Ultrastructure of the siphonaceous green alga Halimeda cuneata, with emphasis on the cytoskeleton

Micron 41 (2010) 598–603

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Ultrastructure of the siphonaceous green alga Halimeda cuneata, with emphasis on the cytoskeleton Zenilda Laurita Bouzon a,b,∗ , Maria Elizabeth Bandeira-Pedrosa c , Éder Carlos Schmidt a a

Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil Central Laboratory of Electron Microscopy, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil c Department of Biology and Botany, Federal University of Rural of Pernambuco 52171-900, CP 476, Recife, PE, Brazil b

a r t i c l e

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Article history: Received 29 January 2010 Received in revised form 1 April 2010 Accepted 1 April 2010 Keywords: Cytoskeleton Halimeda cuneata Microtubules Ultrastructure

a b s t r a c t Cytoplasm streaming is a fundamental process for the transport of molecules and organelles in plant cells. In vegetative filaments of the coenocytic green alga, Halimeda cuneata Hering, the spatial organisation of microtubules in the cytoplasmic layer, was observed under transmission electron microscopy. A cross section of a cortical filament shows a tubular cell wall enclosing a peripheral layer of cytoplasm with numerous chloroplasts, amyloplasts, nuclei, mitochondria and microtubules surrounding a small central vacuole. Towards the thallus medulla the central vacuole enlarges considerably and the cytoplasm becomes gradually reduced to a thin parietal layer, the number of organelles is reduced and the quantity of microtubules increases. Therefore, most of the thallus volume is occupied by the huge central vacuole which extends throughout the coenocytic filaments. Microtubules run longitudinally, being about 0.05 ␮m from each other. Some microtubules were observed in close association to cell organelles. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction The cytoskeleton is responsible for essential functions in plant cells, including cell division, intracellular transport and cell wall construction (Menzel and Elsner-Menzel, 1990; Hasezaka and Nozaki, 1999). The main elements of the cytoskeleton are microtubules (MTs) and microfilaments (MFs), both participating in the processes of cell motility and morphogenesis (Hepler and Palevitz, 1974; Blancaflor, 2000; Collings et al., 2006). Some reports indicate that MTs are probably responsible for the regulation of cytoplasmic currents, amongst other functions (Kuroda and Manabe, 1984; Manabe and Kuroda, 1984; Menzel, 1985, 1987; Collings et al., 1996). Menzel (1985) demonstrated with green alga Chlorodesmis fastigiata (C. Agardh) S.C. Ducker the chloroplasts and amyloplasts exhibit a closely apposed to the surface of the microtubule bundles. These findings suggested that this architecture serves as an efficient differentiation facilitating long range transport along the microtubule bundles. In Bryopsis plumosa (Hudson) C. Agardh, experiments with antimicrotubule agents (colchicine, vinblastine, and griseofulvin)

occurred the blockade the organelles transport, as consequence, appeared morphological anomalies (Mizukami and Wada, 1981, 1983). A number of studies in plant cells have revealed details on cytoplasmic motility, suggesting that the intracellular transport system is co-operative and is supported by interactions between actin and myosin on one hand, and actin-myosin and MTs on the other (Williamson, 1986). There is a wealth of information regarding the structure of siphonaceous green algae, based on observations made at light microscope level. Among these a number have employed light microscopy in combination with immunofluorescence techniques (Menzel, 1986, 1987; La Claire, 1987; Shihira-Ishikawa, 1987; Hayano et al., 1988; McNaughton and Goff, 1989; Mizuta et al., 1991; Liddle et al., 1997). However, very few studies have demonstrated the distribution of MTs based on transmission electron microscopy (TEM). Menzel (1985) is one of these, describing the association of MTs with plastids in Chlorodesmis Harvey and Bailey. The present study reports an ultrastructural investigation of the filaments of Halimeda cuneata K. Hering. Here we show the role of MTs in the mobility of organelles of H. cuneata and sugest a possible role for MTs in filaments architecture. 2. Materials and methods

∗ Corresponding author at: Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, SC Florianópolis, SC, Brazil. Tel.: +55 48 3721 5149. E-mail address: [email protected] (Z.L. Bouzon). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.04.001

Halimeda cuneata were collected from Mar Grande Beach (−12◦ 53 18 and 38◦ 40 43 ) Itaparica-Bahia, Brazil. Small phragments of alive thalli were fixed in the field, with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) with

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Fig. 1. Ligth micrographic and transmission electron microscopy micrographic images of H. cuneata. (A) Ligth micrographic showing the utricule region (arrows) and medullar filaments. (B) Longitudinal section of utricule showing a large quantity of chloroplasts, thick cell wall and large vacuole. Observe a low quantity of amyloplast. A, amiloplast; C, chloroplast; CW, cell wall; MF, medullar filament; U, utricule. Bar in (A) = 10 ␮m; bar in (B) = 2 ␮m.

Fig. 2. Transmission electron microscopy micrographic images of H. cuneata. (A) Detail of transversal sections of a Halimeda utricle showing the cytoplasm filled with a large number of chloroplasts (arrows) and some amiloplats. (B) Detail of transversal sections of a Halimeda between two utricles. Observe the cell wall between them. (C) Detail of chloroplasts. Observe the thylakoid (arrows), amiloplast and plastoglobuli. (D) Longitudinal section of the first part of the medullary region, just below the cortical layer. Note that the cytoplasm, bordered by the large vacuole, is filled with small chloroplasts and nuclei. A, amiloplast; C, chloroplast; CW, cell wall; N, nucleus; Nu, nucleolus; P, plastoglobulus; V, vacuole. Bar in (A) = 2 ␮m; bar in (B) = 0.2 ␮m; bar in (C) = 0.5 ␮m; bar in (D) = 1 ␮m.

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0.2 M sucrose overnight. The material was post-fixed with 1% osmium tetroxide for 4 h, dehydrated in a graded acetone series and embedded in Spurr’s resin. Thin ultrathin sections were stained with aqueous uranyl acetate followed by lead citrate (Reynolds, 1963). The samples were examined and photographed under transmission electron microscope (TEM) Jeol JEM 1011 at 80 kV. 3. Results When observed by light microscopy and by transmission electron microscopy observations show that the coenocytic filaments of H. cuneata are permanently polarised, with a cortical regions (Fig. 1A and B) and distinct medullary (Fig. 1A). This polarisation is structurally expressed by the separation of the cell content in two main zones: the apical, outer region, with vacuoles (Fig. 1B) and a large number of chloroplasts (Fig. 2A–C), and an inner region with vacuole; amyloplasts, nuclei and other organelles are distributed throughout the medullary region (Fig. 2D). The cortical region of the thallus is formed by utricles, which correspond to the expansion of the terminal portion of each fil-

ament. A thick cell wall (Fig. 1B) surrounds the utricle, which is filled with dense cytoplasm, containing mainly elongated chloroplasts (Fig. 2A–C) arranged perpendicularly to the surface. A more detailed examination of this cytoplasmic layer did not reveal the presence of any MTs. However, in the region immediately beneath the chloroplast layer the cytoplasm becomes less dense. In this area, between small vacuoles and sparse organelles, the first MTs began to be observed in small groups in a more or less parallel arrangement, in longitudinal and in cross sections (Fig. 3A–D). Amyloplasts increase considerably in number towards the medullar region, whereas other organelles (chloroplast and mitochondria) decrease in number (Fig. 3A). These amyloplasts were frequently observed aligned with bundles of MTs (Fig. 3D). A more detailed examination of this organelle showed MTs oriented in parallel and linked to the external envelope of the amyloplast (Fig. 3D). As the central vacuole increases below the utricular region the cytoplasm becomes gradually compressed appearing as a thin parietal layer in the medullary filaments and most of the cell volume is occupied by the central vacuole, that drastically reduces the space

Fig. 3. Transmission electron microscopy micrographic images of H. cuneata. (A) Detail of cytoplasm with a large quantity of microtubules. (B) Microtubules seen in longitudinal section (arrows). (C) Microtubules seen in transverse section. (D) Microtubules seen in longitudinal section, adjacent to amyloplasts. A, amiloplast; MTs, microtubules. Bar in (A) = 1 ␮m; bar in (B) = 0.2 ␮m; bars in (C and D) = 0.2 ␮m.

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Fig. 4. Transmission electron microscopy of H. cuneata. (A) Detail of mitochondria associated with microtubules and amyloplast. (B) Choroplast associated with microtubules. A, amiloplast; C, chloroplast; M, mitochondria; MTs, microtubules. Bar in (A and B) = 2 ␮m.

available to organelles. In this region, the regularly spaced MTs are more frequent and disposed into bundles with a greater number of elements. These MTs were occasionally observed near some organelles, such as: amiloplast (Figs. 3A and 4A), mitochondria (Fig. 4A) and chloroplast (Fig. 4B). Sections from deeper medullary filaments show that the central vacuole formed the greater portion of the cell contents, causing the cytoplasm to be restricted to a fine parietal layer. In this region, the parietal cytoplasm was filled by thick bundles of MTs that ran in a parallel manner the whole length of the filament (Fig. 5A). MTs density, in medullary filaments, is around 45 units per 0.25 ␮m2 . In diagonal view, MTs were found to be organised in bundles, apparently connected to the plasma membrane and to the tonoplast (Fig. 5A, B, D, and E). In transverse sections MTs 40-50 nm in diameter (Fig. 5B and C) with an electron-light centre of approximately 22 nm in diameter (Fig. 5B and C). This central region was surrounded by a 7.5-nm-thick electron-dense wall (Fig. 5D). Masses of amorphous material were observed between or in association with MTs (Fig. 5B and C). 4. Discussion The presente study showed relation with cytoskeleton and microtubules of siphanonaceas green alga H. cuneata, demonstrated the interaction of MTs with some organelles (amyloplast, choloroplast and mitocondria). Current evidence suggests that

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microtubules constitute a distinct class of filamentous elements in both plant and animal cells. MTs have been exhaustively studied due to their involvement in a variety of important cell functions such as the production and maintenance of cell asymmetry, the transport of cytoplasmic material and cell division. In the coenocytic filaments of H. cuneata chloroplasts and amyloplasts show a different regional distribution. Chloroplasts are more abundant in the utricular region whereas amyloplasts are present throughout the filament, beneath the utricular region. MTs were not identified in the region where chloroplasts are concentrated. However, towards the medullary region the number of MTs increase considerably. In this area the amyloplasts were observed in close association with bundles of MTs, suggesting that the movement of these organelles might be mediated by these elements of the cytoskeleton. Following the recognition of the important role of the cytoskeleton in cell development, it has become clear that the transport of amyloplasts and other organelles in coenocytic filaments is related to MTs-redundante. It is likely these MTs serve as guides for the directional movement of amyloplasts and other organelles, as well as materials synthesised in the utricular region. The link between MTs and the organelles or other substances to be transported, may be mediated by proteins present on the surface of MTs (Menzel and Elsner-Menzel, 1989). In Caulerpa, a coenocytic algae without siphonous filaments, the chloroplasts are also restricted to the cortical region and are apparently maintained in fixed positions. On the other hand amyloplasts show a more internal distribution and are associated with, and transported by, bundles of MTs (Menzel, 1987; Menzel and Elsner-Menzel, 1989). In another coenocytic alga, Bryopsis, the cortical longitudinal bundles of MTs are extensively associated with actin filaments and both types of bundle are involved in the movement of chloroplasts (Menzel and Schliwa, 1986a,b). This shows that MTs have different distributions among different coenocytic algae. In the pollen tubes of Nicotiana alata Link and Otto MTs were frequently observed, both singly and in groups, arranged in parallel to the longitudinal axis of the cell, but they do not appear to be associated with any particular organelle. MTs have occasionally been observed in association with the nuclear envelope (Lancelle et al., 1987). In Acetabularia, post-division nuclei appear to be orientated by bundles of MTs. In this alga each nucleus is intimately associated with MTs in a three dimensional arrangement which probably interacts with actin filaments in nuclear movement (Menzel, 1986). No such association has been observed in H. cuneata. According to Bulisnki and Gundersen, 1991 and Glimer et al., 1999 MTs enriched with acetylated tubulin are more resistant to depolymerisation. Thus, it may be supposed that the microtubules present in the cytoplasm of H. cuneata filaments may be formed by acetylated tubulin once these filaments are still polymerised after routine fixation procedures, whereas the majority of methods used to study the cytoskeleton employ MTs stabilising buffers. Despite a few ultrastructural studies have been carried out on Halimeda spp. (Wilbur et al., 1969; Palandri, 1972; Borowitzka and Larkum, 1974, 1977; Borowitzka, 1976) there are very few data regarding the cytoskeleton in this genus and no data on the thickness of MTs have been published. In higher plants, MTs appear to be involved in determining cell morphology and the polarity of the cell axis in cell differentiation (Lloyd et al., 1985). MTs, due to their influence over the arrangement of cellulose microfibrils, play an important role in the control of cell shape in plants (Hepler and Palevitz, 1974). Although the presence of a cell wall endows plants with their rigidity, MTs may contribute to the maintenance of cell morphology. A likely reason for the presence of large quantities of MTs in the medullary region of the filaments in Halimeda may be that they are related to an increase in structural stability of the fine cytoplas-

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Fig. 5. Transmission electron microscopy of H. cuneata. (A) Transverse section of the medullary region showing the fine cytoplasm filled with microtubules (arrows) near the cell wall. Spherical body in the vacuole, they may represent a lipidic storage product. (B) Detail of microtubules in the fine cytoplasm of the medullary region seen in transverse section. (C) Magnification of microtubules in transversal sections. (D) Bundles of microtubules in longitudinal section. (E) Detailof bundles of microtubules. CW, cell wall; MTs, microtubules; SB, spherical bodie; V, vacuole. Bar in (A) = 0.5 ␮m; bar in (B) = 200 nm; bars in (B and E) = 50 nm; bar in (D) = 200 nm.

mic layer that surrounds the large central vacuole. Furthermore, closer to the terminal portion of the filaments, the presence of MTs may be related to the maintenance of the spatial distribution of organelles. This suggestion is further supported by the presence of bundles of MTs in close association with chloroplasts and mitochondria. Menzel (1985) demonstrated with green alga C. fastigiata the chloroplasts and amyloplasts exhibit a closely apposed to the surface of the microtubule bundles. These findings suggested that this architecture serves as an efficient differentiation facilitating long range transport along the microtubule bundles. The organelles are transported along cytoplasm strands is the amyloplast, while the majority the chloroplasts remains immobilized in the cortical cytoplasm of the cell (Menzel, 1987). According to Sabnis (1969) the bundles of microtubules serve a cytoskeletal function associated with the development and maintenance of asymmetry in differentiaatin cell and influencing cytoplasmatic streaming and related phenomena.

Acknowledgments The authors would like to thank Professor Eurico C. Oliveira for critical reading of manuscript and the Central Laboratory of Electron Microscopy (LCME), Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil, for the use of their transmission electron microscope.

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