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The development of adult cell form in Pediastrum biradiatum Meyen as revealed by the electron microscope
26
J. ULTRASTRUCTURERESEARCH4, 26-42 (!960)
The Development of Adult Cell Form in Pediastrum biradiatum Meyen as Revealed by the Electron Microscope 1 J. G. MONER and G. B. CHAPMAN2
Department of Zoology, University of Massachusetts, Amherst, Massachusetts, and the Biological Laboratories, Harvard University, Cambridge, Massachusetts Received October 21, 1959
An electron-microscope study was made of thin sections of cells from several developmental stages involved in the transformation of the motile zoospore into the four-pronged adult cell type during asexual reproduction in Pediastrum biradiatum Meyen. The various stages were obtained by separating swarmers from adult colonies using fine nickel screening and allowing progressively longer periods of zoospore change before fixing in OsOa. Early adult stages were obtained by fixing cultures after approximately 6 hours of swarmer production. Chloroplasts are observed in cells from all developmental stages. No grana, typical of the chloroplasts of higher plants, are discernible, but a lamellar structure is present and the chloroplast is limited by a double membrane. The chloroplasts contain low density starch inclusions and small, dense inclusions, similar in texture and density to large, dense, spherical bodies which occupy a considerable proportion of the cell volume. The significance of the latter bodies is unknown, but reference is made to their possible role in cell-wall formation. Pyrenoid bodies are not present in any recognizable form until the adult cell type is achieved and appear to develop from the chloroplast. The pyrenoid appears homogeneous and is nearly surrounded by starch plates in the mature forms. The cell wall develops from a thin, membranous covering into a thickened structure containing a globular network. The latter is found in the early adult stages. A small amount of endoplasmic reticulum is present and a Golgi apparatus which in some instances resembles the Golgi forms of higher animals and in other cases is similar to the dictyosomes of protozoa and invertebrates. The nucleus, which is often nearly rectangular in profile, is enclosed by a double membrane which in some cases appears to have an intimate association with the Golgi apparatus. z Supported in part by funds from NSF Grant 4021. 2 Present address: Department of Anatomy, Comell University Medical College, New York 21, New York.
ADULT CELL FORM IN PEDIASTRUM BIRADIATUM MEYEN
27
The mitochondria are very few in number and possess only a few randomly oriented cristae. Large vacuoles are described which contain secondary double-membrane limited vacuoles in addition to inclusions similar to those found in the cytoplasm. The green alga, Pediastrum, is a colonial organism the coenobia of which commonly consist of 8-16 cells. During asexual reproduction each of the cells in an adult colony undergoes progressive cleavage producing motile zoospores which are released from the mother cell confined within a transparent sac. After a swarming period of approximately 4 minutes, the zoospores cease movement, having formed a flat, round, plate typical of the adult colonies, and then undergo, during the ensuing 8-10 minutes, a series of developmental changes leading to the adult cell type. In P. biradiatum Meyen, this involves the transformation of a biflagellated, oval-shaped zoospore into a flattened, four-pronged, adult cell. The present work was undertaken to study the cytology of different stages in the course of this developmental process. MATERIAL AND METHODS Pure cultures of P. biradiatum Meyen were grown in Erlenmeyer flasks on mineral medium No. 8 according to Chu (3) with 10% soil extract added. The flasks were agitated on a Brunswick rotary shaker under an illumination of 150 foot-candles delivered by four GE cool white fluorescent lamps at a temperature of 22°C. During the swarming or lag phase of growth, cultures were passed, several times, through a Seitz filter containing a fitted disk of nickel lektromesh (200) screen. This procedure removed the newly formed colonies and any of the small adult colonies present. The larger adult colonies left on the screen were resuspended in 30 cc of growth medium, placed in a Seitz filter fitted with a disk of nickel lektromesh (400) screen and constantly agitated by bubbling with air. In a series of four runs, each of which lasted approxinlately 2 hours, the colonies were washed and resuspended three times successively at an interval of 3 minutes. In the first run (stage I) the colonies of each 3-minute aliquot were quickly cooled to 4°C by immersion in a dry ice-cellosolve mixture, centrifuged and fixed in ice-cold 2% OsO~ in growth medium. In the remaining runs the aliquots were allowed to stand, at room temperature, an additional 3 (stage II), 6 (stage III) and 12 (stage IV) minutes respectively after filtering before being fixed as described. This procedure allowed increasingly longer periods of development to take place. Young adult colonies (stage V) were obtained by fixing cultures after approximately 6 hours of swarming had taken place. This gave a mixture of large adult colonies and smaller, newly formed adult colonies. The fixed specimens were washed with two changes of growth medium, dehydrated in a series of ascending concentrations of ethanol and infiltrated with two changes of methacrylate (3 parts N-butyl methacrylate:2 parts ethyl methacrylate, containing 1.5% luperco CDB as catalyst). The colonies were then dispensed from the last change of methacrylate into gelatin capsules and polymerization was effected at 70°C for 16 houCs.
28
J. G. MONER AND G . B . CHAPMAN
The sections were cut on a Porter-Blum microtome and mounted on copper grids coated with collodion. All the micrographs were taken with an RCA EMU-2D electron microscope at initial magnifications of 3960-6800 diameters and were enlarged photographically as necessary. RESULTS
1. Chloroplast It can be seen (Fig. 4) that the chloroplast (C) consists essentially of a double limiting m e m b r a n e (CHM) and lamellae which are generally arranged in parallel arrays. There is no differentiation of the chloroplast internum into definite grana and stroma. The lamellae represent stacked flattened vesicles or disks, the thickened rims of which are evident in Figs. 3 and 6. The chloroplast structure of Pediastrum thus resembles closely that described for the green alga Chlamydomonas by Sager and Palade (17) and differs f r o m that described for the green alga Vaucheria by Greenw o o d (7) in lacking the fenestrations and cross-linking of the latter. Starch grains (MS) can be seen in various stages of maturity in the chloroplasts (Fig. 10). It is interesting to note that as the starch increases in a m o u n t it m a y occupy a large part of the space b o u n d e d by the chloroplast membrane. The starch grains appear limited by a thin, dense m e m b r a n e (SM). This m e m b r a n e is clearly visible in the rather rectangular " m a t u r e " starch grains of the central cell in Fig. 10. It should be noted that, even in chloroplasts wherein the starch has displaced the lamellae to one end of the plastid, the double chloroplast m e m b r a n e continues to enclose the starch grain. In all of the developmental stages, extensive starch synthesis is taking place with starch grains ranging in size f r o m 0.6 x 0.3 # to 1.4 × 0.7 #. In addition to the starch grains, one frequently finds small (50-160 m#), dense, spherical bodies (SB) within the chloroplast (Figs. 4 and 5). These inclusions appear to be of the same texture and density as large, dense, spherical bodies (Figs. 4 and 5, LB) found in the cytoplasm. It is therefore suggested that the large bodies m a y arise within the chloroplast and be released to the cytoplasm. Similar large, dense bodies are
FIG. 1. A section through a colony of zoospores (stage I) showing the lamellar structure of the chloroplast (C), a thin, limiting cell membrane (CM) and the presence of a Golgi body (G). In addition, there are large, dense, spherical bodies present (LB) and a nucleus (N). × 19,000. FIG. 2. A section through a colony of early differentiating zoospores (stage II) showing the presence of two nuclei (N1, N~) and both primary (PV) and secondary (SV) vacuoles. The double nature of the secondary vacuole membrane (SVM) can be seen as well as the apparent single nature of the primary vacuole membrane (PVM). One of the large, dense, spherical body inclusions (LB, arrow) appears to be entering the primary vacuole via a tear in the primary vacuole membrane. In addition, there can also be seen a thin, limiting cell membrane (CM), portions of an endoplasmic reticulum (ER) and other large, dense spherical bodies. The sac (S) enclosing the zoospores is faintly visible in this section, x 19,000.
30
J. G. MONER AND G. B. CHAPMAN
occasionally found within vacuoles. The function of these inclusions is at present unknown.
2. Pyrenoid During the early stages of development, we were unable to find pyrenoids typical of those studied in other algae or those present in the adult stages of Pediastrum. In the stage IV preparation (Fig. 10) there are two structures (PY?) resembling in density the mature pyrenoids of later stages. In Fig. 8, a section of a young adult colony (stage V) shows what appears to be a more advanced stage in the formation of a pyrenoid (PY) (0.6 # in diameter). There are no starch plates present, but a limiting membrane, continuous with that of the adjacent chloroplast, surrounds the structure. In Fig. 9, another pyrenoid is shown with incomplete starch plates (SP) and distinct connections with an adjacent chloroplast. A mature pyrenoid (0.65 # in diameter) is shown in Fig. 11 and once again extensions of an adjacent chloroplast membrane are seen to surround the pyrenoid and the associated starch plates.
3. Nucleus The nuclei (N) frequently appear rectangular in profile (Figs. 1, 2, 6, and 10) measuring 1.5-2.5/z in length and 0.5-1.3 # in width. All the nuclei seen are limited by a double membrane (15-30 m/~ in thickness), the inner component of which is bordered by a fairly thick region of dense, granular material, presumably chromatin (Figs. 2, 4, 6, and 10). Although some of this granular material is found in the center of the nucleus, most of it seems to be located at the periphery. No recognizable nucleolus was present in any of the sections studied. In some cases (Figs. 3 and 4) the outer layer of the nuclear envelope becomes so involved with the Golgi apparatus (G) that it is nearly obscured. In Fig. 2 it can be seen that one of the developing zoospores is unusually large and contains two nuclei. During the process of progressive cleavage involved in the production of zoospores, one of the protoplasts occasionally fails to go through its final cleavage despite the presence of the two nuclei. This protoplast may then develop into an oversized zoospore.
FIG. 3. A section through a colony of early differentiating zoospores (stage II) showing three elements of the cellular membrane system, the Golgi complex (G), the endoplasmic reticulum (ER) and the nuclear membrane (NM). The Golgi complex consists of closely packed cisternae (CI), large vacuoles (V) and smaller vesicles (VS). The nuclear membrane is double in nature and appears to be in very intimate association with the Golgi complex. The endoplasmic reticulum is diffuse and the canaliculi are of an order of size comparable to the cisternae of the Golgi complex. In addition to the membranous components mentioned, the chloroplast (C) is seen to consist of stacked disks, the rims of which show thickenings, x 34,000.
32
J. G. M O N E R A N D G . B .
CHAPMAN
4. Mitochondria The number of mitochondria (M) seen throughout the various developmental stages is small (Figs. 4, 5, and 11). The profiles range in diameter from 0.15 to 0.29 # with elongate, triangular, and branched forms being found. There appear to be two membranes surrounding each mitochondrion. The cristae are, in general, randomly oriented but sometimes lie parallel to the mitochondrial membranes. It should be noted that Pediastrum, in possessing cristae, resembles Chlamydomonas (17) and differs from Vaucheria (7), which appears to have a tubular internum. In Fig. 7, the mitochondrion appears to contain four concentric membranes. This configuration probably represents another instance of the occurrence of a cup-shaped mitochondrion - - a configuration discussed at length by Christensen and Chapman (2).
5. Cytoplasmic membranes Present in all of the stages studied is an endoplasmic reticulum (ER) interspersed throughout the cells as canaliculi and small vesicles. The canaliculi measure approximately 15-30 m# and the vesicles 15-65 m# in diameter. These structures are particularly evident in Figs. 3 and 4. Also found consistently throughout the developmental stages in Pediastrum is the Golgi apparatus (G), which is invariably located near the nucleus. Although the cisternae (15 m# in diameter) are closely packed (Figs. 3, 6, and 10), there are frequently large vacuoles (40 m# in diameter) associated with the apparatus (Figs. 3, 4, and 10). In Fig. 6 the Golgi material is disposed as two distinct dictyosomes with smaller and fewer vacuoles.
6. Other cytoplasmic inclusions In addition to the more commonly occurring inclusions already mentioned, several of the cells contain large vacuoles which appear to be bordered by either a single membrane (Fig. 5) or a double membrane (Figs. 2 and 4). In these vacuoles there are found inclusions ranging from fine vesicles (VS) to large dense bodies (LB). The functional significance of the intravacuolar constituents is unknown. It may be
FIG. 4. A section through a colony of moderately advanced differentiating zoospores (stage III) showing a large primary vacuole (PV) with smaller secondary vacuoles (SV1, SV2). There are small vesicles (VS) present within the primary vacuole which is limited by a single membrane (PVM). One of the secondary vacuoles appears to be limited by a single membrane (SVM) and contains a large, dense spherical body (LB) as an inclusion. The other secondary vacuole is limited by a double membrane (SVM2) and contains a lipid-like inclusion (L). Two cross-sections of mitochondria are shown (M1, M2) with differing shapes and containing randomly oriented cristae. The chloroplast (C) is seen to be limited by a double membrane (CHM) and contains small, dense spherical bodies (SB). The shape of the central cell is elongated indicating the beginnings of prong formation, x 32,000.
I
34
J.G.
M O N E R A N D G. B. C H A P M A N
that some of them are involved (and partially or completely expended) in digestive activities or that they represent material permanently or temporarily stored within the vacuoles. In Fig. 2 (arrow) one can observe what is possibly a stage in the movement of a large, dense body from the cytoplasm into the vacuole. In some of the cells (Figs. 2 and 4) other membrane-limited structures are present within the vacuole. In Fig. 2 a double-membraned secondary vacuole (SV) is found within the primary vacuole, and in Fig. 4 there are two secondary vacuoles present, one single and the other double-membrane limited. The inclusion found within the latter appears to have undergone considerable change, possibly from electron bombardment. The most obvious of the remaining inclusions are the large, dense, spherical bodies (LB) (0.2-0.8 ~ in diameter) already mentioned in connection with the chloroplasts, found at all developmental stages in different parts of the cytoplasm, and in the vacuoles. These bodies seem not to be associated with any particular structures and constitute a considerable proportion of the cytoplasmic bulk.
7. Cell wall During the early stages of development (Figs. 1-5) a thin membrane (CM) is seen to limit the cytoplasm of the zoospores. At a somewhat later stage (Fig. 6) a thicker covering or wall (CW) arises which then develops into a loose, coarser structure (Figs. 8, 9, and 11). Globular bodies (GB) can be seen incorporated within the coarse wall (Figs. 8 and 11). DISCUSSION The developmental stages of Pediastrum represent to a considerable degree the beginning of differentiation as recognized in higher forms. The aggregation of zoospores into an orderly plate-like arrangement, the loss of flagella, and the ensuing development of a four-pronged shape, all point to the more organized forms of higher organisms, despite the apparent lack of any division of labor among the cells. The factors influencing the initiation of the differentiation process are little understood. However, it seems likely that the confining sac (Figs. 2 and 10) may
Fro. 5. A section through a cell of advanced differentiation (stage IV) showing a thin cell membrane (CM) and a large primary vacuole (PV).The primary vacuole contains vesicles (ITS)and large, dense spherical bodies (LB). In addition, a section through a mitochondrion (M) is also shown. An early stage in the formation of prongs (P) can be seen in this section. × 25,000. Fro. 6. A section through an early adult cell (stage V) showing a cell wall (CW), two Golgi bodies (G) resembling dictyosomes and a highly elongated nucleus (N). x 25,000. Fro. 7. A section through an early adult cell (stage V) showing what appears to be a cup-shaped mitochondrion (M). × 25,000.
....... ~ ~i~ ......
~
%
~
~. . . . . . . . . . . .
~ ~
36
J. G. MONER A N D G. B. C H A P M A N
play an important role in preserving the environmental factors necessary for the process to take place. The actual transformation was described earlier by Harper (8, 9, 10, 11) who mentions the fact that the cytoplasm almost divides into two pearshaped halves, the narrowed ends of which give rise to the pronged portion of the cell. What produces this profound change in the cytoplasm or indeed the ensuing events is unknown. F r o m a cytological viewpoint the present study has provided some insight into the more obvious changes taking place during the developmental process. The pyrenoid has long been a structure whose exact origin and function has not been completely understood. Certainly the behavior of this body in different green algae reveals no consistency in function, although it has usually been associated with starch synthesis (6, 18). Among the possible means by which the pyrenoid is believed to be passed on to subsequent cell generations are (1) actual division, (2) the incorporation into one unit of many forming zoospores, and (3) dissolution before division with eventual resynthesis by each of the newly formed zoospores (18). In Pediastrum, the last alternative appears to hold true. Before the formation of zoospores takes place, the pyrenoid breaks down and it is not until the latter part of the developmental process (Stage V) that recognizable pyrenoids reappear. In addition, since starch formation is evidently taking place during the early developmental stages, there can be no relationship between the pyrenoid and chloroplast such as exists in Chlamydomonas (I8). In this organism the pyrenoid is apparently the first organ to form starch, with starch formation taking place secondarily in the chloroplast. Since some algae possess pyrenoids of lamellar structure (12) and are usually closely associated with the chloroplast, it is not unreasonable to suspect that the pyrenoid, in some cases, arises from the chloroplast. If this is correct, it is conceivable, at least in Pediastrum, that the pyrenoid serves a dual function, one of starch synthesis and the other as a reserve of chloroplast material. This idea gains some support from the
FIG. 8. A section through a moderately advanced adult cell (stage V) showing what appears to be an immature pyrenoid (PY) in close association with chloroplast (C) and the accompanying starch (S). The pyrenoid is surrounded by the chloroplast membrane (CHM). In addition, the prongs of the cell are fully developed as is the cell wall (CW). Note the globular bodies (GB) associated with the cell wall. × 25,000. FIG. 9. A section through a moderately advanced adult cell (stage V) showing a pyrenoid (PY) in close association with adjacent chloroplast (C). Note the starch plates (SP) in association with the pyrenoid, x 25,000. Fxo. 10. A section through a colony of advanced differentiation (stage IV) showing two possible pyrenoid formations (PY?) and rectangular granules of mature starch (MS). In addition, there can be seen a Golgi body (G) and elements of the endoplasmic reticulum (ER). The sac enclosing the cells is just visible in this section. × 18,500.
ii ~ii~!~iii~!~!iii~ii~iii!ii~%~i~i~iii~
!~ii~!~ ~ ~ ~?~ ~ ~!~i~iiiiii~i~
38
J.G.
M O N E R A N D G. B. C H A P M A N
fact that certain algae (18) possess pyrenoids which do not form starch plates. In addition, it is known that in Pediastrum, as the cells grow older and larger, the amount of starch increases enormously. Coincident with an increase in cell size is a proportional increase in pyrenoid size and when the starch content is unusually high, there is a decrease in chloroplast material with a resultant yellowing of the cytoplasm (19). Perhaps some of the chloroplast material is incorporated into the pyrenoid body to be utilized in the synthesis of new chloroplast when zoospore formation takes place later on. In addition to the changes which take place with pyrenoid and starch formation, the cell wall undergoes dramatic development during the differentiation period and afterward. It is known from previous work (14) that empty mother cells show a globular network intimately associated with the wall proper. The origin of this wall pattern during the developmental stages is the subject of another paper (15). It should be mentioned, however, that the large, dense, spherical bodies (LB) noted in all the developmental stages may play an important role in the emergence of this cell-wall pattern. In regard to other cytoplasmic components seen in P. biradiatum Meyen, some comments are in order. The chloroplast of this organism is similar in most respects to that of other algal forms (1, 13, 20, 22). There are, of course, no distinct grana present, but the lamellae do seem to exhibit a slight preferential orientation. It is worthy of note that Sager and Palade (17), working with Chlarnydornonas, have found what they consider to be imperfect segmentation into grana. The Golgi apparatus assumes different forms in different Pediastrum cells. The Golgi structures seen in Figs. 3 and 4 show a number of large vacuoles more typical of the Golgi apparatus of higher animal cells, whereas the twin structures of Fig. 6 are more suggestive of the dictyosomes of protozoa and invertebrates (4, 5) with the more compact cisternae and somewhat smaller vacuoles. The fact that the dictyosome-like structures are found in a later stage of development may reflect a difference in metabolic activity between the stages. This would imply that the alternation in the disposition of the Golgi material from a single, rather large Golgi apparatus to several discrete dictyosomes is related to the metabolic state of the cell. Our investigations have not yet considered the possible changes in the metabolic state. The endoplasmic reticulum is quite distinct from the Golgi apparatus, being spread rather diffusely throughout the cell compared to the Golgi body which is always
FIG. 11. A section through a mature adult cell (stage V) showing a well-developedpyrenoid (PY) surrounded by starch plates (SP).In addition, there can be seen a typical adult cell wall (CW) with associated globular bodies (GB), a mitochondrion (M), and Golgi body (G). × 33,000.
i~i ~ i l i ¸¸¸~¸¸¸ ~=ii~ii
Fro. 12. An enlargement of a p~rticn of Fig. 4. The double limiting membrane of the mitochondrion (M) may be seen, as well as sparse cristae. ER designates endoplasmic reticulum; C, chloroplast, with paired membranes; G, Golgi zone; N, nucleus, x 52,000. FIG. 13. An enlargement of a portion of Fig. 11. The pyrenoid (PY) may be seen, as well as its adjacent, and nearly engulfing, starch plates (SP). S denotes a large starch deposit included within the same chloroplast membrane. The double nature of that membrane may l:e seen where it is normally sectioned (arrow). × 59,000.
A D U L T C E L L F O R M I N P E D I A S T R U M B I R A D I A T U M MEYEN
41
found close to the nucleus. Although profiles indicate that canaliculi and vesicles are present and probably interconnected, many of the vesicles are very likely independent entities. In the large vacuoles (Figs. 4 and 5) tiny round inclusions, similar to the vesicles of the cytoplasm, are present. There are no canaliculi visible in these large vacuoles indicating that the vesicles are closed units. This is probably true of many similar vesicles found in the cytoplasm. The outer membrane of the nucleus (Figs. 3 and 4) is seen to be in close relationship with the Golgi apparatus. In the light of Watson's demonstration (21) of the continuity between the outer element of the nuclear envelope and the endoplasmic reticulum, and Palade's hypothesis (16) that the endoplasmic reticulum and Golgi apparatus are simply regional differentiations of the same membranous network, it is not at all surprising to find a close relationship between the nuclear envelope and the Golgi apparatus. This relationship does, however, represent a new aspect of this concept. The large vacuoles found in the cytoplasm are especially interesting due to the varied contents found within them. The small vesicles (VS) seem identical to elements of the endoplasmic reticulum; the large, dense, spherical bodies (LB) are identical to those found in the cytoplasm; the single and double-membrane limited secondary vacuoles (SV) have no cytoplasmic counterparts; nor do the very dense inclusions (L). The significance of the double-membraned secondary vacuoles (Figs. 2 and 4) is not known, nor is that of the dense inclusions in one of these structures (Fig. 4). Its density, however, suggests a lipid nature. We may speculate that the large vacuoles are sites of storage or perhaps storage and modification of some of the cytoplasmic inclusions. This would account for the presence of the vesicular component and perhaps for the confusing configuration indicated by the arrow in Fig. 5. Such a form might represent a partially altered, large, dense, spherical body. In Chlamydornonas (17), the contents of the vacuoles were found to be dense, possibly crystalline, masses and were shown to be metachromatic from staining with azure B. There were, apparently, no inclusions found in the vacuoles of Chlarnydomonas similar to those of the cytoplasm and found in Pediastrum.
REFERENCES 1. 2. 3. 4. 5.
ALBERTSON,P. A. and LEYON, H., Exptl. Cell Research 7, 288 (1954). CHR~STENSEN,A. K. and CHAPMAN, G. B., Exptl. Cell Research 18, 576 (1959). CHU, S. P., J. Ecol. 30, 284 (1942). DALTON,A. J. and FELIX, M. D., J. Biophys. Biochem. Cytol. 2, No. 4, Suppl., 79 (1956). GATENBY,J. B., DALTON,A. J. and FEHx, M. D., Nature 176, 301 (1955).
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J. G. MONER AND G. B. CHAPMAN
6. GRANICK, S., in RUHLAND, W. (Ed.), Encyclopedia of Plant Physiology, Vol. I, p. 535. Julius Springer, Berlin, 1955. 7. GREENWOOD,A. D., J. Exptl. Botan. 10, 55 (1959). 8. HARPER, R. A,, Science 37, 385 (1913). 9. - Mem. N. Y. Botan. Garden 6, 91 (1916). 10. - Proc. Am. Phil. Soc. 57, 375 (1918). 11. - - - - Mem. Torrey Botan. Club 17, 210 (1918). 12. LEVON, H., Exptl. Cell Research 6, 497 (1954). 13. MERCER, F. V., HOD~E, A. J., HOPE, A. B. and McLEAN, J. D., Australian J. Biol. Sci. 8, 1 (1955). 14. MONER, J. G., Am. J. Botany 42, 802 (1955). 15. MONER, J. G. and CHAPMAN, G. B., in preparation. 16. PALADE,G. E., J. Biophys. Biochem. Cytol. 2, No. 4, Suppl., 85 (1956). 17. SAGAR,R. and PALADE, G. E., J. Biophys. Biochem. Cytol. 3, 463 (1956). 18. SMITH, G. M., Fresh-Water Algae of the United States, 2nd ed. McGraw-Hill Book Co., New York, 1950. 19. - Ann. Botany 30, 467 (1916). 20. STEtNMANN,E., Exptl. Cell Research 3, 367 (1952). 21. WATSON, M. L., J. Biophys. Biochem. Cytol. 1, 257 (1955). 22. WOLKEN, J. J. and PALADE, G. E., Ann. N. Y. Acad. Sci. 56, 873 (1953).
J. ULTRASTRUCTURERESEARCH4, 26-42 (!960)
The Development of Adult Cell Form in Pediastrum biradiatum Meyen as Revealed by the Electron Microscope 1 J. G. MONER and G. B. CHAPMAN2
Department of Zoology, University of Massachusetts, Amherst, Massachusetts, and the Biological Laboratories, Harvard University, Cambridge, Massachusetts Received October 21, 1959
An electron-microscope study was made of thin sections of cells from several developmental stages involved in the transformation of the motile zoospore into the four-pronged adult cell type during asexual reproduction in Pediastrum biradiatum Meyen. The various stages were obtained by separating swarmers from adult colonies using fine nickel screening and allowing progressively longer periods of zoospore change before fixing in OsOa. Early adult stages were obtained by fixing cultures after approximately 6 hours of swarmer production. Chloroplasts are observed in cells from all developmental stages. No grana, typical of the chloroplasts of higher plants, are discernible, but a lamellar structure is present and the chloroplast is limited by a double membrane. The chloroplasts contain low density starch inclusions and small, dense inclusions, similar in texture and density to large, dense, spherical bodies which occupy a considerable proportion of the cell volume. The significance of the latter bodies is unknown, but reference is made to their possible role in cell-wall formation. Pyrenoid bodies are not present in any recognizable form until the adult cell type is achieved and appear to develop from the chloroplast. The pyrenoid appears homogeneous and is nearly surrounded by starch plates in the mature forms. The cell wall develops from a thin, membranous covering into a thickened structure containing a globular network. The latter is found in the early adult stages. A small amount of endoplasmic reticulum is present and a Golgi apparatus which in some instances resembles the Golgi forms of higher animals and in other cases is similar to the dictyosomes of protozoa and invertebrates. The nucleus, which is often nearly rectangular in profile, is enclosed by a double membrane which in some cases appears to have an intimate association with the Golgi apparatus. z Supported in part by funds from NSF Grant 4021. 2 Present address: Department of Anatomy, Comell University Medical College, New York 21, New York.
ADULT CELL FORM IN PEDIASTRUM BIRADIATUM MEYEN
27
The mitochondria are very few in number and possess only a few randomly oriented cristae. Large vacuoles are described which contain secondary double-membrane limited vacuoles in addition to inclusions similar to those found in the cytoplasm. The green alga, Pediastrum, is a colonial organism the coenobia of which commonly consist of 8-16 cells. During asexual reproduction each of the cells in an adult colony undergoes progressive cleavage producing motile zoospores which are released from the mother cell confined within a transparent sac. After a swarming period of approximately 4 minutes, the zoospores cease movement, having formed a flat, round, plate typical of the adult colonies, and then undergo, during the ensuing 8-10 minutes, a series of developmental changes leading to the adult cell type. In P. biradiatum Meyen, this involves the transformation of a biflagellated, oval-shaped zoospore into a flattened, four-pronged, adult cell. The present work was undertaken to study the cytology of different stages in the course of this developmental process. MATERIAL AND METHODS Pure cultures of P. biradiatum Meyen were grown in Erlenmeyer flasks on mineral medium No. 8 according to Chu (3) with 10% soil extract added. The flasks were agitated on a Brunswick rotary shaker under an illumination of 150 foot-candles delivered by four GE cool white fluorescent lamps at a temperature of 22°C. During the swarming or lag phase of growth, cultures were passed, several times, through a Seitz filter containing a fitted disk of nickel lektromesh (200) screen. This procedure removed the newly formed colonies and any of the small adult colonies present. The larger adult colonies left on the screen were resuspended in 30 cc of growth medium, placed in a Seitz filter fitted with a disk of nickel lektromesh (400) screen and constantly agitated by bubbling with air. In a series of four runs, each of which lasted approxinlately 2 hours, the colonies were washed and resuspended three times successively at an interval of 3 minutes. In the first run (stage I) the colonies of each 3-minute aliquot were quickly cooled to 4°C by immersion in a dry ice-cellosolve mixture, centrifuged and fixed in ice-cold 2% OsO~ in growth medium. In the remaining runs the aliquots were allowed to stand, at room temperature, an additional 3 (stage II), 6 (stage III) and 12 (stage IV) minutes respectively after filtering before being fixed as described. This procedure allowed increasingly longer periods of development to take place. Young adult colonies (stage V) were obtained by fixing cultures after approximately 6 hours of swarming had taken place. This gave a mixture of large adult colonies and smaller, newly formed adult colonies. The fixed specimens were washed with two changes of growth medium, dehydrated in a series of ascending concentrations of ethanol and infiltrated with two changes of methacrylate (3 parts N-butyl methacrylate:2 parts ethyl methacrylate, containing 1.5% luperco CDB as catalyst). The colonies were then dispensed from the last change of methacrylate into gelatin capsules and polymerization was effected at 70°C for 16 houCs.
28
J. G. MONER AND G . B . CHAPMAN
The sections were cut on a Porter-Blum microtome and mounted on copper grids coated with collodion. All the micrographs were taken with an RCA EMU-2D electron microscope at initial magnifications of 3960-6800 diameters and were enlarged photographically as necessary. RESULTS
1. Chloroplast It can be seen (Fig. 4) that the chloroplast (C) consists essentially of a double limiting m e m b r a n e (CHM) and lamellae which are generally arranged in parallel arrays. There is no differentiation of the chloroplast internum into definite grana and stroma. The lamellae represent stacked flattened vesicles or disks, the thickened rims of which are evident in Figs. 3 and 6. The chloroplast structure of Pediastrum thus resembles closely that described for the green alga Chlamydomonas by Sager and Palade (17) and differs f r o m that described for the green alga Vaucheria by Greenw o o d (7) in lacking the fenestrations and cross-linking of the latter. Starch grains (MS) can be seen in various stages of maturity in the chloroplasts (Fig. 10). It is interesting to note that as the starch increases in a m o u n t it m a y occupy a large part of the space b o u n d e d by the chloroplast membrane. The starch grains appear limited by a thin, dense m e m b r a n e (SM). This m e m b r a n e is clearly visible in the rather rectangular " m a t u r e " starch grains of the central cell in Fig. 10. It should be noted that, even in chloroplasts wherein the starch has displaced the lamellae to one end of the plastid, the double chloroplast m e m b r a n e continues to enclose the starch grain. In all of the developmental stages, extensive starch synthesis is taking place with starch grains ranging in size f r o m 0.6 x 0.3 # to 1.4 × 0.7 #. In addition to the starch grains, one frequently finds small (50-160 m#), dense, spherical bodies (SB) within the chloroplast (Figs. 4 and 5). These inclusions appear to be of the same texture and density as large, dense, spherical bodies (Figs. 4 and 5, LB) found in the cytoplasm. It is therefore suggested that the large bodies m a y arise within the chloroplast and be released to the cytoplasm. Similar large, dense bodies are
FIG. 1. A section through a colony of zoospores (stage I) showing the lamellar structure of the chloroplast (C), a thin, limiting cell membrane (CM) and the presence of a Golgi body (G). In addition, there are large, dense, spherical bodies present (LB) and a nucleus (N). × 19,000. FIG. 2. A section through a colony of early differentiating zoospores (stage II) showing the presence of two nuclei (N1, N~) and both primary (PV) and secondary (SV) vacuoles. The double nature of the secondary vacuole membrane (SVM) can be seen as well as the apparent single nature of the primary vacuole membrane (PVM). One of the large, dense, spherical body inclusions (LB, arrow) appears to be entering the primary vacuole via a tear in the primary vacuole membrane. In addition, there can also be seen a thin, limiting cell membrane (CM), portions of an endoplasmic reticulum (ER) and other large, dense spherical bodies. The sac (S) enclosing the zoospores is faintly visible in this section, x 19,000.
30
J. G. MONER AND G. B. CHAPMAN
occasionally found within vacuoles. The function of these inclusions is at present unknown.
2. Pyrenoid During the early stages of development, we were unable to find pyrenoids typical of those studied in other algae or those present in the adult stages of Pediastrum. In the stage IV preparation (Fig. 10) there are two structures (PY?) resembling in density the mature pyrenoids of later stages. In Fig. 8, a section of a young adult colony (stage V) shows what appears to be a more advanced stage in the formation of a pyrenoid (PY) (0.6 # in diameter). There are no starch plates present, but a limiting membrane, continuous with that of the adjacent chloroplast, surrounds the structure. In Fig. 9, another pyrenoid is shown with incomplete starch plates (SP) and distinct connections with an adjacent chloroplast. A mature pyrenoid (0.65 # in diameter) is shown in Fig. 11 and once again extensions of an adjacent chloroplast membrane are seen to surround the pyrenoid and the associated starch plates.
3. Nucleus The nuclei (N) frequently appear rectangular in profile (Figs. 1, 2, 6, and 10) measuring 1.5-2.5/z in length and 0.5-1.3 # in width. All the nuclei seen are limited by a double membrane (15-30 m/~ in thickness), the inner component of which is bordered by a fairly thick region of dense, granular material, presumably chromatin (Figs. 2, 4, 6, and 10). Although some of this granular material is found in the center of the nucleus, most of it seems to be located at the periphery. No recognizable nucleolus was present in any of the sections studied. In some cases (Figs. 3 and 4) the outer layer of the nuclear envelope becomes so involved with the Golgi apparatus (G) that it is nearly obscured. In Fig. 2 it can be seen that one of the developing zoospores is unusually large and contains two nuclei. During the process of progressive cleavage involved in the production of zoospores, one of the protoplasts occasionally fails to go through its final cleavage despite the presence of the two nuclei. This protoplast may then develop into an oversized zoospore.
FIG. 3. A section through a colony of early differentiating zoospores (stage II) showing three elements of the cellular membrane system, the Golgi complex (G), the endoplasmic reticulum (ER) and the nuclear membrane (NM). The Golgi complex consists of closely packed cisternae (CI), large vacuoles (V) and smaller vesicles (VS). The nuclear membrane is double in nature and appears to be in very intimate association with the Golgi complex. The endoplasmic reticulum is diffuse and the canaliculi are of an order of size comparable to the cisternae of the Golgi complex. In addition to the membranous components mentioned, the chloroplast (C) is seen to consist of stacked disks, the rims of which show thickenings, x 34,000.
32
J. G. M O N E R A N D G . B .
CHAPMAN
4. Mitochondria The number of mitochondria (M) seen throughout the various developmental stages is small (Figs. 4, 5, and 11). The profiles range in diameter from 0.15 to 0.29 # with elongate, triangular, and branched forms being found. There appear to be two membranes surrounding each mitochondrion. The cristae are, in general, randomly oriented but sometimes lie parallel to the mitochondrial membranes. It should be noted that Pediastrum, in possessing cristae, resembles Chlamydomonas (17) and differs from Vaucheria (7), which appears to have a tubular internum. In Fig. 7, the mitochondrion appears to contain four concentric membranes. This configuration probably represents another instance of the occurrence of a cup-shaped mitochondrion - - a configuration discussed at length by Christensen and Chapman (2).
5. Cytoplasmic membranes Present in all of the stages studied is an endoplasmic reticulum (ER) interspersed throughout the cells as canaliculi and small vesicles. The canaliculi measure approximately 15-30 m# and the vesicles 15-65 m# in diameter. These structures are particularly evident in Figs. 3 and 4. Also found consistently throughout the developmental stages in Pediastrum is the Golgi apparatus (G), which is invariably located near the nucleus. Although the cisternae (15 m# in diameter) are closely packed (Figs. 3, 6, and 10), there are frequently large vacuoles (40 m# in diameter) associated with the apparatus (Figs. 3, 4, and 10). In Fig. 6 the Golgi material is disposed as two distinct dictyosomes with smaller and fewer vacuoles.
6. Other cytoplasmic inclusions In addition to the more commonly occurring inclusions already mentioned, several of the cells contain large vacuoles which appear to be bordered by either a single membrane (Fig. 5) or a double membrane (Figs. 2 and 4). In these vacuoles there are found inclusions ranging from fine vesicles (VS) to large dense bodies (LB). The functional significance of the intravacuolar constituents is unknown. It may be
FIG. 4. A section through a colony of moderately advanced differentiating zoospores (stage III) showing a large primary vacuole (PV) with smaller secondary vacuoles (SV1, SV2). There are small vesicles (VS) present within the primary vacuole which is limited by a single membrane (PVM). One of the secondary vacuoles appears to be limited by a single membrane (SVM) and contains a large, dense spherical body (LB) as an inclusion. The other secondary vacuole is limited by a double membrane (SVM2) and contains a lipid-like inclusion (L). Two cross-sections of mitochondria are shown (M1, M2) with differing shapes and containing randomly oriented cristae. The chloroplast (C) is seen to be limited by a double membrane (CHM) and contains small, dense spherical bodies (SB). The shape of the central cell is elongated indicating the beginnings of prong formation, x 32,000.
I
34
J.G.
M O N E R A N D G. B. C H A P M A N
that some of them are involved (and partially or completely expended) in digestive activities or that they represent material permanently or temporarily stored within the vacuoles. In Fig. 2 (arrow) one can observe what is possibly a stage in the movement of a large, dense body from the cytoplasm into the vacuole. In some of the cells (Figs. 2 and 4) other membrane-limited structures are present within the vacuole. In Fig. 2 a double-membraned secondary vacuole (SV) is found within the primary vacuole, and in Fig. 4 there are two secondary vacuoles present, one single and the other double-membrane limited. The inclusion found within the latter appears to have undergone considerable change, possibly from electron bombardment. The most obvious of the remaining inclusions are the large, dense, spherical bodies (LB) (0.2-0.8 ~ in diameter) already mentioned in connection with the chloroplasts, found at all developmental stages in different parts of the cytoplasm, and in the vacuoles. These bodies seem not to be associated with any particular structures and constitute a considerable proportion of the cytoplasmic bulk.
7. Cell wall During the early stages of development (Figs. 1-5) a thin membrane (CM) is seen to limit the cytoplasm of the zoospores. At a somewhat later stage (Fig. 6) a thicker covering or wall (CW) arises which then develops into a loose, coarser structure (Figs. 8, 9, and 11). Globular bodies (GB) can be seen incorporated within the coarse wall (Figs. 8 and 11). DISCUSSION The developmental stages of Pediastrum represent to a considerable degree the beginning of differentiation as recognized in higher forms. The aggregation of zoospores into an orderly plate-like arrangement, the loss of flagella, and the ensuing development of a four-pronged shape, all point to the more organized forms of higher organisms, despite the apparent lack of any division of labor among the cells. The factors influencing the initiation of the differentiation process are little understood. However, it seems likely that the confining sac (Figs. 2 and 10) may
Fro. 5. A section through a cell of advanced differentiation (stage IV) showing a thin cell membrane (CM) and a large primary vacuole (PV).The primary vacuole contains vesicles (ITS)and large, dense spherical bodies (LB). In addition, a section through a mitochondrion (M) is also shown. An early stage in the formation of prongs (P) can be seen in this section. × 25,000. Fro. 6. A section through an early adult cell (stage V) showing a cell wall (CW), two Golgi bodies (G) resembling dictyosomes and a highly elongated nucleus (N). x 25,000. Fro. 7. A section through an early adult cell (stage V) showing what appears to be a cup-shaped mitochondrion (M). × 25,000.
....... ~ ~i~ ......
~
%
~
~. . . . . . . . . . . .
~ ~
36
J. G. MONER A N D G. B. C H A P M A N
play an important role in preserving the environmental factors necessary for the process to take place. The actual transformation was described earlier by Harper (8, 9, 10, 11) who mentions the fact that the cytoplasm almost divides into two pearshaped halves, the narrowed ends of which give rise to the pronged portion of the cell. What produces this profound change in the cytoplasm or indeed the ensuing events is unknown. F r o m a cytological viewpoint the present study has provided some insight into the more obvious changes taking place during the developmental process. The pyrenoid has long been a structure whose exact origin and function has not been completely understood. Certainly the behavior of this body in different green algae reveals no consistency in function, although it has usually been associated with starch synthesis (6, 18). Among the possible means by which the pyrenoid is believed to be passed on to subsequent cell generations are (1) actual division, (2) the incorporation into one unit of many forming zoospores, and (3) dissolution before division with eventual resynthesis by each of the newly formed zoospores (18). In Pediastrum, the last alternative appears to hold true. Before the formation of zoospores takes place, the pyrenoid breaks down and it is not until the latter part of the developmental process (Stage V) that recognizable pyrenoids reappear. In addition, since starch formation is evidently taking place during the early developmental stages, there can be no relationship between the pyrenoid and chloroplast such as exists in Chlamydomonas (I8). In this organism the pyrenoid is apparently the first organ to form starch, with starch formation taking place secondarily in the chloroplast. Since some algae possess pyrenoids of lamellar structure (12) and are usually closely associated with the chloroplast, it is not unreasonable to suspect that the pyrenoid, in some cases, arises from the chloroplast. If this is correct, it is conceivable, at least in Pediastrum, that the pyrenoid serves a dual function, one of starch synthesis and the other as a reserve of chloroplast material. This idea gains some support from the
FIG. 8. A section through a moderately advanced adult cell (stage V) showing what appears to be an immature pyrenoid (PY) in close association with chloroplast (C) and the accompanying starch (S). The pyrenoid is surrounded by the chloroplast membrane (CHM). In addition, the prongs of the cell are fully developed as is the cell wall (CW). Note the globular bodies (GB) associated with the cell wall. × 25,000. FIG. 9. A section through a moderately advanced adult cell (stage V) showing a pyrenoid (PY) in close association with adjacent chloroplast (C). Note the starch plates (SP) in association with the pyrenoid, x 25,000. Fxo. 10. A section through a colony of advanced differentiation (stage IV) showing two possible pyrenoid formations (PY?) and rectangular granules of mature starch (MS). In addition, there can be seen a Golgi body (G) and elements of the endoplasmic reticulum (ER). The sac enclosing the cells is just visible in this section. × 18,500.
ii ~ii~!~iii~!~!iii~ii~iii!ii~%~i~i~iii~
!~ii~!~ ~ ~ ~?~ ~ ~!~i~iiiiii~i~
38
J.G.
M O N E R A N D G. B. C H A P M A N
fact that certain algae (18) possess pyrenoids which do not form starch plates. In addition, it is known that in Pediastrum, as the cells grow older and larger, the amount of starch increases enormously. Coincident with an increase in cell size is a proportional increase in pyrenoid size and when the starch content is unusually high, there is a decrease in chloroplast material with a resultant yellowing of the cytoplasm (19). Perhaps some of the chloroplast material is incorporated into the pyrenoid body to be utilized in the synthesis of new chloroplast when zoospore formation takes place later on. In addition to the changes which take place with pyrenoid and starch formation, the cell wall undergoes dramatic development during the differentiation period and afterward. It is known from previous work (14) that empty mother cells show a globular network intimately associated with the wall proper. The origin of this wall pattern during the developmental stages is the subject of another paper (15). It should be mentioned, however, that the large, dense, spherical bodies (LB) noted in all the developmental stages may play an important role in the emergence of this cell-wall pattern. In regard to other cytoplasmic components seen in P. biradiatum Meyen, some comments are in order. The chloroplast of this organism is similar in most respects to that of other algal forms (1, 13, 20, 22). There are, of course, no distinct grana present, but the lamellae do seem to exhibit a slight preferential orientation. It is worthy of note that Sager and Palade (17), working with Chlarnydornonas, have found what they consider to be imperfect segmentation into grana. The Golgi apparatus assumes different forms in different Pediastrum cells. The Golgi structures seen in Figs. 3 and 4 show a number of large vacuoles more typical of the Golgi apparatus of higher animal cells, whereas the twin structures of Fig. 6 are more suggestive of the dictyosomes of protozoa and invertebrates (4, 5) with the more compact cisternae and somewhat smaller vacuoles. The fact that the dictyosome-like structures are found in a later stage of development may reflect a difference in metabolic activity between the stages. This would imply that the alternation in the disposition of the Golgi material from a single, rather large Golgi apparatus to several discrete dictyosomes is related to the metabolic state of the cell. Our investigations have not yet considered the possible changes in the metabolic state. The endoplasmic reticulum is quite distinct from the Golgi apparatus, being spread rather diffusely throughout the cell compared to the Golgi body which is always
FIG. 11. A section through a mature adult cell (stage V) showing a well-developedpyrenoid (PY) surrounded by starch plates (SP).In addition, there can be seen a typical adult cell wall (CW) with associated globular bodies (GB), a mitochondrion (M), and Golgi body (G). × 33,000.
i~i ~ i l i ¸¸¸~¸¸¸ ~=ii~ii
Fro. 12. An enlargement of a p~rticn of Fig. 4. The double limiting membrane of the mitochondrion (M) may be seen, as well as sparse cristae. ER designates endoplasmic reticulum; C, chloroplast, with paired membranes; G, Golgi zone; N, nucleus, x 52,000. FIG. 13. An enlargement of a portion of Fig. 11. The pyrenoid (PY) may be seen, as well as its adjacent, and nearly engulfing, starch plates (SP). S denotes a large starch deposit included within the same chloroplast membrane. The double nature of that membrane may l:e seen where it is normally sectioned (arrow). × 59,000.
A D U L T C E L L F O R M I N P E D I A S T R U M B I R A D I A T U M MEYEN
41
found close to the nucleus. Although profiles indicate that canaliculi and vesicles are present and probably interconnected, many of the vesicles are very likely independent entities. In the large vacuoles (Figs. 4 and 5) tiny round inclusions, similar to the vesicles of the cytoplasm, are present. There are no canaliculi visible in these large vacuoles indicating that the vesicles are closed units. This is probably true of many similar vesicles found in the cytoplasm. The outer membrane of the nucleus (Figs. 3 and 4) is seen to be in close relationship with the Golgi apparatus. In the light of Watson's demonstration (21) of the continuity between the outer element of the nuclear envelope and the endoplasmic reticulum, and Palade's hypothesis (16) that the endoplasmic reticulum and Golgi apparatus are simply regional differentiations of the same membranous network, it is not at all surprising to find a close relationship between the nuclear envelope and the Golgi apparatus. This relationship does, however, represent a new aspect of this concept. The large vacuoles found in the cytoplasm are especially interesting due to the varied contents found within them. The small vesicles (VS) seem identical to elements of the endoplasmic reticulum; the large, dense, spherical bodies (LB) are identical to those found in the cytoplasm; the single and double-membrane limited secondary vacuoles (SV) have no cytoplasmic counterparts; nor do the very dense inclusions (L). The significance of the double-membraned secondary vacuoles (Figs. 2 and 4) is not known, nor is that of the dense inclusions in one of these structures (Fig. 4). Its density, however, suggests a lipid nature. We may speculate that the large vacuoles are sites of storage or perhaps storage and modification of some of the cytoplasmic inclusions. This would account for the presence of the vesicular component and perhaps for the confusing configuration indicated by the arrow in Fig. 5. Such a form might represent a partially altered, large, dense, spherical body. In Chlamydornonas (17), the contents of the vacuoles were found to be dense, possibly crystalline, masses and were shown to be metachromatic from staining with azure B. There were, apparently, no inclusions found in the vacuoles of Chlarnydomonas similar to those of the cytoplasm and found in Pediastrum.
REFERENCES 1. 2. 3. 4. 5.
ALBERTSON,P. A. and LEYON, H., Exptl. Cell Research 7, 288 (1954). CHR~STENSEN,A. K. and CHAPMAN, G. B., Exptl. Cell Research 18, 576 (1959). CHU, S. P., J. Ecol. 30, 284 (1942). DALTON,A. J. and FELIX, M. D., J. Biophys. Biochem. Cytol. 2, No. 4, Suppl., 79 (1956). GATENBY,J. B., DALTON,A. J. and FEHx, M. D., Nature 176, 301 (1955).
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J. G. MONER AND G. B. CHAPMAN
6. GRANICK, S., in RUHLAND, W. (Ed.), Encyclopedia of Plant Physiology, Vol. I, p. 535. Julius Springer, Berlin, 1955. 7. GREENWOOD,A. D., J. Exptl. Botan. 10, 55 (1959). 8. HARPER, R. A,, Science 37, 385 (1913). 9. - Mem. N. Y. Botan. Garden 6, 91 (1916). 10. - Proc. Am. Phil. Soc. 57, 375 (1918). 11. - - - - Mem. Torrey Botan. Club 17, 210 (1918). 12. LEVON, H., Exptl. Cell Research 6, 497 (1954). 13. MERCER, F. V., HOD~E, A. J., HOPE, A. B. and McLEAN, J. D., Australian J. Biol. Sci. 8, 1 (1955). 14. MONER, J. G., Am. J. Botany 42, 802 (1955). 15. MONER, J. G. and CHAPMAN, G. B., in preparation. 16. PALADE,G. E., J. Biophys. Biochem. Cytol. 2, No. 4, Suppl., 85 (1956). 17. SAGAR,R. and PALADE, G. E., J. Biophys. Biochem. Cytol. 3, 463 (1956). 18. SMITH, G. M., Fresh-Water Algae of the United States, 2nd ed. McGraw-Hill Book Co., New York, 1950. 19. - Ann. Botany 30, 467 (1916). 20. STEtNMANN,E., Exptl. Cell Research 3, 367 (1952). 21. WATSON, M. L., J. Biophys. Biochem. Cytol. 1, 257 (1955). 22. WOLKEN, J. J. and PALADE, G. E., Ann. N. Y. Acad. Sci. 56, 873 (1953).