J. Cell Sti. 3, i-i 6 (1968) Printed in Great Britain HISTOLOGICAL STUDIES ON THE GENUS FUCUS III. FINE STRUCTURE AND POSSIBLE FUNCTIONS OF THE EPIDERMAL CELLS OF THE VEGETATIVE THALLUS MARGARET E. McCULLY Department of Biology, Carleton University, Ottawa, Canada SUMMARY The fine structure of the epidermal cells of the vegetative Fucus thallus has been examined in material fixed with acrolein. These cells are highly polarized, with basal nuclei and chloro- plasts, a hypertrophied perinuclear Golgi system, and a much convoluted wall/plasma membrane interface. Much of the intracellular volume is occupied by single membrane-bounded vesicles containing alginic acid, fucoidin and polyphenols. The chloroplasts were examined by light and electron microscopy and shown to contain structured inclusions not previously described in Fucus plastids. It is suggested en the basis of their morphology that the epidermal cells may be specialized for the absorption of inorganic carbon and sulphate from the outside of the plant and for the secretion of alginic acid, fucoidin and polyphenols. The possible role of these cells in the prevention of desiccation and in osmoregulation is discussed. INTRODUCTION Recently developed techniques of tissue fixation and embedding have facilitated high-resolution light microscopy and histochemistry of the tissues of Fucus (McCully, 1966, 1967). The two major structural polysaccharides of this alga, alginic acid and fucoidin have been localized histochemically and it ha9 been shown that these sub- stances are formed within several cell types of both vegetative and fertile plants and subsequently secreted as macromolecules to the outside of the cells. These secreted polysaccharides form the extensive extracellular matrix of the interior of the thallus and, in the case of fruiting plants, also form the enveloping layers of the gametangia. Fucoidin and alginic acid are present in the cells of the single-layered epidermis which surrounds the vegetative thallus and it has been suggested that the cells secrete these polysaccharides to the outside of the plant. The morphology of these epidermal cells is distinctive. They are columnar, about 15 ft wide and 60 /i deep, and are highly polarized. Both the nucleus and the plastids are in the basal end of the cell, the plastids lying in a cup-shaped formation about the nucleus. Much of the remaining cell volume is occupied by polyphenolic materials and by the deposits of alginic acid and fucoidin. It was considered that a study of the fine structure of the epidermal cells would be 1 Cell Sci. 3 2 M. E. McCully of interest in view of their unusual morphology and their possible role in secretion. There are only a few useful electron-microscopic studies of the mature tissues of the large brown algae, because it is difficult to fix these tissues which are so rich in polysaccharides and polyphenols. Although several recent studies (Bouck, 1965; McCully, 1965; Evans, 1966) have shown that these difficult tissues can be fixed satisfactorily by aldehydes the fine structure of the mature epidermal cells of Fucus has not been described following preparation by the newer methods. In this paper, the fine structure of the epidermal cells of Fucus vesiculosus L. is described following acrolein/osmium tetroxide fixation and the possible functions of these cells are considered. MATERIALS AND METHODS Electron microscopy Mature vegetative thalli of Fucus vesiculosus L. were collected near Bass Rocks, Gloucester, Massachusetts during the winters of 1964-65 and 1965-66. Portions of the upper 2 cm of the thalli were placed immediately into ice-cold fixative and then cut into pieces of about 1 mm8. The fixative used was 10% acrolein in 0-025 M phosphate buffer at pH 6-8. The tissue was fixed for 48 h, thoroughly washed in at least 10 changes of buffer over a 48-h period and post-fixed in 2 % osmium tetroxide in 0*025 M buffer for 24 h. Dehydration was in two 12-h changes of methoxyethanol, followed by two 12-h changes of ethanol. The tissue was then placed in fresh ethanol and propylene oxide was added slowly over several hours until the concentration of propylene oxide was about 75 %. The material was then placed in pure propylene oxide. All the steps of fixation and dehydration up to this stage were done at o °C. The tissue was allowed to remain in the propylene oxide at o °C for about 3 h, then brought to room temperature and given a further 3-h change of propylene oxide. Araldite resin mixture was added slowly over 24 h and the propylene oxide allowed to evaporate. The tissue was infiltrated for 2 weeks, with daily changes of fresh resin mixture. Sections were cut with a diamond knife on a Huxley ultramicrotome. Because of the large amount of tissue components retained by the acrolein fixation it was necessary to cut very thin sections and only those showing grey zero-order interference colours were examined. In many cases, despite the long infiltration period, the polysaccharide matrix material was not completely infiltrated, causing wetting of the block face during cutting. This problem was overcome by floating the sections on a saturated solution of calcium chloride and then washing them thoroughly in several changes of distilled water. Sections were stained for 10 min in uranyl acetate (Watson, 1958) followed by 10 min in lead citrate (Reynolds, 1963) and examined with an RCA EMU3F electron microscope at 50 kV. Fucus epidermal cells 3 Light microscopy Tissue was fixed in 10% acrolein as for electron microscopy but it was post-fixed in 1 % mercuric chloride and dehydrated in a methoxyethanol, ethanol, propanol, butanol series, and embedded in glycol methacrylate. Sections 1-2 fi thick were stained with toluidine blue, acid fuchsin, or by the periodic acid/Schiff (PAS) reaction. Details of these methods have been published previously (McCully, 1966). OBSERVATIONS General features of the cytoplasm There is remarkably little cytoplasmic ground substance in the epidermal cells. Light microscopy shows that it is confined to thin sheaths around the cell periphery, nucleus and plastids, and to narrow threads running between the numerous vacuoles which occupy much of the volume of these cells (see Fig. 1B in McCully, 1966). In low-magnification electron micrographs (Fig. 1) it is especially difficult to distinguish the cytoplasm from the numerous vacuoles containing granular material, which fill the apical ends of the cells. At higher resolution the cytoplasm is seen to be rich in ribosomes (Fig. 18); only rarely is it clear that these are adhering to cisternae of the endoplasmic reticulum (ER), and more often they appear in clusters free in the cytoplasm. Because of the amount of background material retained by the acrolein fixation, however, it is difficult to identify ER cisternae and possibly many of the apparently free ribosome clusters are associated with the ER. There is a great proliferation of the plasma membrane at the apex of each epidermal cell. Numerous small projections protrude into imaginations in the inner portion of the wall (Fig. 1) and, in addition, long, narrow, finger-like projections of the plasmalemma penetrate deeply into the cell (Fig. 1); a few of these canaliculi are also present on the lateral margins of the cells, especially in the regions adjacent to the chloroplasts (Fig. 3). Because of the complexity of the cell contents, it is impossible to determine the full extent of these invaginations. Although some of the minor distortions of the plasma membrane/wall interface may be artifacts, it is unlikely that the deep membrane invaginations could be generated by the preparative pro- cedures. Many of these invaginations show no electron density and their contents are either unstained by heavy metals or not retained by the fixation; however, some of the canaliculi, especially those in the plastid region, contain fine fibrils (Fig. 3). Besides the various invaginations and projections of the plasma membrane, there are frequently large aggregations of strongly stained membranes between the plasma membrane and the cell wall. These are associated occasionally with a very electron- dense body (Fig. 2). These apparently extracellular membranes are seen most frequently against the lateral walls of young, rapidly growing cells close to the thallus apex. However, smaller amounts of membrane, often enclosing various electron-transparent vesicles are also seen between the plasma membrane and the cell wall, especially at the cell apices, and there are lengths of similarly staining membrane-like materials in the inner layers of the outer epidermal wall (see Fig. 7 in McCully, 1965). 4 M. E. McCuUy A peculiar structure bounded by a single membrane is often observed in the cytoplasm just above the nucleus (Figs. i8, 21). This sac-like structure is of irregular shape but always has several projections up to 2 fi in length, and it is readily seen because of the heavy staining of its limiting membrane. This membrane is quite distinct from the rather weakly staining tonoplast enclosing the numerous vacuoles. The sac itself contains many small single membrane-bounded vesicles which vary both in size and in electron transparency. Frequently a number of pieces of such sacs, separated by several microns, were seen in a single section, but since very few adjacent sections were available it was not possible to determine if there are several of these structures per cell or if the various pieces are proliferations of a single sac. No closely similar structures have been reported in other algal cells, although the single membrane- bounded, large multivesiculate bodies which have been observed in diatoms (Drum & Pankratz, 1964; Stoermer, Pankratz & Bowen, 1965) may be homologous structures. Chloroplasts Each cell contains about 25 elongated, discoid chloroplasts. When viewed with the light microscope the plastids of an individual epidermal or cortical cell appear linked together (Fig.