Department of Biology, Carleton University, Ottawa, Canada · of a single lamellar stack Fucus, differs from many other algae, for example, Euglena, in which the thylakoids are so

J. Cell Sti. 3, i- i 6 (1968)

Printed in Great Britain

HISTOLOGICAL STUDIES ON THE

GENUS FUCUS

III. FINE STRUCTURE AND POSSIBLEFUNCTIONS OF THE EPIDERMAL CELLS OFTHE VEGETATIVE THALLUS

MARGARET E. McCULLYDepartment of Biology, Carleton University, Ottawa, Canada

SUMMARY

The fine structure of the epidermal cells of the vegetative Fucus thallus has been examinedin 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 membraneinterface. Much of the intracellular volume is occupied by single membrane-bounded vesiclescontaining alginic acid, fucoidin and polyphenols.

The chloroplasts were examined by light and electron microscopy and shown to containstructured inclusions not previously described in Fucus plastids. It is suggested en the basisof their morphology that the epidermal cells may be specialized for the absorption of inorganiccarbon and sulphate from the outside of the plant and for the secretion of alginic acid, fucoidinand polyphenols. The possible role of these cells in the prevention of desiccation and inosmoregulation is discussed.

INTRODUCTION

Recently developed techniques of tissue fixation and embedding have facilitatedhigh-resolution light microscopy and histochemistry of the tissues of Fucus (McCully,1966, 1967). The two major structural polysaccharides of this alga, alginic acid andfucoidin 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 andsubsequently secreted as macromolecules to the outside of the cells. These secretedpolysaccharides form the extensive extracellular matrix of the interior of thethallus and, in the case of fruiting plants, also form the enveloping layers of thegametangia. Fucoidin and alginic acid are present in the cells of the single-layeredepidermis which surrounds the vegetative thallus and it has been suggested thatthe cells secrete these polysaccharides to the outside of the plant.

The morphology of these epidermal cells is distinctive. They are columnar, about15 ft wide and 60 /i deep, and are highly polarized. Both the nucleus and the plastidsare in the basal end of the cell, the plastids lying in a cup-shaped formation aboutthe nucleus. Much of the remaining cell volume is occupied by polyphenolic materialsand by the deposits of alginic acid and fucoidin.

It was considered that a study of the fine structure of the epidermal cells would be1 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 thelarge brown algae, because it is difficult to fix these tissues which are so rich inpolysaccharides and polyphenols. Although several recent studies (Bouck, 1965;McCully, 1965; Evans, 1966) have shown that these difficult tissues can be fixedsatisfactorily by aldehydes the fine structure of the mature epidermal cells of Fucushas not been described following preparation by the newer methods.

In this paper, the fine structure of the epidermal cells of Fucus vesiculosus L. isdescribed following acrolein/osmium tetroxide fixation and the possible functionsof 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 ofthe upper 2 cm of the thalli were placed immediately into ice-cold fixative and thencut 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 atleast 10 changes of buffer over a 48-h period and post-fixed in 2 % osmium tetroxidein 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 ethanoland propylene oxide was added slowly over several hours until the concentration ofpropylene oxide was about 75 %. The material was then placed in pure propyleneoxide. 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, thenbrought 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 allowedto evaporate. The tissue was infiltrated for 2 weeks, with daily changes of fresh resinmixture.

Sections were cut with a diamond knife on a Huxley ultramicrotome. Because ofthe large amount of tissue components retained by the acrolein fixation it was necessaryto cut very thin sections and only those showing grey zero-order interference colourswere examined. In many cases, despite the long infiltration period, the polysaccharidematrix material was not completely infiltrated, causing wetting of the block faceduring cutting. This problem was overcome by floating the sections on a saturatedsolution of calcium chloride and then washing them thoroughly in several changes ofdistilled water.

Sections were stained for 10 min in uranyl acetate (Watson, 1958) followed by10 min in lead citrate (Reynolds, 1963) and examined with an RCA EMU3F electronmicroscope at 50 kV.

Fucus epidermal cells 3

Light microscopy

Tissue was fixed in 10% acrolein as for electron microscopy but it was post-fixedin 1 % mercuric chloride and dehydrated in a methoxyethanol, ethanol, propanol,butanol series, and embedded in glycol methacrylate. Sections 1-2 fi thick werestained 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 vacuoleswhich occupy much of the volume of these cells (see Fig. 1B in McCully, 1966). Inlow-magnification electron micrographs (Fig. 1) it is especially difficult to distinguishthe cytoplasm from the numerous vacuoles containing granular material, which fillthe apical ends of the cells. At higher resolution the cytoplasm is seen to be rich inribosomes (Fig. 18); only rarely is it clear that these are adhering to cisternae of theendoplasmic reticulum (ER), and more often they appear in clusters free in thecytoplasm. Because of the amount of background material retained by the acroleinfixation, however, it is difficult to identify ER cisternae and possibly many of theapparently free ribosome clusters are associated with the ER.

There is a great proliferation of the plasma membrane at the apex of each epidermalcell. Numerous small projections protrude into imaginations in the inner portionof the wall (Fig. 1) and, in addition, long, narrow, finger-like projections of theplasmalemma penetrate deeply into the cell (Fig. 1); a few of these canaliculi arealso present on the lateral margins of the cells, especially in the regions adjacent tothe chloroplasts (Fig. 3). Because of the complexity of the cell contents, it is impossibleto determine the full extent of these invaginations. Although some of the minordistortions of the plasma membrane/wall interface may be artifacts, it is unlikelythat the deep membrane invaginations could be generated by the preparative pro-cedures. Many of these invaginations show no electron density and their contentsare either unstained by heavy metals or not retained by the fixation; however, someof the canaliculi, especially those in the plastid region, contain fine fibrils (Fig. 3).

Besides the various invaginations and projections of the plasma membrane, thereare frequently large aggregations of strongly stained membranes between the plasmamembrane and the cell wall. These are associated occasionally with a very electron-dense body (Fig. 2). These apparently extracellular membranes are seen most frequentlyagainst the lateral walls of young, rapidly growing cells close to the thallus apex.However, smaller amounts of membrane, often enclosing various electron-transparentvesicles are also seen between the plasma membrane and the cell wall, especially atthe cell apices, and there are lengths of similarly staining membrane-like materialsin 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 thecytoplasm just above the nucleus (Figs. i8, 21). This sac-like structure is of irregularshape but always has several projections up to 2 fi in length, and it is readily seenbecause of the heavy staining of its limiting membrane. This membrane is quite distinctfrom the rather weakly staining tonoplast enclosing the numerous vacuoles. The sacitself contains many small single membrane-bounded vesicles which vary both in sizeand in electron transparency. Frequently a number of pieces of such sacs, separatedby several microns, were seen in a single section, but since very few adjacent sectionswere available it was not possible to determine if there are several of these structuresper cell or if the various pieces are proliferations of a single sac. No closely similarstructures 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 thelight microscope the plastids of an individual epidermal or cortical cell appear linkedtogether (Fig. 7) and at higher resolution each link can be identified as a tenuousthread of cytoplasm, completely surrounded by non-cytoplasmic material (Fig. 22).

When seen in cross-section the mature plastids show the familiar lamellationpattern of algal chloroplasts, with parallel stacks of lamellae traversing the long axisof the organelle (Figs. 4, 10). The unit of construction of each lamellar stack appearsto 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) ofthese units are apparent in a section through a single lamellar stack. Within each stackthe individual thylakoids are separated by a uniform distance of about 150 A inmaterial fixed by the present methods. In h aving a definite space between the thylakoidsof a single lamellar stack, Fucus differs from many other algae, for example, Euglena,in which the thylakoids are so closely appressed that adjacent membranes appearas a single thick membrane except at high resolution (Gibbs, i960, 1962). Similarspaces between thylakoids of an individual lamellar stack have been described inother members of the Phaeophyceae (Evans, 1966) and they appear to be a generalfeature of chloroplast organization in this class.

It has been suggested that the thylakoids of algal plastids are in the form of discswith 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 Fucusplastid where there are very complex interconnexions of the thylakoids (Figs. 4 and8-10). In some cases there are connexions between the lamellar stacks where oneor more thylakoids leave one stack and join a neighbouring one. Sometimes thereappears to be a complete bifurcation of a lamellar stack with the component thylakoidsforming two separate stacks. Furthermore, a constant feature of the cross-sectionalimage 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


observed in a number of other algal species (Mr A. D. Greenwood, personal communi-cation).

So much ground substance is retained in the acrolein-fixed chloroplasts that evenvery thin sections are quite electron-dense (Figs. 4, 10). One of the components ofthis matrix is particulate, resembling ribosomes except that these particles areconsiderably smaller (130 A, compared to 240 A) than cytoplasmic ribosomes inthe 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 thecytoplasm or else their capacity to bind heavy metals is inhibited. An indication thatthis latter suggestion may possibly be correct comes from earlier work (McCully,1966) which showed that Fucus epidermal cell plastids are unstained by toluidineblue 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 littleribonucleic acid, Fig. 14 shows that these plastids are intensely acidophilic whenstained with acid fuchsin (a reaction which indicates a high protein content). Basophilicstaining of the ribosomes of Fucus plastids may thus be prevented by the masking ofphosphate groups of the nucleic acid by proteins, and such masking of the nucleicacid could possibly also account for the low level of heavy-metal staining.

The epidermal cell plastids contain a few osmiophilic droplets of diameter up toabout 1000 A. These bodies show no substructure and are always either uniformlyintensely 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 andin the chloroplasts of many algae (see Greenwood, Leech & Williams, 1963).

In addition to the osmiophilic globules, there are unusual structures between thestacked lamellae of the Fucus plastids. These are much less electron dense than thespherical globules and the staining is heterogeneous, occurring around the peripheryand in the centre of the body, sometimes in an ordered pattern (Figs. 5, 6). Thesestructures appear to be surrounded by a unit membrane and often look like partlycollapsed vesicles (Fig. 4). They occur singly, or bunched together in elongatedgroups. Such structures were not seen in chloroplasts of immature epidermal cellsclose 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), perhapsindicating their localization in the mid-part of the plastid. Rather similar objects havebeen 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 ofepidermal and cortical cell plastids of material fixed with acrolein but not post-fixedin osmium. These are not stained by acid fuchsin, the PAS reaction or toluidineblue but are particularly apparent in sections stained with toluidine blue after a 2-htreatment in chlorous acid (method modified from Rappay & Van Duijn, 1965).After this latter treatment the polyphenolic materials no longer stain green withtoluidine blue and the plastids, mitochondria and nuclei, none of which normallystains with this dye, become basophilic. In addition the vacuoles of the plastidenlarge 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 anomalousbasophilia.

In Araldite-embedded sections of material post-fixed with osmium tetroxide andcut thick for light microscopy these vacuoles are somewhat osmiophilic and can bedistinguished easily from tiny black bodies which are groups of the osmiophilicdroplets. Clearly the chloroplast vacuoles seen in the light microscope are groups ofthe structured inclusions observed with the electron microscope.

These inclusions are not preparation artifacts since similar vacuoles are seen asnon-fluorescing areas within living Fucus plastids in which chlorophyll fluorescencehas been excited by the appropriate wavelengths of light (McCully, unpublished).The staining reactions of these vacuoles indicate that they do not contain any suchproteins, carbohydrates or polyphenols as can be retained by acrolein/mercuricchloride fixation. Their osmiophilia suggests the presence of lipoidal materials and/orpolyphenols which are not fixed in the absence of osmium. It is also possible thatthese vacuoles could serve as storage areas for mannitol, which is an early productof photosynthesis in Fucus (Bidwell, Craigie & Krotkov, 1958), or laminarin, whichthese plants accumulate (see Meeuse, 1962). Neither of these water-soluble materialswould be retained by the fixation procedures used. Certainly the presence of largenumbers of these structures in the plastids of the epidermal and outer cortical cellswhich may be regarded as optimally located for photosynthesis, and their completeabsence from the plastids of the deeply-buried primary filaments and secondaryfibres, 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 the25-30 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 polarizeddistribution of the mitochondria in epidermal cells (Fig. 14). While a few of theseorganelles 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 observedin all except immature epidermal cells within the apical groove of the thallus. Theapical mitochondria are especially apparent in sections cut tangentially through thecell apex (Fig. 13). The periphery of these organelles is always highly convolutedand the cristae are numerous and tubular, resembling those of protozoa. The mito-chondrial matrix is very electron-dense after heavy metal staining and the mitochondriaare acidophilic, staining intensely after as little as 30 sec exposure to dilute acidicacid fuchsin. This strong affinity for the acid dye and for heavy metals suggests a highprotein content.

A few mitochondria are located close to the nucleus and plastids. These differfrom the short rod-like ones of the cell apex in being elongated (Figs. 18, 21) and,


frequently, bifurcated. They also have a lower density of cristae and their matrixmaterial has less affinity for acid fuchsin and heavy metals.

Golgi bodies

There are a few small, isolated Golgi bodies throughout the cytoplasm but inaddition there is a notable population of them around the nucleus (Fig. 11). Unlikethe isolated dictyosomes of higher plants, the bodies appear to be integrated into a Golgisystem, such as is found commonly in cells of animal tissue. In mature epidermal cellseach of the units of this system is hypertrophied but because of the great complexityof the cell contents in this region (see Fig. 12) it is difficult to say with certaintywhich 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 filum and Giffordia (Bouck, 1965)).Manton and her colleagues (Manton et al. 1965 and Manton & Parke, 1965) haveshown that in a variety of unicellular algae the Golgi vesicles contain a remarkablearray of material destined for secretion from the cell.

Nuclei

The nuclei are remarkable in two respects. First, compared to those of mosthigher plants, they have more extensive connexions between the outer membraneof the nuclear envelope and the ER; the cytoplasm forms a thin perinuclear sheathwith narrow fingers running out into the cell from the regions where the ER branchesoff 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 plantsfixed and stained in the same manner (see O'Brien, 1967a). Densely staining massesof chromatin which are present in most of the nuclei of the other thallus cells arenot seen in mature epidermal cell nuclei, where only a slight heterogeneity of stainingis apparent. These nuclei also have peculiar toluidine-blue staining properties. Theystain a distinct pink colour at pH 4-8 (McCully, 1966), a nuclear staining reactionunique not only in the Fucus plant but also in all other plant and animal tissues whichhave 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 theirelectron-microscope image after heavy-metal staining is not clear. Especially puzzlingare 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 usedfor light microscopy of these cells, in spite of the fact that these methods clearlyreveal nucleoli in cells of other Fucus tissue. The uniformity of the heavy metalstaining is especially surprising, since these are the only vegetative Fucus nuclei thatshow a mottled pattern of staining intensity with toluidine blue. Epidermal cellnuclei stain strongly and uniformly with acidic acid-fuchsin (Fig. 14). Thus the patternof 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 reflectingthe distribution of nuclear proteins rather than of the nucleic acids.

A low level of chromatin clumping appears to be characteristic of those algal nucleiof which electron micrographs of aldehyde-fixed material have been published(Manton & Parke, 1965; Barton, 1965; Bouck, 1965). In general, there is littleperipheral chromatin clumping in algal nuclei fixed with either aldehydes or osmiumtetroxide. A clumping of chromatin around the periphery of the nucleus is especiallyprominent in highly differentiated cells of both higher plants and animals (for example,nuclei of companion cells and xylem parenchyma of Avena (O'Brien, 19676), andof the fibrocyte of vertebrates (Porter, 1964)). The apparent absence of large peripheraldeposits of chromatin in the algae may reflect either a masking of chromatin stainingby a high protein content in the nuclei or it may indicate that in these lower plantsa much greater percentage of the genome is continuously active than is the case inhighly differentiated cells of higher organisms, where inactive portions may remainclumped as chromatin.

The nucleoli of the epidermal cells are large (about 3 /i in diameter) and areusually single in each nucleus (two nucleoli are occasionally present). In many casesthey have been seen to contain densely staining bodies about 240 A in diameterwhich resemble cytoplasmic ribosomes (Fig. 21).

Vacuoles

One of the most striking characteristics of the epidermal cells is the varied array ofsingle membrane-bounded vesicles which occupies a large proportion of the total cellvolume. Histochemical tests on sections of material embedded in glycol methacrylateshow that of the four most prominent types of vacuoles, two contain polyphenols,and the other two polysaccharides. These have been identified in electron micrographson 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 palegreen-staining, polyphenol-containing vacuoles so abundant in young epidermal cells(see Fig. 1B of McCully, 1966). The smaller, very dense bodies often seen around thenucleus and at the cell periphery are probably the small turquoise-staining vacuolesmost 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 meta-chromatic, PAS-positive granules which are seen easily in these cells with the lightmicroscope after toluidine-blue staining (see Figs. iB and 5 of McCully, 1966), andwhich probably contain alginic acid. The sulphated polysaccharide fucoidin is mostlikely contained in the very electron-transparent vacuoles which are numerous inthe region of the perinuclear Golgi (Fig. 12). Some of these vacuoles contain finefibrillar material. Similar fibrillar material is seen in some types of mucin-containingvesicles 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. Thosecontaining the fucoidin are most prominent in the perinuclear region (Fig. 12) andit seems probable that they originate from the hypertrophied Golgi in this area.


A similar perinuclear origin of sulphated polysaccharides was observed in thedeveloping oogonium of Fucus (McCully, 1967), and is also described in such animalcells as the goblet cells of the intestinal epithelium (Lane, Caro, Oterovilardebo &Godman, 1964). It is not at all clear where the alginic-acid-containing vesiclesoriginate but, as is the case in the developing oogonia (McCully, 1967) they appearto be produced throughout the cell.

The plastids have long been implicated in the production of the physodes in thePhaeophyta, and Kylin (1918) shows, in a diagram, small physodes originating fromthe chloroplast membrane in Asperococcus. The hypothesis is certainly supportedby the recent work of Wooding & Northcote (1965) which suggests that vacuolescontaining terpene-like resin precursors are pinched off the chloroplast envelopes incells of Pinus. There is, however, no evidence for the occurrence of such a process inthe epidermal cells of Fucus and the origin of the polyphenol-containing vesiclesremains unclear. However, at this point mention should be made of rather unusualstructures composed of concentrically arranged membranes (Fig. 17) which arefrequently seen just above the epidermal cell nucleus. It is not apparent what thesestructures are, but they invariably contain an electron-dense core which couldconceivably be an early stage in the development of a polyphenol vesicle.

Cell walls

Cronshaw, Myers & Preston (1958) showed clearly that the brown algae which theyexamined (including Fucus serratus) had thick cell walls formed of fibrillar materialembedded in amorphous matrix. They considered the microfibrils in these wallsto be randomly oriented and concluded that this construction was characteristic ofthe Phaeophyceae. This generalization has persisted, although it was shown byDawes, Scott & Bowler (1961) that mature cells of a number of brown algaeincluding Dictyota have distinctly oriented fibrils in their walls. It has subsequentlybeen shown (McCully, 1965) that with the exception of the outer epidermal wallsin which the fibrils are less oriented, all the walls in the vegetative thallus of F. vesicu-losus show a high degree of fibrillar orientation. This orientation is particularlyapparent in Araldite-embedded sections which have been etched under vacuumand 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 epidermalcell in which the longitudinally oriented microfibrils can be seen.

The basal pits which join the epidermal cells to each other and to the underlyingparenchyma 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 continuityacross the pits of F. vesiculosus and F. serratus. Bisalputra (1966) has recently confirmedthe presence of plasmodesmata in F. evanescens. The thin-section shadowing techniquewas found useful in demonstrating the plasmodesmata running through the pits ofF. vesiculosus. Figure 23 shows such a section which was cut anticlinally througha lateral pit and shows the plasmodesmata in relief. Shadowed sections cut obliquelythrough 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 contentsof these plasmodesmata are not homogeneous.

DISCUSSION

The morphology of the epidermal cells of the Fucus thallus suggests that they areactively involved in absorption and/or secretion. In animal tissue the morphologicalcharacteristics of elongated cells with polarized location of organelles, especiallymitochondria and highly invaginated apical plasma membrane surfaces are associatedwith 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 physio-logical role has not been clarified experimentally, although they are generally consideredto be glandular. However, in a few cases their morphology and function has beenestablished. For example, the columnar cells of the scutellar epithelium in germinatinggrass 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 possiblyother enzymes) into the endosperm, but they also absorb the mobilized carbohydratesfor use by the developing embryo (see Varner, 1965). A second example is that ofthe glandular cells of insectivorous plants. It has long been known that many of thesecells are columnar, with apical accumulations of secretory granules, basal nuclei anda highly proliferated surface of the apical region of the plasma membrane (Fenner,1904; Schnepf, 1963 a), and Ltittge (1964, 1965) has demonstrated by means ofradioautography that such cells in Nepenthes secrete proteolytic enzymes and reabsorbamino acids liberated from the digested prey.

Unfortunately, no physiological data are yet available to establish with certaintythe relationship between structure and function in Fucus epidermal cells. However,there is some indirect evidence which suggests that these cells both secrete andabsorb materials.

The epidermis as an absorbing organ

Since the morphology of the epidermal cells, especially their proliferated plasmamembrane and canaliculated cytoplasm, suggests that they may be active in absorbingmaterials from the sea water, it is worthwhile to consider what substances are mostlikely to be absorbed. Obviously inorganic carbon must be taken into the plant fromthe 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 thallusabsorb a minimum of 70 % less UCO2 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 desiccatedFucus plants maintain high photosynthetic activity, suggesting that they can, in fact,assimilate C02 directly. Since it is unlikely that either the relatively chlorophyll-poorholdfast or decorticated midribs of older parts of the plant contribute more thana very small fraction of the total photosynthetic assimilation of the whole plant,


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 throughthe thick mucilaginous outer wall, then over long distances either down anticlinalwalls or through the tannin-filled upper parts of the epidermal cells before reachingthe chloroplasts of even the epidermal cell layer. We know little, of course, of therates of diffusion of carbon dioxide or bicarbonate through tannin-rich materials orsubstances which form the walls of Fucus, and it is therefore possible only to speculateon the possibility that the deep imaginations of the plasma membrane might facilitateaccess of assimilatory forms of carbon to the plastids.

In addition to their possible role in the uptake of inorganic carbon the epidermalcells obviously absorb other inorganic ions, and in particular SOf" must be taken upin large quantities for esterification of polysaccharides to form fucoidin, whichmay account for up to 30 % of the dry weight of the plants and which also is probablysecreted in large quantity. Since there is evidence that the sulphated polysaccharidesare present in vesicles most abundant in the region of the perinuclear Golgi, it isprobable that the sulphation of these polysaccharides takes place in the vesicles (seeMcCully, 1967). However, the necessary 'active' sulphate may be formed in or nearthe 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 secretematerials, there is evidence that intact Fucus plants do release substances into theocean. Anyone who has handled these plants, especially during the winter, willattest to their mucilaginous coating, a coating which must be continually sloughed offand replenished. This mucilage has not been analysed chemically, but it is PAS-positiveand stains metachromatically—staining reactions which suggest that it is a mixtureof alginic acid and fucoidin (McCully, 1966). Secretion of alginic acid is also suggestedby the work of Bidwell et al. (1958), which shows that a large amount of radioactivealginic acid is formed when Fucus plants are exposed to 14COa. Since the amount ofradioactive alginate formed in a 30-h period is equal to about one-fifth of the totalalginic acid in a given plant, these authors conclude that this polysaccharide must beundergoing continual breakdown and resynthesis. One could, however, interpret thedata as indicating that alginate is being secreted rather than metabolized.

It is known that many algae other than Fucus secrete large amounts of polysaccharideinto their surrounding medium. While this process has been studied mainly inbrackish water flagellates (see Fogg, 1962) and blue-green algae (Moore & Tischer,1965), metachromatic extracellular polysaccharides have also been demonstrated inthe medium in which Porphyridium cruentum is cultured (Jones, 1962). In addition,although the process does not appear to have been studied, it is generally recognizedthat large brown algae like Laminaria and Macrocystis release mucilage into the ocean.

The presence in the Fucus epidermal cells of numerous membrane-boundedvesicles containing substances with the same histochemical properties as alginic acidand fucoidin suggests that continual secretion of these polysaccharides replenishesthose lost from the plant surface.

12 M.E. McCully

A system in which such a secretion of large polysaccharide molecules occurs hasbeen described for the root-cap cells of Triticum (Northcote & Pickett-Heaps, 1966).Here the polysaccharides are sequestered into vesicles by the Golgi bodies and thesevesicles then migrate through the cytoplasm, fuse with the plasma membrane and, byreverse pinocytosis, empty their contents to the outside of the cell. Clearly themorphology of the Fucus epidermal cells is consistent with such activity. The hyper-trophied Golgi system is located in close proximity to the chloroplasts, organelleswhich produce the initial carbon skeletons for the polysaccharides, and the highlyproliferated plasma membrane with its deep imaginations into the cell may bevisualized as facilitating the excretion process by providing a much-expanded surfacefor vesicle incorporation and by bringing the cell surface closer to the area of synthesis.

In addition to polysaccharide secretion there is good experimental evidence thatintact vegetative plants of Fucus secrete polyphenols into the ocean (Craigie &McLachlan, 1964; McLachlan & Craigie, 1964). While both the epidermal cellsand the underlying cortical cells are filled with large numbers of polyphenol-containingvesicles (McCully, 1966) the epidermal cells are most likely sites of secretion, but itis not clear how this process occurs. Toluidine blue staining, which easily identifiesthe intracellular polyphenols in the cells (McCully, 1966), consistently failed todemonstrate any extracellular polyphenols even in the outer epidermal walls. Thereare at least two possibilities. Phenol release might not be occurring at low tide whenthe material was collected. Alternatively, the acrolein/mercuric chloride fixationmight not retain the secreted form of the phenols. The finding of the strongly osmio-philic bodies outside the cell membrane adjacent to the lateral and outer walls of theepidermal cells after osmium post-fixation suggests that the latter possibility maybe correct and that the secreted form of the polyphenols is retained only if the tissueis treated with osmium.

The possible significance to the organism of the secretion of polyphenols andpolysaccharides is of interest. In the case of polyphenols, the work of Craigie &McLachlan (1964) and McLachlan & Craigie (1964) has shown that these materialshave a selective antibiotic effect on marine flora and that by this means the algaemay exert a profound influence over their environment. The significance to theplants of the polysaccharide secretion, however, is not yet clarified by any experi-mental work.

In the case of Fucus tissue there are several possible functions of the polysaccharidesecretion process. The continuously shifting outer surface produced by such asecretion is undoubtedly a deterrent to colonization by epiphytes, and indeed onlya few types of blue-green algae and bacteria, and one macroscopic alga were seengrowing on these plants. A more fundamental function of the polysaccharide secretion,however, is the prevention of desiccation of the plants during emersion. Underconditions of high temperature and dry winds these plants will suffer a considerablewater stress, the extent of this increasing with the height above mean sea level atwhich they are growing. Zaneveld (1937) studied the desiccation of these plantsrelative to their intertidal position. It was shown that the rate of water loss per unitof surface area from experimentally dried plants was inversely proportional to the


height at which they had been growing in the intertidal area, and examination ofsections showed that the thickness of the walls of the cortical parenchyma cells wasproportional to the height at which they were growing. It appears, therefore, thatthe water-holding capacity of these plants is dependent upon the amount of extra-cellular material present in the thallus matrix.

While the hydrophilic properties of the alginic acid and fucoidin of this matrixare undoubtedly important in preventing desiccation, the outer layer of mucilagemay play an even more important and somewhat different role. It is known thatalginic acid can undergo a salting-out process (Wassermann, 1948), during whichthe pore size of the gel greatly decreases. Such a process could occur in the outsidelayers of the epidermal cells were the salinity of these layers increased by evaporation.The resulting impervious layer would be important in preventing further desiccationsince, despite the hydrophilia of the thallus matrix materials, in the absence of suchan outer layer water stresses in the environment would ultimately be transmittedto the cells.

Percival (1964) has suggested that the polysaccharides forming the extracellularmatrices of the brown algae could have an osmoregulatory function. This is almostcertainly true in the sense that they must act as buffers against sudden changes inthe osmotic concentration of the environment, such as would occur, for example, ifemersed 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-exchangeresin, which is capable of absorbing about 3-0 equivalents of metallic ions (mainlysodium, potassium and calcium) and about i-o equivalent of chloride ion per 1-9equivalents of alginate. It is known that alginic acid comprises up to 25 % of the dryweight of a Fucus plant (Black, 1949). Although the ion-binding capacity of fucoidinis not known it should also be high, and since this polysaccharide also may accountfor a considerable portion of the dry weight of the plant (O'Colla, 1962), it is apparentthat the combined ion-binding capacity of the extracellular polysaccharides is verylarge and that their presence could greatly modify the cellular environment.

The morphology of the epidermal cells suggests that in addition to this passiveosmoregulation by the matrix material there may also be active osmoregulation bythese cells. They resemble to a remarkable degree the chloride cells in the gills ofmarine teleosts (Philpott & Copeland, 1963; Philpott, 1965). These latter cells arepolarized, with a basal nucleus and a highly canaliculized apical region in whichnumerous mitochondria are located. It has been shown by Philpott (1965) that chloridesmove across these cells, probably entering the canaliculi and moving through theminto a large apical vesicle from which they are discharged together with muco-polysaccharides into the sea water. The osmotic concentration of marine teleosts ishypo-osmotic to their environment (Prosser & Brown, 1961) and they absorb saltwater through the gut and actively pump the excess salt back into the ocean bymeans of the chloride cells, the mitochondria adjacent to the canaliculi in the cellsproviding the ATP for the process. Based on purely morphological evidence a similarmechanism 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 tosecreted polysaccharides. The most important site of this secretion would be theepidermis, the cells of which are in good contact with the cytoplasm of the underlyingcells via the large pits. The massive secretion of polysaccharide would provide anexcellent means of removing salt from the plant. The large number of mitochondriapresent in Fucus epidermal cells could supply the ATP needed for an active transportprocess in that region. The existence of such an active osmoregulatory mechanismcan, however, only be speculative until experimental data are available.

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

REFERENCES

BARTON, R. (1965). Electron microscope studies on surface activity in cells of Chora vulgaris.Planta 66, 95-105.

BERKALOFF, C. (1963). Les cellules meiistematiques d'Himanthalia lorea (L.) S. F. Gray.fitude au microscope electronique. J. Microscopie 2, 213-228.

BIDWELL, R. G. S. & CRAIGIE, J. S. (1963). A note on the greatly reduced ability of Fucusvesiculosus to absorb or evolve COt when not submerged. Can. J. Bot. 41, 179-182.

BIDWELL, R. G. S., CRAIGIE, J. S. & KROTKOV, G. (1958). Photosynthesis and metabolism inmarine algae. III. Distribution of C14Oa in Fucus vesiculosus. Can. J. Bot. 581-590.

BISALPUTRA, T. (1966). Electron microscopic study of the protoplasmic continuity in certainbrown algae. Can. J. Bot. 44, 89—93.

BLACK, W. A. P. (1949). Seasonal variation in chemical composition of some of the littoralseaweeds common to Scotland. Part II. Fucus serratus, Fucus vesiculosus, Pclvetia canaliculata.

' J. Soc. chem. Ind., Lond. 68, 183-189.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). BeitrSge zur Kenntnis der Anatomie, Entwicklungsgeschichte und

Biologie der Laubblatter und Driisen einiger Insektivoren. Flora, Jena 93, 335-434.FOGG, G. E. (1962). Extracellular products. In Physiology and Biochemistry of Algae (ed.

R. A. Lewin), pp. 475-489. New York: Academic Press.GIBBS, S. P. (i960). Fine structure of Euglena gracilis with special reference to the chloroplasts

and pyrenoids. J. Ultrastruct. Res. 4, 127-148.GIBBS, S. P. (1962). The ultrastructure of the chloroplasts of algae. J. Ultrastruct. Res. 7,

4i8-435-GREENWOOD, A. D., LEECH, R. M. & WILLIAMS, J. P. (1963). The osmiophilic globules of

chloroplasts. I. Osmiophilic globules as a normal component of chloroplasts and theirisolation and composition in Viciafaba L. Biochim. biophys. Acta 78, 148—162.

HICK, T. (1885). Protoplasmic continuity in the Fucaceae. J. Bot., Lond. 23, 97-102.JONES, R. F. (1962). Extracellular mucilage of the red alga Porphyridium cruentum. J. cell.

comp. Physiol. 60, 61-64.


KYLIN, H. (1918). Ober die Fucosanblasen den Phaeophyseen. Ber. dt. bot. Ges. 36, 10-19.LANE, N., CARO, L., OTEROVILARDEBO, L. R. & GODMAN, G. C. (1964). On the site of sulphation

in colonic goblet cells. J. Cell Biol. 21, 339—351.LANG, N. (1963). Electron microscopy of the Volvocaceae and Astrephomenaceae. Am. J. Bot.

50, 280-300.LEEDALE, G. F., MEEUSE, B. J. D. & PHINGSHEIM, E. G. (1965). Structure and physiology of

Euglena spirogyra. I and II. Arch. Mikrobiol. 50, 68—102.LEYTON, H. & WETTSTEIN, D. VON (1954). Der bei Chromatophoren-Feinbau den Phaeo-

physeen. Z. Naturf. 96, 471-475.LOTTGE, U. (1964). Untersuchungen zur Physiologie der Carnivoren-Drusen. I Mitteilung.

Die an den Verdauungsvorgangen beleitigten Enzyme. Planta 63, 103-117.LUTTGE, U. (1965). Untereuchungen zur Physiologie der Carnivoren-Drusen. II Mitteilung.

Ober die Resorption verschidedener Substanzen. Planta 66, 331-334.MCCULLY, M. E. (1965). A note on the structure of the cell walla of the brown alga Fucus.

Can. J. Bot. 43, 1001-1004.MCCULLY, M. E. (1966). Histological studies on the genus Fucus. I. Light microscopy of the

mature vegetative plant. Protoplasma 6a, 287-305.MCCULLY, M. E. (1967). Histological studies on the genus Fucus. II. Histology of the repro-

ductive tissues. Protoplasma (in the Press).MCLACHLAN, J. & CRAIGIE, J. S. (1964). Algal inhibition by yellow ultraviolet-absorbing

substances from Fucus vesiculosus. Can. J. Bot. 4a, 287-292.MANTON, I. & PARKE, M. (1965). Observations on the fine structure of two species of Platymonas

with special reference to flagellar scales and the mode of origin of the theca. J. mar. biol.Ass. U.K. 45, 743^754-

MANTON, I., RAYNS, D. G. & ETTL, H. (1965). Further observations on green flagellates withscaly flagella: the genus Heteromastix Korshikov. J. mar. biol. Ass. U.K. 45, 241-255.

MASEH, M., O'BRIEN, T. P. & MCCULLY, M. E. (1967). Shadowing of ultrathin sectionsof epoxy embedded tissues as an aid in three-dimensional interpretation. J. Microscopic6, 305-312-

MEEUSE, B. J. D. (1962). Storage products. In Physiology and Biochemistry of Algae (ed.R. A. Lewin), pp. 289-313. New York: Academic Press.

MONTFORD, C. (i937)- Die Trochenresistenz der Gezeitenpflanzen und die Frage der Uberein-8timmig von Standort und Vegetation. Ber. dt. bot. Ges. 55, 85-95.

MOORE, B. G. & TISCHER, R. G. (1965). Biosynthesis of extracellular polysaccharides by theblue-green alga Anabaena flos-aquae. Can. J. Microbiol. 11, 877-885.

NoRTHCOTE, D. H. & PICKETT-HEAPS, J. D. (1966). A function of the Golgi apparatus inpolysaccharide synthesis and transport in the root-cap cells of wheat. Biochem. J. 98, 159—167.

O'BRIEN, J. A. (1942). Cytoplasmic inclusions in the glandular epithelium of the scutellum ofTriticum sativum and Secale cereals. Am. J. Bot. 29, 479-491.

O'BRIEN, T. P. (1967 a). Observations on the fine structure of the oat coleoptile. I. The epidermalcells of the extreme apex. Protoplasma 63, 383-416.

O'BRIEN, T. P. (19676). Observations on the fine structure of the oat coleoptile. II. Correlatedlight and electron microscopy of the vascular system. Protoplasma 63, 417-442.

O'COLLA, P. S. (1962). Mucilages. In Physiology and Biochemistry of Algae (ed. R. A. Lewin),PP- 337-356. New York: Academic Press.

PERCIVAL, E. (1964). Algal polysaccharides and their biological relationships. In Proc. 4thinternatn. Seaweed Symp. (ed. Ad. Davey de Virville & J. Feldmann). Oxford: Pergamon.

PHILPOTT, C. W. (1965). Halide localization in the teleost chloride cell and its identificationby selected area electron diffraction. Protoplasma 60, 7-23.

PHILPOTT, C. W. & COPELAND, D. E. (1963). Fine structure of chloride cells from 3 speciesof Fundulus. J. Cell Biol. 18, 389-404.

PORTER, K. R. (1964). Cell structure and biosynthesis of intercellular macromolecules. InConnective Tissue: Intercellular Macromolecules. Boston: Little Brown and Co.

PORTER, K. R. & BONNEVILLE, M. A. (1964). An Introduction to the Fine Structure of Cells andTissues. Philadelphia: Lea and Febiger.

PROSSER, C. L. & BROWN, F. A. Jr. (1961). Comparative Animal Physiology, 2nd Edn. Phila-delphia: Saundere.

16 M.E. McCullyRAPPAY, G. & VAN DUIJN, P. (1965). Chlorous acid as an agent for blocking tissue aldehydes.

Stain Technol. 40, 275-277.REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in

electron microscopy. J. Cell Biol. 17, 208-212.Ris, H. & PLAUT, W. (1962). Ultrastructure of DNA-containing areas in the chloroplast of

Chlamydomonas. J. Cell Biol. 17, 383-391.SCHNEPF, E. (1963a). Zur Cytologie und Physiologie pflanzlicher Drilsen I. Ober den Fang-

schleim der Insectivoren. Flora, Jena 153, 1—22.SCHNEPF, E. (19636). Golgi-Apparat und Secretbildung in den Drilzenzellen der Schleimgange

von Laminaria hyperborea. Naturwissenschqftm 50, 674.STOERMER, E. F., PANKRATZ, H. S. & BOWEN, C. C. (1965). Fine structure of the diatom

Amphipleura peUucida. II. Cytoplasmic fine structure and frustule formation. Am. J. Bot.52, 1067-1078.

VARNER, J. E. (1965). Seed development and germination. In Plant Biochemistry (ed. J. Bonner& J. E. Varner), pp. 763-792. New York: Academic Press.

WASSERMANN, A. (1948). The continuous swelling of brown algae. Ann. Bot. N.S. 12, 137-145.WASSERMANN, A. (1959). Cation absorption by brown algae: the mode of occurrence of alginic

acid. Am. Bot. N.S. 13, 79-88.WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals.

J. biophys. biochem. Cytol. 4, 475-478.WOODING, F. B. & NORTHCOTE, D. H. (1965). The fine structure of the mature resin canal

cells of Pinus pinea. J. Ultrastruct. Res. 13, 233-244.ZANEVELD, J. S. (1937). The littoral zonation of some Fucaceae in relation to desiccation.

J. Ecol. 25, 431-468.

{Received 15 June 1967)

Fig. 1. Longitudinal section through the apical pole of a mature epidermal cell showingthe thick, outer wall (010) and the lateral wall between two adjacent cells. Theproliferated plasma membrane wall interface is apparent and some of the longinvaginations of the plasma membrane are marked by arrows. Two types of tanninvesicles (tVi) and (tvt) are present, in addition to vesicles (ag) presumed to containalginic 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 andthe lateral wall (Iw). Note the proliferation of smooth membrane outside the plasmamembrane in this area, x 20000.Fig. 3. Longitudinal section of epidermal cell in the plastid region showing a deepinvagination of the plasma membrane (outlined by arrows) into the cell. A mito-chondrion (TO), ribosome-rich cytoplasm, and a portion of a plastid can also be seen,x 30000.

Journal of Cell Science, Vol. 3, No. 1

ow

M. E. McCULLY (Facing p. 16)

Fig. 4. Longitudinal section through the plastid region showing plastids containingosmiophilic droplets (od) and vesicular inclusions (ci). The asterisk marks a largeinvagination 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 showsa group of the vesicular bodies. Figure 6 shows a single vesicular body (a) and anosmiophilic droplet (od). x 50000.Fig. 7. Photomicrograph of a cross-section of mature vegetative thallus, showing thebase of epidermal cells (ep) and parenchyma cells of the cortex (cp). Numerous plastidsare present, those of the epidermis are lying against the lateral walls and those of theparenchyma are scattered throughout the cells. Plastid 'vacuoles' are indicated byarrows. Glycol methacrylate-embedded section. Toluidine blue staining after chlorousacid treatment, x 1900.

Journal of Cell Science, Vol. 3, No.

M. E McCULLY

Journal of Cell Science, Vol. 3, No.

Figs. 8—10. Sections of epidermal cell chloroplasts showing typical arrangement of thelamellae. 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 closelyappressed to a portion of the plastid. Figure 8 is a higher magnification of the areaindicated, 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.

M. E. McCULLY


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

M. E. McCULLY


V

/w

Fig. 13. Transverse section through the apical pole of a mature epidermal cell showingthe apical mitochondria and sections through the proliferated plasma membranesurface. The arrows indicate vesicles with densely staining peripheries, which occurcommonly 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 epidermaland 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.

M. E. McCULLY


For legends see next page.

M. E. McCULLY

Fig. 15. Section through the nucleus of a mature cell showing the narrow envelopeof perinuclear cytoplasm with fingers of cytoplasm running out into the cell betweenthe vacuole areas (v). x 10000.Fig. 16. Sections of nuclear envelope showing nuclear pores and the formation ofa cisterna of the ER by an outpocketting of the outer membrane of the nuclearenvelope, 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 hyper-trophied 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 bodyshown in Fig. 17, showing the large tannin vesicles (tVt) and two other types ofvesicles presumed to contain alginic acid (ag) or fucoidin (Jv). The nature of thelarge, osmiophilic multivesicular body is not known but it appears quite distinctfrom 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). Thearrows 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 wallshowing a narrow strand of cytoplasm typical of those that link together the plastidsin this area. Such strands, which contain numerous ribosomes and a few mito-chondria (m), are often completely surrounded by tannin vesicles (in,) of the typewhich fill the apical portion of the cells, x 35000.


22

M. E. McCULLY


fct .24

Figs. 23-26. Prepared by shadowing thin sections with platinum. The direction ofthe 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 thatin Fig. 23 showing the plasmodesmata in relief. In some plasmodesmata such as theone 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. Theoriented fibrillar structure of this wall can be seen clearly in Fig. 25, which showsa portion of the same wall as shown in Fig. 26. Figure 25, x 42000; Fig. 26, x 20000.

M. E. McCULLY

Documents

Department of Biology, Carleton University, Ottawa, Canada · of a single lamellar stack Fucus, differs from many other algae, for example, Euglena, in which the thylakoids are so