The ultrastructure of Grateloupia filicina (Halymeniaceae, Rhodophyta) pit plugs

Yean-Chang Chen

Department of Aquaculture

National Taiwan Ocean University

Keelung, Taiwan

　

　

　

　

　

　

　

ABSTRACT

Cultured Grateloupia filicina was used as the material for observations on spore development and the ultrastructure of pit plugs. The general characteristics of the pit plugs were the same in the discs, filaments and frond-like thalli of this red alga. The pit plugs were naked (no cap layers) with a cap membrane and a plug core. The matrix was diffuse in the center and dense in the outer portion. However, the sizes and shapes of the pit plugs varied. Pit plugs of the filament-like medullary cells of the frond-like thalli were the largest. These pit plugs were H-shaped with a deep central constriction. Pit plugs of the filament cells, were bead-shaped and were smaller than those of the medullary ones. In contrast, pit plugs of the cortex of the frond-like thalli were the smallest. The pit plugs of disc cell and terminal ball-like structures on filaments were a little larger than the cortex and filament cell pit plugs, and were roughly round-shaped, like those of the cortex. This study confirms that the pit plugs of red algae vegetative cells have taxonomic value, even among the same species in different growth phases.

 

Key index words: Disc, Filament, Grateloupia filicina, Pit plug, Red alga, Ultrastructure.

 

INTRODUCTION

Pit plugs (or pit connections) are a distinctive feature of the cross walls in many red algae, particularly species belonging to the class Florideophyceae. The ultrastructure of the pit plug has been studied by a number of investigators (Ramus 1969, Sommerfeld and Leeper 1970, Lee 1971, Hawkins 1972, Aghajanian and Hommersand 1978, Pueschel 1977, 1979, 1980a, 1980b, 1982, 1987, 1989; Pueschel and Cole 1982, Pueschel and Magne 1987, Maggs and Pueschel 1989). The pit plug is formed by the annular ingrowth of the wall material, leaving an aperture connecting the cytoplasm of the two cells. This aperture is eventually occluded by the deposition of a cylindrical, largely proteinaceous structure, the pit plug (Ramus 1969, 1971, Pueschel 1980a, b).

The distribution of pit plugs among the Rhodophyta provides valuable taxonomic information. Pueschel (1989) examined the pit plugs of about 150 species for systematic significance.

The aim of this study was to observe the pit plugs of varied growth phases in Grateloupia filicina (Lamouroux) C. Agardh, Halymeniaceae, Florideophyceae, Rhodophyta. Grateloupia filicina is the type species of the genus Grateloupia and is distributed worldwide. It has macroscopic, isomorphic tetrasporophytes and gametophytes. The gametophytes are monoecious. The spores (carpospores and tetraspores) germinate to form crusts which will develop further into crusts, filaments or frond-like thalli depending on the culture conditions (Chiang 1993, Chen and Chiang 1994).

 

MATERIALS AND METHODS

Materials. The materials used in this study were the thalli of Grateloupia filicina collected at Keelung, Taiwan, on 12 May, 1995.  Freshly collected algae were wrapped in absorbent paper towels moistened with seawater, put in a plastic bag, and brought back to the laboratory of the Department of Aquaculture, National Taiwan Ocean University in Keelung.

 

Preparation of clean algal material. Selected pieces of vegetative fronds (2 cm²) were thoroughly cleaned in filtered seawater. They were then put into an ultrasonic cleaner (Branson 3200) with two changes of autoclaved seawater for 5 minutes each, to remove animals and epiphytes.  The pieces were then rinsed several times with autoclaved seawater. Finally they were incubated in 100 mL of autoclaved Provasoli’s enriched seawater (PES) (Provasoli 1968) containing 5 mL of antibiotic mixture (Polne-Fuller and Gibor 1984) for 24 hours at 18° C under a photon flux density of 20 μmol photons^.m^-2.s^-1 and 12:12, L:D photoperiod in a plant incubator (Firstek, Taiwan).

 

Induction of spore release. To enhance the release of spores, the cleaned algal thalli were wiped with clean absorbent paper, then laid in a cool and shady place for 5 minutes. Subsequently, they were cultivated in Petri dishes with PES medium in a plant incubator at 24° C, under a light intensity of 100 μmol photons^.m^-2.s^-1 and a photoperiod of 12:12, L:D. These cultures were observed daily with a stereo-microscope (SV-11, Zeiss). When the spores were released, the parent thalli were discarded.

Cultivation and germination of the spores. Light microscopes (LM) (Axioskop and Axiovert 135, Zeiss) were used to observe the development of the released spores. Spores formed disc-like thalli (crusts) and filaments in light intensities of 20 and 100 μmol photons^.m^-2.s^-1 and a photoperiod of 12:12, L:D with PES medium. The discs and filaments were then incubated at 166 μmol photons^.m^-2.s^-1 of light intensity and a photoperiod of 12:12, L:D to form frond-like thalli (Chiang 1993, Chen and Chiang 1994).

Fixation of algal materials for electron microscopic studies. The filaments, discs and frond-like thalli were fixed in 15-mL centrifuge tubes, with gentle shaking, at 4° C for 2 h in PES medium containing 2 % glutaralde­hyde, and 0.2 M sucrose. The filaments and discs were collected by centrifuging at 300 x g for 10 min. All materials were placed in 5 % glutaraldehyde in 0.1 M sodium ca­codylate (pH 6.8) buffer containing 10 mM CaCl₂ and 0.2 M sucrose at 4° C for 2 h. The samples were rinsed four times in 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂, with the sucrose concentration successively reduced to 0.05 M. This treatment was followed by two rinses in sucrose-free 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂.

Post-fixation was performed in a mixture of 2 % osmium tetroxide and 1.1% potassium ferrocyanide with a 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂ for 2h at 4℃.

Thereafter, all materials were rinsed four times with 0.1 M sodium cacody­late buffer containing 10 mM CaCl₂. The samples were rinsed three times with aqueous ethanol (50 %), and dehydrated gradually in ethanol (50, 70, 85, 95 and 100% successively).

Some material was prepared for Transmission Electron Microscopy (TEM) by treatment with three changes of 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 propyl­ene oxide : Spurr's resin to 1:1 of 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) and polymerization of the resin.

Thin-sections were stained with uranyl acetate and lead citrate according to Smith and Croft (1991) and examined on a Jeol 1200EXII or Hitachi HF100 TEMs.

Specimens for Scanning Electron Microscopy (SEM) were dried with a critical-point-drying machine (HITACHI-HCP-1), coated in an ion coater (Jeol, JCF-1100E) for 3 min 40s and examined on a Jeol JSM-6300 or Hitachi S-800 SEMs.

 

RESULTS

Germination and development of spore. Within a week of incubation, the released spore produced a germ tube (Fig. 1) into which the contents of the spore were evacuated (Fig. 1). A septum was formed between the tube and the spore (Figs 1 and 2). The tip of the tube was then repeatedly and rapidly divided to form a radially expanding disc (Fig. 2). The central area of the disc was composed of several layers of cells from which filaments or frond-like thalli (Figs. 3 and 4) developed under further cultivation in a light intensity at 166 μmol photons^.m^-2.s^-1. The layers of the disc gradually decreased from the central area, with the margins consisting of only a simple layer of cells (Figs 2 and 3).

 

SEM observations of the disc and filament. As shown in Figure 5, an erect, cylindrical filament developed from the disc. The filament was primarily composed of a row of cells that were equal in diameter (ca. 6 µm), but varied in length (ca. 6-10 µm). Frequently, a ball-like structure was observed at the tip of the filaments (Fig. 6). When the ball-like structures were attached to substrates, they formed discs.

 

TEM observation of the pit plugs in the filaments, discs and fronds. The main body of the pit plug, the plug core, appeared finely granulated and diffuse in the center. The remaining matrix of the plug core was moderately electron dense (Figs 7a, 7b, 9, 10a, 10b, and 14). The convex ends of the plug core were only covered with a membrane (Figs 7b, 9, 10a and 14) and cap layers were absent (Pueschel 1989). The cap membrane separated the pit plug from the vegetative cell, showing that the pit plug is an extracellular structure (Figs 7b, 9, 10a and 14). However, the pit plugs of this alga with varied forms of thalli were different in size (ranging from 0.3 to 1.4 μm) and shape.

Ultrastructure of pit plugs in the filament. Light microscopy suggested that some of the filaments cells may be empty (Fig. 3) which made it difficult to observe the pit plugs. However, TEM revealed that the filament cells were occupied by large vacuoles that pushed organelles and cytoplasm to the periphery of the cells (Fig. 11), making the cells appear empty under the light microscopy. Cells (Fig. 12) without large inner vacuoles showed a typical red algal cell structure, as did the cells of the disc and frond-like thalli. The nucleus was typically surrounded by several grains of floridean starch (Fig. 12).

As seen in Figure 13, the cells of the ball-like structure were like the cortex and disc cells with many grains of floridean starch inside. The shapes of the cells of the ball-like structure were slightly irregular (Fig. 13) as seen in the SEM (Fig. 6) and were ca. 2x3-4x5 µm in dimensions.

The shapes of the filament pit plugs (Figs 7a and 7b) resembled beads with a deep equatorial constriction and were convex at both ends. The diameter of the filament pit plugs were identical, about 0.5 µm. In addition, the terminal ball-like structures of the filaments also had pit plugs (Fig. 13) with two oval-shaped ends. They were ca. 0.6 µm in diameter, and were larger than those in the filaments. However, the basic structures of the pit plugs, such as the plug core and cap membranes, were identical to those in the filaments.

 

Pit plugs in the disc. In TEM observations, thin-sections showed that the disc cells were cubic or triangular (Fig. 8), and were ca. 3x6-4x7 µm in dimensions, as seen in the SEM (Fig. 5). The pit plugs (Fig. 14) of disc cells were ca. 0.6 µm in diameter, and were smaller than the medullary cells, but similar to that of cortex and ball-like structures. The junctions between the cap membrane and plasmalemma were clear and smooth (Fig. 14). These findings indicate that the cap membrane may be a membrane unit.

 

Pit plugs in the frond-like thallus. The frond-like thalli were composed of several layers of compact and densely colored cortex and a thick and loosely organized layer of filament medullary cells (Fig. 15). The outer layers of the cortex were small and ca. 5-10 μm in diameter. The inner layers of the cortex were bigger and ca. 10-15 μm in diameter. Loosely organized filament-like medullary cells were over 100 μm (ca. 100-140 μm) in length, and ca. 5 μm in width. Two lenticular ends formed the pit plugs (Fig. 9) of the inner and the outer cortex. They were ca. 0.3-0.4 μm in diameter, and were a little smaller than the bead-shaped pit plugs of the filaments. However, they were roughly round-shaped. In contrast, the pit plugs (Figs. 10a and 10b) of the medullary cells were the largest among the algal thalli. They were 1.2-1.4 μm in diameter, which obviously occupied the adjacent area between the filament-like medullary cells (Fig. 10a). The two convex ends were a little flatter with a deep central constriction, making the whole pit plug look H-shaped (Fig. 10a and 10b).

 

DISCUSSION

The frond-like thalli of Grateloupia filicina which included tetrasporophytes and gametophytes, demonstrated the same spore development patterns, from the disc to filament and to the isomorphic tetrasporophyte and gametophyte. The varied thalli growth phases were obtained under culture in this study. The pit plugs of these thalli were identical in associated membranes, cap membrane, the plug core ends, and lack of cap layers, which confirms the value of the taxonomic information. Pueschel (1989) similarly reported that G. filicina pit plugs had no cap layers.

Except for the heteromorphic life cycle, some red algae also show different growth patterns reflecting on diverse environmental factors. The spores of Grateloupia filicina only germinated to form discs and filaments while they were kept under low light intensity. When transferred to 166 μmol photons^.m^-2.s^-1 of light intensity, filament and disc cells formed erect, frond-like thalli. It is rare that a paper reports on the ultrastructure of pit plugs of the same algal species with such different thallus forms. Pueschel (1989) examined the possibility of differences in plug structure in different phases of the heteromorphic life histories, and found this theory to be without support. In Pueschel’s study the names of the algal species examined were not detailed. However, this study confirms that a species with different phases still exhibits the same types of pit plugs, even though this involved examination of an isomorphic life history.

In this study, cap membranes of the medullary cell pit plugs were obvious. This may be due to the larger size of the pit plugs (1.2-1.4 µm in diameter) (Figs. 10a, and 10b). Pueschel (1977) reported that cap membranes look, stain, fracture and extract like membranes, but the junction with the plasmalemma is anomalous. There was no evidence of its presence before or during deposition of the plug core. Therefore, it is questionable that the cap membrane is a true unit membrane. However, the findings in this study showed that the cap membrane junctions between the plasmalemma were clear and smooth (Fig.14). The cap membrane may be a true unit membrane.

Kugrens and Delivopoulos (1985) found that large septal plugs interconnect the gonimoblast cells and auxiliary cells of Faucheocolax attenuata Setch. These plugs are small when first formed but increase dramatically in size during carposporophyte development. Broadwater and Scott (1982) found that the female system of Polysiphonia harveyi Bailey varied in the presence of cap membranes. The pit plugs without cap membranes were more readily dismantled after fertilization. In contrast, neither the size of the pit plugs changed significantly nor was its structure altered during the vegetative growth of the varied thalli phases in this study. However, only mature algae have those limits and changeable pit plugs. The structural changes or disappearance of the pit plugs may involve the transference of nutrients to the reproductive cells (Broadwater and Scott 1982).

In conclusion, the vegetative cells of the red alga Grateloupia filicina have different pit plugs sizes and shapes due to the diverse cell patterns composing its varied thalli. Despite the diverse appearance and size of these pit plugs, the ultrastructure of the pit plugs among the varied thalli in Grateloupia filicina are entirely identical. Therefore, the characteristics of the vegetative cell pit plugs appear to have a stable taxonomic value.

 

ACKNOWLEDGMENTS

Financial support from the National Science Council (NSC-87-2313-B-019-031; NSC-88-2313-B-019-046; NSC-89-2313-B-019-032) and Council of Agriculture (COA-88-AST-1.4-FID-02[12-2]) of the Republic of China is highly appreciated.

Accepted 12 June 1999

 

REFERENCES

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Chen, Y. C. and Y. M. Chiang. 1994. Development of protoplasts from Grateloupia sparsa and G. filicina (Halymeniaceae, Rhodophyta). Bot. Mar. 37: 361-366.

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Hawkins, E. 1972. Observation on the developmental morphology and fine structure of pit connections in red algae, Cytologia 37: 759-768.

Kugrens, P. and S. G. Delivopoulos. 1985. Ultrastructure of auxiliary and gonimoblast cells during carposporophyte development in Faucheocolax attenuata (Rhodophyta). J. Phycol. 21:240-249.

Lee, R. E. 1971. The pit connections of some lower red algae: ultrastructure and phylogenetic significance. Br. Phycol. J. 6: 29-38.

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Provasoli, L. 1968. Media and products for the cultivation of marine algae. In: (Watarabe, A. and A. Hattori, eds.): Cultures and Collections of Algae. Jap. Soc. Plant Physiol., Tokyo.pp.63-75.

Pueschel, C. M. 1977. A freeze-etch study of the ultrastructure of red algal pit plugs. Protoplasma 91: 15-30.

Pueschel, C. M. 1979. Ultrastructure of tetrasporogenesis in Palmaria palmata (Rhodophyta). J. Phycol. 15: 409-24.

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Pueschel, C. M. 1980b. A reappraisal of the cytochemical properties of rodophycean pit plugs. Phycologia 19: 210-7.

Pueschel, C. M. 1982. Ultrastructural observations of tetrasporangia and conceptacles in Hildenbrandia (Rhodophyta). Br. Phycol. J. 17: 333-41.

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FIGURE LEGENDS

Abbreviations: BL: Ball-like structure, CH: Chloroplast, Cm: Cap membrane, Co: Plug Core, Ct: Cytoplasm in GT, CW: Cell wall, D: Disc, F: Filament, FLM: Filament-like medullary cell, Fs: Floridean starch, FT: Frond-like thallus, GT: Germ tube, IC: Inner cortex, Mi: Mitochondrion, Nu: Nucleolus, OC: Outer cortex, PP: Pit plug, Sp: Septum, V: Vacuole.

 

Figs 1-4: Observations by light microscopy. Fig. 1. The germination of spores of Grateloupia filicina within a week of culture in PES medium at 24° C, 100 μmol photons^.m^-2.s^-1 light intensity and a photoperiod of 12:12 L:D. The spore firstly produced a germ tube (GT), then transferred its cytoplasm into this tube, subsequently the spore became empty with the newly formed septum between the spore and tube (arrowheads: empty spores). Fig. 2. The freshly formed discs which developed at the end of the germ tube. Several layers of cells composed the central area of the disc, from which the layers decreased to one layer of cells at the margins (arrowheads: empty spores). Culture conditions were 24° C, 20 μmol photons^.m^-2.s^-1 light intensity and a photoperiod of 12:12 L:D. Fig. 3. Filaments developed from the discs. When the ends of filaments attached to the substrate, they formed discs. A few of the intervening filaments (arrowheads) were composed of two or three cells. Culture conditions were 24°C, 100 μmol photons^.m^-2.s^-1 light intensity and photoperiod of 12:12 L:D. Fig. 4. A frond-like thallus developed from the disc. Culture conditions were 24° C, 166 μmol photons^.m^-2.s^-1 light intensity and a photoperiod of 12:12 L:D.

Figs 5-6: S. E. M. Fig. 5. The fine structure of a disc and a filament. The cells of the disc were cubic or triangle-shaped. A row of cylinder-shaped cells composed the filaments. Fig. 6. The fine structure of the filament and its terminal ball-like structure. Shapes of the cells of the surface of ball-like structure were irregular.

Figs 7a-10: T. E. M. Figs 7a and 7b. Ultrastructure of bead-shaped pit plugs (PP) between the filament cells. Fig. 8. Ultrastructure of three roughly round-shaped pit plugs between disc cells. They were ca. 0.6 μm in diameter. Fig. 9. Ultrastructure of pit plugs between cortex cells of the frond-like thallus. They were the smallest at 0.3-0.4 μm in diameter. Fig. 10a. Ultrastructure of H-shaped pit plugs between filament-like medullary cells of the frond-like thallus. They were the largest and were 1.2-1.4 μm in diameter. Fig. 10b. Two H-shaped of pit plugs between three filament-like medullary cells.

Figs 11-14: T. E. M. Figs 11 and 12. Ultrastructure of the transverse thin-sections of filaments. Fig. 11. A large vacuole occupies the inside of the cell of a filament. Fig. 12. Ultrastructure of typical red algal cells of the filament. Floridean starch granules typically surround the nucleus. Fig. 13. Ultrastructure of pit plugs between the cells of the ball-like structure of the filament. Their shapes were similar to those of cortex and were ca. 0.6 μm in diameter. Fig. 14. Ultrastructure of the pit plug between the disc cells. The junctions were clear and smooth.

Fig. 15. Vertical section of frond-like thallus, observed under the light microscope. Several layers of cortex composed of two densely colored regions between which were thick and loosely organized layers of filament-like medullary cells, 5x100-140 μm in dimensions. The cells of the outer cortex were compactly laid out and were ca. 5-10 μm in diameter. Those located between the outer cortex and the filament-like medullary cells, the inner cortex, were ca. 10-15 μm in diameter.