Unusual dictyosome morphology and vesicle formation in tetrasporangia of the marine red alga Polysiphonia denudata

JOURNAL OF ULTRASTRUCTURE RESEARCH 58, 2 8 9 - 2 9 8 (1977)

Unusual Dictyosome Morphology and Vesicle Formation in Tetrasporangia of the Marine Red Alga Polysiphonia denudata C. D. ALLEY1 AND J. L. SCOTT Department of Biology, College of William and Mary, Williamsburg, Virginia 23185 Received July 26, 1976 Dictyosomes in differentiating tetrasporangia of the red alga Polysiphonia denudata undergo extraordinary changes in size and morphology. In early stages of development the dictyosomes are approximately 0.5 tzm in size and are structurally and functionally similar to dictyosomes in most other plants. However, in later stages of sporangial development, the dictyosomes become hypertrophied and exhibit anomalous, densely staining, laminated midregions which are formed due to close apposition of adjacent cisternae. In addition, the manner of secretory vesicle formation changes in mature sporangia such that only a single vesicle is formed by the direct transformation of a single cisterna. Deviating from the classical models proposed for plants and animals, these two aspects of dictyosome morphology and activity are a further demonstration of unique ultrastructural features of the red algae.

Morphological, cytochemical, and bio- published ultrastructural accounts of spochemical investigations have contributed rogenesis in certain species of macroscopic greatly to interpretations of Golgi appara- red algae that several characteristics of tus structure and function. As a result, the Golgi apparatus deviate from the several models have been presented which "classical" models (Peyriere, 1970; Tripodi, take into account not only dictyosome mor- 1971, 1974; Kugrens and West, 1972, 1973, phology, but also the dynamic interrela- 1974; Chamberlain and Evans, 1973; Scott tionship of this organelle with other com- and Dixon, 1973; Konrad Hawkins, ponents of the endomembrane system 1974a,b; Wetherbee and West, 1976). This (Mollenhauer and Morr6, 1966; Morr~ and paper is concerned with electron microMollenhauer, 1971; Morr~ et al., 1971; scope observations of anomalous features Northcote, 1971). In most higher plant of dictyosomes in tetrasporangia of the cells, the Golgi apparatus consists of sev- marine red alga Polysiphonia denudata. eral to numerous dictyosomes composed of Preliminary results of this study have usually polarized stacks of cisternae. Cen- been presented elsewhere (Alley and tral portions of adjacent cisternae are typi- Scott, 1972). cally separated by an intercisternal space MATERIALS AND METHODS of a consistent width. Variable numbers of Matre, healthy plants of Polysiphonia denudata vesicles are released from the dictyosome's (Rhodomelaceae, Rhodophyta) were collected from maturing face and function in the cell's the York River near Yorktown, Virginia. Apical filaments bearing tetrasporangia were immediately secretory activities. fixed at ambient temperature for 2 h in a 3% formalStudies on most eukaryotic algae have dehyde-3% glutaraldehyde mixture in 0.1 M phosdemonstrated that the Golgi apparatus in phate buffer at pH 6.6 with 0.15 M sucrose. Followthese organisms is essentially similar to ing a brief to overnight buffer rinse, the material models proposed for other plants and ani- was postfixed in similarly buffered 1% OsO4 for 2 hr, mals. However, it is evident from recently dehydrated in acetone, and embedded in Epon. Thin sections were cut with a Dupont diamond knife on both Sorvall MT-2B and LKB III ultramicrotomes and stained with uranyl acetate and lead citrate. Most material, including that presented in this paper, was prestained during the dehydration series in

1Present address: Department of Anatomy, Health Sciences Division, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298. 289 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

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2% uranyl acetate in 70% acetone for 16-24 hr at 4°C and was poststained in lead citrate only. Sections were examined with a Zeiss EM 9S-2 electron microscope. RESULTS AND DISCUSSION The red algae are a morphologically diverse assemblage of primitive eucaryotic plants totally lacking motile gametes and spores. Reproductive differentiation and the life history sequence in most species are comparatively complicated. However, in most macroscopic forms haploid gametophyte generations bear small, diploid carposporophyte generations which are formed by the fertilization and retention of specialized egg cells. Liberated carpospores develop into free-living diploid sporophytes which typically produce four spores by meiosis in a structure known as a tetrasporangium. Tetraspores subsequently give rise to gametophyte generations. Carpospore and tetraspore formation, liberation, and development during the early stages of germination are nearly identical (Fritsch, 1945). In addition, most ultrastructural features accompanying sporogenesis are remarkably similar in the investigated species (Peyriere, 1970; Tripodi, 1971, 1974; Kugrens and West, 1972, 1973, 1974; Chamberlain and Evans, 1973; Scott and Dixon, 1973; Konrad Hawkins, 1974a,b; Wetherbee and West, 1976). One feature which has attracted much attention is the remarkable activity of the Golgi apparatus. Dictyosomes comprising the Golgi apparatus appear to change greatly in number, size, and morphology during spore d i f f e r e n t i a t i o n . These changes occur in an uninterrupted sequence and can be related to aspects of spore development observable with the light microscope. The functions generally attributed to dictyosome activity in red

algae include the production of large quantities of complex carbohydrates which are instrumental during sporangial wall formation, spore liberation, maintenance of a capsule or ~wall" of liberated spores, and subsequent spore attachment and germination. Although these functional aspects have been studied in some detail, red algae are usually difficult to satisfactorily prepare for electron microscopy and the quality of resolution necessary to adequately document the temporal changes in dictyosome structure and function has generally been lacking. It is believed that our present work on well-preserved tetrasporangia of P. denudata will clarify several of these earlier observations.

Dictyosomes of Immature Sporangia Figure 1 shows a portion of a tetrasporangium at an early stage of development. The single diploid nucleus is centrally located. Ribosomes are abundant but floridean starch, ER, and chloroplasts are present only in reduced quantity. Dictyosomes have already increased considerably in numbers by this time and manifest their consistent association with mitochondria, a feature which has been observed repeatedly in the higher taxonomic forms of red algae (Scott, 1972). Persistent associations of mitochondria with dictyosomes are also found in Vaucheria and Saprolegnia (Ott and Brown, 1974). Each dictyosome is approximately 0.5 t~m in width and usually consists of five to seven cisternae. The morphology, size, and manner of secretory vesicle formation of dictyosomes at this stage are quite comparable to that found in actively growing vegatative cells of P. denudata and in most other algae, fungi, and higher plants. Figure 2 shows a single dictyosome and its juxtaposed mitochondrion at higher mag-

Fia. 1. Early developmental stage of a tetrasporangium showing single nucleus and mitochondriadictyosome association. × 18 000. FIG. 2. Typical form of dictyosome and its associated mitochondrion during early sporangial development. x 46 000.

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nification. Note both the degree to which the cisternae are separated by a consistent intercisternal space and the minimal peripheral cisternal dilation.

Dictyosornes of Older Sporangia As time proceeds, the tetrasporangia increase greatly in size and the nucleus eventually divides meiotically. Concomitant with nuclear division, cytokinesis is initiated by annular ingrowing cleavage furrows (Fig. 3). The cytoplasm now contains large quantities of starch, ER, and large fibrillar vacuoles (FV) which are derived from dictyosome activity and are reported by most workers to contain complex polysaccharides. The chloroplasts are abundant and are located at the sporangium's periphery. Three of the four haploid nuclei are shown still located in the central region of the cytoplasm and are surrounded by numerous mitochondria. Changes in dictyosome morphology are first noticed just prior to and concurrent with sporangial cell division. The dictyosomes at this time exhibit extreme hypertrophy. However, this is not limited only to modifications in the size and number of cisternae. Figure 4 shows several dictyosomes (arrows) observed in a section cut slightly tangential to two of the four haploid nuclei in a sporangium. The number of cisternae constituting the dictyosomes, which are 2-3 t~m in size, is quite variable. Usually 6 to 15 cisternae are observed, but occasionally 20 or more can be present. The most striking features seen at this time, however, are the changes in appearance of the middle and peripheral regions of each dictyosome. The peripheral portions are extremely dilated or inflated while the midregions are compressed and densely laminated. This last-mentioned

characteristic is clearly demonstrated in Fig. 5. Elements of the ER can be seen along the dictyosome's forming face, except immediately between the mitochondrion and the first-formed cisterna. Darkly staining primary or transition vesicles are found here also and appear to be derived from the ER (Figs. 5, 7, and 8). With the exception of the invariable presence of the mitochondrion, this aspect of endomembrane association in P. denudata is compatible with that observed commonly in other cells (Morr~ and Mollenhauer, 1971; Morr~ et al., 1971). However, the dense lamellae of the dictyosome's midregions are phenomena which are unique among all observations on plant and animal Golgi apparatus. Excluding the first-formed cisterna, all adjacent cisternae are closely appressed, thereby obliterating most of the intercisternal space. This myelin-like appearance does not repr e s e n t an especially tight fusion of membranes, but is apparently due to loose binding or fusion of the outer leaflets of the adjacent cisternal membranes (a single cisternal membrane is 5.5-6.5 nm thick while the dimensions of the densely staining lamellae are 15-17 nm). Our first thoughts, especially when observing low magnification micrographs, were t h a t the lamellae represented membranes tightly bound by a visible intercisternal cementing substance, somewhat comparable to the material recently demonstrated in higher plants (Mollenhauer et al., 1973). However, observations at higher magnification of several transversely sectioned areas of the lamellae reveal occasional, thin, less dense regions of intercisternal separation (arrows, Fig. 5). This unusual modification of the dictyosomes in P. denudata has been noticed in

FIG. 3. Partially cleaved, postmeiotic tetrasporangium. Note the large, dictyosome-derivedfibrous vacuoles (FV), the peripheral chloroplasts (C), and three of the four nuclei. × 2300. FIG. 4. Dictyosomes (arrows) of postmeiotic sporangia demonstrating dilated peripheral and densely staining, laminated, midregions. Two nuclei (N) are tangentially sectioned. × 7400.

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FIG. 7. This dictyosome is in a cell at a later developmental stage than the one in Fig. 6, but at an earlier stage than the one in Fig. 8. Apparently only a few vesicles are formed from each cisterna after it detaches from the dictyosome. Arrow, potential remnant of cisternal midregion. × 72 000. s p o r a n g i a of o t h e r species of r e d a l g a e we h a v e e x a m i n e d as well as b y o t h e r a u t h o r s ( P e y r i e r e , 1970; Tripodi, 1971, 1974; K u g r e n s a n d West, 1972, 1973, 1974; C h a m b e r l a i n a n d E v a n s , 1973; Scott a n d Dixon, 1973; K o n r a d H a w k i n s , 1974a,b; W e t h e r bee a n d West, 1976). We c a n c u r r e n t l y offer no e x p l a n a t i o n to a c c o u n t for t h e close cisternal apposition.

F i g u r e s 6-8 a r e a s e r i e s of m i c r o g r a p h s r e p r e s e n t i n g g r a d u a l m o r p h o l o g i c a l modif i c a t i o n s of h y p e r t r o p h i e d d i c t y o s o m e s in tetrasporangia at progressively later s t a g e s of d e v e l o p m e n t . E v e n t h o u g h all three dictyosomes exhibit densely staining m i d r e g i o n s , t h e size a n d n u m b e r s of vesicles p r o d u c e d by e a c h a r e n o t i c e a b l y different. T h e d i c t y o s o m e s in Figs. 6 a n d 7 a r e

FIG. 5. The densely staining, laminated, midregions of the dictyosomes are formed by the close apposition of adjacent cisternae. The intercisternal space is thereby obliterated except between the number 1 and number 2 cisternae. The distalmost cisterna is almost completely released from the cisternal stack, being attached only at one small area (arrows). x 160 000. FIG. 6. The dictyosomes in the next three figures are found in cells at progressively later stages of development. In this figure, numerous vesicles can be seen among several remnants (arrows) of the dictyosome's midregion. × 29 000.

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DICTYOSOMES IN TETRASPORANGIA OF POLYSIPHONIA i n t e r p r e t e d as being involved in the elaboration of either n u m e r o u s (Fig. 6) or several (Fig. 7) secretory vesicles which coalesce to form the large fibrous vacuoles shown in Fig. 3. The secretory vesicles a p p e a r to be formed after the d e t a c h m e n t of an entire cisterna from the m a t u r a t i o n or distal face of the dictyosome. Following the removal of a cisterna, e i t h e r n u m e r o u s or a few vesicles are released but r e m a i n associated with r e m n a n t s of the midregions of the m a t u r e cisternae (arrows, Figs. 6 and 7). The r e m n a n t s closely correspond to the nonsecretory portions of m a t u r e cisternae which M o l l e n h a u e r reports as being sloughed from dicytosomes in maize cells at the same time t h a t secretory vesicles are formed (Mollenhauer, 1971). One other n o t e w o r t h y morphological feature is depicted in Fig. 8, which we believe represents the culmination of secretory activity in P. d e n u d a t a tetrasporagnia. At this time, cisternae at the maturation face of the dictyosome dissociate from the compact midregion and assume a spherical configuration. As a result, only a single, large secretory vesicle is formed from the release and direct t r a n s f o r m a t i o n of a single cisterna. M e m b r a n e in excess of w h a t is necessary to enclose a sphere appears to be pinched off at the m a r g i n of the nascent secretory vesicle (Fig. 8, arrows). However, we cannot disallow the possibility t h a t such observations instead represent the continued addition of cisternal m e m b r a n e and/or secretory m a t e r i a l to be utilized in vesicle production. The large secretory vesicles are approxim a t e l y 1 tLm in d i a m e t e r and contain a m o d e r a t e l y to densely staining central core of m a t e r i a l (Figs. 8 and 9). The cen-

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tral core appears to r e s u l t from a progressive centripetal a c c u m u l a t i o n of fibrous or g r a n u l a r intracisternal substance as the cisternae advance from the forming to the m a t u r a t i o n face of the dictyosome (Fig. 8). Electron microscopic c y t o c h e m i s t r y has shown t h a t the dark-cored vesicles are mucopolysaccharides (Peyriere, 1970) or glycoproteins ( C h a m b e r l a i n and Evans, 1973) and, following exocytosis, function in spore protection or adhesion subsequent to spore liberation. One a u t h o r (Konrad Hawkins, 1974b), however, believes t h a t the vesicles can function similar to lysosomes. After dark-cored vesicle production has ceased, the dictyosomes r e v e r t to the vegetative or premeiotic size and morphology and a p p a r e n t l y are only m i n i m a l l y involved in secretory activity. F i g u r e 9 also shows several dark-cored vesicles and a portion of one of the m a n y large fibrous vacuoles which are present in fully cleaved t e t r a s p o r a n g i a prior to spore liberation. The vesicles can e i t h e r r e m a i n intact or fuse with each other or with the large fibrous vacuoles. L i b e r a t e d or newly settled spores are n e a r l y identical to m a t u r e spores still r e t a i n e d within the old sporangial walls (unpublished micrographs) both in r e g a r d to the conditions of the darkcored vesicles and to dictyosome size and morphology. This u n u s u a l sequence of vesicle formation, coupled with the a p p e a r a n c e of densely-staining, appressed midregions of the dictyosomes, has been observed in carposporangia and t e t r a s p o r a n g i a of n u m e r ous red algae we have e x a m i n e d and is either evident to some e x t e n t or briefly alluded to in the published micrographs Of several different authors. The conversion

FIG. 8. Culmination of dictyosome activity in cell at late stage of sporogenesis. A single vesicle is formed by the transformation of a single cisterna after the release of the cisterna from the dictyosome. Two of the many small, peripheral regions of the cisternae which appear to be detaching are indicated by the arrows, x 60 000. FIa. 9. Dictyosome (arrow) in a mature tetrasporangium prior to spore liberation. It has reverted to the premeiotic size and morphology now that secretory activity is greatly diminished. Several dark-colored vesicles and a portion of a large fibrous vacuole are visible. × 30 000.

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of a s i n g l e c i s t e r n a i n t o a s i n g l e s e c r e t o r y vesicle deviates from the usual method of Golgi-mediated secretory vesicle product i o n ( M o l l e n h a u e r a n d MorrO, 1966; M o r r ~ a n d M o l l e n h a u e r , 1971; M o r r ~ et al., 1971; N o r t h c o t e , 1971). T h e o n l y o b s e r v a t i o n s which appear fairly similar would be those on t h e G o l g i a p p a r a t u s b o t h i n s c a l e p r o duction in some unicellular algae (Mant o n , 1966, 1967; B r o w n , 1969) a n d m u c o complex secretion in Malpighian tubules of c e r t a i n h o m o p t e r a n i n s e c t s ( M a r s h a l l , 1974). W e t h e r b e e a n d W e s t (1976) h a v e rep o r t e d on a u n i q u e t y p e o f s t r i a t e d v e s i c l e produced by the Golgi apparatus in carpos p o r a n g i a o f a d i f f e r e n t s p e c i e s of P o l y s i phonia. Their report, our present work, a n d e a r l i e r s t u d i e s ( D o d g e , 1973) c o n t r i b u t e f u r t h e r to t h e e v i d e n c e t h a t r e d a l g a e are not only an unusual group of plants at the organismal level, but are also unique w i t h r e g a r d to v a r i o u s a s p e c t s o f t h e i r ultastructure. The authors thank Jewel Thomas for her excellent technical assistance during the study and Sandra Deaton for contributing Fig. 5. REFERENCES ALLEY, C. D., AND SCOTT, J. L. (1972) J. Phycol. 8, 7. BROWN, R. M., JR. (1969) J. Cell Biol. 41, 109. CHAMBERLAIN, A. H. L., AND Evans, L. V. (1973) Protoplasma 76, 139. DODGE, J. D. (1973) The Fine Structure of Algal

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