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Chloroplast continuity during the formation of the tetraspore in antithamnion subulatum Burton, Arthur Hugh Scott 1971

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CHLOROPLAST CONTINUITY DURING THE FORMATION OF THE TETRASPORE IN ANTITHAMNION SUBULATU.M by Arthur Hugh Scott Burton B.Sc, University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biology We accept this thesis as confirming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada i i ABSTRACT The development of the tetrasporangium of Antithamnion subulatum (Harvey) J.G. Agardh was studied using light and electron microscopy in order to elucidate the origin of proplastids, and the continuity of chloro-plasts during the production of the tetraspore. The results show that proplastids arise through a "blebbing" process of the mature chloroplasts. This results in the production of proplastids which are identical to those found free in the cytoplasm of the tetra-sporangial mother c e l l , and are in most respects similar to proplastids observed by others in the apical regions of other red algae. The inclusion of a single DNA-containing genophore within the forming proplastids strongly suggests that each of the scattered genophores in the mature chloroplasts contains at least one complete genome. Division of mature chloroplasts was not seen within the tetra-sporangial mother c e l l .or the tetrasporangial i n i t i a l . However, within the young tetrasporangium the mature chloroplasts appear to undergo several simultaneous divisions resulting in numerous smaller discoid plastids. It is these plastids which, through a succession of growth and division phases, make the major contribution to the continuity of chloroplasts in the form-ation of the tetraspore, rather than the proplastids which have been produced in low numbers throughout i t s development. The colourless nature of the young tetrasporangium is not due to the presence of a high concentration of proplastids, but rather is related i i i to the high frequency of chloroplast d i v i s i o n , r e s u l t i n g i n membrane pro-duction being much more rapid than phycobilisome formation. iv TABLE OF CONTENTS Page LIST OF PLATES AND FIGURES v ACKNOWLEDGEMENTS v i INTRODUCTION 1 MATERIALS AND METHODS 5 OBSERVATIONS I. ORIGIN OF PROPLASTIDS 6 A. Light Microscopy 6 B. Electron Microscopy 9 1. The Mature Chloroplast 9 2. The Proplastid 12 3. Relations Between Proplastid-like Structures and Mature Chloroplasts 13 II. ONTOGENY OF CHLOROPLASTS DURING FORMATION OF THE TETRASPORE. 16 DISCUSSION I. ORIGIN OF PROPLASTIDS 21 II. ONTOGENY OF CHLOROPLASTS DURING FORMATION OF THE TETRASPORE. 28 PLATES AND EXPLANATIONS 35 BIBLIOGRAPHY 58 V LIST OF PLATES AND FIGURES Page PLATE I Diagrammatic thallus of Antithamnion. 36 PLATE II Light micrographs of tetrasporangium formation. 37 PLATE III Figures 8, 9 Phase contrast micrographs of mother c e l l . 38 Figure 10 Low power electron micrograph of mother c e l l . 38 PLATE IV Chloroplasts i n mother c e l l . 39 PLATE V Chloroplasts in mother c e l l . 40 PLATE VI Genophores in chloroplasts. 41 PLATE VII Figure 20 High magnification of DNA in chloroplast. 42 Figure 21 Chloroplast with subterminal swelling. 42 PLATE VIII Proplastids 43 PLATE IX Proplastids and proplastid-like structures closely associated with chloroplasts. 44 PLATE X ' Serial sections of proplastid-like structure joined to chloroplast 45 PLATE XI Stage C i n i t i a l with proplastid-like structure joined to chloroplast 46 PLATE XII Serial sections of proplastid-like structure joined to chloroplast. Cross-over of peripheral thylakoid and plastid envelope. 47 PLATE XIII Proplastid-like structure joined to chloroplast with clear membrane continuity. 48 PLATE XIV Diagrams of proplastid origin. 49 PLATE XV Stage A and B i n i t i a l s and plastids. 50 I v i Page PLATE XVI Stage C i n i t i a l with chloroplast details. 51 PLATE XVII Figures 55-56 Stage C chloroplasts. 52 Figures 57-58 Single-celled stage; proplastid. 52 PLATE XVIII Dividing chloroplasts i n single-celled stage. 53 PLATE XIX Dividing Chloroplasts in single-celled stage. 54 PLATE XX Chloroplasts of 2-celled tetrasporangium. 55 PLATE XXI Chloroplasts of 4-celled tetrasporangium. 56 PLATE XXII Diagram of chloroplast ontogeny. 57 ACKNOWLEDGEMENTS I wish to thank Dr. Thana Bisalputra for his advice and guidance during the course of this study. I would also li k e to express my appreciation to Dr. Janet Stein and Dr. I. Taylor for their constant ava i l a b i l i t y and assistance in writing this thesis, and Dr. Gilbert Hughes for his advice and encouragement. INTRODUCTION Meyer and Schimper (1883) proposed that chloroplasts are derived from previously existing chloroplasts. Since then the study of plastid continuity has become an active f i e l d of research. According to our present knowledge, there are two ways in which chloroplasts may be formed. The chloroplasts may divide more or less equally, thus producing two new, but smaller plastids. Growth of the plastid usually follows division, so that the chloroplast volume, as well as number, is maintained in actively growing tissue. This method obviously provides a line of direct continuity from c e l l to c e l l and generation to generation. The other way in which chloro-plasts may be formed is through the growth, division and differentiation of a population of structurally and physiologically simple proplastids. Since the origin of these remains uncertain, they cannot be said to provide for direct continuity. Chloroplast continuity may be followed easily in green and brown algae, and appears to be mainly through the division of mature chloroplasts. For example, a thorough and convincing study of chloroplast division in the green alga, N i t e l l a was made by Green (1964) using time-lapse cinematography. The result shows the elongation and division of numerous mature chloroplasts. Bisalputra and Bisalputra (1970) have combined phase microscopy with electron microscopy to trace the division of the chloroplasts of the brown alga, Sphacelaria sp. In this work particular emphasis was placed on the replic-ation and transmission of the DNA-containing genophore. 2 However, variation in the mode of chloroplast formation has been reported in the green alga, Acetabularia. Puiseiix-Dao (1970) reported division of Acetabularia chloroplasts to be characteristically nearly equal, but Boloukere (1970) described the budding of mature chloroplasts and the rearrangement of internal thylakoids, which produced a "proplastid-like" structure. Budding and proplastid formation both occur at the onset of nuclear division, which i n i t i a t e s gamete formation. Boloukere suggests that such ac t i v i t i e s of the chloroplasts may be in response to the require-ment for rapid production of new chloroplasts prior to gamete formation. Both types of chloroplast continuity apparently occur in higher plant tissues. The meristematic regions have been shown by electron microscopy to contain a variable number of proplastids. It i s the repeated divisions of these proplastids, or of the immature and partly differentiated chloro-plasts, which are responsible for the'maintenance of chloroplast numbers in these rapidly growing regions. Mature chloroplasts are formed in the tissues derived from the meristems by differentiation of proplastids through the elaboration of internal membranes, and the organization of these membranes into grana and stroma lamellar systems (26). Division of higher plant chloroplasts has been observed by several authors at the ultrastructural level. However according to Honda, et a l • (1971) light microscope observations of chloroplast divisions have only been well documented once, by Kusanoki and Kawasaki in 1936. Honda presents s t a t i s t i c a l evidence, based on direct observation of size classes of chloro-plasts, that continuity is maintained through the division of members of a sub-population of small but functionally mature chloroplasts. Gantt and 3 Arnott (1963) reported in their ultrastructural study of chloroplast division in the fern Matteuccia struthiopteris, two additional references of this process occurring in other higher plants. Division of mature chloroplasts in higher plant cells would indicate a direct continuity. However, where mature chloroplasts are derived from proplastids, the story of chloroplast continuity w i l l not be complete u n t i l the origin of proplastids is understood. Recently, attempts have been made to trace the origin of proplastids in higher plants, and have resulted in several hypotheses. Lance-Nougarede (1960) followed the a c t i v i t i e s of the plastids of Chrysanthemum segetum during the i n i t i a t i o n of f l o r a l parts. Her findings indicate that mature chloroplasts undergo a series of divisions, but a growth phase i s lacking between each division. The result is a progressive diminution and simplification, ending with the production of a number of proplastids similar to those of meristematic zones (28). Bell and Muhlethaler (1962) proposed a de novo origin for chloroplasts in the egg c e l l of Pteridium. On the basis of ultrastructural observation, they suggested that one complete generation of mitochondria and chloroplasts degenerated as the egg c e l l matured. This phenomenon was followed by a regeneration of these organelles through a process which involved the "blebbing" of the nuclear envelope. This contention, however, was later dismissed by Diers (14). Maltzhan and Muhlethaler (1962) reported the presence of "proplastid-l i k e " bodies in regenerating moss leaf tissue. These structures were seen 4 both free in the cytoplasm and occasionally attached to the mature chloro-plast via a narrow isthmus. The authors concluded that this phenomenon was not a method of proplastid production, on the grounds that such an occurrence had never been observed in normal, vegetatively growing tissue, and therefore was probably an activity restricted to the regenerative process. There has been relatively l i t t l e ultrastructural work done on the red algae, and only a small proportion of this is related to chloroplast conti-nuity. However, the studies of Mitakos (1960), Bouck (1962) and Brown and Weir (1968, 1970) indicated that the maintenance of chloroplast numbers may be similar to that reported in higher plants. Light microscopic investigations on red algae l i f e histories within the subclass Floridiophycidae indicate that subapical cells are deeply pig-mented, while apical c e l l s , spermatia, carpogonial filaments, carpogonia and young tetrasporangia may be colourless. Therefore, from a development point of view, the duality of chloroplast origin should be of extreme significance. The purpose of this study is to trace the origin of the proplastids in Antithamnion subulatum (Harvey) J.G. Agardh and to investigate the rela-tive importance of each type of chloroplast development in providing for chloroplast continuity during the differentiation of the tetraspore. A study of the origin of proplastids in the red algae could be made using the early developmental stages of either carpogonial or tetrasporangia, since both of these structures may be colourless, even though the mature cells from which they are derived contain f u l l y differentiated and pigmented 5 chloroplasts. There are, however, distinct advantages to using tetra-sporangial development rather than carpogonia formation for such a study: tetrasporangia are produced in far greater numbers on the thallus than are carpogonia; tetrasporangia in a number of red algal species are completely exposed, while carpogonia are mostly obscured by vegetative filaments throughout their development; furthermore, tetrasporangia do not require f e r t i l i z a t i o n to ensure continuation of normal development and they do not undergo the complex sequence of events which follows f e r t i l i z a t i o n in most of the higher red algae. MATERIALS AND METHODS Specimens of Antithamnion subulatum (Harvey) J.G. Agardh epiphytic on Nereocytis luetkeana (Mertens) Postels and Ruprecht were collected at Rosario Beach, Washington, U.S.A. The material was fixed in the f i e l d for 4 hrs. with 5% gluteraldehyde neutralized over CaCO^ and buffered to pH 7.2. with .07 M phosphate buffer. A post-fixation of 2% hr. was carried out in the laboratory using 2% OsO^ diluted with equal parts of the buffer described above. Both fixations were carried out at ca_. 0 C. The temper-ature was allowed to rise slowly to room temperature during the subsequent washing. Dehydration was carried out using a graded ethanol series. The material was then put through an i n f i l t r a t i o n series of Spurr's medium (41) and 100% ethanol in the ratios 1:3, 1:1 and f i n a l l y 3:1. The material was l e f t for 30 min. in the f i r s t two concentrations, and overnight in the 3:1 mixture. A small amount of material was removed at this stage for study with the light microscope. I n f i l t r a t i o n in the 3:1 mixture was followed 6 by two changes in 100% Spurr's medium for a total of 3 hr. The material was transferred to f l a t aluminum embedding pans f i l l e d to a depth of 0.5 cm with fresh Spurr's medium. The plastic was cured in a vacuum oven at 70 C for 10 hr. Sections were cut with a Dupont diamond knife using either a Porter-Blum MT-1 or a Reichert 0MU3 ultramicrotome, and collected a Formvar coated 200 mesh copper grids. The sections were stained for electron microscope observation using uranyl acetate and Reynold's lead citrate (38), and then examined on a Zeiss EM 9A electron microscope. A l l light microscopy was done on gluteraldehyde and osmium fixed material, using a Zeiss Photomic-roscope. OBSERVATIONS I Origin of Proplastids A. Light Microscopy The tetrasporangia of Antithamnion subulatum arise as a result of renewed growth in a group of cells termed tetrasporangial mother c e l l s . These mother cells usually comprise only the f i r s t few cells of the lateral branchlets adjacent to a major or secondary axis. In this species the mother cells give rise directly to the tetrasporangial i n i t i a l and no stalk c e l l or short branchlet is formed. Development of the tetrasporangia is sequential along two axes, as shown in Fig. 1. The youngest i n i t i a l or tetrasporangium on any one branchlet is normally found on the mother c e l l farthest from the axis, while the youngest tetrasporangia on the thallus are found on the late r a l branchlets of the apical region. This means that nearly a complete range of developmental stages may be obtained on any one 7 thallus, not only ensuring a l l material used may be given identical treatment, but also f a c i l i t a t i n g study of chloroplast continuity. Since, as previously indicated, the tetrasporangial i n i t i a l s and young tetrasporangia are v i r t u a l l y colourless in Antithamnion subulatum, the study of the origin of proplastids was restricted to an investigation of a series of a r b i t r a r i l y chosen stages, beginning with the mother c e l l and ending with the young single-celled tetrasporangium. The light micrographs, Fig. 2, 3, 4, represent stages A; B and C; and the young single-celled stage, respectively. Stage A, represented in Fig. 2, i s typified by a slight swelling of the mother c e l l at the di s t a l end, indicating the beginning of the formation of the i n i t i a l . The height of this swelling is from 10 to 15% of the length of the mother c e l l . The cytoplasm of the entire c e l l i s granular, due to a high concentration of floridean starch. In the fixed material the presence of this starch almost completely obscures the peripherally oriented chloro-plasts and the centrally located nucleus. This condition is also true in l i v i n g f i e l d material. The i n i t i a l i s designated stage B when i t s length is 25-30% of the mother c e l l (upper c e l l , Fig. 3). This stage also is characterised by the beginnings of a regional differentiation of the cytoplasm. Though the cyto-plasm of the mother c e l l remains f i l l e d with the starch grains and the nucleus remains diffuse and centrally located, the cytoplasm of the i n i t i a l becomes denser and more or less homogeneous. It is also possible to see the decreased pigmentation of the outgrowing i n i t i a l in l i v i n g material at this stage. 8 Stage C is defined by the growth of the i n i t i a l to 65-100% of the length of the mother c e l l , as shown by the lower c e l l in Fig. 3. There are several changes apparent at this stage. The upper portion of the cytoplasm of the i n i t i a l remains extremely dense and appears almost completely homo-geneous in both l i v i n g and fixed material. It can be shown with light microscopy that there is an intrusion of some of the components of the mother c e l l cytoplasm into the basal part of the growing i n i t i a l (Fig. 3). Here the axial cytoplasm of the i n i t i a l contains the dark granules of floriden starch extending in a continuous line from the body of the mother c e l l . The nucleus of the mother c e l l becomes enlarged and more clearly defined, and the nucleolus i s prominent. Part of the increase of nuclear definition at this stage is due to the decrease of cytoplasmic density in the perinuclear region, accompanied by the reorientation of the starch grains to form a thin shell surrounding the nucleus. Shortly after this stage the nucleus under-goes division, with the i n i t i a l receiving one daughter nucleus and the mother c e l l the other. Immediately following this event the i n i t i a l i s cut off from the mother c e l l by septation (upper tetrasporangium, Fig. 4). At f i r s t the single-celled tetrasporangium has a homogeneous cytoplasm similar to that of the i n i t i a l from which i t arises (Fig. 4, upper sporangium). The tetrasporangial nucleus does not become evident u n t i l further growth has proceeded (Fig. 4, middle sporangium). The nucleus, when i t does become evident, i s located slightly toward the apex of the sporangium. It is nearly always spherical and contains a large, well defined nucleolus. The apical region of the tetrasporangium retains i t s dense, homogeneous cytoplasm, while the basal region becomes a steadily more granular (Fig. 5). It is presumably the apical portion of the tetrasporangium which is most actively growing, since 9 the nucleus eventually takes on a basal position relative to the length of the tetrasporangium although the distance from the nucleus to the sporangial base remains nearly the same. The small, very dense starch grains are located in a position similar to that in the mother c e l l of stage C; that i s , they become oriented around the nucleus in a thin sheath-like formation. Almost a l l of the starch contained in the tetrasporangium remains in the basal cyto-plasm. Following this the l i v i n g tetrasporangium becomes noticeably pigmented, although the individual chloroplasts remain indistinct in light microscopy. The two subsequent stages are illustrated in Fig. 6, 7. In Fig. 6 both a f u l l y mature and a 2-celled tetrasporangium may be seen, while Fig. 7 shows a group of three 4-celled tetrasporangia, one of which (right hand side) is immature. These three stages are characterised by steadily increasing pigmentation, which in the fixed material is revealed by higher OsO^ staining. The general morphology of the chloroplasts of the tetrasporangial mother c e l l may be seen particularly clearly following growth in a prolonged dark period when the starch content of the c e l l is greatly reduced. When viewed with phase contrast illumination the chloroplasts appear as ribbon shaped, somewhat lobed, and deeply pigmented organelles with a marked peri-pheral orientation within the c e l l . Their size varies considerably within any one mother c e l l , and their average size is dependent upon the size of the c e l l in which they are found. Their general morphology is illustrated in Fig. 8, 9. B. Electron Microscopy (a) The Mature Chloroplast. Low power electron micrographs of longi-tudinal and tangential sections of the mother c e l l (Fig. 10, 11), showing 10 portions of mature chloroplasts, demonstrate the characteristic close inter-locking of ribbon chloroplasts, as observed with phase (Fig. 8, 9). This interlocking results in almost a continuous photosynthetic area in the periphery of the c e l l . The numbers of chloroplasts in this region are sufficient to exclude a l l other organelles, with the exception of a few interspersed mitochondria. The size is dependent upon c e l l size. In the median longitudinal section (Fig. 10) the nearly completely peripheral orientation of the chloroplasts i s shown. A few general ultrastructural features of the chloroplasts may also be seen in low power electron micro-graphs. Particularly obvious in Fig. 11, 12 are the numerous plastoglobuli and the scattered genophores, both of which appear to l i e between the thyla-koids. The surfaces of the thylakoids as seen in Fig. 11-13, 15 have numerous granules, similar to the phycobilosomes reported by Gantt and Conti (1966). At higher magnification (Fig. 14, 15) the chloroplasts of the mother c e l l may be seen to be similar to the chloroplasts of other Florideae pre-viously studied (3, 9, 10, 11, 5). There are two distinct systems of thyla-koids. The f i r s t consists of a single thylakoid which runs parallel to and i s confluent with the chloroplast envelope. The distance between the envelope and this thylakoid is highly constant, being approximately 320A. The second system consists of thylakoids which are distributed singly throughout the stroma, internal to the f i r s t , or peripheral system. An interthylakoid distance of 0.08 - O.l/i appears to be relatively constant, particularly in the larger chloroplasts (Fig. 14, 15). This spacing, however, is not com-pletely regular, since the thylakoids do not always run the f u l l length of the chloroplast. Most commonly these shorter thylakoids terminate at the edge of a genophore (arrows, Fig. 14, 15). Branching of these internal 11 thylakoids may occasionally be noted (white arrows, Fig. 14), but connections between the peripheral and internal systems have not been definitely estab-lished in mature chloroplasts. Phycobilisomes with an average diameter of 270 A occur irregularly on the outer surfaces of the thylakoid membranes of both the internal and peripheral systems. The stroma appears dense and finely granular (Fig. 13, 15, 20). The scattered genophores may appear anywhere in the chloroplast stroma inside the peripheral thylakoid. This has been noted previously by Yokomura (1967) and Bisalputra and Bisalputra (1967). They most frequently occur between the thylakoids, although they may terminate a thylakoid as described above (Fig. 14, 15 arrows) or occasionally they may interrupt the continuity of a thylakoid (dashed arrow, Fig. 14). It is noted that a number of geno-phores may be seen to l i e in close proximity to the peripheral thylakoid. These genophores may be either laterally or terminally oriented in the chlo-roplast, and both of these conditions may occur in any chloroplast simul-taneously (Fig. 16, 17). Occasionally i n tangential sections of the c e l l , which show the lobed nature of the chloroplasts, a genophore may be seen associated with the peripheral thylakoid i n an area of the apex of a lobe (Fig. 18). In Fig. 19, 20 genophores which are closely associated with the peripheral and internal systems in the terminal position of the chloroplast are shown. The association between the thylakoid membranes and the DNA f i b r i l s of the genophore is particularly evident in Fig. 19, 20. (arrows) In the tetrasporangial mother c e l l there is nearly a pure population of mature chloroplasts. However, occasionally profiles of smaller chloro-plasts are found which have a less dense stroma, conspicuous genophores and 12 very few thylakoids (Fig. 12, 13, 21). In a l l other respects these chloro-plasts resemble the structural plan of the mature chloroplasts. In some of these, rearrangement of the internal thylakoids is noticeable and con-nections between the internal and peripheral systems have been noted (Fig. 21, white arrows). These are considered to be immature chloroplasts. (b) The Proplastid. The proplastids of Antithamnion subulatum are found both in the tetrasporangial mother c e l l and in the i n i t i a l s of a l l of the developmental stages studied. Several of these are evident i n the phase contrast micrographs of the tetrasporangial mother c e l l and axial c e l l shown in Fig. 8, 9. There appears to be an increase in their numbers within the tetrasporangial mother c e l l during Stages B and C, but they are never more than one or two found in the i n i t i a l s , irrespective of stage. In most instances the proplastids observed free in the cytoplasm were closely associated with the mature chloroplasts (Fig. 15, 22, 23, 28). One exception to this i s il l u s t r a t e d by Fig. 25-27. This series of sequential sections shows a proplastid more closely associated with the nucleus, but there is no connection between the proplastid envelope and the nuclear envelope. The proplastids have a simple structure, similar to those of other Florideae (9, 10, 29). They are either spherical or ovoid, and range from 0.5 - 1.0/x at their widest point. They are limited by a double membrane envelope, 180 A in width, which is identical to that of the mature chloro-plasts (Fig. 23, 24). Paralleling this envelope and separated from i t by a distance of 320 A is the peripheral thylakoid. This thylakoid appears to stain more deeply than the proplastid envelope and is 180 A in width. A similar phenomenon 13 is evident in the thylakoid membranes of mature chloroplasts. Another simi-l a r i t y between the thylakoids of the mature chloroplasts and the peripheral thylakoid of the proplastid i s the association of granules with i t s outer surfaces. These granules are similar in density and size to the phycobili-somes found on the outer surfaces of the thylakoids of the mature chloroplasts, though there appear to be fewer of them on the outer surfaces of the pro-plastid peripheral thylakoid. Some proplastids have in addition to the single peripheral thylakoid, one and sometimes two internal thylakoids, as reported by Brown and Weier (1968). There do not appear to be granules associated with the outer sur-faces of these membranes at this stage of development. The number of these membranes and their relationship to the ontogeny of the proplastids w i l l be referred to later. Examples of proplastids containing an internal thylakoid are given in Fig. 24, 28 and 37. Most of the proplastids observed are seen to have a single genophore which contains DNA. The finest of these f i b r i l s corresponds with the 25 -30 A f i b r i l s typical of the naked chloroplast DNA described by other authors (3, 4, 42) and found i n the genophores of the mature chloroplasts of this alga. Examples of proplastid genophores are shown in Fig. 22, 23, 24 and 28. The size of these genophores corresponds to those seen in the mature chloroplasts. (c) Relations Between Proplastid-like Structures and Mature Chloro-plasts. Since the origin of proplastids has not been definitely determined before, (13) the small spherical structures seen attached to mature chloro-plasts w i l l be referred to as "proplastid-like structures" u n t i l they can 14 be accurately definied. As indicated earlier in this paper, close associa-tions between the proplastids and the mature chloroplasts are frequently found (Fig. 15, 22, 28). Sometimes these associations are close enough to make i t exceedingly d i f f i c u l t to distinguish between the membranes of the proplastid and those of the mature chloroplast (Fig. 28). In addition to these very close associations, a number of observations and structures which resemble proplastids in size and mophology. This phenomenon is illustrated in Fig. 29-36, 38, 43, 44. Similar configurations are indicated in the phase contrast micrographs (Fig. 8, 9, arrows). The continuity between a portion of the envelope of a mature chloro-plast and that of a proplastid-like structure is shown in Fig. 29 (arrows). It also may be seen that this proplastid-like structure contains a peripheral thylakoid which conforms to the contour of i t s envelope, and which in Fig. 30 may be seen to pass into the mature chloroplast in continuity with the peripheral thylakoid of the latter (Fig. 30, arrows). It contains a distinct electron transparent area, assumed to be a genophore. The matrix of this proplastid-like structure is similar to the stroma of the mature chloroplast to which i t is attached. There is no evidence of an internal thylakoid in either section (Fig. 29, 30). Several granules, similar to those described elsewhere in this paper as occurring on the peripheral thylakoid of the proplastids, may be seen on the outer surface of this peripheral membrane system. Because of this similarity they are assumed to be phycobilisomes. From the orientation of the internal thylakoids of the mature chloroplast i t appears that the connection occurs in a subterminal position. A similar connection is exemplified in Fig. 37-38. As can be seen 15 from these low power micrographs, the connection between the proplastid-lik e structure and the mature chloroplast in this case occurs within the axial cytoplasm of a stace C tetrasporangial i n i t i a l . The actual connection (shown in Fig. 38 arrow) is with the lateral aspect of the mature chloroplast. These two sections and the two intervening sections are shown at higher mag-nificati o n in Fig. 39-42. There i s one noticeable difference between this proplastid-like structure and that of the one just described, this being presence of an internal thylakoid in addition to the peripheral system. Phycobilisomes do not appear to be present on this internal membrane. Due to the tangential plane of sectioning through the connection, or isthmus, the continuity of the bounding membranes of the mature chloroplast and the proplastid-like body is not clear. The continuity of the stroma, however, is evident (Fig. 41, 42, arrows). One further interesting observation may be made concerning the membranes of the proplastid-like body. In the two ser i a l sections (Fig. 39, 40) the peripheral thylakoid can be seen to branch, with one branch continuing on as the peripheral system, while the other be-comes continuous with the envelope. This area in each of the micrographs is enlarged in the inserts in Fig. 39, 40. The DNA f i b r i l s may be seen quite clearly i n the genophore of the proplastid-like structure in Fig. 41, 42. A similar connection between a proplastid-like body and a mature chloroplast i s shown in six ser i a l sections from Fig. 31-36. This sequence passes almost entirely through the isthmus which joins these two structures, thus giving an indication of i t s size. Assuming an average thickness of 650 A, this gives the connecting isthmus an approximate width of 0.4^ . 16 Figure 33 shows particularly clearly the phycobilisomes (white arrows) associated with the peripheral system of the attached structure. Fig. 31, 32 give an indication of the proximity of the genophore of the mature chloroplast to that of the proplastid-like body. In each of the above examples of connections between the mature chlo-roplast and the proplastid-like body, the continuity of the membranes between the two structures has been indistinct. However, in the example shown in Fig. 43, 44 (arrows) the envelope of the mature chloroplast may be seen to be continuous with the envelope of the proplastid-like structure. The two peripheral systems, however, appear to be separate. Tangential views of two membranes are evident in the interior of the proplastid-like body (Fig. 43, 44) and i n the s e r i a l section a single genophore is included. Phyco-bilisomes are evident on both the outer surfaces of the peripheral thylakoid and the surface of the tangentially cut internal membranes. An osmophilic granule similar to the plastoglobuli seen in both mature chloroplasts and proplastids may also be seen close to the isthmus, within the proplastid-l i k e structure. (Fig. 43, 44) Once again, a peripherally oriented geno-phore in the mature chloroplast may be seen to l i e close to the junction. II Ontogeny of Chloroplasts During Formation of the Tetraspore As indicated previously, the tetrasporangial mother c e l l contains both mature chloroplasts and proplastids throughout a l l of the developmental stages of the tetrasporangial i n i t i a l up to and including the young single-celled tetrasporangium. The size, shape, content, and orientation of these organelles within the tetrasporangial mother c e l l has already been described. For the sake of brevity i t may be said here that these factors are retained 17 vi r t u a l l y unchanged within the mother c e l l throughout a l l the stages up to and including stage B. However, even in stage A there are changes in the orientation of the mature chloroplast within the expanding i n i t i a l , which continue throughout stages B and C. Beginning in the latter stage and continuing into the early mature chloroplast within the expanding i n i t i a l development of the single celled stage, these orientation changes are accompanied by structural changes. The following descriptions are concerned with the a c t i v i t i e s involving the mature chloroplasts and the proplastids, which are related to the development of the i n i t i a l , the single-celled, the 2-celled and 4-celled stages. A. Stages A and B As the i n i t i a l begins to grow outward, the mature chloroplasts, which in the mother c e l l are peripherally oriented, may be seen to follow the contour of the swelling which defines stage A (Fig. 47), As growth of the i n i t i a l continues into stage B (Fig. 48) those mature chloroplasts which l i e immediately adjacent to the outgrowth appear to flow into i t at a rate which is commensurate with the rate of growth of the i n i t i a l . Mature chloro-plasts seen in both cross section and oblique section may be seen in Fig. 48. An unusual connection may be seen in Fig. 49 and at higher magnification in Fig. 50. Serial sections which were obtained show what appears to be a lobe of a large chloroplast which is in the process of being pinched off (arrows). Otherwise there is no evidence of mature chloroplasts undergoing division. Proplastids may also be seen in sections of the stage B i n i t i a l (Fig. 51). The distribution of starch revealed in Fig. 47, 48 correspond with the light micrographs Fig. 2, 3. Also, the dense homogeneous cytoplasm 18 occurring in the tip of stage B i n i t i a l s can be seen in the electron micro-graphs to be due to the presence of many chloroplasts. B. Stage C During the early part of stage C there i s l i t t l e change, with the mature chloroplasts continuing to move outward i n keeping with the growth of the i n i t i a l (Fig. 37, 38). However, in addition to these mature chloroplasts, proplastid-like structures (Fig. 37-42) and immature chloroplasts which seem to be undergoing division may occasionally be found in the i n i t i a l (Fig. 53, 54). Chloroplasts which appear to be undergoing division are usually found in the apex of the i n i t i a l . In addition, proplastids and immature chloro-plasts are seen (Fig. 55, 56) closely associated with the base of the i n i t i a l . In these adjacent sections, what appears to be the division of one of the immature chloroplasts may be seen (Fig. 56). Figure 52 shows a section taken through the axis of a late stage C i n i t i a l just prior to septation, which w i l l result in the formation of the single-celled stage. The f i r s t few sections obtained from this i n i t i a l indicate that septation i s just beginning, as shown in insert (b), Fig. 52. It may be seen that the chloroplasts are concentrated in the apical region, and that starch grains are absent from this region. The distribution of starch and chloroplasts seen in this micrograph corresponds exactly with the light micrograph (lower c e l l , Fig. 3). Insert (a) in Fig. 52 is a higher magnification of the apex of the i n i t i a l , showing the details of chloroplast structure. It may be seen that there is very l i t t l e structural change in the mature chloroplasts during the development of the i n i t i a l which are not related to changes in orientation. 19 u n t i l the last part of development i n stage C. Phycobilisomes may be found on the internal and peripheral thylakoids of the mature chloroplasts through-out a l l of the stages. Orientation of the thylakoids and genophores within the chloroplasts remains consistent with that seen in the mother c e l l ; size and morphology also appear to remain constant. Constructions in mature chloroplasts are rarely seen, and proplastids and immature chloroplasts occur very infrequently in the i n i t i a l s , regardless of the stage, between A and C. C. The Single-celled Tetrasporangium The occurrence of a proplastid in the single-celled tetrasporangium has only been observed once (Fig. 57, 58). This sporangium was adjacent to a late stage C on a l a t e r a l branchlet and was therefore probably newly formed. Proplastids have not been observed i n any subsequent stage in tetraspore formation. Fig. 59, 60 i l l u s t r a t e young and intermediate tetra-sporangial stages. In both, constrictions in the mature chloroplasts can be seen. In the younger tetrasporangium (Fig. 59) there are several constrictions i n a mature chloroplast, (arrows) which could potentially result in the production of six daughter chloroplasts. The highest chloro-plast concentration is s t i l l in the apical region. As indicated earlier i n reference to light microscopy (Fig. 5) the distance between the nucleus and the apex of the c e l l increases, indicating a more rapid growth of this region. It i s interesting to note that the chloroplasts of this area are more discoid, smaller, and that two adjacent chloroplasts are apparently undergoing division (Fig. 60, arrows). Only slightly later in the develop-ment of the single-celled tetrasporangium, almost a l l of the chloroplasts appear to be in some stage of division, and the large band-shaped chloro-20 plasts which were s t i l l evident up u n t i l the end of stage C have been reduced to a smaller discoid state (Fig. 61-64). There are practically no phyco-bilisomes present on the peripheral or internal thylakoids of these dividing chloroplasts. This is evident on both tangential and cross sectional views of the chloroplast membranes. The genophores, however, are very numerous and obvious. In some of the dividing chloroplasts a reorientation of the internal thylakoids is evident (Fig. 63, 64). D. The Two-celled Tetrasporangium A low magnification of the 2-celled tetrasporangium, shown in Fig. 65, ill u s t r a t e s the high concentration of small discoid chloroplasts i n this stage. Fig. 66 is a s e r i a l section at higher magnification, showing the two dividing chloroplasts which are labelled in Fig. 65. In one of these the division is unequal. Figures 67, 68 show the slightly increased concent-ration of phycobilisomes on the membranes and connections between the perip-heral and internal thylakoids (Fig. 67, arrows) and between the envelope and peripheral thylakoid i n Fig. 68 (arrows). Genophores appear to be much less numerous in the chloroplasts of this stage. In Fig. 68 the DNA f i b r i l s of a genophore may be seen to be closely associated with the peripheral thylakoid. E. The Four-celled Tetrasporangium The chloroplasts of the 4-celled stage are similar in shape and size to those of the previous stage. However, the outer surfaces of the thyla-koids are much more densely covered with phycobilisomes. Definite continua-tions of the peripheral thylakoid with the internal thylakoids may be seen (Fig. 70 arrows). Some branching of the internal thylakoids appears to be 21 possible (Fig. 71, 72 arrows). Although divisions of the chloroplasts appear to be reduced, occasional division profiles may be encountered. A chloro-plast undergoing a multiple division is shown in Fig. 71. The number of genophores seems to be increased in the chloroplasts of this stage, as compared to the noticeable lack of them in the chloroplasts of the previous stage. DISCUSSION I Origin of Proplastids The mature chloroplasts of Antithamnion subulatum which make up the greatest percentage of the plastid types in the tetrasporangial mother c e l l are similar in most structures to those of other Floridiophycidae described previously (3, 9, 10, 32, 34, 35). With respect to the arrangement and distribution of photosynthetic membranes and the genophores, these chloro-plasts resemble closely the chloroplasts of Laurencia spectabilis (3), G r i f f i t h s i a sp. (35) and Lomentaria baileyana (9). There are differences in the organization and the number of the internal thylakoids, since the chloroplasts of Antithamnion subulatum are elongate and lobed structures, rather than lenticular, as are those of Laurencia and G r i f f i t h s i a . The term "outer limiting disc" which Bouck (1962) used to describe the thylakoid running para l l e l to the chloroplast envelope is not used here, as i t implies that this membrane may act as a barrier, similar to the chloro-plast envelope. It is true that in Antithamnion subulatum, (as in other red algae which have been described as having this single outer thylakoid), the genophores and internal thylakoids are never found outside this membrane system. However, since i t may be discontinuous in places, i t provides no 22 barrier to the continuity of the stroma from the center of the chloroplast to the inner membrane of the envelope. Therefore, this thylakoid w i l l be referred to as a peripheral thylakoid to distinguish i t from the internal photosynthetic membranes, or thylakoids. As shown, both the peripheral and internal thylakoids of Antithamnion  subulatum have on their outer surfaces numerous small granules which are assumed to be phycobilisomes. The concentration of these on the membrane surfaces is much lower than the concentration reported for Porphyridium  cruentum (21), I \ aerugineum (22) or Griffthsia flosculosa (34, 35). In these, the phycobilisomes are arranged regularly and are separated by a regular distance, whereas in A. subulatum they are irregularly scattered, similar to those of Laurencia spectabilis (3). Also, the phycobilisomes observed i n A. subulatum appear to be smaller i n size, averaging 270A, whereas those reported by Gantt and Conti in P_. cruentum are approximately 320 A. Observations (12) on cultured material of A. glanduliferum Kylin and A. pacificum (Harvey) Kylin revealed much higher concentrations of phycobilisomes, and sizes that are closer to those found by Gantt and Conti (21.) This leads to the speculation that f i e l d material may have fewer and smaller phycobilisomes than has cultured material. The genophore of the mature chloroplasts of A. subulatum are similar in size and distribution to those observed in Porphyra tenera (42) Polysi- phonia elongata (42), and Laurencia spectabilis (3). The dimensions of the DNA contained within the genophores conform to previous reports (3, 42). What has not been reported before with respect to chloroplast DNA in red algae, but what is observed here, is the connection between the DNA f i b r i l s 23 and both the peripheral and internal thylakoids (Fig. 19-21, 68). Connections between chloroplast and bacterial DNA and their respective membranes have been reported previously using both thin sectioned material and material spread by the Kleinschmidt method (4, 5, 6, 16). Such connections between DNA and membranes are thought to be important in segregation of the newly replicated DNA molecules. Proplastids have been observed in a number of species of red algae which have been studied with the electron microscope (9, 10, 29, 37). These studies indicate that the proplastids occur in the apical c e l l s , which i s consistent with early investigations with the light microscope (18) which showed that many red algae have almost colourless apical c e l l s . They may occur in combination with a few immature chloroplasts (9, 10), while in some species they appear to comprise a pure population (29) . Ultrastructural investigation also indicates that proplastids may be more or less restricted to members of the Florideophycidae. Thus far there is only one report of proplastids in a member of the Bangiophycidae (33). Most authors (9, 10, 11, 29) describe the proplastid i n i t s simplest form as being a small spherical (with a diameter ranging from 0.5 - 1.2^u ) to an irregularly ovoid body which is bounded by a double membrane envelope. Immediately inside is a second double membrane system, the peripheral thy-lakoid, which runs para l l e l to the envelope. Although this peripheral thylakoid i s present at some stage in the proplastids described, Bouck (1962) suggests that in the simplest form, proplastids of Lomentaria baileyana lack this membrane system, and that i t i s derived during the very early different-iation of the proplastid, from an invagination of the internal membrane of the envelope. On the other hand, the proplastids of red algae described 24 by Brown and Weier (1968), Ramus (1969) and Lichtle and Giraud (1969) a l l apparently have the peripheral thylakoid from the start. A great deal of degeneration of the mitochondria is evident in the permanganate fixed material of Lomentaria, and i t is here considered possible that some of the very small structures (averaging 0.2 p. in diameter) which Bouck (1962) defines as proplastids without a peripheral thylakoid, may in fact be vesi-cular components of broken down mitochondria. Certainly the size reported by Bouck f a l l s considerably below the average size indicated above. Genophores have been shown to occur in the proplastids of Polysiphonia  elongata (29) and i n the very young chloroplasts of Pseudogloiophloea confusa (37), but the presence of phycobilisomes associated with any of the poten-t i a l l y photosynthetic membranes of the proplastids has not been reported to date. The proplastids of Antithamnion subulatum observed in the present study are in most respects similar to others referred to above. They appear to have the peripheral thylakoid from the beginning, and most of those observed contain a genophore. However, in addition, phycobilisomes have been observed on the outer surfaces of the peripheral thylakoids. Each of the studies referred to above has been concerned at least partly with the development of proplastids into the mature chloroplasts. However, to my knowledge, there has been no study directed toward tracing the origin of these proplastids in the red algae. There are a number of p o s s i b i l i t i e s which may account for their presence. F i r s t , they may be present in a l l cells of these algae in very small numbers, only reaching high concentrations in the apical cells which are the actively growing and 25 dividing cells of the red algal thallus (18). Such a situation would pose no serious problem to their continuity from c e l l to c e l l or from generation to generation. It means that most of those proplastids in the apical system would have to undergo growth, division, and differentiation continuously to maintain the characteristic chloroplast number per c e l l , while a small r e s i -dual number might remain temporarily static and be transmitted to the sub-apical portion of the thallus during cytokinesis of the apical c e l l . Their transmission from generation to generation requires that one or more be included in the reproductive unit, and a return to a rapid division rate among these proplastids during formation of the unit and i t s subsequent germination would guarantee the continuity of chloroplasts, and reestablish the apical system of the next generation. This seems a plausible system, for red algae, except for the evidence presented by Manganot (31) from light microscope studies. He indicates that carpogonia of Lemanea become colour-less only after a v i s i b l e fragmentation of the f u l l y pigmented plastids. A problem is also presented in those algae which have recognizable chloroplast in the apical c e l l from the start, but which s t i l l produce colourless re-productive structures. A second po s s i b i l i t y , though an unlikely one, is that proplastids are formed jie novo, through blebbing of the nuclear envelop as suggested by Bell and Muhlethaler (1). Finally, the proplastids may be derived in some manner from the mature chloroplasts. There are two reports in which structures that resemble proplastids have been observed in contact with mature chloroplasts; one is that described earlier (Maltzhan and Muhlethaler, 1962). In the other, Nichols, Ridgeway and Bold (33) report connections between proplastid-like structures and mature chloroplasts in the red alga, Compsopogon. However, they do not discuss the significance 26 of such a phenomenon. The proplastid-like structures described in the observations have so many features i n common with the proplastids observed free i n the tetras-porangial mother c e l l and tetrasporangial i n i t i a l s of A. subulatum, as well as those described by other authors, that they can be defined as forming proplastids. It can therefore be concluded that in A. subulatum, proplastids are formed in the tetrasporangial mother c e l l and tetrasporangial i n i t i a l from mature chloroplasts. The following discussion of the formation of proplastids i s il l u s t r a t e d diagrammatically in Fig. 45, 46. There is a set of requirements which must be f u l l f i l l e d during the formation of a proplastid from a parent structure: a complete genome must be transmitted from the parent structure which w i l l guarantee the capacity of the proplastid to grow and differentiate; there must also be present in the proplastid the molecular machinery which i s capable of translating and transcribing the transmitted information. The process observed here f u l f i l l s these requirements, at least at a physical level. Based on the results of this study and two previous works (30,33) i t is suggested that proplastids may originate through a localized blebbing of the mature chloroplasts. This involves the extension of the mature chloroplast envelope, the peripheral thylakoids, possibly the inclusion of one or two internal thylakoids, as well as a peripherally oriented genophore and a small amount of stroma. However, several questions remain unanswered; i t is not known for certain whether the DNA is attached to the peripheral thylakoid, and whether this attachment is required to guarantee the inclusion 27 of the genome in the developing proplastid. Although i t is possible that the genophore could be carried passively with the intrusion of the stroma from the mature chloroplast, an attachment to the peripheral membrane i s assumed on the basis of a strong similarity between the observations here, and those of other works reviewed elsewhere in this thesis (4, 5). It is also not known whether the bleb results from a localized overgrowth of the envelope and peripheral thylakoid in the vi c i n i t y of a peripheral genophore; or whether i t may occur randomly over the surface of the chloroplast. In most of the developing proplastids examined, a genophore occurred in the mature chloroplast adjacent to the connection, as well as within the body of the forming proplastid. This organization is suggestive of a segregation process following the replication of the DNA within the genophore. It seems, therefore, that during this process the attachment of DNA to the membranes of the internal and peripheral thylakoids could become significant. The inclusion of a single genophore per developing proplastid suggests that the genophores of mature chloroplasts are identical, and each genophore contains enough information for the differentiation and functioning of a new chloro-plast . The way in which the internal thylakoids arise in these developing proplastids i s also uncertain. It is possible that once the forming pro-plastid i s separated from the parent chloroplast, the new internal thylakoids are derived from the peripheral thylakoid as described previously by Bouck (1962), Brown and Weier (1968), Nichols et al( 1,966) and Lichtle and Giraud (1969). However, there i s some indication that these internal thylakoids seen within the developing and s t i l l attached proplastid may, in some cases, 28 already contain phycobilisomes (Fig. 43,44). This suggests the inclusion of short terminal portions of the internal thylakoids of the mature chloro-plast, as indicated diagrammatically in Fig. 46. The way in which this might occur i s presented diagrammatically in Fig. 46, which is based on the chloroplast shown in Fig. 21. The observations made in this study show that proplastids, at least in A. subulatum, are derived from mature chloroplasts and therefore provide a direct continuity. This further supports the original hypothesis of Meyer and Schimper in 1883 that a l l chloroplasts are derived from previously exist-ing chloroplasts. However, this must be considered a preliminary investiga-tion, and much work at the ultrastructural and biochemical levels i s required to c l a r i f y the points of uncertainty raised by this investigation. II Ontogeny of Chloroplasts During the Development of the Tetraspore Bold (1951) has pointed out that the transmission of chloroplasts into reproductive structures has been inadequately studied. This type of study i s particularly intriguing in those genera in which the reproductive unit is at some point in i t s development colourless. In reference to Kylin's work on Fucus serratus, Bold (1951) reports that the antheridial i n i t i a l contains a small number of pale plastids which apparently become completely colourless by the 8-celled stage. This colourless condition persists un t i l the 64-celled stage, when pigmentation is restored, and each nucleus becomes associated with a single small plastid. Manganot (1922) reports a similar occurrence in the developing carpogonia of Lemanea, and Drew (1951) indicates that the tetrasporangial i n i t i a l of the red algae is most frequently colour-less. These observations suggest that proplastids may be formed during the 29 early development of these reproductive structures, and that they provide the continuity of the chloroplasts from generation to generation in at least some algae. The results of the present study on A. subulatum indicate that pro-plastids are formed from the mature chloroplasts throughout the development of the tetrasporangium. Since proplastids are found in the tetrasporangial mother c e l l , even prior to the production of the tetrasporangial i n i t i a l , i t i s probable that their production commences before the onset of any v i s i b l e growth of the i n i t i a l . Whether this indicates that the process is continuous i n a l l of the mature c e l l s , or perhaps only the potential mother c e l l s , i s impossible to say from our present knowledge. Proplastids are apparently also produced by the mature chloroplasts within the tetrasporangial i n i t i a l at least in stage C, and i t is quite l i k e l y that this occurs inter-mittently throughout the development up to and including the very early single-celled tetrasporangium. Immature chloroplasts, some of which appear to be undergoing division, have been observed in, as well as closely asso-ciated with,-the stage C i n i t i a l . The study also shows that during the last part of stage C (which occurs just before the developing i n i t i a l i s cut off from the mother cell) and during the young single-celled stage, there is a division of at least some of the large mature chloroplasts which have been forced into the i n i t i a l during i t s growth. As shown, the divisions of these chloroplasts may be multiple, with as many as five constrictions having been seen per chloroplast. This indicates that at least six small discoid plastids may be derived from one mature chloroplast. This type of division has been reported earlier by Nichols, et a l (1966) in the red alga Compsopogon 30 corelius. Observations such as these suggest that there may be a mixed method for providing chloroplast continuity between the tetrasporophyte and the gametophyte stages in Antithamnion. The results thus far obtained show that proplastids and forming proplastids occur much more frequently in the tetra-sporangial mother c e l l than in the tetrasporangial i n i t i a l . This does not necessarily mean that division of the mature chloroplasts i s the major contri-butor to the increase i n chloroplast numbers which is seen in the 2-celled and 4-celled tetrasporangium. The results of previous works on the growth and differentiation of proplastids indicate that this process is probably quite rapid (3, 9, 29) and that divisions of the immature chloroplasts in the apical cells are probably both rapid and frequent (29). Further investigation of a finely graded series of stages between stage B and the formation of the tetra-sporangium might reveal the relative importance of these two methods of chloroplast formation i n the continuity of plastids through tetraspore formation. One fact that has been shown by this study is that the pale colour-^ ation of the i n i t i a l and colourless condition of the young tetrasporangium is not due to the presence of proplastids, but rather i s related to a decrease i n density of the phycobilisomes on the thylakoids of the chloro-plasts. The results indicate the presence of phycobilisomes on the thylakoids of the chloroplasts. The results indicate the presence of phycobilisomes on the thylakoids of the chloroplasts within the stage C i n i t i a l and even in the earliest single-celled stage. However, in the rapidly dividing chloro-plasts of the nearly mature tetrasporangium phycobilisomes are nearly absent. 31 Investigation of subsequent stages (i.e. the 2-celled and 4-celled tetra-sporangia) with the light microscope reveals a considerable increase in pigmentation. These observations are borne out ultrastructurally by the reappearance of the phycobilisomes just prior to the 2-celled stage, and their increased numbers on the surface of the thylakoids in the 4-celled stage. Peyriere (35) has shown that there i s an increase of size in the chloroplasts and in the number of internal thylakoids per chloroplast during the growth of the tetrasporangium of G r i f f i t h s i a from the juvenile to the mature stage. However, from the observations made in the present study, i t would seem that d i f f i c u l t i e s could be encountered i f emphasis were placed on the significance of thylakoid number in the chloroplasts of the developing tetrasporangium. The reason for this is that a f a i r l y high percentage of these small discoid plastids found in the tetrasporangium are derived from the division of the mature chloroplasts which were included in the i n i t i a l ; this means that the derived plastids have, in most cases, a number of internal thylakoids which is very close to that of the parent plastids. As previously described (10, 11, 12) this number i s i t s e l f variable, since the chloroplasts of the tetrasporangial mother cells found lowest on the thallus have the highest number of internal thylakoids. Therefore, observations on numbers of thylakoids are only significant i f chloroplasts of the tetrasporangium are derived through the differentiation of a relatively pure population of proplastids, or i f tetrasporangia from comparable regions of the thallus are observed. Peyriere (35) also reports the presence of phycobilisomes in low concentration on the thylakoids of chloroplasts of G r i f f i t h s i a , and a f a i r l y high number of division figures among the chloroplast population within the 32 tetrasporangium. The tetrasporangia of G_. flosculosa and A. subulatum are both sessile upon the mother c e l l s , and in comparable stages the ultra-structural aspects of their development seems to be practically identical. The exception i s the presence of phycobilisomes in the chloroplasts of G r i f f i t h s i a in both the juvenile and the nearly mature tetrasporangium. However, since the period of lowest pigmentation during the development of the tetrasporangium is of short duration (probably only a matter of a few hours), i t is possible that the stages examined in G r i f f i t h s i a did not f a l l within this time range, though i t is also possible that the method of plastid continuity i s not the same. The question is what is the cause of the disappearance of the phyco-bilisomes evident i n the developing tetrasporangium of Antithamnion? Two explanations seem feasible: the f i r s t is that phycobilisome production may be suppressed i n favor of the high production of new membrane proteins which must be coincident with the rapid chloroplast division of this stage. The second po s s i b i l i t y i s that the same rate of production is maintained, but that this production i s at a low enough rate in comparison to the rate of growth of' the membranes that a considerable dilution occurs, resulting in a low phycobilisome distribution per unit area of membrane. This imbalance would be gradually n u l l i f i e d as the division and interdivisional growth rate of the chloroplasts slows down in the 2-celled and 4-celled stages. There is some indication within this study that the latter p o s s i b i l i t y i s the more probable. The immature chloroplasts described earlier were seen to have some phycobilisomes associated with the thylakoids. In this case the lower concentration of phycobilisomes on the membranes of the dividing 33 chloroplasts within the tetrasporangium would be related to their higher frequency of division and rate of membrane growth. One further observation has been made which is highly relevant to chloroplast ontogeny. A considerable increase in the number of genophores per chloroplast and in the size of the individual genophores has been noted in the dividing chloroplasts of the nearly mature tetrasporangium. On the other hand, sections of material taken through the 2-celled stage show very few genophores per plastid, although the size remains f a i r l y constant. There appears to be nothing remarkable about the genophore in the 4-celled stage. It has been shown that bacterial chromosomes may be replicated at a higher rate i n an actively growing culture with the result that a single c e l l may contain several genomes, and that a balance between number of genomes and cells i s restored as the division rate of the cells f a l l s off (25). The occurrence of genophores in greater numbers and of increased size within these chloroplasts of the nearly mature single-celled stage i s considered by this author to be the vis i b l e result of an increased rate of DNA replica-tion in response to the increased division rate. Presumably the DNA replic-ation rate increases to i t s highest point prior to the onset of the rapid chloroplast division, and then drops off quite quickly, so that during the subsequent divisions there i s a segregation of the newly formed genophores to the daughter chloroplasts. This would result in the lowest number of genophores to the daughter chloroplasts. This would result in the lowest number of genophores being found in the chloroplasts just after the decline in chloroplast divisions. This stage corresponds to the 2-celled stage. By the 4-celled stage the chloroplast density i s reaching i t s peak, with the 34 result that the division rate f a l l s off toward zero, and the genophores may then attain their characteristic number relative to the size of the chloro-plast. An overview of the events related to chloroplast continuity during the formation of the tetraspore indicates that probably both the proplastids which are formed in , and during the outgrowth of the i n i t i a l , and the mature chloroplasts which undergo a multiple division provide the chloroplast conti-nuity between the tetrasporophytic and gametophytic generations. A summary of these events i s presented in Fig. 7 3 . From present observations, the mature chloroplasts seem to be most important in this role, but further work is required to c l a r i f y the relative values of each of these methods of chloro-plast formation during tetraspore development. It may be added here as an incentive to further study that the significance of proplastid formation may not be in tetraspore formation, but rather in i t s germination. This function may be much more significant during the production of such repro-ductive structures as carpogonia and spermatia. A number of the questions which have been raised by this study and which would be d i f f i c u l t to answer using tetrasporophytic material might well be answered using carpogonia material. 35 PLATES AND EXPLANATIONS LEGEND a = apical c e l l os ax = axial c e l l p c = mature chloroplast pe dc = dividing mature chloroplast pg DNA = DNA f i b r i l s ph fp = forming proplastid pis ge = genophore pt i c = immature chloroplast s i t = internal thylakoid sp lb = la t e r a l branchlet t n = nucleus t i ne = nuclear envelope tmc nu = nucleolus w osmophilic granule proplastid plastid envelope plastoglobuli phycobilisome proplastid-like structure peripheral thylakoid starch grains septation tetrasporangium tetrasporangial i n i t i a l tetrasporangial mother c e l l wall 36 Plate I Figure 1. Diagrammatic representation of the thallus of Antithamnion subulatum. showing sequential production of tetrasporangia. 37 Plate II Figure 2. Light micrograph of stage A tetrasporangialinitial (ti) x 1200 Figure 3. Light micrograph of stage B tetrasporangial i n i t i a l ( t i , top cell) and stage C ( t i , lower c e l l ) . Note that the apical cytoplasm of the i n i t i a l s i s denser and more homo-genous than that of the mother c e l l . x 1200 Figure 4. Light micrograph of three early stages in the development of the tetrasporangium. Septation is incomplete i n the upper c e l l , and the nucleus has not yet become evident. x 520 Figure 5. Light micrograph of nearly mature one--celled tetrasporangium. Nucleus with conspicuous nucleolus (jm) is evident. Apical cytoplasm remains homogeneous. x 1300 Figure 6. Light micrograph of mature tetrasporangium and 2-celled tetrasporangium. Note even distribution of very granular cytoplasm in the mature tetrasporangium. x 530 Figure 7. Light micrograph of three, 4-celled tetrasporangia. Lower right is the youngest of the three. Note retention of granular cytoplasm. 38 Plate III Figure 8. Phase contrast micrograph showing the morphology of the mature chloroplast (c) in the tetrasporangial mother c e l l . Numerous spherical structures assumed to be proplastids (p) may be seen toward the base of the c e l l . In several places connections between these and the chloroplast may be seen (arrows). x 1500 Figure 9. Phase contrast micrograph showing similar chloroplast morphology in c e l l of a lateral branchlet. Several pro-plastids are evident, as are constrictions of the mature chloroplasts. x 1500 Figure 10. Low^  power electron micrograph of median longitudinal section of mother c e l l . Axial cytoplasm is f i l l e d with starch grains (s). Portions of the larger mature chloroplasts (c) are seen in the periphery of the c e l l . x 4800 Low power electron micrograph of tangential section of tetrasporangial mother c e l l , showing the tight packing of the chloroplasts (c). Numerous scattered genophores (ge) are clearly evident. x 16,400 Tangential section of mother c e l l showing a portions of two mature chloroplasts (c) and one immature chloroplast ( i c ) . Plastoglobuli may be seen in both types of chloro-plast (pg). A lobe free of internal thylakoids and contain-ing a genophore may be seen (arrow) at the periphery of one of the mature chloroplasts. x 19,500 Higher power micrograph of another immature chloroplast (ic) and small portion of mature chloroplast (c). Phyco-bilisomes are evident on the outer surface of internal (it) and peripheral thylakoids (pt) of both chloroplasts. x 29,100 40 Plate V Figure 14. Portions of mature chloroplasts showing relationships between the chloroplast envelope (pe), the peripheral thylakoid (pt) and internal thylakoids ( i t ) . Branching of internal thyla-koids is evident (white arrows). Genophores (ge) are seen between the internal thylakoids, interrupting the continuity of internal thylakoids (solid arrows) and crossing an i n -ternal thylakoid (dotted arrows). x 24,000 Figure 15. A small portion of the tetrasporangial mother c e l l showing a proplastid (p) closely associated with a large peripher-al l y oriented chloroplast (c). Further examples of genophores terminating internal thylakoids may be seen (arrows). Al Plate VI Figure 16. Part of mature chloroplast (c) showing genophores (ge) closely associated with the peripheral thylakoid in the latera l aspect of the chloroplast. x 31,600 Figure 17. Portions of two chloroplasts showing genophores (ge) asso-ciated with the peripheral thylakoid (pt) both lat e r a l l y and terminally. x 31,000 Figure 18. A genophore in the apex of a lobe of a mature chloroplast. Association between genophore (ge) and peripheral thylakoid (pt) i s again evident. x 18,600 Figure 19. Higher power micrograph showing one complete genophore (ge) closely associated (arrow) with the peripheral thylakoid (pt) as well as internal thylakoids ( i t ) . x 51,000 42 High power electron micrograph showing genophore (ge) in terminal portion of mature chloroplast. Some of the finest 25-30 A. DNA f i b r i l s may be seen attached to the internal thylakoids ( i t ) , seen here i n oblique section, and to the peripheral thylakoid (lower arrows). x 72,200 A longitudinal section of an immature chloroplast (ic) containing a large genophore (ge) and showing reorientation of internal thylakoids associated with subterminal swelling. A connection between the internal and peripheral thylakoids may also be seen (white arrow). The mature chloroplast above (c) also shows a subterminal swelling containing a genophore (black arrow). x 55,400 43 Plate VIII Figure 22. A proplastid (p) closely associated with mature chloroplast (c) adjacent to stage A i n i t i a l within mother c e l l cyto-plasm. x 22,300 Figure 23. High power view of same proplastid shoving relationship of peripheral thylakoid to plastid envelope, and slightly eccentric genophore containing DNA f i b r i l s . Phycobilisomes may be seen on the surfaces of the peripheral thylakoid. x 50,000 Figure 24. A single proplastid containing a single internal thylakoid (it) and genophore (ge) which i s closely associated with the peripheral thylakoid (pt). Phycobilisomes (ph) are again evident. x 49,500 Figures 25, Adjacent sections showing a proplastid (p) lying close to 26, 27. the nucleus (n). Figure 25 x 49,000 Figure 26, 27 x 52,000 44 Plate IX Figure 28. Proplastid (p) containing single internal thylakoid and genophore (ge) is shown to be closely appressed to the mature chloroplast (c) in top half of micrograph. x 50,700 Figure 29. High power micrograph of the connection between a pro-plastid-like-structure (pis) and a mature chloroplast (c). The structure contains a peripheral thylakoid (pt), phyco-bilisomes (ph) and a genophore (ge). Continuity between the proplastid-like-structure and the mature chloroplast may be seen (arrows). x 53,200 Figure 30. Serial section of connection shown in Figure 29, showing continuity of peripheral thylakoid with that of mature chloroplast (arrows). x 52,500 45 Plate X Figures 36-36 Serial sections taken through the connection between a proplastid-like structure (pis) and a mature chloroplast (c), showing that four sections are required to pass through the genophore (ge) of the proplastid-like-structure and mature chloroplast. Note phycobilisomes in Figure 33 (white arrows). x 45,300 46 Plate XI Figure 37. Lower micrograph of stage C i n i t i a l showing included mature chloroplast (c) and single proplastid-like-structure, in axial cytoplasm, (pis), containing a single internal thyla-koid and genophore. x 15,600 Figure 38. Adjacent section of a stage C i n i t i a l , showing the attach-ment (arrow) of the proplastid-like structure (pis) to the latera l aspect of the mature chloroplast (c). x 15,600 47 Plate XII Figure 39-42. Sequence of sections of the proplastid-like-structure (pis) in a stage C i n i t i a l showing the connection with the mature chloroplast. The connection i s slightly oblique through the point of connection; hence the membrane continuity i s not clear in the connecting piece (arrows, Figs. 41 and 42. Fine f i b r i l s of DNA in the genophore are clearly evi-dent in Figures 41 and 42. Figure 39 x 42,700 Figure 40 x 43,200 Figure 41 x 44,600 Figure 42 x 44,600 Inserts, Figures 39 and 40. High power micrograph showing se r i a l sections of connection between outer membrane of peripheral thylakoid and chloroplast envelope. x 84,000 48 Plate XIII Figure 43. Section of a proplastid-like-structure (pis) connected to a mature chloroplast (c). Note continuity of plastid envelope (arrows). Proplastid-like-structure contains two internal thylakoids. x 53,000 Figure 44. Serial section of Figure 43. Note proximity of mature chloroplast genophore to connection, and inclusion of geno-phore in proplastid-like structure. x 54,400 49 Plate XIV Figure 45. Diagrammatic representation of proplastid formation. Figure 46. Diagrammatic representation of proplastid formation, showing how internal thylakoid may be included in the proplastid. 50 Plate XV Figure 47, Figure 48. Figure 49. Figure 50. Figure 51. Electron micrograph of slightly oblique, median longitudi-nal section of stage A tetrasporangial i n i t i a l . Chloroplasts (c) follow contours of outgrowth of i n i t i a l ( t i ) . Starch grains (s) are present in the mother c e l l cytoplasm. A small portion of the nucleus (n) is also evident. x 5,700 Stage B i n i t i a l , f i l l e d with mature chloroplasts (c). Starch (s) at this stage seems to be confined to the mother c e l l cytoplasm. Note the large nucleus (n) and conspicuous nucleolus (nu). x 4,900 Apex of the stage B i n i t i a l shown above, showing similarity of mature chloroplasts to those found in mother c e l l . Note the dense cytoplasm of the apex. x 15,300 Lobe of mature chloroplast which i s being cut off in stage B i n i t i a l . Note constriction, indicated by arrows. x 16,000 Proplastid observed in stage B i n i t i a l . x 27,500 51 Plate XVI Figure 52. Longitudinal section of late stage C i n i t i a l . Note the continuing high concentration of chloroplasts (c) in apex. Starch (s) is found in the axial cytoplasm. x 5,100 Insert (a). Enlargement of boxed area i n Figure 52 showing details of mature chloroplasts in late stage C i n i t i a l . Phycobilisomes (ph) are evident on outer surface of thyla-koids . x 51,800 Insert (b). Beginning of septation of late stage C i n i t i a l evident in f i r s t few tangential cuts of this i n i t i a l . x 13,800 Figures 53 Serial sections of a dividing immature chloroplast (ic) and 54. observed in the apex of a stage C i n i t i a l . Note the small number of thylakoids and their reorganization. Numerous genophores are present (ge). 52 Plate XVII Figures 55 and 56. Figure 57. Figure 58. Adjacent sections of a portion of the tetrasporangial mother c e l l at the base of a stage C i n i t i a l . Two immature chloro-plasts (ic) are evident, one of which i s dividing. A pro-plastid (p) i s also evident. Figure 55 x 16,200 Figure 56 x . 26,000 Very young single celled stage showing high density of chloro-plasts (c) and centrally oriented starch grains (s). Pro-plastid visible in boxed area. x 5,900 High power of boxed area showing proplastid (p). x 41,000 53 Plate XVIII Figure 59. Young tetrasporangium (t). Multiple division of mature chloroplast i s evident on the l e f t hand side of the c e l l (see arrows indicating constrictions). Apical cytoplasm of the tetrasporangium remains dense and is f i l l e d with chloroplasts (c). Numerous starch grains (s) surround the conspicuous, centrally located nucleus (n). x 3,600 Figure 60. Slightly older tetrasporangium. Most of the plastids are discoid and several of these appear to be undergoing d i v i -sions (see arrows at constrictions). x 5,200 Figures 61 Chloroplasts of nearly mature tetrasporangium undergoing and 62. division. Division plane may be with or at right angles to. long axis of the chloroplast. Figure 61 x 21,600 Figure 62 x 34,500 54 Plate XIX Figures 63 Higher magnification of dividing chloroplasts from nearly and 64. mature tetrasporangium, showing division planes, reorienta-tion of internal thylakoids and numerous genophores. Note that phycobilisomes are scarce and indistinct, (ph). Figure 63 x 40,000 Figure 64 x 38,800 55 Plate XX Figure 65. Low power electron micrograph of longitudinal section of 2-celled tetrasporangium. Note wall separating the 2 spores (w). Almost a l l chloroplasts are discoid and several within the plane of sectioning, marked (c) are undergoing division. x 4,500 Figure 66. Higher power micrograph of boxed area in Figure 65., showing details of the dividing chloroplasts. Division is unequal in the chloroplast upper right. x 13,900 Figure 67. A single chloroplast from a 2-celled tetrasporangium. Con-nections between the internal thylakoids and peripheral thylakoid are clearly discernable (arrows). x 27,600 Figure 68. A portion of a chloroplast from a 2-celled tetrasporangium. The section is tangential to the surface of an internal thylakoid (it) upon which phycobilisomes (ph) may be seen. A genophore (ge) is evident, associated with the peripheral thylakoid (pt) . A possible connection also exists between the outer membrane of the peripheral thylakoid and the inner membrane of the chloroplast envelope. (arrow) x 40,900 56 Plate XXI Figure 69. Portion of a four celled tetrasporangium. Note the relatively thick wall (w) which separates the four tetraspores. Phyco-bilisomes are obvious on the thylakoids of a l l micrographs of this stage (ie. Figs. 69-72). x 15,000 Figure 70. Longitudinal and cross sections of chloroplasts are present in this micrograph. Continuity of internal and peripheral thylakoids is evident (arrow) in cross sectional view. x 35,800 Figure 71. Multiple division of chloroplast. A portion of another chloro-plast undergoing division may be seen in the lower right of this micrograph. x 24,000 Figure 72. Longitudinal section of chloroplasts of four celled tetra-sporanium. Branching of an internal thylakoid i s seen (arrow). x 28,600 57 Plate XXII Figure 73. Diagram i l l u s t r a t i v e of chloroplast ontogeny during the form-ation of tetrasporangia. Two types of chloroplast continuity are i l l u s t r a t e d : (a) Through formation of proplastids, occurring in a l l stages up to and including the single-celled stage, (b) through division of mature chloroplasts, occuring after stage C. 58 BIBLIOGRAPHY 1. B e l l , P.R. and K. Muhlethaler, 1962. The fine structure of the cells taking part i n oogenesis in Pteridium aquilinum (L.) Kuhn. J. Ultrastructure Res. 7: 452. 2. Bird, Robert and Karl G. Lark. 1968. Initiation and termination of DNA replication after amino acid starvation of _E. c o l i 15T. In Cold Spring Harbor Symps. Quant. Biol. 33: 802-808. 3. 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