Ultrastructure of cell wall regeneration from isolated protoplasts of Grateloupia sparsa (Halymeniaceae, Rhodophyta)

Yean-Chang Chen and Young-Meng Chiang

Institute of Oceanography National Taiwan University Taipei,

Taiwan, Republic of China

Abstract

Ultrastructural observations were conducted on viable protoplasts of Grateloupia sparsa isolated from the thallus by enzymatic degradation of the cell wall. Various stages (from 0 h to 168 h in culture) of protoplasts were examined under a SEM and a TEM. From the first six hours after isolation, the protoplasts showed a deposition of an amorphous matrix on the surface of the protoplasts. This substance is apparently produced in the endoplasmic reticulum, and is directly and continuously released to the surface. The protoplasts were fully covered by the amorphous matrix after one day in culture, as thereafter no obvious alteration was found on the surface of protoplasts before cell division was observed under SEM. Under TEM, the cell membrane of protoplasts was seen as smooth and intact without particular alteration before 6 h in culture; thereafter vesicles were excreted on the membrane. Those vesicles formed an increasingly thick amorphous matrix; after about four days in culture, the cell membrane became irregular. At the same time, randomly arranged microfibrils were regenerated from this irregular membrane, these extended and later formed a thin layer of cell wall. The cell walls were continuously deposited, and at about day 7 in culture, the microfibrils became increasingly thick and ordered into arrays, and became involved with large amounts of terminal complex-like granules, before gradually forming a new complete cell wall.

Key index words: Amorphous matrix deposition; Cell wall regeneration; Fine structure; Grateloupia sparsa; Microfibrils deposition; Protoplasts.

Introduction

Because the organization of the cell wall and the chemical composition of the Rhodophyta differ significantly from those of all other plants (Craigie, 1990), this phylum has attracted much research (Myers and Preston, 1959a, b; Dawes et al., 1960, 1961; Evans et al., 1974; Gordon-Mills and McCandless, 1975, 1977, 1978; Gretz et al., 1980,1982, 1984, 1986; Cole et al., 1985, 1986; Kloareg and Quatrano, 1988; Tsekos and Reiss, 1994.) on the structure and composition of the rhodophycean cell wall. Regeneration of the cell wall in isolated protoplasts of muticellular red algae has been less investigated. Cheney et al. (1986) and Le Gall et al. (1990) with calcofluor staining, and Waaland et al. (1990) with transmission-electron microscopy reported that isolated protoplasts of a few red algal species developed new cell walls. Liu et al. (1992) made detailed observations of cell wall regeneration of isolated protoplasts of Palmaria palmata (L.) Kuntze; elements of the endo-membrane system are involved in the synthesis of the cell wall. Our study on formation of the cell wall of isolated protoplasts of Grateloupia sparsa (Okamura) Chiang with electron microscopy confirmed the findings of Liu et al. (1992).

Here we describe the formation of the cell wall by isolated viable protoplasts from the marine, multicellular red alga G. sparsa.

Grateloupia sparsa is a red alga common along coasts of northern Taiwan during spring, and is easily collected. This alga is annual and has macroscopic thalli, isomorphic gametophytes and tetrasporophytes.

Materials and Methods

Material

The materials used in this study were young thalli of Grateloupia sparsa obtained from protoplasts of the alga (Chen and Chiang, 1994), cultivated in the phycological laboratory, Institute of Oceanography, National Taiwan University, for about five months.

Isolation of protoplasts

Protoplasts were isolated according to the method described previously (Chen and Chiang, 1994). The algal thalli were cleaned with sea water containing KI-I₂, then treated with an antibiotic mixture. After being cleaned, the thalli were cut into small pieces of about 0.5 mm². The protoplasts were isolated with commercial enzymes (4% cellulase, Onozuka R-10, Yakult Honsha Co. Ltd.; 2% macerozyme R-10, Yakult Honsha Co. Ltd.; 50U/mL agarase, Sigma A-6300, and 2% papain, Sigma P-3375) dissolved in 0.6 M mannitol-seawater (35?) The protoplasts were filtered through a 59µm nylon mesh, and then centrifuged at 150 xg (HERMLE Z320) for 20 min.

Fixation of protoplasts for electron microscopic studies

Some purified protoplasts were fixed immediately (as 0-h old protoplasts), whereas others were cultured in six 15 mL centrifuge tubes containing 10 mL 0.7 M mannitol-Provasoli's (Provasoli, 1968) enriched seawater medium (mannitol-PES) for 6, 24, 48, 72, 96 and 168 hours followed by fixation. Protoplasts were collected by centrifuging gently (HERMLE Z320) at 120 xg for 20 min at 18 °C. Zero and 6-h old protoplasts were fixed at 4°C for 1 h in PES medium containing 2 % glutaraldehyde, and 0.4M sucrose with gentle shaking. The protoplasts were collected by centrifuging at 300 xg for 10 min. The pellets were then re-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 rinsed with 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂ four times, with the sucrose concentration successively reduced to 0.05 M. This treatment was followed by two rinses in pure (sucrose-free) 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂.

The protoplasts of 24 h or older, were fixed like those of 0 h and 6 h, except that the duration of fixing was extended to 2 h and the concentration of sucrose was 0.2M for the first fixation.

Post-fixation was done with 2 % osmium tetroxide in 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂ for 1h at 4°C.

Thereafter, all materials were rinsed four times with 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂. Then they were rinsed thrice with aqueous ethanol (50 %), and dehydrated gradually in ethanol (50, 70, 85, 95 and 100% successively). During this dehydration, protoplasts generally gathered to form small clumps. These clumps (0, 6, 24, 48, 72, 96 and 168 h-old protoplasts) were prepared for transmission electron microscopy (TEM) by rinsing thrice with transitional solvent (propylene oxide) for 30 min each time, followed by infiltration in propylene oxide-Spurr's resin at a decreasing ratio from 2:1 (2 parts propylene oxide :1 part Spurr's resin) to 1:1 each for 4 h, then in pure Spurr's resin for two days at 4°C in darkness before embedding in Spurr's resin (Spurr, 1969).

When the embedded resin had polymerized to form blocks, the latter were trimmed with a razor blade (Corrux) and then cut with an ultra-microtome (Reichert Ultracuts, Leica). The thin-sections were stained with uranyl acetate and lead citrate according to the methods described by Smith and Croft (1991).

Protoplast clumps (0, 6 and 24-h old protoplasts) were also prepared for scanning electron microscopy (SEM). They were suspended in pure ethanol for two days with gentle shaking (two cycles per min.) on a rotary disc (Firstech). This procedure provided protoplasts extricated from clumps. The protoplast-ethanol suspension was gently dropped on specimen holders; then they were dried with a critical-point drying machine (HITACHI-HCP-1). Finally the individual protoplasts were coated on an ion coater (JOEL, JCF-1100E) for 3 min 40s.

Results

SEM observation

Under the scanning electron microscope, a freshly isolated protoplast (about 20 min old) was spherical with amorphous, film-like matrices irregularly distributed on its surface (Fig. 1). Matrices seemed to be produced continuously to form bulges of many sizes on the surface of the protoplast (Fig. 2). This phenomenon was especially notable on the surface of the 6-h protoplast. As protoplasts aged the matrix layer thickened. Then the bulges joined to form a smooth surface in 24 h (Fig. 3) due to the continuously increased matrix. Thereafter, no particular alteration of the protoplast surface was observed.

TEM observation

Electron micrographs of ultra-thin sections of freshly isolated protoplasts showed no cell wall residue (Figs 4, 5). Most freshly isolated protoplasts appeared not much different from cells in the tissue of origin, except that they had become spherical and that their volume expended. However, viable protoplasts showed an increased type and quantity of cell organelles and more numerous endoplasmic reticula (ER) at the periphery (Figs 4, 5).

The predominant element in almost all cultured protoplasts was the ER, distributed throughout the cytoplasm (Fig. 4). Layers of ER were seen to lie parallel to the plasma membrane, generally near the plasma membrane (Figs 4, 5) with the trait of appearance of small vacuoles located between the long ER and plasmalemma. Some portions of ER were swollen (Fig. 4), which was evidence that ER apparently produced vesicles in the early stage of cell wall regeneration. The smooth ER had varied lengths (Figs 4, 5); those that had secreted small vesicles (Fig. 4) were much longer than those ( Fig. 5) that did not. Golgi bodies were rarely seen in the thin sections. Many small vesicles were evident just outside the smooth plasma membrane surface, in most protoplasts after culture for 6 h (Fig. 6).

Cell walls did not regenerate uniformly on the protoplasts; the cell membrane of the protoplast remained smooth and intact from freshly isolated until cultured for four days; then the membrane became irregular and some granules and precursors were underneath it (Fig. 7). Those granules and precursors are assumed to be proteins. The patchy microfibrils regenerated from the irregular membrane were randomly deposited into the thick amorphous matrix layer and becoming loosely organized (Fig. 7), then formed random arrays composed of thin microfibril layers (thickness ca. 140 nm) (Fig. 8) after culture for four days. The microfibrils later lengthened from about 100-200 nm to 300 nm and were arranged more orderly, between which some terminal complex-like granules were found (Fig. 9). Most surviving protoplasts had regenerated a thin layer of new wall material after culture for four days.

After culture for four to seven days, the thin wall (microfibril layer) increasingly thickened; the cell walls of protoplasts (Fig. 10) were almost the same as the normal inner walls of vegetative cells of G. sparsa, but less compact than normal. In these inner cell walls many terminal-like granules frequently appeared (Fig. 10). Arrays of microfibrils (Fig. 10) were more orderly than those (Figs 8, 9) of protoplasts aged four days. The thickness of the microfibril layer of protoplasts aged seven days reached about 370-500 nm (Fig. 10). The protoplasts then gradually formed a complete cell wall. Those protoplasts with new walls developed to form radially expanded discs with meristematic marginal portion of one-celled layer, subsequent the discs regenerated either leafy thalli or filaments in further cultivation (Chen and Chiang 1994).

Discussion

Regenerating protoplasts are ideal tools for understanding the structure and formation of cell wall, and several authors love used algal models (Mizuta and Wada, 1981; Itoh et al., 1984, 1986; Mizuta, 1985; Mizuta et al., 1985; Waaland et al., 1990; Liu et al., 1992). Most algae have complex, multilayered, microfibrillar walls with an interspersed amorphous matrix (Millner et al., 1979) that is useful to understand the sequential nature of wall formation. For most algae, including the red alga in this study, the ultrastructure of the protoplast is better defined than in vegetative cells, in which the walls commonly interfere with the penetration of fixative (Berliner, 1981). Observations of fine structure of the interior of protoplasts have served largely to show their similarity to vegetative cells in the tissue from which they are isolated (Burgess et al., 1977). Therefore, the main interest of algal protoplasts may lie in making cell wall formation accessible for study.

According to observation of protoplasts with a transmission-electron microscope (TEM), the enzymatic removal of the cell wall appears not to alter substantially the normal internal structure, nor does the osmoticum have more than a transient effect (Burgess et al., 1977; Berliner, 1981). In the present ultrastructural study, also no difference was found between vegetative cells and freshly isolated protoplasts of this alga, except for volume expansion which occurs when protoplasts were isolated. The expansion of volume facilitates observation of organelles of protoplasts.

According to our observations with the scanning-electron microscope (SEM), freshly isolated protoplasts excreted an amorphous matrix to their surface (Fig. 1), and become fully covered by the matrix after culture for six hours (Fig. 2). This result is consistent with the results with a fluorescent stain of our previous work (Chen and Chiang, 1994) in which protoplasts of G. sparsa had a tendency to sink to the bottom of the flask and continuously excreting the amorphous matrix to adhere to the substrate. With calcofluor white stains, most protoplasts were found to secrete an amorphous matrix when they were isolated. Almost all showed a bright-blue stain color after culture for 6 h. The chemical nature of this amorphous substance remains unknown. Kloareg and Quatrano (1988) indicated that the components of outer cell walls of Grateloupia are considered polymers intermediary between carrageenans and agars, but, the polymers are ß1,4-galactans.

The SEM results of this study and the previous fluorescent stain observation (Chen and Chiang, 1994) showed that protoplasts of G. sparsa deposited a thick layer of amorphous substance on their surface within 6 h of the isolation, such deposition seems to continue while the protoplasts are alive. Therefore, the use of TEM to observe the deposition inside the walls and the organelle behavior during continuous excretion of the amorphous substance and deposition of the cellulose walls is necessary.

Further observation of freshly isolated protoplasts with TEM showed several long endoplasmic reticula parallel to the plasmalemma, and many vesicles were located between the reticula themselves and the reticula and plasmalemma (Fig. 4). Some fractions of the two-layer membrane of the endoplasmic reticula become swollen (Fig. 4). Conspicuous development of endoplasmic reticula and their localization just below the plasmalemma (Fig. 4) at which microfibrils were observed (Fig. 7) indicates that endoplasmic reticulum is involved in regeneration of cell wall of protoplasts of G. sparsa; we deduced that the amorphous substance was apparently produced from endoplasmic reticula that were directly excreted near the plasmalemma.

According to the observation of regeneration of cell walls of G. sparsa protoplasts, microfibrils were regenerated from the irregular plasmalemma (Fig. 7) and were subsequently deposited to form the microfibril layer underneath the thick amorphous matrix layer. Hence the site of regeneration of cellulose microfibril was the plasmalemma. Synthesis of cellulose occurs at or near the plasmalemma (Villemez et al., 1968). Even though precursors may be formed within the cell, assembly of microfibrils occurs at the cell surface (Robinson, 1977).

Biochemical tests on the synthesizing locus of the cell wall suggest that synthesis of the amorphous matrix is mediated by Golgi bodies (Northcote and Pickett-Heaps, 1965) and that synthesis of cellulose occurs within plasmalemma (Villemez et al., 1968). The dynamic alteration of ultrastructure of plasmalemma may be accompanied by increasingly active synthesis or transport of polysaccharides to the cell wall (Mori et al., 1983). Deposition of microfibrils in a liverwort Marchantia berteroana L. is apparently caused by activity in the Golgi body; incorporation of their vesicles into rapidly growing walls was reported by Fowke and Pickett-Heaps (1972). However, in this study on regeneration of cell walls of the red algal Grateloupia sparsa protoplasts, no development of Golgi bodies was observed during deposition of the amorphous matrix layer and the inside microfibril layer, but the endoplasmic reticula apparently lengthened and swelled. Mori et al., (1983) also reported that protoplasts of Marchantia polymorpha L. had no Golgi bodies during the early stage of cell wall formation. Liu et al. (1992) found that the endoplasmic reticula of protoplasts of the red alga Palmaria palmata (L.) Kuntze discharged electron-dense bodies outside the plasmalemma as protein precursors of microfibrils. Direct secretion of cell wall materials by the endoplasmic reticulum possibly allows rapid regeneration of the cell wall in these red algal protoplasts; this phenomenon was also reported by Mori et al. (1983) and by Liu et al. (1992).

There are no published reports on wall deposition on regenerating protoplasts in Grateloupia sparsa or even in red algal protoplasts. However, the altered ultrastructure of the protoplast during early periods of cultivation seems important in wall regeneration. The alteration from a smooth plasmalemma (Figs 4, 5, 6) to an irregular one (Fig. 7) was observed for the protoplast of G. sparsa, consistent with results on protoplasts of tomato fruit (Pojnar et al., 1967) and of Marchantia polymorpha (Mori et al., 1983). This irregular plasmalemma may be a sign that cell wall synthesis occurs (Mori et al., 1983).

In this study the microfibrils lengthened, joining to the fine granules on their terminal end (Fig. 9), and then forming ordered arrays (Fig. 10). This increased length of microfibrils may be caused by rearrangement of microfibril arrays from random to ordered ones. In two dimensional thin sections (thickness ca. 50-60 nm) the length of microfibrils of random arrays is less than that of ordered ones when seen under the TEM. The orientation behavior (rearrangement) of microfibrils is discussed in regeneration of the cell wall of a green algal protoplast of Boergesenia forbesii (Harvey) Feldmann (Mizuta et al. 1985). The fine granules located in lamellae of new microfibril layers of Grateloupia sparsa protoplasts may play the same role as the terminal complex binding on the microfibril end of protoplasts of Boergesenia forbesii. Mizuta et al. (1985) reported that protoplasts of B. forbesii rapidly developed an amorphous matrix layer on the outer surface of the plasmalemma and then regenerated a randomly oriented fibril layer underneath the former. When the matrix-rich layers thickened, microfibril-rich layers formed underneath them. Cellulose synthesizing enzyme complexes (terminal complexes) were observed at the ends of the microfibrils. Orientation of cellulose microfibrils was assumed to be induced by the altered direction of the complexes.

In conclusion, regeneration of cell walls of protoplasts of Grateloupia sparsa directly involves the endoplasmic reticula, which resembles those of higher plants (Pojnar et al., 1967), liverworts (Mori et al., 1983) and red algae (Liu et al., 1992). By this strategy the freshly isolated protoplast may rapidly adapt to the osmotic stress. Although no further information about regeneration of cell walls of red macroalgal protoplasts can be discussed, this process for G. sparsa protoplasts is similar to that of the green alga Boergesenia forbesii (Mizuta et al., 1985).

Acknowledgements

We 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 support.

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Legends of figures.

Abbreviations used in figures:

Am(l): amorphous matrix (layer); B: bulge; Chl: chloroplast; Cm: cell membrane; Chr: chromatin; D: transparent area; Er: endoplasmic reticulum; Fs: Floridean starch granule; Mf(l): Microfibril (layers); Mi: mitochondrion; Np: nucleus pole; Nu: nucleus; P: peroxisome; PP: protein-like precursors and granules. Osm: osmiphilic granules; Tc: terminal complex-like granule; V: vacuole.

Figs 1-3. Protoplasts observed under SEM. Fig. 1. Freshly isolated protoplast, its surface is already covered by some of the excreted amorphous matrix. Fig. 2. Protoplast of six hrs old and fully covered with amorphous matrix that forms bulges. Fig. 3. Protoplast of one day old. The matrix layer has become increasingly thick, so that made some areas have fused together to form a smooth surface (arrow head).

Figs 4-5. Thin-section of freshly isolated protoplast. Fig. 4. A large number of vacuoles present between the long endoplasmic reticula themselves and the cell membrane. Some fractions of the two-layer endoplasmic reticula are swollen (* ). The nucleus pole is conspicuous. The cell membrane is intact and recognizable. Fig. 5. The short endoplasmic reticula has no obvious of vacuole. The nucleus is surrounded by the typical floridean starch granules.

Fig. 6. Thin-section of protoplast of six hrs old. The vacuoles are outside the cell membrane (arrow heads), the chloroplast is dividing by a middle partition, the end (* ) of the mitochondrion is swollen by an unknown cause. The cell membrane is intact and recognizable.

Figs 7-9. Thin-section of protoplasts of four days old. Fig. 7. The microfibrils appear in the irregular cell membrane and are deposited into the amorphous matrix layer. The inside cell of protein-like precursors and granules are underneath the cell membrane. The length of microfibrils is about 100-200nm. Fig. 8. The microfibrils have deposited to form a thin layer that is about 140 nm in thickness, the terminal complex-like (Tc) granules frequently appear between them. Fig. 9. The microfibrils are lengthened to about 100-200 to 300 nm, and show the rearrangement of microfibrils. Their terminus is generally bonded with the Tc-granules.

Fig. 10. Thin section of protoplast of seven days old whose microfibril layer has become thicker (ca. 375-500 nm). The number of the Tc-granules (double arrow heads) is apparently increased and the array of the microfibril layer is more ordered than in those of four days old, following gradually complete cell wall formation.