Microbodies of protoplasts of Grateloupia sparsa (Halymeniaceae, Rhodophyta)  

Yean-Chang Chen¹ and Young-Meng Chiang²  

¹Institute of Aquaculture National Taiwan Ocean University

Keelung, Taiwan, Republic of China and ²Institute of Oceanography National Taiwan University Taipei, Taiwan, Republic of China  

ABSTRACT

Cytochemical reagent diaminobenzidine (DAB) was used with an electron microscope to locate microbodies in protoplasts of the multicellular red alga Grateloupia sparsa. Protoplasts of freshly isolated and of age 6 h generally contain 1-3 peroxisomes per protoplast. The peroxisomes have a roughly spherical shape and a diameter of 0.5-1 m m. A strong positive reaction to the DAB cytochemical test was found in the peroxisomes, and inhibition of the DAB reaction with standard reagents produced the same results. Those results indicated that catalase may be absent, thereby making the staining of microbodies due to peroxidase. No peroxisomes were found in protoplasts aged 1 to 4 days. The disappearance of microbodies of protoplasts on increasing age of protoplasts from 0-6 h to 1-4 days is assumed to be a growth behavior of viable protoplasts of G. sparsa.

(Key words: DAB (diaminobenzidine), Grateloupia sparsa, Peroxisome, Protoplast, Red alga)

Abbreviations: Chl chloroplast; Cm cell membrane; Fs Floridean starch granule; Mi mitochondrion; Nm nucleus membrane; Nu nucleus; P peroxisome; Osm osmiophilic granules; V vacuole.  

INTRODUCTION

Microbodies are ubiquitous and essential organelles in diverse plant and animal cells. Microbodies are morphologically characterized as being bounded with a single membrane, having a uniform granular matrix and occasionally a crystalline core (Oakley and Dodge, 1974). The term microbody was originally restricted to structures visible on electron micrographs. Therefore, organelles can be referred to as microbodies without any previous knowledge regarding their enzymatic capabilities (Gross, 1993). Microbodies seem to present a bag into which almost any enzyme reaction or pathway can be compartmentalized, if necessary. In higher plants, three types of peroxisomes are recognized by their locations and are diverse in metabolism pathways. In fat-storing tissue of germinating seeds, they are also termed glyoxysomes that are involved in fatty-acid metabolism. In photosynthetically active cells, so-called leaf peroxisomes are involved in photorespiration. In other plant tissues, a third type has been found and is generally referred to as “unspecialized” peroxisome.

Far less is known about the microbody in algae than in higher plants, despite the fact that these organisms contribute more than half of the world’s biomass production. However, there is an enormous variability on algal metabolism. Therefore, examining the microbody in algae seem to be a worthwhile task. An illustrative example of the diversity in algae is the function of peroxisomes (Gross, 1993).

Applications of DAB cytochemistry to discover the morphological and cytochemical status of peroxisomes in algae (Silverberg and Sawa, 1973; Oakley and Dodge, 1974; Silverberg, 1975a, 1975b; Hornung et al., 1977; Stabenau et al., 1984, 1989) are as important as in higher plants. However, information about these cytochemical properties of microbodies of red algal protoplasts is scarce because protoplast research in seaweeds is a relatively new field (Chen and Chiang, 1994).

Protoplasts of Grateloupia sparsa were isolated, and those protoplasts quickly formed new cell walls (Chen and Chiang, 1995), then to form discs, which subsequently erected fronds or filaments after further cultivation (Chen and Chiang, 1994). Previous studies of protoplasts regenerated system of G. sparsa (Chen and Chiang, 1994, 1995), indicated that the protoplasts contain microbodies.

This investigation is undertaken to elucidate the distribution of microbodies in regenerated protoplasts of Grateloupia sparsa (Okamura) Chiang (1970).

  MATERIALS AND METHODS

Materials

Young thalli of Grateloupia sparsa with broad, leafy branches were used in this study. These thalli were regenerated from protoplasts of this alga (Chen and Chiang, 1994), and were cultivated in the laboratory of the Institute of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan, R. O. C.

Isolation of protoplasts

Viable protoplasts were isolated according to the method described previously (Chen and Chiang, 1994). Cleaned, vegetative materials of Grateloupia sparsa were cut into small pieces of 0.3-0.5 mm² with a sterilized razor blade on a clean bench. Next, five hundred milligrams (fresh weight) of the material were incubated with 10 mL of enzyme solution (4% cellulase Onozuka R-10 Yakult Honsha Co. Ltd., 2% macerozyme R-10 Yakult Honsha Co. Ltd., 50 U/mL agarase Sigma A-6300 and 2% papain Sigma P-3375, 0.7 M mannitol in 10 mL 35‰ seawater) and rotated with an orbital shaker at 50 rpm. All procedures for isolation were carried out in a cabinet with a laminar flow of air in darkness for 18 h at 18°C.

Incubation and fixation of protoplasts of various ages

Incubation of protoplasts

Protoplasts were incubated according to the method described previously (Chen and Chiang, 1994) with slight modifications. Freshly isolated protoplasts (ca. 7x10⁸ protoplasts or equal to 2500 protoplasts per area 0.2 mm²) were cultured in 10 mL of Provasoli's enriched seawater (35‰) (PES) (Provasoli, 1968) medium with 0.7M of mannitol immediately. Protoplasts were incubated at 18°C in darkness for the first three days to promote sinking, and were then cultured under a photon flux density 166 µmol.m^-2s^-1 and a photoperiod 12:12 L:D at the same temperature in a culture room.

Fixation of protoplasts for electron microscopy study

During incubation, cultured protoplasts in six groups of varied age were accumulated at 0, 6, 24, 48, 72 and 96 h of culture, respectively. A centrifuge (HERMLE Z320) was used to accumulate those protoplasts that were centrifuged at 120 Xg for 20 min. As soon as the mannitol-PES medium was poured off, protoplasts aged 0 and 6 h were fixed separately for 1 h with 2 % glutaraldehyde in PES medium containing 2 % NaCl and 1 % sucrose at 4° C with gentle shaking. The protoplasts were centrifuged for 10 min at 300 Xg. The pellets were fixed with 5% glutaraldehyde in 0.1 M sodium cacodylate (pH 6.8) buffer containing 10 mM CaCl₂ and 0.2 M sucrose for 1 h at 4° C. They were then rinsed four times with 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂, of which the concentration of sucrose varied in decrements 0.05M. This operation was followed by two rinses in pure (sucrose-free) 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂.

The protoplasts aged 24 h or older were fixed separately similar to the protoplasts aged 0 h and 6 h. However, the duration of fixation was extended to 2 h, with NaCl omitted and the concentration of sucrose decreased to 0.2 M during the first fixation. All protoplasts in the six groups of varying ages (0, 6, 24, 48, 72 and 96 h) were then treated for cytochemical tests.

Cytochemical testing of protoplasts

(Localization of catalase and peroxidase)

The fixed protoplasts of varying ages were rinsed in a 0.2 M sodium cacodylate buffer. The protoplasts were placed in incubation media as follows:

(1) H₂O₂ (1%, 0.2 mL) and 3,3' diaminobenzidine tetrahydrochloride (20 mg, 0.2%) in 2-amino-2-methyl-1,3-propanediol buffer (0.05M, 10 mL, pH adjusted to 9.0) at room temperature;

(2) as (1) but without H₂O₂;

(3) as (1) but pretreated for 30 min with AT (3-amino-1,2,4- triazole) (0.02 M) in propanediol buffer (0.05M);

Solutions prepared according to (2) and (3) served as control groups to those from (1).

Post fixation and embedding the protoplasts

All protoplasts were post-fixed with 2 % osmium tetroxide in 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂ for 1 h at 4° C.

Thereafter, they were rinsed four times with 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂ and then rinsed three times with 50 % ethanol, dehydration in ethanol of concentration increased through 50%, 70%, 85% and 95% to 100%. The dehydrated protoplasts were rinsed with transitional solvent (propylene oxide) three times each for 30 min, followed by infiltration in propylene oxide-Spurr's resin with decreased ratio from 2:1 (2 parts propylene oxide : 1 part Spurr's resin) to 1:1 each for 4 h, and to pure Spurr's resin for 2 days at 4ºC in darkness before embedding in Spurr's resin (Spurr, 1969).

All trimmed blocks were cut with an ultra-microtom (Reichert Ultracuts, Leica) and Diamond knife (DDK,U.S.A.); the thin-sections were stained with uranyl acetate and with lead citrate according to the description by Smith and Croft (1991).

  RESULTS

The protoplasts of freshly isolated and aged 6 h showed a heavy deposition following incubation in the complete DAB reaction medium (Figs. 1 and 3). This reaction is oxidation and reduction between DAB and peroxidase and/or catalase of peroxisomes and subsequent polymerization with osmium tetroxide. The reaction was so strong in most instances that the limiting membrane was obscured. The peroxisomes were defined, however, according to their size and location. Those protoplasts, generally contained several roughly spherical peroxisomes of a diameter about 0.25-1 µM, maximum 1.5 µM (Fig. 1). Although in this cytochemical test, in which the inhibitor (AT, triazole) of catalase was added and in another in which no H₂O₂ was added, they still showed a positive reaction with DAB (Fig. 2). This result may be due to peroxisomes containing peroxidase but no catalase. According to morphological observations, the peroxisome of G. sparsa protoplasts associated not only with chloroplast but also with mitochondrion (Fig. 3).

After one day in culture, the chloroplast of the protoplast contained many osmiophilic granules that were orderly arrayed inside the lamella of chloroplast (Fig. 4) with the disappearance of peroxisome. The disappearance of peroxisomes was also show in protoplasts aged 4 days.

  DISCUSSION

According to ultrastructural observations, algae contain microbodies morphologically similar to those in higher plants (Frederick et al., 1975; Silverberg, 1975 a, b). Results in this study demonstrated that Grateloupia sparsa protoplasts less than 6 h old contain several roughly spherical peroxisomes (Fig. 1), i.e., morphologically similar to those of higher plants, such as those having a diameter of 0.15-0.5µM (Frederick et al., 1968). The peroxisomes of these red algal protoplasts may be related to photosynthesis. They are generally associated with chloroplasts and mitochondria (Figs. 1-3). This phenomenon is common in leaf peroxisomes of higher plants (Stabenau et al., 1989). In some green algae, microbodies are found to be in close proximity to chloroplasts (Silverberg, 1975a). These observations have been taken as evidence for an exchange of metabolites similar to higher plants. However, they are different from those of Carpomitra cabrerae (Clemente) Kutzing (Motomura et al., 1985). The peroxisomes of this brown alga C. cabrerae associate only with mitochondrion.

DAB test results indicate that peroxisomes of G. sparsa protoplasts of this study may contain peroxidase but no catalase. Therefore, this cytochemical result demonstrated that enzyme components of this red algal protoplasts differ somewhat from those in higher plants. However, this result is similar to that of Oakley and Dodge (1974), who suggested that peroxisomes of red unicellular alga Porphyridium contain peroxidase but no catalase. This result differs from that of Stabenau et al. (1989), who investigated microbodies such as organelles of Prasinophycean algae Platymonas, Heteromastix, and Pedinomonas. Their investigations indicated that these algae contain peroxisome-like organelles more similar to glyoxysome involved in the b -oxidation of fatty acids. They suggested that those green algae are representative of the evolutionary line towards higher plants. However, although the morphology and localization of the peroxisome of G. sparsa protoplasts are similar to those of higher plants, enzymatical characteristics of the former are not quite similar to those of green algae described by Stabenau et al. (1989), and from those of higher plants.

Microbodies, the peroxisomes, exist in protoplasts of various ages that were freshly isolated or aged 6 h and disappeared in the protoplasts aged 1-4 days. This phenomenon is assumed to be a growth behavior of G. sparsa protoplasts because peroxisomes are involved in photosynthesis (Nadakavukaren and McCracken, 1985). The reason that protoplasts freshly isolated and 6 h old contain peroxisomes is that they may retain the ability of photosynthesis, similar to their mother algae body. However, when they were cultured for more than one day, they may alter their photosynthetic ability because of an absence of peroxisomes. However, the ability of growth is demonstrated. However, our previous work showed that those protoplasts divided at four to seven days in culture (Chen and Chiang, 1994).

Most studies involving the enzymatic capabilities of microbodies in algae have been restricted to the green algae and Euglena. So far, peroxisomes have been investigated in only two primitive red algae. Both species, Cyanidium caldarium and the closely related Galdieria sulphuraria, contain peroxisomes that house the enzymes catalase, glycolate oxidase, and glyoxylate-glutamate aminotransferase (Gross and Beevers, 1989; Seckbach et al., 1992).

An attempt is made here to use the vegetable tissue of G. sparsa for material to assay the enzyme component. However, this attempt failed since the abundant layers of cell walls (b -carrageenan) obstructed the procedures of organelle separation and enzyme fraction. Cell fraction was applied successfully only to green algal species and Euglena (Huang et al., 1983). Although we had a yield 10⁸ protoplasts per gram fresh mass of tissue (Chen and Chiang, 1994), such a yield compared to vegetable tissue, is too small to be the material for enzyme fraction. Another method must be found to assay the enzyme components of microbodies of this red alga and its protoplasts.

Enzymatically digesting the cell walls of multicellular red alga is relatively difficult, especially the more complex form of red algae, such as the agar- or carrageenan-producing algae, to obtain a sufficient viable protoplasts and, subsequently, culture them for cytochemical localization and definitions of their microbodies. The microbodies of protoplasts have received little attention. Therefore, to our knowledge, this is the first investigation in microbodies in plant protoplast research.

  ACKNOWLEDGEMENTS

The authors would like thank the National Science Council (NSC-84-2621-B-002A-002) and the Council of Agriculture (84-AST-1.1-FAD-62(29)) of the Republic of China for financial support of this work.

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LEGENDS OF FIGURES

Figs. 1-2. Peroxisomes of freshly isolated protoplasts. Fig. 1. The peroxisomes of protoplast associate with mitochondrion and chloroplast respectively. They are positive reaction with DAB and show deep black color which strongly contract to the floridean starch granules which show white appearance inside with black crystal core. Fig. 2. Protoplast pretreated with the inhibitor (AT, triazole) of catalase. The peroxisomes of the protoplast are obviously recognizable. They associate with chloroplast and still show positive reaction to DAB, in which evident the peroxisome may contain peroxidase but not catalase.

Fig. 3. The peroxisome of protoplast aged 6 h shows strong reaction with DAB. It associate with both mitochondrion and chloroplast. This phenomenon is common in leaf peroxisome of higher plant.

Fig. 4. The chloroplast of protoplast aged 1 day shows many osmiophilic granules that were orderly arrayed inside the lamella of chloroplast.