Histological Studies on the Genus Fucus Iii. Fine Structure and Possible Functions of the Epidermal Cells of the Vegetative Thallus

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 chloroplasts, 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 substances 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 procedures. 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. 7) and at higher resolution each link can be identified as a tenuous thread of cytoplasm, completely surrounded by non-cytoplasmic material (Fig. 22). When seen in cross-section the mature plastids show the familiar lamellation pattern of algal chloroplasts, with parallel stacks of lamellae traversing the long axis of the organelle (Figs. 4, 10). The unit of construction of each lamellar stack appears to be a flattened sac or thylakoid which is delimited by a single unit membrane. An individual thylakoid is about 150 A thick and usually 3 (occasionally 4 or 5) of these units are apparent in a section through a single lamellar stack. Within each stack the individual thylakoids are separated by a uniform distance of about 150 A in material fixed by the present methods. In h aving a definite space between the thylakoids of a single lamellar stack, Fucus differs from many other algae, for example, Euglena, in which the thylakoids are so closely appressed that adjacent membranes appear as a single thick membrane except at high resolution (Gibbs, i960, 1962). Similar spaces between thylakoids of an individual lamellar stack have been described in other members of the Phaeophyceae (Evans, 1966) and they appear to be a general feature of chloroplast organization in this class. It has been suggested that the thylakoids of algal plastids are in the form of discs with each lamellar stack made up of a number of adjacent piles of these discs (Gibbs, i960, 1962). This interpretation does not appear correct in the case of the Fucus plastid where there are very complex interconnexions of the thylakoids (Figs. 4 and 8-10). In some cases there are connexions between the lamellar stacks where one or more thylakoids leave one stack and join a neighbouring one. Sometimes there appears to be a complete bifurcation of a lamellar stack with the component thylakoids forming two separate stacks. Furthermore, a constant feature of the cross-sectional image of the Fucus plastid is the continuation of at least one lamellar stack around the 'nucleoid' region at the edge of the plastid. Such a complex lamellation has been Fucus epidermal cells 5 observed in a number of other algal species (Mr A. D. Greenwood, personal communication). So much ground substance is retained in the acrolein-fixed chloroplasts that even very thin sections are quite electron-dense (Figs. 4, 10). One of the components of this matrix is particulate, resembling ribosomes except that these particles are considerably smaller (130 A, compared to 240 A) than cytoplasmic ribosomes in the same cells and they are much less strongly stained by heavy metals (Fig. 10). If these particles are ribosomes either they lack some component of those of the cytoplasm or else their capacity to bind heavy metals is inhibited. An indication that this latter suggestion may possibly be correct comes from earlier work (McCully, 1966) which showed that Fucus epidermal cell plastids are unstained by toluidine blue at pH 4-8 while those of higher plants prepared in the same way are basophilic. Whereas absence of basophilia may indeed indicate the presence of relatively little ribonucleic acid, Fig. 14 shows that these plastids are intensely acidophilic when stained with acid fuchsin (a reaction which indicates a high protein content). Basophilic staining of the ribosomes of Fucus plastids may thus be prevented by the masking of phosphate groups of the nucleic acid by proteins, and such masking of the nucleic acid could possibly also account for the low level of heavy-metal staining. The epidermal cell plastids contain a few osmiophilic droplets of diameter up to about 1000 A. These bodies show no substructure and are always either uniformly intensely osmiophilic or lightly stained in the middle with a dark periphery (Figs. 4, 6 and 10); they appear similar to those occurring in higher plant chloroplasts and in the chloroplasts of many algae (see Greenwood, Leech & Williams, 1963). In addition to the osmiophilic globules, there are unusual structures between the stacked lamellae of the Fucus plastids. These are much less electron dense than the spherical globules and the staining is heterogeneous, occurring around the periphery and in the centre of the body, sometimes in an ordered pattern (Figs. 5, 6). These structures appear to be surrounded by a unit membrane and often look like partly collapsed vesicles (Fig. 4). They occur singly, or bunched together in elongated groups. Such structures were not seen in chloroplasts of immature epidermal cells close to the thallus apex or in those of the primary filaments and fibres of the thallus, nor were they seen in all sections of epidermal cell chloroplasts (Fig. 10), perhaps indicating their localization in the mid-part of the plastid. Rather similar objects have been observed in glutaraldehyde-fixed plastids of Bifurcaria and Dictyota (Dr L. V. Evans, personal communication). Numerous small 'vacuoles' can be seen with the light microscope in sections of epidermal and cortical cell plastids of material fixed with acrolein but not post-fixed in osmium. These are not stained by acid fuchsin, the PAS reaction or toluidine blue but are particularly apparent in sections stained with toluidine blue after a 2-h treatment in chlorous acid (method modified from Rappay & Van Duijn, 1965). After this latter treatment the polyphenolic materials no longer stain green with toluidine blue and the plastids, mitochondria and nuclei, none of which normally stains with this dye, become basophilic. In addition the vacuoles of the plastid enlarge somewhat and are easily seen as unstained areas (Fig. 7). It is not clear why 6 M. E. McCulIy the chlorous acid treatment produces swelling of the vacuoles and the anomalous basophilia. In Araldite-embedded sections of material post-fixed with osmium tetroxide and cut thick for light microscopy these vacuoles are somewhat osmiophilic and can be distinguished easily from tiny black bodies which are groups of the osmiophilic droplets. Clearly the chloroplast vacuoles seen in the light microscope are groups of the structured inclusions observed with the electron microscope. These inclusions are not preparation artifacts since similar vacuoles are seen as non-fluorescing areas within living Fucus plastids in which chlorophyll fluorescence has been excited by the appropriate wavelengths of light (McCully, unpublished). The staining reactions of these vacuoles indicate that they do not contain any such proteins, carbohydrates or polyphenols as can be retained by acrolein/mercuric chloride fixation. Their osmiophilia suggests the presence of lipoidal materials and/or polyphenols which are not fixed in the absence of osmium. It is also possible that these vacuoles could serve as storage areas for mannitol, which is an early product of photosynthesis in Fucus (Bidwell, Craigie & Krotkov, 1958), or laminarin, which these plants accumulate (see Meeuse, 1962). Neither of these water-soluble materials would be retained by the fixation procedures used. Certainly the presence of large numbers of these structures in the plastids of the epidermal and outer cortical cells which may be regarded as optimally located for photosynthesis, and their complete absence from the plastids of the deeply-buried primary filaments and secondary fibres, suggest that they serve as some sort of storage area for assimilated material. A distinct membrane-free area occurs at the end of each plastid (Figs. 4, 9 and 10). These areas which were described as 'vacuoles' by Leyton & von Wettstein (1954) contain a few fibrils about 25 A in diameter which probably correspond to the 25-30 A fibrils of DNA in the chloroplasts of Chlamydomonas (Ris & Plaut, 1962). Similar ' nucleoids' are found at each end of the plastids of the brown alga Chorda (Bouck, 1965).

Mitochondria Light microscopy of sections stained with acid fuchsin reveals a markedly polarized distribution of the mitochondria in epidermal cells (Fig. 14). While a few of these organelles are scattered throughout the cells they are mostly clustered to form a closely- packed, single-layered cap over the apical end of each cell. This cap was observed in all except immature epidermal cells within the apical groove of the thallus. The apical mitochondria are especially apparent in sections cut tangentially through the cell apex (Fig. 13). The periphery of these organelles is always highly convoluted and the cristae are numerous and tubular, resembling those of protozoa. The mito- chondrial matrix is very electron-dense after heavy metal staining and the mitochondria are acidophilic, staining intensely after as little as 30 sec exposure to dilute acidic acid fuchsin. This strong affinity for the acid dye and for heavy metals suggests a high protein content. A few mitochondria are located close to the nucleus and plastids. These differ from the short rod-like ones of the cell apex in being elongated (Figs. 18, 21) and, Fucus epidermal cells 7 frequently, bifurcated. They also have a lower density of cristae and their matrix material has less affinity for acid fuchsin and heavy metals.

Golgi bodies There are a few small, isolated Golgi bodies throughout the cytoplasm but in addition there is a notable population of them around the nucleus (Fig. 11). Unlike the isolated dictyosomes of higher plants, the bodies appear to be integrated into a Golgi system, such as is found commonly in cells of animal tissue. In mature epidermal cells each of the units of this system is hypertrophied but because of the great complexity of the cell contents in this region (see Fig. 12) it is difficult to say with certainty which of the numerous vesicles are produced by the Golgi apparatus. Hypertrophied Golgi systems are characteristic components of many algal cells (Berkaloff, 1963; Schnepf, 19636; Manton, Rayns&Ettl, 1965; Manton & Parke, 1965; Leedale, Meeuse & Pringsheim, 1965) and a number of these systems are perinuclear (for example in Astrephomena (Lang, 1963), Chorda filuman d Giffordia (Bouck, 1965)). Manton and her colleagues (Manton et al. 1965 and Manton & Parke, 1965) have shown that in a variety of unicellular algae the Golgi vesicles contain a remarkable array of material destined for secretion from the cell.

Nuclei The nuclei are remarkable in two respects. First, compared to those of most higher plants, they have more extensive connexions between the outer membrane of the nuclear envelope and the ER; the cytoplasm forms a thin perinuclear sheath with narrow fingers running out into the cell from the regions where the ER branches off the nuclear envelope (Fig. 15). Secondly, there is a greater homogeneity of heavy- metal staining of these nuclei (Figs. 11, 15) compared with those of higher plants fixed and stained in the same manner (see O'Brien, 1967a). Densely staining masses of chromatin which are present in most of the nuclei of the other thallus cells are not seen in mature epidermal cell nuclei, where only a slight heterogeneity of staining is apparent. These nuclei also have peculiar toluidine-blue staining properties. They stain a distinct pink colour at pH 4-8 (McCully, 1966), a nuclear staining reaction unique not only in the Fucus plant but also in all other plant and animal tissues which have been similarly fixed and stained (unpublished observations, and Dr N. Feder, personal communication). The relation between the absence of a normal basophilia of these nuclei and their electron-microscope image after heavy-metal staining is not clear. Especially puzzling are the nucleoli, one or two of which are clearly seen with the electron microscope (Fig. 21), although they are not distinguished by any of the staining methods used for light microscopy of these cells, in spite of the fact that these methods clearly reveal nucleoli in cells of other Fucus tissue. The uniformity of the heavy metal staining is especially surprising, since these are the only vegetative Fucus nuclei that show a mottled pattern of staining intensity with toluidine blue. Epidermal cell nuclei stain strongly and uniformly with acidic acid-fuchsin (Fig. 14). Thus the pattern of electron density after heavy-metal staining seems to correspond more closely 8 M. E. McCuUy to the pattern of acid fuchsin staining than to that produced with toluidine blue. In other words, the electron density of the heavy-metal staining is perhaps reflecting the distribution of nuclear proteins rather than of the nucleic acids. A low level of chromatin clumping appears to be characteristic of those algal nuclei of which electron micrographs of aldehyde-fixed material have been published (Manton & Parke, 1965; Barton, 1965; Bouck, 1965). In general, there is little peripheral chromatin clumping in algal nuclei fixed with either aldehydes or osmium tetroxide. A clumping of chromatin around the periphery of the nucleus is especially prominent in highly differentiated cells of both higher plants and animals (for example, nuclei of companion cells and xylem parenchyma of Avena (O'Brien, 19676), and of the fibrocyte of vertebrates (Porter, 1964)). The apparent absence of large peripheral deposits of chromatin in the algae may reflect either a masking of chromatin staining by a high protein content in the nuclei or it may indicate that in these lower plants a much greater percentage of the genome is continuously active than is the case in highly differentiated cells of higher organisms, where inactive portions may remain clumped as chromatin. The nucleoli of the epidermal cells are large (about 3 /i in diameter) and are usually single in each nucleus (two nucleoli are occasionally present). In many cases they have been seen to contain densely staining bodies about 240 A in diameter which resemble cytoplasmic ribosomes (Fig. 21).

Vacuoles One of the most striking characteristics of the epidermal cells is the varied array of single membrane-bounded vesicles which occupies a large proportion of the total cell volume. Histochemical tests on sections of material embedded in glycol methacrylate show that of the four most prominent types of vacuoles, two contain polyphenols, and the other two polysaccharides. These have been identified in electron micrographs on the basis of their size, frequency, and distribution (see Figs. 1, 12).

The large, moderately dense, granular bodies (tvv Fig. 1) are most likely the pale green-staining, polyphenol-containing vacuoles so abundant in young epidermal cells (see Fig. 1B of McCully, 1966). The smaller, very dense bodies often seen around the nucleus and at the cell periphery are probably the small turquoise-staining vacuoles most prominent in older epidermal cells (Fig. 1A of McCully, 1966). The less dense, but quite granular, small bodies (ag) appear to correspond to the strongly metachromatic, PAS-positive granules which are seen easily in these cells with the light microscope after toluidine-blue staining (see Figs. iB and 5 of McCully, 1966), and which probably contain alginic acid. The sulphated polysaccharide fucoidin is most likely contained in the very electron-transparent vacuoles which are numerous in the region of the perinuclear Golgi (Fig. 12). Some of these vacuoles contain fine fibrillar material. Similar fibrillar material is seen in some types of mucin-containing vesicles of higher animal cells (for example, see Fig. 14 in Fawcett, 1966). The origin of the various vacuoles of the epidermal cells is of interest. Those containing the fucoidin are most prominent in the perinuclear region (Fig. 12) and it seems probable that they originate from the hypertrophied Golgi in this area. Fucus epidermal cells 9 A similar perinuclear origin of sulphated polysaccharides was observed in the developing oogonium of Fucus (McCully, 1967), and is also described in such animal cells as the goblet cells of the intestinal epithelium (Lane, Caro, Oterovilardebo & Godman, 1964). It is not at all clear where the alginic-acid-containing vesicles originate but, as is the case in the developing oogonia (McCully, 1967) they appear to be produced throughout the cell. The plastids have long been implicated in the production of the physodes in the Phaeophyta, and Kylin (1918) shows, in a diagram, small physodes originating from the chloroplast membrane in Asperococcus. The hypothesis is certainly supported by the recent work of Wooding & Northcote (1965) which suggests that vacuoles containing terpene-like resin precursors are pinched off the chloroplast envelopes in cells of Pinus. There is, however, no evidence for the occurrence of such a process in the epidermal cells of Fucus and the origin of the polyphenol-containing vesicles remains unclear. However, at this point mention should be made of rather unusual structures composed of concentrically arranged membranes (Fig. 17) which are frequently seen just above the epidermal cell nucleus. It is not apparent what these structures are, but they invariably contain an electron-dense core which could conceivably be an early stage in the development of a polyphenol vesicle.

Cell walls Cronshaw, Myers & Preston (1958) showed clearly that the brown algae which they examined (including Fucus serratus) had thick cell walls formed of fibrillar material embedded in amorphous matrix. They considered the microfibrils in these walls to be randomly oriented and concluded that this construction was characteristic of the Phaeophyceae. This generalization has persisted, although it was shown by Dawes, Scott & Bowler (1961) that mature cells of a number of brown algae including Dictyota have distinctly oriented fibrils in their walls. It has subsequently been shown (McCully, 1965) that with the exception of the outer epidermal walls in which the fibrils are less oriented, all the walls in the vegetative thallus of F. vesiculosus show a high degree of fibrillar orientation. This orientation is particularly apparent in Araldite-embedded sections which have been etched under vacuum and shadowed with platinum (for details of method see Maser, O'Brien & McCully, 1967). Figs. 25 and 26 show such a section of a lateral wall of a mature epidermal cell in which the longitudinally oriented microfibrils can be seen. The basal pits which join the epidermal cells to each other and to the underlying parenchyma cells are very much bigger than even the largest pits of higher plants (about 20 /£ compared to 5 /t in diameter). It was first demonstrated by Hick (1885) by observations on alkali-swollen sections that there was protoplasmic continuity across the pits of F. vesiculosus and F. serratus. Bisalputra (1966) has recently confirmed the presence of plasmodesmata in F. evanescens. The thin-section shadowing technique was found useful in demonstrating the plasmodesmata running through the pits of F. vesiculosus. Figure 23 shows such a section which was cut anticlinally through a lateral pit and shows the plasmodesmata in relief. Shadowed sections cut obliquely through a pit (Fig. 24) show that a Fucus plasmodesmata has a small central core io M.E. McCully that is depressed in relation to the surrounding material and it is clear that the contents of these plasmodesmata are not homogeneous.

DISCUSSION The morphology of the epidermal cells of the Fucus thallus suggests that they are actively involved in absorption and/or secretion. In animal tissue the morphological characteristics of elongated cells with polarized location of organelles, especially mitochondria and highly invaginated apical plasma membrane surfaces are associated with absorption or secretion (Bloom & Fawcett, 1962; Porter & Bonneville, 1964; Fawcett, 1966). Examples of similar cell types in plants are fewer and in many cases their physiological role has not been clarified experimentally, although they are generally considered to be glandular. However, in a few cases their morphology and function has been established. For example, the columnar cells of the scutellar epithelium in germinating grass seeds have a basal nucleus and mitochondria localized above it (O'Brien, 1942). It has now been established that these cells not only secrete a-amylase (and possibly other enzymes) into the endosperm, but they also absorb the mobilized carbohydrates for use by the developing embryo (see Varner, 1965). A second example is that of the glandular cells of insectivorous plants. It has long been known that many of these cells are columnar, with apical accumulations of secretory granules, basal nuclei and a highly proliferated surface of the apical region of the plasma membrane (Fenner, 1904; Schnepf, 1963 a), and Ltittge (1964, 1965) has demonstrated by means of radioautography that such cells in Nepenthes secrete proteolytic enzymes and reabsorb amino acids liberated from the digested prey. Unfortunately, no physiological data are yet available to establish with certainty the relationship between structure and function in Fucus epidermal cells. However, there is some indirect evidence which suggests that these cells both secrete and absorb materials.

The epidermis as an absorbing organ Since the morphology of the epidermal cells, especially their proliferated plasma membrane and canaliculated cytoplasm, suggests that they may be active in absorbing materials from the sea water, it is worthwhile to consider what substances are most likely to be absorbed. Obviously inorganic carbon must be taken into the plant from the environment, although it is not clear in what form this carbon is assimilated. Bid well & Craigie (1963) have shown that moist, emersed pieces of Fucus thallus U absorb a minimum of 70 % less CO2 than they do when submerged in sea water. The authors conclude that Fucus absorbs carbon probably as the bicarbonate ion, although it has already been shown by Montford (1937) that even quite desiccated Fucus plants maintain high photosynthetic activity, suggesting that they can, in fact, assimilate C02 directly. Since it is unlikely that either the relatively chlorophyll-poor holdfast or decorticated midribs of older parts of the plant contribute more than a very small fraction of the total photosynthetic assimilation of the whole plant, Fucus epidermal cells 11 most of the carbon assimilated must pass into the plant via the epidermal cells. Whatever the form in which it is taken up, the carbon compound must diffuse through the thick mucilaginous outer wall, then over long distances either down anticlinal walls or through the tannin-filled upper parts of the epidermal cells before reaching the chloroplasts of even the epidermal cell layer. We know little, of course, of the rates of diffusion of carbon dioxide or bicarbonate through tannin-rich materials or substances which form the walls of Fucus, and it is therefore possible only to speculate on the possibility that the deep imaginations of the plasma membrane might facilitate access of assimilatory forms of carbon to the plastids. In addition to their possible role in the uptake of inorganic carbon the epidermal cells obviously absorb other inorganic ions, and in particular SOf" must be taken up in large quantities for esterification of polysaccharides to form fucoidin, which may account for up to 30 % of the dry weight of the plants and which also is probably secreted in large quantity. Since there is evidence that the sulphated polysaccharides are present in vesicles most abundant in the region of the perinuclear Golgi, it is probable that the sulphation of these polysaccharides takes place in the vesicles (see McCully, 1967). However, the necessary 'active' sulphate may be formed in or near the apical mitochondria, near the site of uptake of sulphate and a ready source of ATP.

The epidermis as a secretory organ While there is no direct evidence that the epidermal cells themselves secrete materials, there is evidence that intact Fucus plants do release substances into the ocean. Anyone who has handled these plants, especially during the winter, will attest to their mucilaginous coating, a coating which must be continually sloughed off and replenished. This mucilage has not been analysed chemically, but it is PAS-positive and stains metachromatically—staining reactions which suggest that it is a mixture of alginic acid and fucoidin (McCully, 1966). Secretion of alginic acid is also suggested by the work of Bidwell et al. (1958), which shows that a large amount of radioactive 14 alginic acid is formed when Fucus plants are exposed to COa. Since the amount of radioactive alginate formed in a 30-h period is equal to about one-fifth of the total alginic acid in a given plant, these authors conclude that this polysaccharide must be undergoing continual breakdown and resynthesis. One could, however, interpret the data as indicating that alginate is being secreted rather than metabolized. It is known that many algae other than Fucus secrete large amounts of polysaccharide into their surrounding medium. While this process has been studied mainly in brackish water flagellates (see Fogg, 1962) and blue-green algae (Moore & Tischer, 1965), metachromatic extracellular polysaccharides have also been demonstrated in the medium in which Porphyridium cruentum is cultured (Jones, 1962). In addition, although the process does not appear to have been studied, it is generally recognized that large brown algae like Laminaria and Macrocystis release mucilage into the ocean. The presence in the Fucus epidermal cells of numerous membrane-bounded vesicles containing substances with the same histochemical properties as alginic acid and fucoidin suggests that continual secretion of these polysaccharides replenishes those lost from the plant surface. 12 M.E. McCully A system in which such a secretion of large polysaccharide molecules occurs has been described for the root-cap cells of Triticum (Northcote & Pickett-Heaps, 1966). Here the polysaccharides are sequestered into vesicles by the Golgi bodies and these vesicles then migrate through the cytoplasm, fuse with the plasma membrane and, by reverse pinocytosis, empty their contents to the outside of the cell. Clearly the morphology of the Fucus epidermal cells is consistent with such activity. The hypertrophied Golgi system is located in close proximity to the chloroplasts, organelles which produce the initial carbon skeletons for the polysaccharides, and the highly proliferated plasma membrane with its deep imaginations into the cell may be visualized as facilitating the excretion process by providing a much-expanded surface for vesicle incorporation and by bringing the cell surface closer to the area of synthesis. In addition to polysaccharide secretion there is good experimental evidence that intact vegetative plants of Fucus secrete polyphenols into the ocean (Craigie & McLachlan, 1964; McLachlan & Craigie, 1964). While both the epidermal cells and the underlying cortical cells are filled with large numbers of polyphenol-containing vesicles (McCully, 1966) the epidermal cells are most likely sites of secretion, but it is not clear how this process occurs. Toluidine blue staining, which easily identifies the intracellular polyphenols in the cells (McCully, 1966), consistently failed to demonstrate any extracellular polyphenols even in the outer epidermal walls. There are at least two possibilities. Phenol release might not be occurring at low tide when the material was collected. Alternatively, the acrolein/mercuric chloride fixation might not retain the secreted form of the phenols. The finding of the strongly osmiophilic bodies outside the cell membrane adjacent to the lateral and outer walls of the epidermal cells after osmium post-fixation suggests that the latter possibility may be correct and that the secreted form of the polyphenols is retained only if the tissue is treated with osmium. The possible significance to the organism of the secretion of polyphenols and polysaccharides is of interest. In the case of polyphenols, the work of Craigie & McLachlan (1964) and McLachlan & Craigie (1964) has shown that these materials have a selective antibiotic effect on marine flora and that by this means the algae may exert a profound influence over their environment. The significance to the plants of the polysaccharide secretion, however, is not yet clarified by any experimental work. In the case of Fucus tissue there are several possible functions of the polysaccharide secretion process. The continuously shifting outer surface produced by such a secretion is undoubtedly a deterrent to colonization by epiphytes, and indeed only a few types of blue-green algae and bacteria, and one macroscopic alga were seen growing on these plants. A more fundamental function of the polysaccharide secretion, however, is the prevention of desiccation of the plants during emersion. Under conditions of high temperature and dry winds these plants will suffer a considerable water stress, the extent of this increasing with the height above mean sea level at which they are growing. Zaneveld (1937) studied the desiccation of these plants relative to their intertidal position. It was shown that the rate of water loss per unit of surface area from experimentally dried plants was inversely proportional to the Fucus epidermal cells 13 height at which they had been growing in the intertidal area, and examination of sections showed that the thickness of the walls of the cortical parenchyma cells was proportional to the height at which they were growing. It appears, therefore, that the water-holding capacity of these plants is dependent upon the amount of extracellular material present in the thallus matrix. While the hydrophilic properties of the alginic acid and fucoidin of this matrix are undoubtedly important in preventing desiccation, the outer layer of mucilage may play an even more important and somewhat different role. It is known that alginic acid can undergo a salting-out process (Wassermann, 1948), during which the pore size of the gel greatly decreases. Such a process could occur in the outside layers of the epidermal cells were the salinity of these layers increased by evaporation. The resulting impervious layer would be important in preventing further desiccation since, despite the hydrophilia of the thallus matrix materials, in the absence of such an outer layer water stresses in the environment would ultimately be transmitted to the cells. Percival (1964) has suggested that the polysaccharides forming the extracellular matrices of the brown algae could have an osmoregulatory function. This is almost certainly true in the sense that they must act as buffers against sudden changes in the osmotic concentration of the environment, such as would occur, for example, if emersed plants were suddenly soaked by rain water after several hours of drying. It was shown by Wasserman (1959) that alginic acid is an excellent ion-exchange resin, which is capable of absorbing about 3-0 equivalents of metallic ions (mainly sodium, potassium and calcium) and about i-o equivalent of chloride ion per 1-9 equivalents of alginate. It is known that alginic acid comprises up to 25 % of the dry weight of a Fucus plant (Black, 1949). Although the ion-binding capacity of fucoidin is not known it should also be high, and since this polysaccharide also may account for a considerable portion of the dry weight of the plant (O'Colla, 1962), it is apparent that the combined ion-binding capacity of the extracellular polysaccharides is very large and that their presence could greatly modify the cellular environment. The morphology of the epidermal cells suggests that in addition to this passive osmoregulation by the matrix material there may also be active osmoregulation by these cells. They resemble to a remarkable degree the chloride cells in the gills of marine teleosts (Philpott & Copeland, 1963; Philpott, 1965). These latter cells are polarized, with a basal nucleus and a highly canaliculized apical region in which numerous mitochondria are located. It has been shown by Philpott (1965) that chlorides move across these cells, probably entering the canaliculi and moving through them into a large apical vesicle from which they are discharged together with muco- polysaccharides into the sea water. The osmotic concentration of marine teleosts is hypo-osmotic to their environment (Prosser & Brown, 1961) and they absorb salt water through the gut and actively pump the excess salt back into the ocean by means of the chloride cells, the mitochondria adjacent to the canaliculi in the cells providing the ATP for the process. Based on purely morphological evidence a similar mechanism can perhaps be postulated for Fucus. By such a mechanism sea water could diffuse into any part of the plant. The excess H M. E. McCuUy salt would be removed from the cells mainly as sodium and chloride ions bound to secreted polysaccharides. The most important site of this secretion would be the epidermis, the cells of which are in good contact with the cytoplasm of the underlying cells via the large pits. The massive secretion of polysaccharide would provide an excellent means of removing salt from the plant. The large number of mitochondria present in Fucus epidermal cells could supply the ATP needed for an active transport process in that region. The existence of such an active osmoregulatory mechanism can, however, only be speculative until experimental data are available.

The material presented in this paper forms part of a doctoral dissertation presented to Harvard University.

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BLOOM, W. & FAWCETT, D. W. (1962). A Textbook of Histology. Philadelphia: Saunders. BOUCK, G. B. (1965). Fine structure and organelle associations in brown algae. J. Cell Biol. 36, 523-537- CRAIGIE, J. S. & MCLACHLAN, J. (1964). Excretion of coloured ultraviolet-absorbing substances by marine algae. Can. J. Bot. 42, 23-33. CRONSHAW, J., MYERS, A. & PRESTON, R. D. (1958). A chemical and physical investigation of the cell walls of some marine algae. Biochim. biophys. Acta 27, 89-103. DAWES, C. J., SCOTT, F. M. & BOWLER, E. (1961). A light- and electron-microscopic survey of algal cell walls. I. Phaeophyta and Rhodophyta. Am. J. Bot. 48, 925-934. DRUM, R. W. & PANKRATZ, H. S. (1964). Pyrenoids, raphes, and other fine structure in diatoms. Am. J. Bot. 51, 405-418. EVANS, L. V. (1966). Distribution of pyrenoids among some brown algae. J. Cell Set. 1, 449-454. FAWCETT, D. S. (1966). An Atlas of Fine Structure. The Cell. Philadelphia: Saunders. FENNER, C. A. (1004). 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Fig. 1. Longitudinal section through the apical pole of a mature epidermal cell showing the thick, outer wall (010) and the lateral wall between two adjacent cells. The proliferated plasma membrane wall interface is apparent and some of the long invaginations of the plasma membrane are marked by arrows. Two types of tannin vesicles (tVi) and (tvt) are present, in addition to vesicles (ag) presumed to contain alginic acid, x 10000. Fig. 2. Longitudinal section through epidermal cell in the region of the plastids (p) showing what is probably a tannin vesicle (tvt) between the plasma membrane and the lateral wall (Iw). Note the proliferation of smooth membrane outside the plasma membrane in this area, x 20000. Fig. 3. Longitudinal section of epidermal cell in the plastid region showing a deep invagination of the plasma membrane (outlined by arrows) into the cell. A mitochondrion (TO), ribosome-rich cytoplasm, and a portion of a plastid can also be seen, x 30000. Journal of Cell Science, Vol. 3, No. 1

M. E. McCULLY (Facing p. 16) Fig. 4. Longitudinal section through the plastid region showing plastids containing osmiophilic droplets (od) and vesicular inclusions (ci). The asterisk marks a large invagination of the plasma membrane. The oriented fibrillar nature of the lateral wall (liv) can be seen, x 25000. Figs. 5, 6. Higher magnification views of the chloroplast inclusions. Figure 5 shows a group of the vesicular bodies. Figure 6 shows a single vesicular body (a) and an osmiophilic droplet (od). x 50000. Fig. 7. Photomicrograph of a cross-section of mature vegetative thallus, showing the base of epidermal cells (ep) and parenchyma cells of the cortex (cp). Numerous plastids are present, those of the epidermis are lying against the lateral walls and those of the parenchyma are scattered throughout the cells. Plastid 'vacuoles' are indicated by arrows. Glycol methacrylate-embedded section. Toluidine blue staining after chlorous acid treatment, x 1900. Journal of Cell Science, Vol. 3, No.

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Figs. 8—10. Sections of epidermal cell chloroplasts showing typical arrangement of the lamellae. The ribosome-like particles of the plastid matrix are apparent. The ribosome- rich cytoplasm (c) can be distinguished easily from the vacuole (v) which is closely appressed to a portion of the plastid. Figure 8 is a higher magnification of the area indicated, showing a lamellar stack. Figure 9 shows details of the 25-A fibrils of the 'nucleoid'. The continuation of thylakoids around the plastid periphery is apparent. Figures 8, 9, x 50000; Fig. 10, x 33 000.

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Fig. 11. Section through the nucleus of a young epidermal cell. Portions of the perinuclear Golgi complex are indicated by arrows, x 35000. Fig. 12. Hypertrophied Golgi system in the perinuclear region and in close proximity to the plastids (p) of a mature epidermal cell. The numerous vesicles apparently derived from this Golgi system possibly contain fucoidin. x 35000.

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Fig. 13. Transverse section through the apical pole of a mature epidermal cell showing the apical mitochondria and sections through the proliferated plasma membrane surface. The arrows indicate vesicles with densely staining peripheries, which occur commonly in this region. The fibrillar nature of the lateral walls (liv) is apparent, x 38000. Fig. 14. Photomicrograph of cross-section of mature thallus, showing epidermal and cortical cells with apical mitochondria (TO) and basal plastids (p) and nuclei (n). Epiphytes (e) are apparent on the surface of the thallus. Glycol methacrylate- embedded section. Acidic acid-fuchsin staining, x 10 000.

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For legends see next page.

M. E. McCULLY Fig. 15. Section through the nucleus of a mature cell showing the narrow envelope of perinuclear cytoplasm with fingers of cytoplasm running out into the cell between the vacuole areas (v). x 10000. Fig. 16. Sections of nuclear envelope showing nuclear pores and the formation of a cisterna of the ER by an outpocketting of the outer membrane of the nuclear envelope, x 38000. Fig. 17. Section showing structures which are seen frequently in the epinuclear region. These appear as membranes arranged concentrically around a dense core, x 20000. Fig. 18. Section showing dense cytoplasmic area adjacent to the nucleus (n). A hypertrophied Golgi body and several mitochondria are apparent. Part of a vesicular body (vb) can also be seen, x 25000. Fig. 19. Section through region of epidermal cell just above the area of the Golgi body shown in Fig. 17, showing the large tannin vesicles (tVt) and two other types of vesicles presumed to contain alginic acid (ag) or fucoidin (Jv). The nature of the large, osmiophilic multivesicular body is not known but it appears quite distinct from the type of vesicular body shown in Fig. 18. x 30000.

Fig. 20. Details of a nuclear pore in a nucleus (n) similar to that shown in Fig. 21. Fine fibrillar material, occasionally present in the nuclear pores, is apparent, x 40000. Fig. 21. Section showing nucleus with large nucleolus (nu) which contains ribosome- like particles. A large vesicular body is present in addition to a mitochondrion (m). The arrows indicate sections of tubular elements within the vesicular body, x 35000. Fig. 22. Section from the lower portion of an epidermal cell near a lateral wall showing a narrow strand of cytoplasm typical of those that link together the plastids in this area. Such strands, which contain numerous ribosomes and a few mitochondria (m), are often completely surrounded by tannin vesicles (in,) of the type which fill the apical portion of the cells, x 35000. Journal of Cell Science, Vol. 3, No. 1

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.24 fct

Figs. 23-26. Prepared by shadowing thin sections with platinum. The direction of the arrow indicates the direction of the shadowing in all the figures. Fig. 23. Longitudinal section through a lateral wall pit showing the plasmodesmata. x 20000. Fig. 24. Higher magnification of an oblique section through a pit similar to that in Fig. 23 showing the plasmodesmata in relief. In some plasmodesmata such as the one within the circle, an inner core can be seen which appears as a depression, x 40000. Figs. 25, 26. Longitudinal sections of epidermal cell showing a lateral wall. The oriented fibrillar structure of this wall can be seen clearly in Fig. 25, which shows a portion of the same wall as shown in Fig. 26. Figure 25, x 42000; Fig. 26, x 20000.

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