-factor of IAA oxidation on the presence of acidic (most probably cell-wall ionically by the Prunus peroxidase functioning as an IAA oxidase, bound) peroxidases which oxidize phenolic compounds to the same catalase-like activity is expressed. Such a catalaseradicals which will be added to the growing lignin polymer like activity agrees with the high catalase activity observed [12]. There is no lignification process in Acetabularia, but in our unpublished experiments and by J. Brachet and a thickening of the basal part of the mannose-containing H. Chantrenne (personal communication) in Acetabularia. stalk occurs in the course of development [ll, 331. Since However, the kinetics of the ‘‘catalase activity”, examined the basal part displays the highest peroxidase content, these by classical methods, cannot be explained by the presence enzymes may be involved in a thickening process which of a catalase enzyme (unpublished results with M.F. Couprovides a mechanical reinforcement of the wall. In support of this suggestion is the fact that the very young cap stains mans). blue in the region that serves as an attachment zone between Acknowledgements. We are grateful to Dr. H. Alexandre for the the stalk and cap. photographs of the benzidine-stained algae. We thank Mr. L. LaClearly, the elucidation of the possible role of peroxiteur for cultivating the large quantities of Acetabularia required dases in the metabolism of auxin-like substances and thickin this research and Mr. D. Franckx for the preparation of the ening of the cell wall calls for future research. final photographs of the figures. We also thank Miss M. Bouchet Peroxidases have been observed, mainly at the ultrafor technical help in the first experiments. structural level, by Menzel, both in the stalk [23] and the cap [24]. Menzel used classical fixation and postfixation methods which probably released the peroxidase observed References in his research from the cell wall. The absence of pretreat1. Barbotin JN, Thomas D (1 974) Electron microscopic and kinetment with H,O, might also have played a part in the differic studies dealing with an artificial membrane. Application to ence between the results. 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Received March 1984 / Accepted in revised form April 1984