Studies in the brown alga Ectocarpus in culture: ultrastructural localization of enzymic activities

LUIS OLIVEIRA~ AND THANA BISALPUTRA~ Departrtlent of Botcrtzy, University qfBritish Coirtmbirr, ~~~~~~~~~~~, British Colrrnlbirr V6T I W.5

Received November 13, 19744

OLIVEIRA, L., and T. BISALPUTRA. 1976. Studies in the brown alga Ectoc~crrprrs in culture: ultrastructural localization of enzymic activities. Can. J. Bot. 54: 913-922.

The localization of enzymic activities (catalase. peroxidase. adenosinetriphosphatase) in veg- etative cells of Ectocarplrs sporophytes was carried out at the ultrastructural level.

Catalase (EC I . 1 1.1.6) was only found in organelles recognizable as rnicrobodies. Peroxidase (EC 1.11.1.7) was present in the cell wall, but the deposition of reaction product was also observed in the perinuclear space, endoplasmic reticulum, dictyosomes, and paramural space. Adenosinetriphosphatase (ATPase) (EC 3.6.1.3) was found associated with mitochondria, thylakoids, and the plasma membrane.

OLIVEIRA, L., et T. BISALPUTRA. 1976. Studies in the brown alga Ecfocarpus in culture: ultrastructural localization of enzymic activities. Can. J. Bot. 54: 913-922.

L'activite de la catalase, de la peroxydase et de I'adenosine-triphosphatase (ATPase) dans les cellules vegetatives de sporophytes d'Ectocarpus a ete localisee au niveau ultrastructural.

La catalase (EC 1.1 1.1.6) se rencontre uniquement dans les organites identifies des 'mic- robodies.' La peroxydase (EC 1.11.1.7) est presente dans la paroi cellulaire mais les produits de reaction ont Cgalernent etC observes dans I'espace perinucleaire, le reticulum endoplasmique, les dictyosornes et I'espace paramural. L'ATPase (EC 3.6.1.3) est associee aux mitochondries, aux thylakoi'des et a la membrane plasrnique.

[Traduit par le journal]

Introduction In previous communications the cellular

morphology of young, aging, and necrotic vegetative cells of Ectocarpus was studied in de- tail (Oliveira and Bisalputra 1973, 1976a, 19766). By conventional electron microscopical methods the interpretation of ultrastructural changes as- sociated with the complex aging process is ex- tremely difficult. Cytochemical studies at the electron microscope level would greatly assist in the better understanding of this process. The localization of various enzymes in young, aging, and senescent cells would provide useful infor- mation on the cellular activities of these cells so that they could be correlated with the ultra- structural data obtained in previous work. Re- sults regarding the localization of acid phos- phatase were reported elsewhere (Oliveira and Bisalputra 1976a, 19766). In this work, the following enzyme systems will be studied:

- 'Project supported by National Research Council of

Canada grant no. A2288. 2 0 n leave from the Institute of Botany, The University

of Porto, Portugal. 3Address correspondence to this author. 4Revised manuscript received January 21, 1976.

catalase (EC 1.1 1.1.6), peroxidase (EC 1.1 1.1.7), and adenosinetriphosphatase (ATPase) (EC 3.6.1.3).

Materials and Methods Ectocarpus sp. was obtained from the culture collection

of algae, Indiana University (culture LB 1433). The cul- ture conditions were as reported in a former paper and so was the standard handling of the material for electron microscopic studies (Oliveira and Bisalputra 1973).

For the ultrastructural localization of enzymic activi- ties, the material was briefly fixed (20 min, at room tem- perature) with a 4 z (vlv) solution of purified glutaralde- hyde (Electron Microscope Sciences, Fort Washington, D.C.) in 0.1 M sodium cacodylate buffer (pH 7.0), to which sucrose was added to make a final concentration of 7 . 5 z (w/v). After a brief rinsing, the material was incubated in the appropriate incubation media in- dicated below. It was then quickly rinsed and refixed for a further 2-h period with glutaraldehyde. The material was then postfixed in Os04, dehydrated, and embedded as in normal procedures.

Catalase The composition of the incubation medium was 10 mg

diaminobenzidene tetrahydrochloride (DAB), 5 ml of 0.05 M propanediol buffer (pH 10.0), and 1 ml of 3 z H 2 0 2 . The final pH of the incubation medium was ad- justed to 9.0 (Fredrick and Newcomb 1969), and the in- cubation was carried out at 37 "C for 1 h. Control ma- terial was incubated without H 2 0 2 or preincubated for

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30 min with propanediol buffer containing 0.02 M of aminotriazole. In another test, the material was prein- cubated in propanediol buffer containing 0.01 M KCN, followed by incubation in standard DAB medium con- taining 0.01 M KCN. The material was also boiled for 5 min in propanediol buffer before incubation in standard DAB medium.

Peroxidase Media for incubation and controls were as indicated

for catalase except that the final pH was adjusted to 7.6. Incubations were carried out at room temperature for 1 h.

Adenosinetriphosphatase (ATPase) Three standard media (adapted from Henrikson (1971))

were used in studying ATPase localization. Medium A: Tris5-maleate, 80 m M (pH 7.2); I0 m M MgS0,; 3 m M ATP; 3 m M lead nitrate; 220 m M sucrose. Medium B: Tris5-maleate, 80 m M (pH 7.2); 10 m M MgS0,; 5 m M ATP; 100 m M NaCI; 30 m M KCl; 3 m M lead nitrate; 220 m M sucrose. Medium C : Similar to medium B but without MgSO,.

Incubations were carried out for 1 h at 37 "C. Control material was incubated in standard media without ATP or boiled in standard media before second fixation in glu- taraldehyde.

Stained (lead citrate, Reynolds (1963)) and unstained sections, cut on a Reichert OMU3 ultramicrotome equip- ped with a DuPont diamond knife, were viewed with a Zeiss EM 9A electron microscope.

Results Catalase

In young cells, catalase activity was detected in single membrane-bound microbody-like struc- tures (Figs. 1, 2). Such an organelle possesses a fine granular matrix (Figs. 3 and 6). As a rule, it is round or slightly ovoid in profile and measures about 0.2-0.4 microns (p) in diameter. In some cases, a non-crystalline core is also apparent (Fig. 6, arrow). The reaction product is located in the peripheral zone of the organelle, but when

the core is present, it seems to be preferentially associated with this structure (Fig. 2, arrow).

Control preparations of aminotriazole-treated material showed no deposition of reaction pro- duct in these single-membrane-bound organel- les (Fig. 3). However, both in test conditions (Fig. 4) and in aminotriazole-treated material (Fig. 3), deposits of reaction products are always found within the mitochondria. In KCN-treated material, the deposition of reaction products in microbody-like organelles is greatly reduced. Material incubated without hydrogen peroxide or boiled for 5 min in propanediol buffer before incubation in the standard DAB medium gives negative reaction.

In aging cells, microbody-like structures were also detected. One microbody closely associated with a mitochondrion is shown in Fig. 5 (arrow). We failed, however, to detect microbodies in the final stages of aging.

The chloroplast endoplasmic reticulum (ER), particularly the part surrounding the pyrenoid, also shows deposition of reduced products (Fig. I), but similar results are observed in amitrol- control preparations (Fig. 3). In this case, de- position of reaction product is most likely to re- sult from a reaction between DAB and osmium or other enzyme activities with affinity for DAB. It should be noted that chloroplast ER outside of the pyrenoidal region shows negative reaction (Figs. 3, 4).

Associations of microbodies and mitochondria are of relatively common occurrence (Figs. 1, 2, 3, and 5).

Peroxidase In young cells peroxidase activity was found

associated with the outer portion of the cell wall (Figs. 9, 10) and the paramural space (Fig. 10,

.. . . . . . , . . . . . . . . . I.. . _ . . . . , . . - . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . .

. . . . . . ~ . . .. . . . ...,....

KEY OF SYMBOLS: Ch, chloroplast; cw, cell wall; D, dictyosome; rr7, microbody; M, mitochondrion (a); N, nucleus; pm, paramural space; Py, pyrenoid.

FIGS. 1 and 2. Young material incubated for catalase activity. Microbody-like organelles (m) show deposition of reaction product. Note the close association between the microbody (m) and the mito- chondrion (M). Fig. 1, x 30 000. Fig. 2. Arrow points to a core-like structure. x 36000. FIG. 3. Catalase control (aminotriazole incubation), young cell. N o reaction product is observed in the microbodies (m). x 22 000. FIG. 4. Young material incubated for the identification of catalase activity. Reaction product is observed in the mitochondrion (M). x 30 000. FIG. 5. Material from early stage of aging and in- cubated for the identification of catalase activity. Arrow indicates a zone of deposition of reaction product. Note the close association between the microbody where deposition of reaction product occurs and the mitochondrion (M). x 30 000. FIG. 6. Typical morphology of an Ectocarpus micro- body. Arrow points to a core-like structure. x 36 000. FIG. 7. Material incubated for the identification of peroxidase activity (early stage of aging). x 30 000. FIG. 8. Material incubated for the identification of peroxidase activity (early stage of aging). Control preparation. x 30 000.

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arrows). Heavy depositions of reaction products were also detected in the perinuclear space, endoplasmic reticulum, and Golgi complex (Fig. 11). KCN treatment eliminated or con- spicuously reduced the overall deposition of reaction product (Fig. 13). Incubation in the absence of hydrogen peroxide (Fig. 14) and the boiling of material before prefixation of the material yielded negative results. Aminotriazole treatment showed no effect in the deposition of reaction product.

In aging cells peroxidase reactions could not be determined with confidence. This was due to the characteristics of the ultrastructure of the cells (Oliveira and Bisalputra 1976a), which made the results difficult to interpret. Figures 7 and 8 show peroxidase localization and control experi- ments, respectively; slight depositions of pro- ducts can be detected in the cell wall region. In

Incubation in a medium containing Na+-K+- ATP without Mg2+ gave no noticeable deposi- tion of reaction product in any of the above- mentioned situations.

The Mg2+-ATP-incubated material displays, in young cells, reaction products within the mitochondrial intermembrane space and cristae (Fig. 16), on the plasma membrane, in the paramural space, and along the thylakoids (Fig. 18). On the whole, depositions of reaction pro- ducts do not seem to be as intense as in the case of the NA+-K+-Mg2+-ATP-incubated mater- ial. There is no reaction when the material is incubated in a medium without ATP (Fig. 19). Slight but positive reaction is detectable in very early stages of aging, especially in mitochondria (Fig. 17). In more advanced stages, the localiza- tion of reaction products becomes very difficult to determine because of the characteristics of the

senescent cells we failed to detect any signs of cells. In senescent cells no deposition of reaction peroxidase activity. products could be detected.

Adenosinetriplrosphatase (ATPase) In young cells, incubation in a medium con-

taining K+-Na+-Mg2+-ATP yields conspicu- ous depositions of reaction products on the plasma membrane (Fig. 12), the mitochondria (Fig. 15), paramural bodies, and along the thylakoids (Fig. 12). Figure 20 depicts a control treatment. The absence of reaction products is apparent.

Similar patterns of deposition of reaction pro- ducts were also observed in aging cells (Fig. 21). However, the reaction is not as distinct as in the younger cells because of the abundance of cellu- lar inclusions and alteration of cell organelles (Oliveira and Bisalputra 1976a), which tend to obscure the results. The absence of reaction pro- ducts from control preparations is evident in Fig. 22. ATPase activity could not be detected in senescent cells.

Discussion

Catalase Biochemical, cytochemical, and ultrastructural

studies have, during the past few years, provided sufficient evidence for the identification in plant cells of a class of organelles usually described as microbodies (Tolbert 1971). Bisalputra et al. (1971) reported, on the basis of fine structural studies, the presence of microbodies in the zygote of Lanzinaria. It is possible that the crystal- containing bodies described by Bouck (1965) in Fucus and G~ffordia are also microbodies. Organelles whose ultrastructural features (single- membrane boundary, fine granular matrix with occasional core-like differentiations) and dimen- sions resembling those of the so-called micro- bodies were found in all sporophytic cells of Ectocarpus, with the exception of the necrotic

FIGS. 9, 10, and 11. Young material incubated for peroxidase activity. Fig. 9. Peroxidase activity in the cell wall. x 16 000. Fig. 10. Peroxidase activity in the cell wall ( cw) and paramural space (arrows). x 13 000. Fig. 11. Peroxidase activity in the perinuclear space, dictyosome cisternae (D), and dictyosome-derived vesicles. x 26 000. FIG. 12. ATPase activity (Na+-K+-MgZ+-dependent sys- tem) in young cell. Deposition of reaction product is conspicuous a t the plasmalemma and thyalkoids. x 16 000. FIGS. 13 and 14. Peroxidase activity (control preparations; KCN incubation), young cells. Note the absence of reaction product from the different cell parts. Fig. 13, x 12 000; Fig. 14, x 20 000. FIG. 15. ATPase activity (Na+-K+-dependent system), young cell. Deposition of reaction product in the mitochondrial cristae and envelope. x 32 000. FIG. 16. Young cell, ATPase activity (MgZ+- dependent system). Deposition of reaction product is apparent in the mitochondrial cristae and envelope. Unstained preparation. x 40 000. FIG. 17. ATPase activity (Mgz+-dependent system; early stage of aging), showing the reaction product in the mitochondrion (arrow), x 12 000.

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stages. The identification of these organelles was confirmed cytochemically by the positive reac- tion upon diaminobenzidene (DAB) incubation. Under these conditions heavy deposition of reac- tion products was found inside the organelles. Control reaction using incubation in amino- triazole indicates the presence of catalase (Margoliash and Novogrodsky 1958), an enzyme considered to be a marker for microbodies (Tolbert 1971). The results are corroborated by other parameters of incubation, i.e., optimum pH in the alkaline range and high temperature of incubation (37 "C) (e.g. Roels and Wisse 1973). The boiling experiment indicates that the reac- tion is definitely enzymatic in nature.

In plant cells, microbodies are subdivided into glyoxysomes and peroxisomes on the basis of the enzyme content and their involvement in either the glyoxylate cycle or the glycolate pathway, respectively (Tolbert 1971). I t is inter- esting to note that a consistent and close spatial association was found between microbodies and mitochondria in Ectocarpus. Associations with chloroplasts, although detectable, were not so frequent. However, without biochemical evi- dence, it is impossible to make any definite con- clusion as to the nature of microbodies in Ectocarpus.

DAB reaction products were also found in mitochondria. Although catalase has been re- ported to be present in mitochondria (Herzog and Fahimi 1972), the explanation for the mito- chondria] DAB reaction is still debatable. Several possible causes were advanced to explain this reaction (Rothman 1968; Seligman et al. 1968; Beard and Novikoff 1969; Novikoff and Goldfischer 1969 ; Gerhardt and Berger 197 1 ; Poux 1972). Of all these, cytochrome oxidase and (or) cytochrome c seem to be the more likely (Seligman et al. 1968; Beard and Novikoff 1969; Novikoff and Goldfischer 1969; Gerhardt and Berger 1971 ; Poux 1972).

The positive reaction with DAB in the chloro- plast ER around the pyrenoid and the negative reaction in the area outside the pyrenoid seem to indicate functional differences and heterogeneity in this membrane system.

Peroxidase Both the total activity and the isoenzyme poly-

morphism of peroxidase were shown to increase with the increase in maturity of plant cells (see Bredemeijer (1973) for a review) and were also reported to be characteristic of the aging process (e.g. Galston and Davies 1969). In necrotic cells of Ectocarpus no peroxidase activity was de- tected. It is possible that peroxidase activity is present in the early stages of aging, but the over- all characteristics of the cells (Oliveira and Bisalputra 1976~) make it difficult to evaluate the pattern and the intensity of the reaction. There- fore, any comparison with the data available for the young cells stage is not possible.

In young cells deposition of reaction product was detected in the paramural space, cell wall, perinuclear-space ER, and in dictyosomes. That the reaction is enzymic in nature can be inferred from the boiling control. The assumption that the deposition of reaction product is due to peroxidase activity is confirmed by the experi- mental conditions used (e.g. Poux 1972; Roels and Wisse 1973), as well as from the results of the following control experiments: (1) aminotriazole incubation does not inhibit the deposition of reaction product, and (2) potassium cyanide strongly reduces the deposition of reaction pro- duct (Strum and .Karnovsky 1970; Poux 1972).

Existing evidence indicates that peroxidases, although ubiquitous in distribution, possess a high degree of heterogeneity (e.g. Shannon 1968; Delincte and Radola 1970; Radola and Drawert 1970). Riicker and Radola (1971) have shown that in tobacco tissue cultures, a number of peroxidase isoenzymes occur which could be

FIG. 18. Young cell ATPase activity (Mgz+-dependent system). Deposition of reaction product in association with the plasmalemma and thylakoids. Notice, however, that the reaction is not as intense as in the case of the Na+-K+-MgZ+-dependent system (Fig. 12). x 12 000. FIG. 19. ATPase activity, young cell (Mgz+-dependent system; control preparation). No deposition of reaction product can be detected. x 30 000. FIG. 20. ATPase activity (Na+-K+-MgZ+-dependent system; control preparation, young cell). Notice the absence of deposition of reaction product. x 18 000. FIGS. 21 and 22. ATPase activity (Na+-K+-MgZ+-dependent system; early stages of aging). Fig. 21. Deposition of reaction product in association with the plasmalemma and thylakoids. x 30 000. Fig. 22. Control preparation. Note the absence of reaction product. x 20 000.

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separated into three groups on the basis of their isoelectric points. Hall and Sexton (1972) have studied the effect of pH on peroxidase activity and found a wide variation in pH-dependent responses. These facts and a recent study by Poux (1972) show that different isoperoxidases may require individual incubation parameters to be demonstrated. This is in agreement with our findings that visualization of peroxidase activity in endomembranes (dictyosomes, endoplasmic reticulum) requires special care, namely in the temperature of incubation. Although there has been increasing interest in the biochemical and physiological aspects of peroxidases in recent years, little is known of their real functions. Ridge and Osborne (1970) have suggested that wall peroxidases might facilitate hydroxylation of proline in wall proteins, hence affecting the extensibility of the cell wall (Ridge and Osborne 1971). However, any similar conclusion as to the roles of peroxidase in Ectocarpus has to be post- poned until better knowledge of cell wall com- position in this alga becomes available.

Adenosinetriphosphatase (ATPase) Recently a number of papers have appeared

dealing with plant ATPases (e.g. Hall and Davie 1971 ; Coulomb and Coulomb 1972; Cronshaw and Gilder 1972; Hodges et al. 1972; Lai and Thompson 1972a, 1972b ; Maier and Maier 1972; Sundberg et al. 1973; Edwards and Hall 1973). Coulomb and Coulomb (1972) and Maier and Maier (1972) detected the existence of a M ~ ~ + - dependent ATPase along the plasma membrane. Other authors reported that ATPase activity is activated by monovalent ions but inhibited (under certain conditions) by divalent ions (Atkinson and Polya 1967). A pH-dependent, K+-stimulated ATPase was detected in the plasma membrane of the green alga Mougeotia (Sundberg et al. 1973). There is also evidence for the occurrence of monovalent-ion-stimulated ATPases on the plasma membrane of other plants, but the system was reported to be dif- ferent from the Na+-K+-ATPase-activated systems (e.g. Hodges et al. 1972; Ratner and Jacoby 1973). Lai and Thompson (1972~) pro- vided evidence for the existence of a Na+-K+- activated ATPase associated with the plasma membrane of plant cells. Bowling et al. (1972) reported a similar situation and concluded that the plant ATPase studied by them has different

properties from the N ~ + - K + -ATPase systems of animal cells (see also Baker and Hall (1973)).

The study of a Mg2+- and a N ~ + - K + - M ~ ~ + - ATPase-dependent activity is, hence, of interest, especially because both systems were reported to occur in young cells of the brown alga Sphace- laria (Kalley, personal communication). In Ectocarpus the pattern of distribution of reac- tion products is similar for both systems. How- ever, in the presence of monovalent ions (K+, Na'), deposition of reaction products along both the plasma membrane and the thylakoids seems to have been increased.

Involvement of monovalent-ion-dependent ATPases in stimulating ion transport has been emphasized in plant cells (e.g. Hall and Davie 1971 ; Coulomb and Coulomb 1972; Maier and Maier 1972). Ratner and Jacoby (1973) found in their material that monovalent salt effects on ATPase were not cation specific and are there- fore not related to the cell potential for cation absorption. Whether or not the enhancement of reaction product deposition by monovalent ions in Ectocarpus reflects cation specificity remains to be determined. Our results seem, however, to agree in most part with those of other authors (Fisher and Hodges 1969; Fisher et 01. 1970; Leonard and Hanson 1972) which show that ~ g ~ + is required for plant ATPases to be stimulated by monovalent ions. Indeed, in the absence of Mg2+, the ions K + and Na+ gave no significant deposition of reaction product in our material.

The ATPase reaction in mitochondria is prob- ably due to the ~ g ~ + - a c t i v a t e d system only, since deposition of reaction product is heavy either in the presence or absence of monovalent ions. Novikoff et al. (1958) also reported that Mg2+ ions preferentially stimulated the ATPase system of mitochondria1 fractions.

ATPase reactions were easily detected in young cells and during the early stages of aging, but not in more advanced stages. The apparent lack of ATPase from more advanced stages leads one to conclude that those cells are no longer functional. The absence of ATPase activity at the plasma membrane level indicates a drastic change in the physiological properties of this membrane, although ultrastructurally the membrane seems to remain intact (see Fig. 8 of Oliveira and Bisalputra (19766)). It has been reported that as senescence proceeds, changes in permeability are

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observed in ,the cells (e.g. Eilam 1965; Sacher 1967; Fergusson and Simon 1973), while there is no detectable change in the integrity of the membrane.

In conclusion, the absence of all enzymic activities from the final stages of senescence (necrotic stages) is not surprising in face of the level of disorganization attained by the cells. The results concerning ATPase activities are of par- ticular interest since it is clear that these enzvmes cease to be functional long before any change in the physical integrity of membranes can be ob- served. Since ATPases are regarded as respon- sible for active transport, their absence may be taken as indicative of cell death. Therefore, in Ectocarpus, cells at the basal portions of the filaments that stained intensely with OsO, (Oliveira and Bisalputra 1976a), or those that lost OsO, affinity (Oliveira and Bisalputra 1976b), are indeed non-functional. The living portion of the filaments is, hence, restricted to the first 8-15 cells below the meristematic apical cells.

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