CYTOLOGICAL STUDIE OSN PHYSODES IN THE VEGETATIVE … · STRICTA SAUVAGEAU (PHAEOPHYTA, FUCALES) LILIANE PELLEGRINI Institut de Cytologie et de Biologie Cellulaire, LA CNRS 179, de

J. Cell Set. 41, 209-231 (1980) 20QPrinted in Great Britain © Company of Biologists Limited 1980

CYTOLOGICAL STUDIES ON PHYSODES IN

THE VEGETATIVE CELLS OF CYSTOSEIRA

STRICTA SAUVAGEAU (PHAEOPHYTA,

FUCALES)

LILIANE PELLEGRINI

Institut de Cytologie et de Biologie Cellulaire, LA CNRS 179, Faculti de Luminy,70 route Lion Lacliamp, 13288 Marseille Cedex 2, France

SUMMARY

Physodes have been recognized in meristodermic and promeristematic cells by correlatedlight- and electron-microscope investigations using different fixation procedures. They arevesicles which contain an osmiophilic material of phenolic nature. Their content changes inappearance according to the fixative used. Osmiophilic deposits are often associated with coiledand disturbed lamellar formations.

It has been possible to distinguish feveral ultrastructural stages which occur during thesecretion of the content of the physodes, namely: a chloroplast accumulation and exudation,and a reticular transport to accumulation vacuoles where materials undergo evolution orhydrolysis. Inside plastids, osmiophilic granules are found in close association with thylakoidstacks. They may contain the polyphenolic precursors of physodes, though this has not yetbeen proved by electron-microscope procedures. They are expelled from plastids to thechloroplast endoplasmic reticulum. The mechanism of transfer through the chloroplastenvelope remains to be elucidated. Lytic activities have been reported inside physodes whichmight thus act in the same way as the secondary lysosomes of animals and higher plants.Occasionally, the physode content seems to be excreted from the cytoplasm to the cell wallsby exocytosis after the probable fusion of plasmalemma and tcnoplast.

These cytological changes, observed in the vegetative apex of a brown alga, recall someultrastructural characteristics of the secretory processes described in various glandular tissuesof higher plants and which consist of the synthesis, the transport and the elimination of anexudate of flavonic, terpenic or lipophenolic nature.

INTRODUCTION

One distinguishing feature of the brown algal cell is possession of several refractiveand irregularly shaped bodies called 'fucosan vesicles' or, more usually, 'physodes'.

Small light-refracting inclusions, now known to be physodes, were referred to asearly as 1847 by Nilgeli. Many phycologists have since studied them by light micro-scopy and light cytochemistry in an attempt to find out their nature and physiologicalrole (see Ragan, 1976). Nowadays, it is generally agreed that vegetative and repro-ductive cells of Phaeophyceae contain phloroglucinol compounds and other tannins,most of which are included in the physodes. However, many biochemical and cyto-logical problems remain unsolved. As pointed out by Ragan, physodes have not beenisolated and characterized by modern biochemical techniques, so that one does notknow whether all the physodes enclose phloroglucinol and whether phloroglucinolderivatives are (or are not) associated therein with other compounds. Moreover,

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electron-microscope studies have not clarified the nature of brown algal physodesand their biogenesis is still problematical.

This account concerns correlated light- and electron-microscope observationsmade on the physodes present in the promeristematic and meristodermic cells of thevegetative apex of Cystoseira. Particular attention has been given to the origin of thephysode content and to the ultrastructural characteristics of physode secretion.

MATERIAL AND METHODS

Plants of Cystoseira stricta Sauvageau were collected throughout the vegetative growthperiod from the rocky shores of Provence between Marseilles and Toulon. They were eitherdirectly prepared for examination by light and electron microscopy or placed in tanks containingseawater with added mineral salts and left at 18 °C in complete darkness for several days, asdescribed previously (Pellegrini, 1976).

Light microscopy

Living tips of main axes were observed after vital staining in neutral red and cresyl bluediluted in seawater. Other apices were fixed by various procedures using (a) the fixative ofKarpechenko modified by Papenfuss (1946) for 15 to 17 h, (b) the fixative of Johansen (1940)for 48 h, or (c) the fixative of Dalton (1955) for 5 h. Then they were washed, embedded inparaffin wax and sectioned for conventional light microscopy. Most of the sections wereobserved directly without staining.

The chemical nature of physodes was investigated using various specific reactions of phenoliccompounds, especially (a) 1 % caffeine in distilled water: the appearance of white globulesproves the presence of tannins (Laurent, 1966), (b) 1 % vanillin dissolved either in a freshlymixed 9:1 solution of 95° ethanol and concentrated hydrochloric acid (Ragan & Craigie, 1976),or in 70 % aqueous sulphuric acid (Forrest & Bendall, 1969), (c) diazotized benzidine: the reagentconsists of a freshly mixed solution v/v of 05 % benzidine in 0-2 N hydrochloric acid and 10 %aqueous sodium nitrite (Jensen & Haug, 1952; Gaillard, 1962). The 2 last reagents are specificfor condensed tannins. Fresh sections of apices were immersed for a few minutes in thesevarious stains and observed directly. Sections of fixed apices were also treated with diazotizedbenzidine.

Electron microscopy

The main branches about 1-2 cm long were cut from young plants. They were quicklyimmersed in the chosen fixative and, after 15 min, 1-2 mm apical regions were excised andprepared for observation by electron microscopy. Different procedures of fixation, madebetween 5 and 10 °C, were used: (a) osmium tetroxide fixations for 60—90 min, with either 2 or4% osmium tetroxide in acetate veronal buffer (pH 7-4), sometimes with o-oi % calciumchloride added, or with the chrome—osmium mixture of Dalton (1955); and (b) glutaraldehydefixations followed by postfixations. Material was fixed for 8 h in 5-6 % glutaraldehyde inphosphate buffer (005 or 01 M, pH 7-3). It was rinsed in six 30-min changes of the same buffer.Then, it was postfixed either in 2 % osmium tetroxide (same buffer) for 7 h or in 2 % potassiumpermanganate in veronal buffer for 1 h. Sometimes, 1-5 % potassium ferrocyanide was addedto the osmium tetroxide postfixation solution according to Endress & Thomson (1976). Then,fixed apices were rapidly washed in several changes of the same fixation buffer, dehydrated for15 min each through an acetone or ethanol series, embedded either in Araldite M or in a mixtureof Epon and Araldite (Mollenhauer & Totten, 1971) and sectioned on a Porter Blum MT2ultramicrotome. Some thin sections were viewed directly using a Philips EM-300 electronmicroscope at 80 kV. Others were prestained with 2 % aqueous potassium permanganate. Theperiodic acid-thiocarbohydrazide-silver proteinate method of Thiery (1967) was sometimesused to obtain a better contrast of cytomembranes: 25 min oxidation in 1 % aqueous periodicacid, 2-5 h in thiocarbohydrazide, 30 min in silver proteinate in darkness. Staining in phospho-

Physodes in vegetative cells 211

tungstic acid-chromic acid (PTAc) was also employed according to Roland, Lembi & Morre(1972): 25 min oxidation in 1 % aqueous periodic acid, 30-50 min in 1 % phosphotungstic acidin 10 % chromic acid.

Acid phosphatase localization

Small apical regions from main branches were prefixed for 3-4 h at 4 °C in the normalbuffered glutaraldehyde solution plus 3 or 4 drops of hydrogen peroxide according to Perracchia& Mittler (1972). They were rinsed in a series of citrate buffers of decreasing pH (7-6-5-6-5-5-5) to remove calcium salts. They were then incubated at 4 °C for 2 h and 37 °C for 1 h in thestandard medium with sodium /?-glycerophosphate as substrate and lead nitrate as coupler.After the incubation wash, specimens were osmicated, dehydrated and embedded normally.Controls were (a) medium with substrate omitted, or (6) complete medium containing 001 Msodium fluoride as inhibitor. Thin sections were examined without post-staining.

RESULTS

The different regions of a vegetative apex of Cystoseira stricta can be seen in alongitudinal section (Fig. 1). As in other Fucales, the apical cell (a) is located at thetip of the axis at the bottom of a groove which is filled with mucilage. Its segmentationgives rise to a parenchymatous construction, the ' promeristem' (pm) of Moss (1967).Medullar and cortical cells of the adult thallus result from the differentiation of pro-meristematic cells. The most external cell layer of promeristem and cortex is the'meristoderm' (m) (Fritsch, 1945). Meristodermic cells divide to accommodatethickening of the thallus.

Observations on physodes in light microscopy

The physodes appear as inclusions of various sizes. In fresh sections, they stain redwith neutral red and turquoise with cresyl blue, differing from the neighbouringiridescent bodies which are refractive formations unstained by these dyes (Fig. 2).

In sections of material fixed in the solutions of Karpechenko (Fig. 1), Dalton(Fig. 3) and Johansen (Fig. 4), phenolic-containing vesicles are more easily distin-guished since these fixatives contain various substances - chromic acid, potassiumbichromate, ferrous salts or osmium tetroxide - which give black or dark-brownprecipitates with tannoids. Such coloured inclusions are located preferentially at theexternal pole of meristodermic cells and around the nucleus in promeristematic cells(Figs. 3, 4), and are larger in lateral branches (Fig. 1, /).

These inclusions are clearly equivalent to physodes. Moreover, their contentsdarken with the chrome—osmium fixative (Fig. 3) and, in living material, precipitateas large white globules with caffeine solution (Fig. 5), stain red with vanillin-acid(Fig. 6) and diazotized benzidine (Fig. 7). Therefore, they contain condensed tannins.

Observations on physodes in electron microscopyGeneral aspect. Physodes have been located, unambiguously, at the ultrastructural

level after correlated studies of sections prepared for light microscopy and for electronmicroscopy. They lie at the external pole of meristodermic cells and scatteredthroughout the cytoplasm of promeristematic cells (Figs. 8, 9). They are vesicles

L. Pellegrini


bounded by a thin unit membrane. The fine structure of their contents seems tochange according to the fixative used. Comparative observations of promeristematiccells have therefore been made with different fixation procedures.

In cells fixed with buffered osmium tetroxide (Fig. 8), physodes contain anelectron-transparent or finely granular matrix and an osmiophilic material. Thelatter may be a number of small particles or may fill the whole vesicle. It may bealveolate in material kept in darkness for 10 days (Fig. 10). The various aspects ofphysode content may be observed in one and the same cell, where the physodes canbe grouped or confluent (Fig. 8).

The fine structure of physode content fixed according to Dalton reveals strikinganalogies with the preceding description (Fig. 9). Compounds have reacted withosmium tetroxide and chrome salts in such a way that the resulting micrographssuggest fluid or semifluid contents which have taken various forms in response to thefixative.

Inside cells fixed with glutaraldehyde-osmium tetroxide (Fig. 11), the cytoplasmstill contains large or small vesicles with an electron-transparent or finely granularmatrix and osmiophilic deposits. However, the appearance of the latter dense materialhas changed to globular compact masses associated with membrane configurations.With modified postfixation, either by replacing osmium tetroxide with potassiumpermanganate (Figs. 12, 13) or by adding potassium ferrocyanide to osmium tetroxidesolution (Figs. 14, 15), the aspect of the physodes is similar.

Origin and evolution of the physode content. Plastids of meristodermic and pro-meristematic cells sometimes contain electron-dense inclusions reaching about 0-5 /xvalong (Fig. 16). These bodies seem not to be surrounded by a membrane. They arefrequently in close association with thylakoid stacks. Their central area appearselectron transparent, in some cells, probably due to loss of material during fixationand inclusion. The largest and most numerous of them are found in material fixedduring spring. They are also present inside the plastids of algae which have been indarkness for several days (Fig. 10).

These dense formations become protruded from the plastids and move either intothe chloroplast endoplasmic reticulum (Figs. 17, 18) or directly into the physodesintimately associated with the plastids (Fig. 19). The chloroplast endoplasmic

Fig. 1. Apical region in longitudinal section showing the general distribution ofphysodes in a young lateral branch (/) and in the apical (a), promeristematic (pnt)and meristodermic (m) cells of the main axis. Karpechenko fixation, benzidinereaction, x 125.Fig. 2. Physodes (ph) vital stained by cresyl blue are clearly distinct from unstainediridescent bodies («') in promeristematic cells, x 800.Figs. 3, 4. Longitudinal sections of apices fixed according to Dalton (Fig. 3) andJohansen (Fig. 4). Localization of physodes (ph). m, meristoderm. No staining.Fig. 3, x 680; Fig. 4, X640.Figs. 5-7. Cytochemical reactions on fresh sections with solutions of caffeine (Fig. 5),vanillin-sulphuric acid (Fig. 6) and diazotized benzidine (Fig. 7) proving the phenolicnature of physodes. x 650.

214 L. Pellegrini

8Fig. 8. Portion of a longitudinal section of material fixed in buffered osmiumtetroxide. Physodes (ph) are located beneath the cuticle (c) of the meristodermiccells (m). Different aspects are illustrated in the promeristematic cells (pm). ci, iri-descent body; d, dictyosome; mi, mitochondrion, n, nucleus;£, plastid. No staining.X3500.


Fig. 9. Portion of a longitudinal section of material fixed in chrome—osmium mixture.The content of physodes (ph), in varying quantities, shows different aspects, c, cuticle;ci, iridescent body; d, dictyosome; mi, mitochondrion; n, nucleus; p, plastid. Nostaining, x 3350.

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11Fig. io. Meristodermic cell after io days in total darkness showing iridescent bodies(et), a physode (ph) and a plastid (p) with osmiophilic granules. Fixation: osmiumtetroxide. Thiery test, x 31400.Fig. 11. A glutaraldehyde—osmium tetroxide-fixed promeristematic cell with variousphysodes (ph). Stain: potassium permanganate, x 21 500.

Physodes in vegetative cells

15Figs. 12-15. Aspects of physodes (ph.) in material fixed in glutaraldehyde-potassiumpermanganate (Figs. 12, 13) and in glutaraldehyde-osmium tetroxide-potassiumferrocyanide (Figs. 14, 15). The arrows point to lamellar configurations ci, iridescentbody; n, nucleus; p, plastid. No staining.Fig. 12. Meristematic cell, x 22600. Fig. 13, promeristematic cell, x 37000. Fig. 14,promeristematic cell, x 22600. Fig. 15, promeristematic cell, x 36900.j< CKL 41

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> *

P

mp

I0-5

Figs. 16-18. Sections of plastids (p) in promeristematic cells showing osmiophilicbodies (06) closely associated with thylakoid stacks. The thin arrow on Fig. 16 pointsto a protrusion of dense material. The thick ones on Figs. 17 and 18 show densematerial lodged between the 2 chJoroplast endoplasmic reticulum membranes (cer).mp, plastidial membrane. Fixation: glutaraldehyde—osmium tetroxide. Fig. 16. po-tassium permanganate staining, x 51 300. Fig. 17, Thiery test, x 64000. Fig. 18,Thiery test, x 80000.

18

Physodes in vegetative cells 2 I Q

Figs. 19-22. Cytochemical localization of acid phosphatases in promeristematic cells.The arrows on Fig. 20 indicate 2 deposits of lead phosphate inside the dense materialof a physode. ci, iridescent body; er, endoplasmic reticulum; p, plastid; ph, physode;vl, lysosome-like vesicle. No staining. Fig. 19, x 39900; Fig. 20, x 28000; Figs. 21,22, x 31400.

15-2

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26


reticulum connects with the smooth endoplasmic reticulum and the physode envelope

(Fig- 23)-The cytochemical test for localization of acid phosphatases allows better under-

standing of the evolution of the physode content (Figs. 19-22). Lysosome-likevesicles empty into the physodes (Fig. 21) and the enclosed material is slowly lysed.Micrograph 20 shows 2 aggregates of lead phosphate precipitates inside a physodedense deposit, indicating the localized presence of lytic enzymes. The intraplastidialosmiophilic formations do not give a positive reaction to the cytochemical test(Fig. 21). Compounds ejected from the plastids become positive only when they areincluded inside the vesicles (Figs. 19, 21). There, the products of lysis appear ascompact masses and/or unfurled membrane configurations (Fig. 22).

However, the physode content may also be excreted from the cell towards the cellwalls (Figs. 24, 25). The physode envelope probably fuses with the plasmalemma whichimplies a similar configuration of the 2 cytomembranes. These latter clearly show thesame positive reaction with the PTAc test (Fig. 26). After the membrane fusion, thephysode content may then be discharged against the walls where it lies on both sidesof the middle lamella (Fig. 25).

DISCUSSION

Notion of physodes

In the light microscope, brown algal physodes are distinguished by their reactivitywith neutral red, cresyl blue, caffeine, vanillin-hydrochloric acid and diazotizedbenzidine. The first 2 compounds are vacuolar vital stains; the last 3, precipitatephenolics. This is evidence that physodes are phenolic-storing vacuoles.

In the electron microscope, the identification of physodes is more difficult. Indeed,one cannot adapt light-microscopic methods for they profoundly alter cell contents.However, a few workers have tried to locate phenolic compounds in higher plants atthe ultrastructural level either by fluorometry (Charriere-Ladreix, 1976) or by addingto fixatives potassium bichromate (Dumas, 1975), ferric sulphate (Endress &Thomson, 1976), caffeine (Mueller & Greenwood, 1978) or else ferric chloride(Robb, Brisson, Busch & Lu, 1978). Tannins have also been located by their strongaffinity for osmium (Baur & Walkinshaw, 1974; Chafe & Durzan, 1973). In Cystoseira,the localization of physodes has been realized on the basis of comparative fixation

Fig. 23. Relation between the chloroplast endoplasmic reticulum (cer), a physode (ph),an iridescent body (a) and the smooth endoplasmic reticulum (er) in a promeristematiccell. Fixation: glutaraldehyde-osmium tetroxide. Stain: potassium permanganate,x 30100.

Figs. 24, 25. Excretion of physodes (ph) from cytoplasm to cell walls (pc) of pro-meristematic cells. Fixation: osmium tetroxide (Fig. 24) added to calcium chloride(Fig. 25). Stain: potassium permanganate. Fig. 24, x 25000; Fig. 25, x 20 500.Fig. 26. Staining with PTAc test showing the reactivity of plasmalemma (pi) and thephysode envelope (mph) in a promeristematic cell. Fixation: osmium tetroxide—calcium chloride, x 39900.

222 L. Pellegrini

techniques in light and electron microscopy by adding to fixatives chemicals whichreact with phenolics.

Electron-opaque vesicles are confined to the external pole of meristodermic cellsand around the nucleus of promeristematic cells. They occupy the same location asthe phenolic vacuoles which stain vitally with neutral red and cresyl blue and whosecontents precipitate with caffeine, vanillin-hydrochloric acid and diazotized benzidine.It is clear that these vesicles are the physodes. The present account shows thereforethat physodes of Cystoseira cells are really equivalent to higher plant tannin vacuoles.It agrees with the ideas of Mangenot (1930) who implied the same from light-cytological investigations on several Phaeophyceae.

Genesis of physodes

The development of higher plant vacuoles involves several successive stages:(a) formation of provacuoles, (b) fusion of provacuoles into small vacuoles, and(c) extension of the vacuoles. Sometimes, other processes may occur during the last2 stages: (a) association with reticular profiles, (b) intervention of lysosomal activities,and (c) accumulation of various substances, for example, tannins.

The genesis of physodes in Cystoseira must involve these processes, some of themrelated to the vacuole proper and the others more especially to the secretion productwhich is enclosed therein. The origin and development of vacuoles in Cystoseira havebeen described in another paper (Pellegrini, 1978). They involve the sequestration ofcytoplasmic materials by formations originating probably from GERL as in somehigher plants (Buvat, 1977; Buvat & Robert, 1979; Marty, 1974, 1978). Some of thesevacuoles may evolve into physodes after accumulation of phenolics. The secretion ofthe physode content consists of several successive stages: plastidial accumulation andexudation, reticular transport to accumulation vacuoles, and excretion into cell walls.A few of these processes were suggested as early as 1974 (Pellegrini, 1974a, b).

Plastidial accumulation. The first phase of physode secretion is seen inside theplastids. These osmiophilic inclusions are rarely observed in osmium tetroxide-fixedmaterial, except when the algae live in darkness for a long time. They may often beseen inside cells fixed by glutaraldehyde and postfixed by osmium tetroxide orpotassium permanganate. However, they do not always seem to be present in allmaterials collected during 1 year's growth. This suggests that physode activity maychange according to the season. In several Cystoseira species of the Adriatic, thehighest amounts of reducing compounds (e.g. polyphenols) are measured duringwinter and during spring, with a maximum in March (Munda, 1962). Large intra-plastidial inclusions are most numerous in Cystoseira stricta apices collected in March.They are irregularly shaped formations, not surrounded by a membrane.

Plastidial bodies, more or less identical in appearance, have been reported (a) inother Phaeophyceae (McCully, 1968; Evans, 1968; Magne, 1971; Bisalputra, Shields& Markham, 1971; Feldmann & Guglielmi, 1972; Evans & Holligan, 1972; Bisal-putra, 1974; Oliveira & Bisalputra, 1977 a, c; Forbes & Hallam, 1978) and (b) indifferent higher plant cells which elaborate terpene-like resins (Wooding & Northcote,1965; Carde, 1976), essential oils (Amelunxen & Arbeiter, 1967; Amelunxen &


Gronau, 1969), flavonoids (Charriere-Ladreix, 1973, 1976, 1978; Charriere-Ladreix &Dumas, 1975) and lipo-polyphenolic compounds (Dumas, 1975, 1977; Charriere-Ladreix & Dumas, 1975).

The chemical nature of the dense inclusions in brown algal plastids has beeninterpreted in various ways (Magne, 1971; Feldmann & Guglielmi, 1972; Evans &Holligan, 1972; Bisalputra, 1974; Oliveira & Bisalputra, 1977a). As in Evans &Holligan's study, the present investigation suggests, though does not likewise proveby electron-cytochemical procedures, that they may contain the polyphenolic pre-cursors of physodes. Several papers would seem to confirm this suggestion, althoughmost of them refer only to higher plants. Indeed, phenolics are considered as usualcomponents of angiosperm plastids and their amounts may fluctuate according to theseason (Monties, 1969). Flavonoids have been detected by fluorometry inside theplastids of Populus nigra (Charriere-Ladreix, 1976). Concerning Phaeophyceae,Davies, Ferrier & Johnston (1973) wrote that biochemical studies made in theirlaboratory 'indicate that polyphenol precursors appear to be derived from plastids,and fractionation studies provide evidence for the presence of enzymes involved inthe metabolism of phloroglucinol-containing phenolics'. Our attempts to locatepolyphenoloxidases inside Cystoseira plastids by electron cytochemistry are still un-successful. However, Davies et al. added: 'the degree of polymerization achieved inor on leaving the plastid may vary, and similarly any interaction with other metabolitesand the species involved'. This might explain difficulties in obtaining positive cyto-chemical results in some cases.

Plastidial exudation. The dense globules of Cystoseira plastids are ejected into thechloroplast endoplasmic reticulum (Figs. 17, 18). Transfer through the chloroplastenvelope has not still been elucidated.

Similar protrusions of portions of plastids have been mentioned in Dictyota (Evans& Holligan, 1972) and in Ectocarpus (Oliveira & Bisalputra, 1977a). Plastidial exud-ations of osmiophilic material into the endoplasmic reticulum surrounding plastidsalso occur during secretory processes in various higher plants, for example in matureresin canal cells of Pinus picea (Wooding & Northcote, 1965), in oil cells of Acoruscalamus (Amelunxen & Gronau, 1969), in transfer cells of Pinus pineaster leaves(Carde, 1973), in glandular cells of Forsythia intermedia stigma (Dumas, 1975, 1977)and in glandular tissue of Populus nigra buds (Charriere-Ladreix, 1973, 1978).

Reticular transport. Electron-opaque material has been shown inside the chloroplastendoplasmic reticulum and then inside vacuoles. Two types of association must there-fore assist the transport, one between plastids and the chloroplast reticulum, the otherbetween this reticulum and physodes.

Brown algal plastids are surrounded by a reticular profile: the 'chloroplast endo-plasmic reticulum' (see Gibbs, 1962, 1970; Dodge, 1973; Bisalputra, 1974).

Associations between plastids and the endoplasmic reticulum have also been foundamong higher plants, in various secretory cells or tissues but always in materials witheffective metabolic functions (see Dumas, 19746 for a review) where they would beinvolved in the synthesis and transport of several compounds (Galatis, Apostolakos &Hatzopoulou, 1974; Dumas, 19746, 1975; Whatley, 1977).

224 L. Pellegrini

In Phaeophyceae, Bouck hypothesized as early as 1965 the participation of chloro-plast endoplasmic reticulum during the transport of elaborated products. Thisintervention might concern (a) carbohydrate transport where the chloroplast endo-plasmic reticulum would play an important part during the transport of alginic acidprecursors from plastids to dictyosomes (Leppard, 1973, 1974), and (b) transport ofpolyphenolic precursors to the physodes. Dumas (19746) discussed the role of plastid-ER associations in phenolic synthesis during stigmatic secretion in Forsythia andVerbascum. He suggested 2 pathways. In one, precursors would be synthesized insideplastids and then collected by the periplastidial reticulum. In the other, this reticulummight bring essential precursors of polyphenolic biogenesis towards the plastids. Itseems likely that the first Dumas' possibility may occur in Cystoseira. We cannot, ofcourse, rule out the second, but Fig. 16 suggests more an exudation than an intrusionof dense material into the plastid. This material is clearly lodged between the 2endoplasmic reticulum membranes (Figs. 17, 18). In Dictyota, a process of extrusionof bodies from the chloroplast matrix had also been seen but Evans & Holligan (1972)thought that these bodies were 'probably surrounded by a membrane' and that thechloroplast endoplasmic reticulum was not involved in the transport of these' physodes'. However, it is difficult to imagine the mode of transfer of a vesicle throughmembranes whereas the crossing of material only assumes a particular permeabilityof these membranes to certain molecules.

Electron-dense bodies have also been reported closely associated with either theperiplastidial reticulum or with the plastids in Laminaria (Davies et al. 1973), Fucus(Brawley, Quatrano & Wetherbee, 1977) and Cutleria (La Claire & West, 1978).Membrane continuities between the periplastidial reticulum and the chloroplastenvelope have been described in Ectocarpus by Oliveira & Bisalputra (1973, 19776).Similar continuities have been observed in Cystoseira but only with a low frequency,so that the possibility of fixation artifacts must also be considered (Pellegrini, 1978).It is evident that these membrane contacts might facilitate substance exchanges.

The present observations closely resemble the plastidial origin and reticularintervention during the processes of synthesis and transport of the terpene-like resinprecursors in Pinus secretory cells (Wooding & Northcote, 1965; Carde, 1976). Theyalso agree with the suggestions of Mueller & Beckman (1974) about polyphenolicsynthesis in specialized cells of banana. It will be of interest to find if a similar path-way of physode^formation takes place during cell differentiation of other Phaeophyceae.

In Cystoseira, the chloroplast endoplasmic reticulum connects the smooth endo-plasmic reticulum and the physode tonoplast. These latter associations are probablytemporary for they are seldom observed. They may facilitate exchanges betweencellular compartments.

Some connexions between the endoplasmic reticulum and vesicles have beenreported (a) in 3 other brown algae: Dictyota (Feldmann & Guglielmi, 1972), Ecto-carpus (Oliveira & Bisalputra, 1973) and Laminaria (Davies et al. 1973), and (b) invarious higher plants (Berjak, 1972; Fineran, 1973; Dumas, 1974a, 1975; Baur &Walkinshaw, 1974; Endress & Thomson, 1976; Carde, 1976; Ramsey & Berlin, 1976;Kristen, 1977; Coulomb, 1978). In particular, these ER-vacuole contacts might


function during the tanniferous synthesis in banana (Mueller & Beckman, 1974) orPinus elliotti cells (Baur & Walkinshaw, 1974) and during the lipo-polyphenolicsecretion of Forsythia stigma (Dumas, 1975, 1977)- They would also allow a regulationof the synthesis of terpene hydrocarbons in Pinus pinaster (Carde, 1976).

Vacuolar accumulation. Dense material is carried to physodes where it is accumu-lated sparsely or in plentiful quantities. Sometimes, amorphous electron masses areassociated there with membrane configurations.

Most ultrastructural studies on higher plants confirm that phenolics are storedinside vacuoles where they appear either as dense globules or as lamellar bodies whichmay or may not be enclosed inside osmiophilic and amorphous deposits. The presenceof lamellar configurations raises some problems. They have been reported in animalcells and in cells of higher and lower plants. Their significance is not clear and variousinterpretations are possible. Firstly, lamellar formations might result from modifi-cations of membranar systems, especially the ER and the tonoplast. That view hassometimes been taken into consideration to explain the origin of myelin-like materialincluded inside brown algal vesicles or physodes (Feldmann & Guglielmi, 1972;Oliveira & Bisalputra, 1973; Loiseaux, 1973; Brawley, Wetherbee & Quatrano, 1976;Forbes & Hallam, 1978). Secondly, lamellar formations might represent some partiallylipid aggregates which would be more or less hydrated (Curgy, 1968; Marty, 1974).Recently, Mollenhauer, Morre & Jelsema (1978) proposed lamellar bodies as inter-mediates in endoplasmic reticulum biogenesis from seed reserves. They reportedthat these myelin-like figures would be produced by the transformation of some proteinbodies. These different suggestions do not seem to justify the origin of the lamellarcontent in brown algal physodes because no lipid and no protein have been detectedthere by modern cytochemical or biochemical procedures (see Ragan, 1976). Thirdly,lamellar formations might occur in relation to cellular lysis as Coulomb & Buvat (1968)and Coulomb (1972) considered to be the case for vacuoles of higher plants. It wouldseem that the last interpretation would be reasonable for Cystoseira physodes wheremyelin-like arrangements are often disturbed, which would involve the interventionof a lytic process.

Destiny of the secretion product. Lytic enzymes have been located inside physodeswhich could thus act in the same way as the secondary lysosomes of animals and higherplants. The appearance of their contents would change according to the intensity oflytic processes which occur therein. These variations of aspect have been reportedin several works (Chadefaud, 1932, 1934, 1935; Ando, 1951, 1958; McCully, 1966,1968; Feldmann & Guglielmi, 1972; Rawlence, 1973). However, the present accountalso suggests that the various appearances of physode content observed in Cystoseiracells may relate to several stages of an evolutionary process: the phenolic secretionconsisting of a synthesis and a transformation of phenolics. On the other hand, theseobservations do not exclude the possibility of the presence of several biochemicalclasses of physodes both within brown algal orders and within distinctive species.

In relation to the above, it is important to consider whether the phenomenon ofhydrolysis is compatible with the usually accepted ideas on tannoid metabolism inplant cells. None of the cytochemical and biochemical investigations on Phaeophyceae

226 L. Pellegrini

provide evidence on this but several studies of higher plant cells may do so. Inparticular, Vaudois & Laurent (1976) have reported, during cell differentiation of thefern prothallus, the existence of a morphological evolution of vacuoles. This evolutionwas associated with concomitant variations of the appearance and amounts of tannoidsincluded in the vacuoles. The phenolic content underwent successively an impoverish-ment, which came from the preparation of materials, and a synthesis which includedbiogenesis of monomers, their condensation and finally a hydrolysis of tannoids.Hence, the tannins of higher plants are currently considered to be both waste productsand substances which might provide some metabolic requirements. Similarly, it islikely that turnover or chemical evolution of phenolics may occur inside brown algalphysodes. The presence of oligomers and polymers of phloroglucinol has been reportedin extracts of Halidrys (Glombitza & Sattler, 1973), Bifurcaria (Glombitza & Rosener,1974) and Fucus (Ragan & Craigie, 1976). As pointed out by Ragan (1976), thepolymerization of phloroglucinol units might explain the 'aging process' of thephysodes mentioned by a few early cytologists. Biochemical and developmentinvestigations of several Phaeophyceae should help to clarify the situation.

The physode content in Cystoseira cells may be excreted by a typical process ofexocytosis which takes place after the probable fusion of plasma membrane andphysode envelope. There may exist a mechanism of membrane flow similar to thatdescribed by Vian & Roland (1972) and Prat, Vian, Reis & Roland (1977) which wouldcontribute to plasmalemma enlargement and produce the synthesis of wall components.An excretion of physodes has been reported by Chadefaud as early as 1935 in light-microscopic studies. At the ultrastructural level, Loiseaux (1973) observed, in thedeveloping zoospores of Elachista and Pylaiella, physodes which were expelled fromthe cytoplasm, then located between the plasmalemma and the cell wall and finallyintegrated into the cell walls. Caram (1977) reported diffusion of physodes through thewalls in the female gametangia of Cutleria adspersa. La Claire & West (1978) alsonoted during the gametogenesis of Cutleria hancockii a secretion of osmiophilicmaterial into the extracytoplasmic spaces and finally into the medium. The presenceof phenolic substances in the sea, excreted from various brown algae, has also beenmentioned in several biochemical works (see Hellebust, 1974).

CONCLUSION

It is becoming evident that physodes are equivalent to the specialized vacuolesstoring phenolics in higher plants but there has been only one previous ultrastructuralstudy on the origin of their contents, Evans & Holligan (1972) having described aplastidial origin of physodes in Dictyota. The present account confirms the partici-pation of plastids in the secretion of physode contents in Cystoseira but also allows theelucidation of some further cytological stages of the secretion which have not beenreported before. This secretory process must consist of several successive stages.Firstly, there is a phase of plastidial secretion in which osmiophilic material is foundin close association with the thylakoids. This material is secreted more actively duringthe spring than during other seasons of the annual growth cycle. Next, it is expelled


from the plastids by an unknown mechanism which must concern the chloroplastenvelope and the chloroplast endoplasmic reticulum and which implies a particularpermeability of these membranes. Thirdly, there is a phase of intracellular secretion.The material from the plastids is carried by the chloroplast endoplasmic reticulum andthen by the endoplasmic reticulum to vacuoles where it is accumulated and mayundergo an evolution. Lytic processes occur inside physodes which thus function inthe same way as the secondary lysosomes of animals and higher plants. Fourthly,a phase of extracellular secretion is occasionally observed. The release of physodecontent is facilitated by a typical exocytosis.

The infrastructural characters of the physode secretion in Cystoseira recall somepeculiarities which have been mentioned during glandular processes in higher plants,,especially the role of plastids in the elaboration of secretory products which will thenbe exuded and the role of the endoplasmic reticulum in the transport of these productsto vacuoles. These comparisons strongly suggest that the physodes of Cystoseira arenot special formations. It also suggests that their biogenesis fits into the general,pattern of secretory phenolic activities in plants.

I am greatly indebted to Professor R. Buvat (Institut de Cytologie et de Biologie CellulaireFaculty de Marseille-Luminy) for his continued interest and support of this work and toProfessor G. F. Leedale (Department of Plant Sciences; University of Leeds, U.K.) forcritically reading the manuscript. I would like also to thank Ginette Robert and MichelBerthoumieux for excellent technical assistance in the preparation of the manuscript.

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(Received 9 July 1979)

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CYTOLOGICAL STUDIE OSN PHYSODES IN THE VEGETATIVE … · STRICTA SAUVAGEAU (PHAEOPHYTA, FUCALES) LILIANE PELLEGRINI Institut de Cytologie et de Biologie Cellulaire, LA CNRS 179, de