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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 B r i t i s h Columbia  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n 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 p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference and  study.  I further agree that permission for extensive copying of this thesis for scholarly purposes may by his representatives.  be granted by the Head of my  Department or  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 B r i t i s h Columbia Vancouver 8, Canada  ii  ABSTRACT  The development of the tetrasporangium of Antithamnion subulatum (Harvey) J.G.  Agardh was  studied using l i g h t and electron microscopy i n  order to elucidate the o r i g i n of proplastids, and the continuity of chloroplasts during the production  The  of the  tetraspore.  r e s u l t s show that proplastids a r i s e through a "blebbing"  of the mature chloroplasts.  This r e s u l t s i n the production  process  of proplastids  which are i d e n t i c a l to those found free i n the cytoplasm of the t e t r a sporangial mother c e l l , and are i n most respects s i m i l a r to proplastids observed by others i n the a p i c a l regions of other red algae.  The i n c l u s i o n  of a single DNA-containing genophore within the forming proplastids strongly suggests that each of the scattered genophores i n the mature chloroplasts contains at l e a s t one  complete genome.  D i v i s i o n of mature chloroplasts was  not seen within the t e t r a -  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 d i v i s i o n s r e s u l t i n g i n numerous smaller d i s c o i d p l a s t i d s . i s these p l a s t i d s which, through a succession  It  of growth and d i v i s i o n phases,  make the major contribution to the continuity of chloroplasts i n the formation of the tetraspore, rather than the proplastids which have been produced i n low numbers throughout i t s development.  The  colourless nature of the young tetrasporangium i s not due  the presence of a high concentration  to  of proplastids, but rather i s related  iii  to t h e h i g h f r e q u e n c y o f c h l o r o p l a s t  d i v i s i o n , r e s u l t i n g i n membrane p r o -  d u c t i o n b e i n g much more r a p i d than p h y c o b i l i s o m e  formation.  iv  TABLE OF CONTENTS  Page LIST OF PLATES AND FIGURES ACKNOWLEDGEMENTS  v vi  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 P r o p l a s t i d  3.  Relations Between P r o p l a s t i d - l i k e Structures and Mature Chloroplasts  II.  ONTOGENY OF CHLOROPLASTS DURING FORMATION OF THE TETRASPORE.  12  13 16  DISCUSSION I. II.  ORIGIN OF PROPLASTIDS  21  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 t h a l l u s of Antithamnion.  36  PLATE I I  Light micrographs of tetrasporangium formation.  37  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 i n mother c e l l .  40  PLATE VI  Genophores i n chloroplasts.  41  Figure 20  High magnification  42  Figure 21  Chloroplast with subterminal swelling.  42  PLATE VIII  Proplastids  43  PLATE IX  Proplastids and p r o p l a s t i d - l i k e structures c l o s e l y associated with chloroplasts.  44  S e r i a l sections of p r o p l a s t i d - l i k e structure joined to chloroplast  45  Stage C i n i t i a l with p r o p l a s t i d - l i k e structure joined to chloroplast  46  S e r i a l sections of p r o p l a s t i d - l i k e structure joined to chloroplast. Cross-over of peripheral thylakoid and p l a s t i d envelope.  47  PLATE I I I  PLATE VII  PLATE X PLATE XI PLATE XII  PLATE XIII  '  of DNA i n chloroplast.  P r o p l a s t i d - l i k e structure joined to chloroplast with c l e a r membrane continuity.  48  PLATE XIV  Diagrams of p r o p l a s t i d o r i g i n .  49  PLATE XV  Stage A and B i n i t i a l s and p l a s t i d s .  50  I  vi Page  PLATE XVI  Stage C i n i t i a l with chloroplast d e t a i l s .  51  Figures 55-56  Stage C chloroplasts.  52  Figures 57-58  S i n g l e - c e l l e d stage; p r o p l a s t i d .  52  PLATE XVIII  Dividing chloroplasts i n s i n g l e - c e l l e d stage.  53  PLATE XIX  Dividing Chloroplasts  54  PLATE XX  Chloroplasts of 2-celled tetrasporangium.  55  PLATE XXI  Chloroplasts of 4-celled tetrasporangium.  56  PLATE XXII  Diagram of chloroplast ontogeny.  57  PLATE XVII  i n s i n g l e - c e l l e d stage.  ACKNOWLEDGEMENTS  I wish to thank Dr. Thana Bisalputra f o r his advice and guidance during  the course of t h i s study.  I would also l i k e to express my appreciation  to Dr. Janet Stein  and Dr. I. Taylor for t h e i r constant a v a i l a b i l i t y and assistance i n w r i t i n g t h i s t h e s i s , and Dr. G i l b e r t Hughes for his advice and encouragement.  INTRODUCTION  Meyer and Schimper (1883) proposed that chloroplasts are from previously e x i s t i n g chloroplasts.  Since then the study of p l a s t i d  continuity has become an active f i e l d of research.  According to our present  knowledge, there are two ways i n which chloroplasts may chloroplasts may  derived  be formed.  divide more or less equally, thus producing two new,  smaller p l a s t i d s .  The but  Growth of the p l a s t i d usually follows d i v i s i o n , so that  the chloroplast volume, as well as number, i s maintained i n a c t i v e l y growing tissue.  This method obviously provides a l i n e of d i r e c t continuity from  c e l l to c e l l and generation p l a s t s may  to generation.  The other way  i n which chloro-  be formed i s through the growth, d i v i s i o n and d i f f e r e n t i a t i o n  of a population  of s t r u c t u r a l l y and p h y s i o l o g i c a l l y simple p r o p l a s t i d s .  Since the o r i g i n of these remains uncertain, they cannot be said to provide for  d i r e c t continuity.  Chloroplast continuity may  be followed e a s i l y i n green and brown  algae, and appears to be mainly through the d i v i s i o n of mature chloroplasts. For example, a thorough and convincing green alga, N i t e l l a was  study of chloroplast d i v i s i o n i n the  made by Green (1964) using time-lapse cinematography.  The r e s u l t shows the elongation and d i v i s i o n of numerous mature chloroplasts. Bisalputra and Bisalputra (1970) have combined phase microscopy with electron microscopy to trace the d i v i s i o n of the chloroplasts of the brown alga, Sphacelaria  sp.  In this work p a r t i c u l a r emphasis was  ation and transmission  placed on the r e p l i c -  of the DNA-containing genophore.  2 However, v a r i a t i o n i n the mode of chloroplast formation has been reported i n the green alga, Acetabularia.  Puiseiix-Dao  (1970) reported  d i v i s i o n of Acetabularia chloroplasts to be c h a r a c t e r i s t i c a l l y nearly  equal,  but Boloukere (1970) described  the  the budding of mature chloroplasts and  rearrangement of i n t e r n a l thylakoids, which produced a " p r o p l a s t i d - l i k e " structure.  Budding and p r o p l a s t i d formation both occur at the onset of  nuclear d i v i s i o n , which i n i t i a t e s gamete formation. that such a c t i v i t i e s of the chloroplasts may ment for rapid production  of new  Boloukere suggests  be i n response to the require-  chloroplasts p r i o r to gamete formation.  Both types of chloroplast continuity apparently tissues.  occur i n higher plant  The meristematic regions have been shown by electron microscopy  to contain a v a r i a b l e number of p r o p l a s t i d s .  I t i s the repeated d i v i s i o n s  of these p r o p l a s t i d s , or of the immature and p a r t l y d i f f e r e n t i a t e d chlorop l a s t s , which are responsible for the'maintenance of chloroplast numbers i n these r a p i d l y growing regions.  Mature chloroplasts are formed i n the  tissues derived from the meristems by d i f f e r e n t i a t i o n of proplastids through the elaboration of i n t e r n a l membranes, and the organization  of  these membranes into grana and stroma lamellar systems (26).  D i v i s i o n of higher plant chloroplasts has been observed by authors at the u l t r a s t r u c t u r a l l e v e l . (1971) l i g h t microscope observations  However according  several  to Honda, et a l •  of chloroplast d i v i s i o n s have only  been well documented once, by Kusanoki and Kawasaki i n 1936. s t a t i s t i c a l evidence, based on d i r e c t observation  Honda presents  of s i z e classes of chloro-  p l a s t s , that continuity i s maintained through the d i v i s i o n of members of a sub-population of small but f u n c t i o n a l l y mature chloroplasts.  Gantt and  3  Arnott  (1963) reported  i n t h e i r u l t r a s t r u c t u r a l study of chloroplast d i v i s i o n  i n the fern Matteuccia s t r u t h i o p t e r i s , two  a d d i t i o n a l references  of t h i s  process occurring i n other higher plants.  D i v i s i o n of mature chloroplasts i n higher plant c e l l s would indicate a d i r e c t continuity.  However, where mature chloroplasts are derived from  p r o p l a s t i d s , the story of chloroplast continuity w i l l not be complete u n t i l the o r i g i n of proplastids i s understood.  Recently, attempts have been made  to trace the o r i g i n of proplastids i n higher plants, and have resulted i n several hypotheses.  Lance-Nougarede (1960) followed the a c t i v i t i e s of the p l a s t i d s 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 d i v i s i o n s , but a growth phase i s lacking between each d i v i s i o n . diminution  The  result i s a  and s i m p l i f i c a t i o n , ending with the production  progressive  of a number of  proplastids s i m i l a r to those of meristematic zones (28).  B e l l and Muhlethaler (1962) proposed a de novo o r i g i n for chloroplasts i n the egg c e l l of Pteridium.  On the basis of u l t r a s t r u c t u r a l observation,  they suggested that one complete generation degenerated as the egg c e l l matured. regeneration "blebbing" dismissed  of these organelles  This phenomenon was  chloroplasts  followed by a  through a process which involved  of the nuclear envelope. by Diers  of mitochondria and  This contention, however, was  the later  (14).  Maltzhan and Muhlethaler (1962) reported l i k e " bodies i n regenerating  moss l e a f t i s s u e .  the presence of " p r o p l a s t i d These structures were seen  4 both free i n the cytoplasm and occasionally attached to the mature chloroplast v i a a narrow isthmus. was  The authors concluded that t h i s phenomenon  not a method of p r o p l a s t i d production, on the grounds that such an  occurrence had never been observed i n normal, vegetatively growing t i s s u e , and therefore was  probably an a c t i v i t y r e s t r i c t e d to the regenerative  process.  There has been r e l a t i v e l y l i t t l e u l t r a s t r u c t u r a l work done on the red algae, and only a small proportion nuity.  of t h i s i s related to chloroplast c o n t i -  However, the studies of Mitakos (1960), Bouck (1962) and Brown and  Weir (1968, 1970)  indicated that the maintenance of chloroplast numbers  be s i m i l a r to that reported  may  i n higher plants.  Light microscopic investigations on red algae l i f e h i s t o r i e s within the subclass  Floridiophycidae  indicate that subapical c e l l s are deeply p i g -  mented, while a p i c a l c e l l s , spermatia, carpogonial and young tetrasporangia  may  be colourless.  filaments,  carpogonia  Therefore, from a development  point of view, the d u a l i t y of chloroplast o r i g i n should be of extreme significance.  The purpose of t h i s study i s to trace the o r i g i n of the i n Antithamnion subulatum (Harvey) J.G.  Agardh and  proplastids  to investigate the  t i v e importance of each type of chloroplast development i n providing chloroplast continuity during  the d i f f e r e n t i a t i o n of the  relafor  tetraspore.  A study of the o r i g i n of proplastids i n the red algae could be made using the early developmental stages of either carpogonial since both of these structures may  or  tetrasporangia,  be colourless, even though the mature  c e l l s from which they are derived contain f u l l y d i f f e r e n t i a t e d and pigmented  5 chloroplasts.  There are, however, d i s t i n c t advantages to using t e t r a -  sporangial development rather than carpogonia formation for such a study: tetrasporangia are produced i n f a r greater numbers on the t h a l l u s than are carpogonia; tetrasporangia i n a number of red a l g a l species are  completely  exposed, while carpogonia are mostly obscured by vegetative filaments throughout t h e i r 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 i n 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 c o l l e c t e d at  Rosario Beach, Washington, U.S.A. 4  The material was  fixed i n the f i e l d f o r  hrs. with 5% gluteraldehyde neutralized over CaCO^ and buffered to pH  7.2. with .07 M phosphate b u f f e r . out i n the laboratory using 2% OsO^ described above.  A p o s t - f i x a t i o n of 2% hr. was  carried  d i l u t e d with equal parts of the buffer  Both f i x a t i o n s were carried out at ca_. 0 C.  The temper-  ature was  allowed to r i s e slowly to room temperature during the subsequent  washing.  Dehydration was  material was  c a r r i e d out using a graded ethanol s e r i e s .  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 i n the ratios 1:3,  1:1 and f i n a l l y 3:1.  The material was  l e f t f o r 30 min. i n the f i r s t two concentrations, and overnight i n the mixture.  The  A small amount of material was  with the l i g h t microscope.  3:1  removed at this stage for study  I n f i l t r a t i o n i n the 3:1 mixture was  followed  6 by two changes i n 100% transferred to f l a t  The material was  aluminum embedding pans f i l l e d to a depth of 0.5  fresh Spurr's medium. 10 hr.  Spurr's medium for a t o t a l of 3 hr.  The p l a s t i c was  cm with  cured i n a vacuum oven at 70 C for  Sections were cut with a Dupont diamond knife using e i t h e r a Porter-  Blum MT-1  or a Reichert 0MU3 ultramicrotome,  200 mesh copper g r i d s .  and c o l l e c t e d a Formvar coated  The sections were stained for electron microscope  observation using uranyl acetate and Reynold's lead c i t r a t e (38), and then examined on a Zeiss EM 9A electron microscope.  A l l l i g h t microscopy  was  done on gluteraldehyde and osmium fixed material, using a Zeiss Photomicroscope.  OBSERVATIONS I  O r i g i n of P r o p l a s t i d s A.  Light Microscopy The tetrasporangia of Antithamnion subulatum a r i s e as a r e s u l t of  renewed growth i n a group of c e l l s termed tetrasporangial mother c e l l s . These mother c e l l s usually comprise only the f i r s t few c e l l s of the l a t e r a l branchlets adjacent to a major or secondary axis.  In this species the  mother c e l l s give r i s e d i r e c t l y to the tetrasporangial i n i t i a l and no s t a l k c e l l or short branchlet i s formed.  Development of the tetrasporangia i s  sequential along two axes, as shown i n F i g . 1. tetrasporangium  on any one branchlet i s normally  The youngest i n i t i a l or found on the mother c e l l  farthest from the a x i s , while the youngest tetrasporangia on the thallus are found on the l a t e r a l branchlets of the a p i c a l region. nearly a complete range of developmental stages may  This means that  be obtained on any  one  7 t h a l l u s , not only ensuring  a l l material used may  be given i d e n t i c a l 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 i n Antithamnion subulatum, the study  of the o r i g i n of proplastids was  r e s t r i c t e d to an i n v e s t i g a t i o n 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 s i n g l e - c e l l e d tetrasporangium. 4, represent  The  l i g h t micrographs, F i g . 2,  3,  stages A; B and C; and the young s i n g l e - c e l l e d stage, respectively.  Stage A, represented i n F i g . 2, i s t y p i f i e d by a s l i g h t swelling of the mother c e l l at the d i s t a l end, of the i n i t i a l .  i n d i c a t i n g the beginning of the formation  The height of t h i s swelling i s from 10 to 15% of the  of the mother c e l l . high concentration  The  cytoplasm of the e n t i r e c e l l i s granular, due  of f l o r i d e a n starch.  the c e n t r a l l y located nucleus.  living field  to a  In the fixed material the presence  of t h i s starch almost completely obscures the p e r i p h e r a l l y oriented p l a s t s and  length  chloro-  This condition i s also true i n  material.  The i n i t i a l i s designated stage B when i t s length i s 25-30% of the mother c e l l  (upper c e l l , F i g . 3).  This stage also i s characterised by  beginnings of a regional d i f f e r e n t i a t i o n of the cytoplasm.  Though the  plasm of the mother c e l l remains f i l l e d with the starch grains and  the cyto-  the  nucleus remains d i f f u s e and c e n t r a l l y located, the cytoplasm of the i n i t i a l becomes denser and more or less homogeneous.  I t i s also possible to see  the  decreased pigmentation of the outgrowing i n i t i a l i n l i v i n g material at this stage.  8 Stage C i s 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 i n F i g . 3. several changes apparent at this stage. of  There are  The upper portion of the cytoplasm  the i n i t i a l remains extremely dense and appears almost completely homo-  geneous i n both l i v i n g and fixed material.  I t can be shown with l i g h t  microscopy that there i s an i n t r u s i o n 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  ( F i g . 3).  Here the a x i a l cytoplasm of the i n i t i a l contains the dark granules of f l o r i d e n starch extending i n a continuous l i n e from the body of the mother c e l l .  The  nucleus of the mother c e l l becomes enlarged and more c l e a r l y defined, and the nucleolus i s prominent.  Part of the increase of nuclear d e f i n i t i o n at  t h i s stage i s due to the decrease of cytoplasmic density i n the perinuclear region, accompanied by the reorientation of the starch grains to form a thin s h e l l surrounding the nucleus.  Shortly a f t e r this stage the nucleus under-  goes d i v i s i o n , 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 o f f from  the mother c e l l by septation (upper tetrasporangium, F i g . 4).  At f i r s t  the s i n g l e - c e l l e d tetrasporangium has a homogeneous cytoplasm  s i m i l a r to that of the i n i t i a l from which i t arises ( F i g . 4, upper  sporangium).  The tetrasporangial nucleus does not become evident u n t i l further growth has proceeded  ( F i g . 4, middle sporangium).  The nucleus, when i t does become  evident, i s located s l i g h t l y toward the apex of the sporangium. always s p h e r i c a l and contains a large, w e l l defined nucleolus.  It i s nearly The a p i c a l  region of the tetrasporangium retains i t s dense, homogeneous cytoplasm, while the basal region becomes a s t e a d i l y more granular ( F i g . 5).  I t i s presumably  the a p i c a l portion of the tetrasporangium which i s most a c t i v e l y growing, since  9 the nucleus eventually takes on a basal p o s i t i o n r e l a t i v e 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  i n a p o s i t i o n s i m i l a r to that i n the mother c e l l of stage C; that i s , they become oriented around the nucleus i n a thin sheath-like formation.  Almost  a l l of the starch contained i n the tetrasporangium remains i n the basal cytoplasm.  Following this the l i v i n g tetrasporangium becomes noticeably pigmented,  although the i n d i v i d u a l chloroplasts remain i n d i s t i n c t i n l i g h t microscopy.  The two subsequent stages are i l l u s t r a t e d i n F i g . 6, 7. both a f u l l y mature and a 2-celled tetrasporangium may  In F i g . 6  be seen, while F i g . 7  shows a group of three 4-celled tetrasporangia, one of which (right hand side) i s immature.  These three stages are characterised by s t e a d i l y increasing  pigmentation, which i n the fixed material i s revealed by higher OsO^ s t a i n i n g . The general morphology of the chloroplasts of the tetrasporangial mother c e l l may  be seen p a r t i c u l a r l y c l e a r l y following growth i n a prolonged  dark period when the starch content of the c e l l i s greatly reduced.  When  viewed with phase contrast i l l u m i n a t i o n the chloroplasts appear as ribbon shaped, somewhat lobed, and deeply pigmented organelles with a marked p e r i pheral o r i e n t a t i o n within the c e l l .  Their s i z e varies considerably within  any one mother c e l l , and t h e i r average s i z e i s dependent upon the s i z e of the c e l l i n which they are found. i n F i g . 8,  B.  Their general morphology i s i l l u s t r a t e d  9.  Electron Microscopy (a)  The Mature Chloroplast.  Low  power electron micrographs of l o n g i -  tudinal and tangential sections of the mother c e l l (Fig. 10, 11), showing  10  portions of mature chloroplasts, demonstrate the c h a r a c t e r i s t i c close i n t e r locking of ribbon chloroplasts, as observed with phase ( F i g . 8, 9). i n t e r l o c k i n g r e s u l t s i n almost a continuous photosynthetic periphery  of the c e l l .  area i n the  The numbers of chloroplasts i n t h i s region  s u f f i c i e n t to exclude a l l other organelles, with the exception interspersed mitochondria.  This  are  of a  The s i z e i s dependent upon c e l l s i z e .  few In the  median l o n g i t u d i n a l section ( F i g . 10) the nearly completely peripheral o r i e n t a t i o n of the chloroplasts i s shown. features of the chloroplasts may graphs. and  A few general u l t r a s t r u c t u r a l  also be seen i n low power electron micro-  P a r t i c u l a r l y obvious i n F i g . 11, 12 are the numerous p l a s t o g l o b u l i  the scattered genophores, both of which appear to l i e between the t h y l a -  koids.  The  granules,  surfaces of the thylakoids as seen i n F i g . 11-13, 15 have numerous  s i m i l a r to the phycobilosomes reported by Gantt and Conti (1966).  At higher magnification c e l l may  be seen to be s i m i l a r to the chloroplasts of other Florideae pre-  v i o u s l y studied koids.  ( F i g . 14, 15) the chloroplasts of the mother  The  (3, 9, 10, 11, 5).  There are two d i s t i n c t systems of t h y l a -  f i r s t consists of a s i n g l e thylakoid which runs p a r a l l e l to and  i s confluent with the chloroplast envelope.  The distance between the envelope  and t h i s thylakoid i s highly constant, being approximately 320A.  The  second  system consists of thylakoids which are d i s t r i b u t e d singly throughout the stroma, i n t e r n a l to the f i r s t , or peripheral system. distance of 0.08  An i n t e r t h y l a k o i d  - O.l/i appears to be r e l a t i v e l y constant, p a r t i c u l a r l y i n  the larger chloroplasts ( F i g . 14, 15).  This spacing, however, i s not com-  p l e t e l y 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, F i g . 14, 15).  Branching of these i n t e r n a l  11  thylakoids may  occasionally be noted (white arrows, F i g . 14), but connections  between the peripheral and i n t e r n a l systems have not been d e f i n i t e l y establ i s h e d i n mature chloroplasts.  Phycobilisomes with an average diameter of  270 A occur i r r e g u l a r l y on the outer surfaces of the thylakoid membranes of both the i n t e r n a l and peripheral systems. f i n e l y granular  The  (Fig. 13, 15,  The stroma appears dense and  20).  scattered genophores may  inside the peripheral thylakoid.  appear anywhere i n the chloroplast stroma  This has been noted previously by Yokomura  (1967) and B i s a l p u t r a and Bisalputra (1967). between the thylakoids, although they may  They most frequently occur  terminate a thylakoid as  above (Fig. 14, 15 arrows) or occasionally they may of a thylakoid (dashed arrow, F i g . 14). phores may  interrupt the continuity  I t i s noted that a number of geno-  be seen to l i e i n close proximity  These genophores may  described  to the peripheral thylakoid.  be e i t h e r l a t e r a l l y or terminally oriented i n the chlo-  r o p l a s t , and both of these conditions may taneously (Fig. 16, 17).  occur i n any chloroplast  simul-  Occasionally i n tangential sections of the  which show the lobed nature of the chloroplasts, a genophore may  cell,  be seen  associated with the peripheral thylakoid i n an area of the apex of a lobe (Fig.  18).  In F i g . 19, 20 genophores which are c l o s e l y associated with the  peripheral and i n t e r n a l systems i n the terminal p o s i t i o n of the chloroplast are shown.  The association between the thylakoid membranes and  f i b r i l s of the genophore i s p a r t i c u l a r l y evident  i n F i g . 19, 20.  In the tetrasporangial mother c e l l there i s nearly a pure of mature chloroplasts.  the  DNA  (arrows)  population  However, occasionally p r o f i l e s of smaller  chloro-  p l a s t s are found which have a less dense stroma, conspicuous genophores and  12  very few thylakoids  ( F i g . 12, 13, 21).  In a l l other respects these chloro-  plasts resemble the s t r u c t u r a l plan of the mature chloroplasts.  In some  of these, rearrangement of the i n t e r n a l thylakoids i s noticeable and  con-  nections between the i n t e r n a l and peripheral systems have been noted (Fig. 21, white arrows).  (b)  These are considered  The P r o p l a s t i d .  to be immature chloroplasts.  The proplastids of Antithamnion subulatum are  found both i n the tetrasporangial mother c e l l and i n 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 a x i a l c e l l shown i n F i g . 8, 9.  There appears to be an increase i n t h e i r numbers within  the  tetrasporangial mother c e l l during Stages B and C, but they are never more than one or two instances  found i n the i n i t i a l s , i r r e s p e c t i v e of stage.  In most  the proplastids observed free i n the cytoplasm were c l o s e l y  associated with the mature chloroplasts ( F i g . 15, 22, 23, 28). to t h i s i s i l l u s t r a t e d by F i g . 25-27.  One  This series of sequential  exception  sections  shows a p r o p l a s t i d more c l o s e l y associated with the nucleus, but there i s no connection between the p r o p l a s t i d envelope and the nuclear  envelope.  The proplastids have a simple structure, s i m i l a r to those of other Florideae 0.5  - 1.0/x  (9, 10, 29).  They are e i t h e r spherical or ovoid, and range from  at t h e i r widest point.  They are l i m i t e d by a double membrane  envelope, 180 A i n width, which i s i d e n t i c a l to that of the mature chloroplasts ( F i g . 23,  24).  P a r a l l e l i n g t h i s envelope and separated from i t by a distance of 320 i s the peripheral thylakoid.  This thylakoid appears to s t a i n more deeply  than the p r o p l a s t i d envelope and i s 180 A i n width.  A s i m i l a r phenomenon  A  13 i s evident i n 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 p r o p l a s t i d i s the association of granules with i t s outer surfaces.  These granules are s i m i l a r i n density and s i z e to the p h y c o b i l i -  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 prop l a s t i d peripheral thylakoid.  Some proplastids have i n addition to the single peripheral thylakoid, one and sometimes two i n t e r n a l 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 t h i s stage of development.  The number of these  membranes and t h e i r r e l a t i o n s h i p to the ontogeny of the proplastids w i l l be referred to l a t e r .  Examples of proplastids containing an i n t e r n a l thylakoid  are given i n F i g . 24, 28 and 37.  Most of the proplastids observed are seen to have a single genophore which contains DNA.  The f i n e s t of these f i b r i l s corresponds with the 25 -  30 A f i b r i l s t y p i c a l of the naked chloroplast DNA described by other authors (3, 4, 42) and found i n the genophores of the mature chloroplasts of t h i s alga. 28.  Examples of p r o p l a s t i d genophores are shown i n F i g . 22, 23, 24 and The s i z e of these genophores corresponds to those seen i n the mature  chloroplasts.  (c) plasts. before,  Relations Between P r o p l a s t i d - l i k e Structures and Mature ChloroSince the o r i g i n of proplastids has not been d e f i n i t e l y determined  (13) the small spherical structures seen attached  to mature chloro-  plasts w i l l be referred to as " p r o p l a s t i d - l i k e structures" u n t i l they can  14 be accurately d e f i n i e d .  As indicated e a r l i e r i n t h i s paper, close associa-  tions between the proplastids and found ( F i g . 15, 22, 28).  the mature chloroplasts are  frequently  Sometimes these associations are close enough to  make i t exceedingly d i f f i c u l t to d i s t i n g u i s h between the membranes of the p r o p l a s t i d and those of the mature chloroplast (Fig. 28). these very close associations, a number of observations resemble proplastids i n s i z e and mophology. i n F i g . 29-36, 38, 43, 44.  In addition to  and structures which  This phenomenon i s i l l u s t r a t e d  Similar configurations are indicated i n the  phase contrast micrographs ( F i g . 8, 9, arrows).  The continuity between a portion of the envelope of a mature chlorop l a s t and that of a p r o p l a s t i d - l i k e structure i s shown i n F i g . 29 It also may  (arrows).  be seen that t h i s p r o p l a s t i d - l i k e structure contains a peripheral  thylakoid which conforms to the contour of i t s envelope, and which i n F i g . 30 may  be seen to pass into the mature chloroplast i n continuity with the  peripheral thylakoid of the l a t t e r ( F i g . 30, arrows).  I t contains a d i s t i n c t  electron transparent  The matrix of t h i s  area, assumed to be a genophore.  p r o p l a s t i d - l i k e structure i s s i m i l a r to the stroma of the mature chloroplast to which i t i s attached.  There i s no evidence of an i n t e r n a l thylakoid i n  e i t h e r section ( F i g . 29, 30).  Several granules,  s i m i l a r to those described  elsewhere i n t h i s paper as occurring on the peripheral thylakoid of the p r o p l a s t i d s , may system.  be seen on the outer surface of t h i s peripheral membrane  Because of t h i s s i m i l a r i t y they are assumed to be phycobilisomes.  From the o r i e n t a t i o n of the i n t e r n a l thylakoids of the mature chloroplast i t appears that the connection occurs i n a subterminal p o s i t i o n .  A s i m i l a r connection i s exemplified  i n F i g . 37-38.  As can be seen  15 from these low power micrographs, the connection between the p r o p l a s t i d l i k e structure and the mature chloroplast i n this case occurs within the a x i a l cytoplasm of a stace C tetrasporangial i n i t i a l .  The actual connection  (shown i n F i g . 38 arrow) i s with the l a t e r a l aspect of the mature chloroplast. These two sections and the two intervening sections are shown at higher magn i f i c a t i o n i n F i g . 39-42.  There i s one noticeable difference between t h i s  p r o p l a s t i d - l i k e structure and that of the one j u s t described,  t h i s being  presence of an i n t e r n a l thylakoid i n addition to the peripheral system. Phycobilisomes do not appear to be present on t h i s i n t e r n a l 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 p r o p l a s t i d - l i k e body i s not c l e a r . i s evident  (Fig. 41, 42, arrows).  The continuity of the stroma, however, One further i n t e r e s t i n g observation may  be made concerning the membranes of the p r o p l a s t i d - l i k e body. s e r i a l sections  In the two  ( F i g . 39, 40) the peripheral thylakoid can be seen to branch,  with one branch continuing on as the peripheral system, while the other becomes continuous with the envelope. i s enlarged  This area i n each of the micrographs  i n the i n s e r t s i n F i g . 39, 40. The DNA f i b r i l s may be seen  quite c l e a r l y i n the genophore of the p r o p l a s t i d - l i k e structure i n F i g . 41, 42.  A s i m i l a r connection between a p r o p l a s t i d - l i k e body and a mature chloroplast i s shown i n s i x s e r i a l sections from F i g . 31-36.  This sequence  passes almost e n t i r e l y through the isthmus which j o i n s these two structures, thus giving an i n d i c a t i o n of i t s s i z e .  Assuming an average thickness of  650 A, t h i s gives the connecting isthmus an approximate width of 0 . 4 ^ .  16 Figure 33 shows p a r t i c u l a r l y c l e a r l y the phycobilisomes (white arrows) associated with the peripheral system of the attached 32 give an i n d i c a t i o n of the proximity  structure.  F i g . 31,  of the genophore of the mature  chloroplast to that of the p r o p l a s t i d - l i k e body.  In each of the above examples of connections between the mature chloroplast and the p r o p l a s t i d - l i k e body, the continuity of the membranes between the two Fig.  structures has been i n d i s t i n c t .  43, 44  However, i n the example shown i n  (arrows) the envelope of the mature chloroplast may  be seen to  be continuous with the envelope of the p r o p l a s t i d - l i k e structure. peripheral systems, however, appear to be separate. two membranes are evident  i n the i n t e r i o r of the p r o p l a s t i d - l i k e body (Fig. Phyco-  on both the outer surfaces of the peripheral thylakoid  and the surface of the t a n g e n t i a l l y cut i n t e r n a l membranes.  An  osmophilic  granule s i m i l a r to the p l a s t o g l o b u l i seen i n both mature chloroplasts proplastids may l i k e structure.  and  also be seen close to the isthmus, within the p r o p l a s t i d ( F i g . 43, 44)  Once again, a p e r i p h e r a l l y oriented geno-  phore i n the mature chloroplast may  II  two  Tangential views of  43, 44) and i n the s e r i a l section a s i n g l e genophore i s included. bilisomes are evident  The  be seen to l i e close to the junction.  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 s i n g l e c e l l e d tetrasporangium.  The s i z e , shape, content, and o r i e n t a t i o n 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 v i 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 i n stage A there are changes i n the o r i e n t a t i o n of the mature chloroplast within the expanding i n i t i a l , which continue throughout stages B and C.  Beginning i n the l a t t e r stage and continuing into the early  mature chloroplast within the expanding i n i t i a l development of the s i n g l e c e l l e d stage, these o r i e n t a t i o n changes are accompanied by s t r u c t u r a l changes. The following descriptions are concerned with the a c t i v i t i e s involving the mature chloroplasts and the p r o p l a s t i d s , which are related to the development of the i n i t i a l , the s i n g l e - c e l l e d , the 2-celled and 4-celled  A.  stages.  Stages A and B As the i n i t i a l begins to grow outward, the mature chloroplasts, which  i n the mother c e l l are p e r i p h e r a l l y 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 ( F i g . 48) those mature chloroplasts which l i e immediately adjacent to the outgrowth appear to flow into i t at a rate which i s commensurate with the rate of growth of the i n i t i a l . p l a s t s seen i n both cross section and oblique section may An unusual connection may Fig.  50.  Mature chloro-  be seen i n F i g . 48.  be seen i n F i g . 49 and at higher magnification  in  S e r i a l sections which were obtained show what appears to be a lobe  of a large chloroplast which i s i n the process of being pinched o f f  (arrows).  Otherwise there i s no evidence of mature chloroplasts undergoing d i v i s i o n . Proplastids may  The  also be seen i n sections of the stage B i n i t i a l  ( F i g . 51).  d i s t r i b u t i o n of starch revealed i n F i g . 47, 48 correspond with  the l i g h t micrographs F i g . 2, 3.  Also, the dense homogeneous cytoplasm  18 occurring i n the t i p of stage B i n i t i a l s can be seen i n the electron micrographs 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 the i n i t i a l  to move outward i n keeping with the growth of  ( F i g . 37, 38). However, i n addition to these mature chloroplasts,  p r o p l a s t i d - l i k e structures ( F i g . 37-42) and immature chloroplasts which seem to be undergoing d i v i s i o n may occasionally be found i n the i n i t i a l 54).  (Fig. 53,  Chloroplasts which appear to be undergoing d i v i s i o n are usually found  i n the apex of the i n i t i a l .  In addition, proplastids and immature chloro-  p l a s t s are seen ( F i g . 55, 56) c l o s e l y associated with the base of the i n i t i a l . In these adjacent sections, what appears to be the d i v i s i o n of one of the immature chloroplasts may be seen ( F i g . 56).  Figure 52 shows a section taken through the axis of a l a t e stage C i n i t i a l j u s t p r i o r to septation, which w i l l r e s u l t i n the formation of the s i n g l e - c e l l e d stage.  The f i r s t few sections obtained from t h i s i n i t i a l  indicate that septation i s j u s t beginning, as shown i n i n s e r t (b), F i g . 52. It may be seen that the chloroplasts are concentrated i n the a p i c a l region, and that starch grains are absent from t h i s region.  The d i s t r i b u t i o n of  starch and chloroplasts seen i n t h i s micrograph corresponds exactly with the l i g h t micrograph (lower c e l l , F i g . 3).  Insert (a) i n F i g . 52 i s a higher magnification i n i t i a l , showing the d e t a i l s of chloroplast structure.  of the apex of the I t may be seen that  there i s very l i t t l e s t r u c t u r a l change i n the mature chloroplasts during the development of the i n i t i a l which are not related to changes i n o r i e n t a t i o n .  19 u n t i l the l a s t part of development i n stage C.  Phycobilisomes may be found  on the i n t e r n a l and peripheral thylakoids of the mature chloroplasts throughout a l l of the stages.  Orientation of the thylakoids and genophores within  the chloroplasts remains consistent with that seen i n the mother c e l l ; s i z e and morphology also appear to remain constant.  Constructions  i n mature  chloroplasts are rarely seen, and proplastids and immature chloroplasts occur very infrequently i n the i n i t i a l s , regardless of the stage, between A and C.  C.  The S i n g l e - c e l l e d Tetrasporangium The occurrence of a p r o p l a s t i d i n the s i n g l e - c e l l e d tetrasporangium  has only been observed once ( F i g . 57, 58). This sporangium was adjacent to a l a t e stage C on a l a t e r a l branchlet formed.  Proplastids have not been observed i n any subsequent stage i n  tetraspore formation. sporangial stages. be seen.  and was therefore probably newly  F i g . 59, 60 i l l u s t r a t e young and intermediate  tetra-  In both, c o n s t r i c t i o n s i n the mature chloroplasts can  In the younger tetrasporangium ( F i g . 59) there are several  c o n s t r i c t i o n s i n a mature chloroplast, (arrows) which could p o t e n t i a l l y r e s u l t i n the production p l a s t concentration reference  of s i x daughter chloroplasts.  i s s t i l l i n the a p i c a l region.  The highest  chloro-  As indicated e a r l i e r i n  to l i g h t microscopy (Fig. 5) the distance between the nucleus and  the apex of the c e l l increases, i n d i c a t i n g a more rapid growth of t h i s region.  I t i s i n t e r e s t i n g to note that the chloroplasts of t h i s area are  more d i s c o i d , smaller, and that two adjacent chloroplasts are apparently undergoing d i v i s i o n ( F i g . 60, arrows).  Only s l i g h t l y l a t e r i n the develop-  ment of the s i n g l e - c e l l e d tetrasporangium, almost a l l of the chloroplasts appear to be i n some stage of d i v i s i o n , 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 d i s c o i d state ( F i g . 61-64).  There are p r a c t i c a l l y no phyco-  bilisomes present on the peripheral or i n t e r n a l thylakoids of these d i v i d i n g chloroplasts.  This i s evident on both tangential and cross s e c t i o n a l views  of the chloroplast membranes. and obvious.  The genophores, however, are very numerous  In some of the d i v i d i n g chloroplasts a reorientation of the  i n t e r n a l thylakoids i s evident  D.  (Fig. 63,  The Two-celled Tetrasporangium A low magnification  of the 2-celled tetrasporangium, shown i n F i g . 65,  i l l u s t r a t e s the high concentration stage. two  64).  of small d i s c o i d chloroplasts i n t h i s  F i g . 66 i s a s e r i a l section at higher magnification,  d i v i d i n g chloroplasts which are l a b e l l e d i n F i g . 65.  the d i v i s i o n i s unequal.  showing the  In one of these  Figures 67, 68 show the s l i g h t l y increased  concent-  r a t i o n of phycobilisomes on the membranes and connections between the p e r i p heral and i n t e r n a l thylakoids  ( F i g . 67, arrows) and between the envelope  and peripheral thylakoid i n F i g . 68 (arrows).  Genophores appear to be much  less numerous i n the chloroplasts of t h i s stage. of a genophore may  In F i g . 68 the DNA  fibrils  be seen to be c l o s e l y associated with the peripheral  thylakoid. E.  The Four-celled Tetrasporangium The  chloroplasts of the 4-celled stage are s i m i l a r i n shape and s i z e  to those of the previous stage.  However, the outer surfaces of the t h y l a -  koids are much more densely covered with phycobilisomes.  Definite  tions of the peripheral thylakoid with the i n t e r n a l thylakoids may (Fig. 70 arrows).  continuabe seen  Some branching of the i n t e r n a l thylakoids appears to be  21  possible ( F i g . 71, 72 arrows).  Although d i v i s i o n s of the chloroplasts appear  to be reduced, occasional d i v i s i o n p r o f i l e s may  be encountered.  p l a s t undergoing a multiple d i v i s i o n i s shown i n F i g . 71.  A chloro-  The number of  genophores seems to be increased i n the chloroplasts of t h i s stage, as compared to the noticeable lack of them i n the chloroplasts of the previous stage.  DISCUSSION  I  O r i g i n of Proplastids The mature chloroplasts of Antithamnion subulatum which make up  the  greatest percentage of the p l a s t i d types i n the tetrasporangial mother c e l l are s i m i l a r i n most structures to those of other Floridiophycidae previously  (3, 9, 10, 32, 34, 35).  d i s t r i b u t i o n of photosynthetic  described  With respect to the arrangement and  membranes and the genophores, these chloro-  p l a s t s resemble c l o s e l y the chloroplasts of Laurencia G r i f f i t h s i a sp. (35) and Lomentaria baileyana  (9).  s p e c t a b i l i s (3),  There are differences  i n the organization and the number of the i n t e r n a l thylakoids, since the chloroplasts of Antithamnion subulatum are elongate and lobed rather than l e n t i c u l a r , as are those of Laurencia  The  and  structures,  Griffithsia.  term "outer l i m i t i n g d i s c " which Bouck (1962) used to  describe  the thylakoid running p a r a l l e l to the chloroplast envelope i s not used here, as i t implies that this membrane may p l a s t envelope.  act as a b a r r i e r , s i m i l a r to the chloro-  I t i s true that i n Antithamnion subulatum, (as i n other red  algae which have been described  as having this single outer thylakoid), the  genophores and i n t e r n a l thylakoids are never found outside this membrane system.  However, since i t may  be discontinuous  i n places, i t provides no  22 b a r r i e r 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 d i s t i n g u i s h i t from the i n t e r n a l photosynthetic membranes, or thylakoids.  As shown, both the peripheral and i n t e r n a l thylakoids of Antithamnion subulatum have on t h e i r outer surfaces numerous small granules which are assumed to be phycobilisomes.  The concentration of these on the membrane  surfaces i s much lower than the concentration reported f o r Porphyridium cruentum (21), I \  aerugineum (22) or G r i f f t h s i a flosculosa (34, 35).  these, the phycobilisomes  are arranged  In  regularly and are separated by a  regular distance, whereas i n A. subulatum they are i r r e g u l a r l y scattered, s i m i l a r to those of Laurencia s p e c t a b i l i s (3). observed  Also, the  phycobilisomes  i n A. subulatum appear to be smaller i n s i z e , averaging 270A,  whereas those reported by Gantt and Conti i n P_. cruentum are 320 A.  Observations  approximately  (12) on cultured material of A. glanduliferum K y l i n  and A. pacificum (Harvey) K y l i n revealed much higher concentrations of phycobilisomes, and sizes that are c l o s e r to those found by Gantt Conti (21.)  and  This leads to the speculation that f i e l d material may  fewer and smaller phycobilisomes  have  than has cultured m a t e r i a l .  The genophore of the mature chloroplasts of A. subulatum are s i m i l a r i n s i z e and d i s t r i b u t i o n to those observed  i n Porphyra tenera (42) P o l y s i -  phonia elongata (42), and Laurencia s p e c t a b i l i s (3). DNA  The dimensions of the  contained within the genophores conform to previous reports (3, 42).  What has not been reported before with respect to chloroplast DNA  i n red  algae, but what i s observed here, i s the connection between the DNA  fibrils  23  and both the peripheral and i n t e r n a l thylakoids  (Fig. 19-21, 68). Connections  between chloroplast and b a c t e r i a l DNA and t h e i r respective membranes have been reported previously using both t h i n sectioned material and material spread by the Kleinschmidt  method (4, 5, 6, 16). Such connections between  DNA and membranes are thought to be important i n segregation of the newly r e p l i c a t e d DNA molecules.  Proplastids have been observed i n 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 i n the a p i c a l c e l l s , which i s consistent with early investigations with the l i g h t microscope (18) which showed that many red algae have almost colourless a p i c a l c e l l s .  They may  occur i n combination with a few immature chloroplasts (9, 10), while i n some species they appear to comprise a pure population  (29) . U l t r a s t r u c t u r a l  i n v e s t i g a t i o n also indicates that proplastids may be more or l e s s r e s t r i c t e d to members of the Florideophycidae.  Thus far there i s only one report of  proplastids i n a member of the Bangiophycidae (33).  Most authors (9, 10, 11, 29) describe the p r o p l a s t i d i n i t s simplest form as being a small s p h e r i c a l (with a diameter ranging from 0.5 - 1.2^u ) to an i r r e g u l a r l y ovoid body which i s bounded by a double membrane envelope. Immediately i n s i d e i s a second double membrane system, the peripheral thyl a k o i d , which runs p a r a l l e l to the envelope. thylakoid i s present  Although this peripheral  at some stage i n the proplastids described, Bouck (1962)  suggests that i n the simplest form, proplastids of Lomentaria baileyana  lack  t h i s membrane system, and that i t i s derived during the very early d i f f e r e n t i a t i o n of the p r o p l a s t i d , from an invagination of the i n t e r n a l membrane of the envelope.  On the other hand, the proplastids of red algae  described  24  by Brown and Weier (1968), Ramus (1969) and L i c h t l e and Giraud (1969) a l l apparently have the peripheral thylakoid from the s t a r t .  A great deal of  degeneration of the mitochondria i s evident i n the permanganate fixed material of Lomentaria,  and i t i s here considered possible that some of  the very small structures (averaging 0.2 p. i n diameter) which Bouck (1962) defines as p r o p l a s t i d s without a peripheral thylakoid, may cular components of broken down mitochondria.  i n fact be v e s i -  Certainly the s i z e reported  by Bouck f a l l s considerably below the average s i z e indicated above.  Genophores have been shown to occur i n the proplastids of Polysiphonia elongata (29) and i n the very young chloroplasts of Pseudogloiophloea (37),  confusa  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 p r o p l a s t i d s of Antithamnion  subulatum observed i n the present  study are i n most respects s i m i l a r to others referred to above.  They appear  to have the p e r i p h e r a l thylakoid from the beginning, and most of those observed contain a genophore.  However, i n 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  p a r t l y with the development of proplastids into the mature c h l o r o p l a s t s . However, to my knowledge, there has been no study directed toward tracing the o r i g i n of these proplastids i n the red algae. p o s s i b i l i t i e s which may  account  f o r t h e i r presence.  There are a number of F i r s t , they may  be  present i n a l l c e l l s of these algae i n very small numbers, only reaching high concentrations i n the a p i c a l c e l l s which are the a c t i v e l y growing and  25 d i v i d i n g c e l l s of the red a l g a l t h a l l u s (18).  Such a s i t u a t i o n would pose  no serious problem to t h e i r continuity from c e l l to c e l l or from to generation.  generation  I t means that most of those proplastids i n the a p i c a l system  would have to undergo growth, d i v i s i o n , and d i f f e r e n t i a t i o n continuously  to  maintain the c h a r a c t e r i s t i c chloroplast number per c e l l , while a small r e s i dual number might remain temporarily  s t a t i c and be transmitted  to the  a p i c a l portion of the t h a l l u s during cytokinesis of the a p i c a l c e l l . transmission  from generation  included i n the reproductive  to generation  subTheir  requires that one or more be  u n i t , and a return to a rapid d i v i s i o n rate  among these proplastids during formation of the unit and i t s subsequent germination would guarantee the continuity of chloroplasts, and reestablish the a p i c a l system of the next generation. for  This seems a p l a u s i b l e system,  red algae, except for the evidence presented by Manganot (31) from l i g h t  microscope studies.  He indicates that carpogonia of Lemanea become colour-  l e s s only a f t e r a v i s i b l e fragmentation of the f u l l y pigmented p l a s t i d s . problem i s also presented i n those algae which have recognizable  A  chloroplast  i n the a p i c a l c e l l from the s t a r t , but which s t i l l produce colourless reproductive  structures.  A second p o s s i b i l i t y , though an u n l i k e l y one,  is  that proplastids are formed jie novo, through blebbing of the nuclear as suggested by B e l l and Muhlethaler (1).  envelop  F i n a l l y , the proplastids may  derived i n some manner from the mature chloroplasts.  There are two  be  reports  i n which structures that resemble proplastids have been observed i n contact with mature chloroplasts; one i s that described e a r l i e r (Maltzhan and Muhlethaler, 1962).  In the other, Nichols, Ridgeway and Bold  (33)  report  connections between p r o p l a s t i d - l i k e structures and mature chloroplasts i n the red alga, Compsopogon.  However, they do not discuss the s i g n i f i c a n c e  26 of such a phenomenon.  The p r o p l a s t i d - l i k e structures described  i n the observations  have so  many features i n common with the proplastids observed free i n the t e t r a s 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.  I t can therefore be concluded that i n A. subulatum, proplastids  are formed i n the tetrasporangial mother c e l l and tetrasporangial from mature chloroplasts.  The  initial  following discussion of the formation of  proplastids i s i l l u s t r a t e d diagrammatically i n F i g . 45,  46.  There i s a set of requirements which must be f u l l f i l l e d during formation of a p r o p l a s t i d from a parent structure: be transmitted  the  a complete genome must  from the parent structure which w i l l guarantee the  capacity  of the p r o p l a s t i d to grow and d i f f e r e n t i a t e ; there must also be present i n the p r o p l a s t i d the molecular machinery which i s capable of t r a n s l a t i n g and transcribing the transmitted  information.  The process observed here f u l f i l l s  these requirements, at l e a s t at a physical l e v e l . Based on the r e s u l t s of t h i s study and two previous works (30,33) i t i s suggested that proplastids may of the mature chloroplasts.  originate through a l o c a l i z e d blebbing  This involves the extension  of the mature  chloroplast envelope, the peripheral thylakoids, possibly the i n c l u s i o n of one or two  i n t e r n a l thylakoids, as well as a p e r i p h e r a l l y oriented genophore  and a small amount of stroma.  However, several questions remain unanswered;  i t i s not known for c e r t a i n whether the DNA  i s attached  to the peripheral  thylakoid, and whether t h i s attachment i s required to guarantee the i n c l u s i o n  27 of the genome i n the developing p r o p l a s t i d .  Although i t i s possible that  the genophore could be c a r r i e d passively with the i n t r u s i o n of the stroma from the mature chloroplast, an attachment to the peripheral membrane i s assumed on the basis of a strong s i m i l a r i t y between the observations  here,  and those of other works reviewed elsewhere i n this thesis (4, 5). I t i s also not known whether the bleb r e s u l t s from a l o c a l i z e d overgrowth of the envelope and peripheral thylakoid i n the v i c i n i t y of a peripheral genophore; or whether i t may occur randomly over the surface of the chloroplast. most of the developing proplastids examined, a genophore occurred  In  i n the  mature chloroplast adjacent to the connection, as w e l l as within the body of the forming p r o p l a s t i d .  This organization i s suggestive of a segregation  process following the r e p l i c a t i o n of the DNA within the genophore.  I t seems,  therefore, that during t h i s process the attachment of DNA to the membranes of the i n t e r n a l and peripheral thylakoids could become s i g n i f i c a n t . The i n c l u s i o n of a s i n g l e genophore per developing p r o p l a s t i d suggests that the genophores of mature chloroplasts are i d e n t i c a l , and each genophore contains enough information  f o r the d i f f e r e n t i a t i o n and functioning of a new chloro-  plast .  The way i n which the i n t e r n a l thylakoids a r i s e i n these developing proplastids i s also uncertain.  I t i s possible that once the forming pro-  p l a s t i d i s separated from the parent chloroplast, the new i n t e r n a l thylakoids are derived from the peripheral thylakoid as described previously by Bouck (1962), Brown and Weier (1968), Nichols et al( 1,966) and L i c h t l e and Giraud (1969).  However, there i s some i n d i c a t i o n that these i n t e r n a l thylakoids  seen within the developing and s t i l l attached  p r o p l a s t i d may, i n some cases,  28 already contain phycobilisomes  ( F i g . 43,44).  This suggests the i n c l u s i o n  of short terminal portions of the i n t e r n a l thylakoids of the mature chlorop l a s t , as indicated diagrammatically  i n F i g . 46.  might occur i s presented diagrammatically  The way  i n which this  i n F i g . 46, which i s based on the  chloroplast shown i n F i g . 21.  The observations made i n this study show that p r o p l a s t i d s , at least i n A. subulatum, are derived from mature chloroplasts and therefore provide a direct continuity. and Schimper i n 1883 ing  This further supports  the o r i g i n a l hypothesis of Meyer  that a l l chloroplasts are derived from previously e x i s t -  c h l o r o p l a s t s . However, this must be considered a preliminary i n v e s t i g a -  t i o n , and much work at the u l t r a s t r u c t u r a l and biochemical l e v e l s i s required to  II  c l a r i f y the points of uncertainty raised by t h i s i n v e s t i g a t i o n .  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 p a r t i c u l a r l y i n t r i g u i n g i n those genera i n which the reproductive unit i s at some point i n i t s development colourless.  In reference to Kylin's  work on Fucus serratus, Bold (1951) reports that the a n t h e r i d i a l  initial  contains a small number of pale p l a s t i d s which apparently become completely colourless by the 8-celled stage.  This colourless condition p e r s i s t s u n t i l  the 64-celled stage, when pigmentation associated with a s i n g l e small p l a s t i d . occurrence  i n the developing carpogonia  i s restored, and each nucleus becomes Manganot (1922) reports a s i m i l a r of Lemanea, and Drew (1951) indicates  that the tetrasporangial i n i t i a l of the red algae i s most frequently colourless.  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  i n at l e a s t  some algae.  The  r e s u l t s of the present study on A. subulatum indicate that pro-  p l a s t i d s are formed from the mature chloroplasts throughout the development of the tetrasporangium.  Since proplastids are found i n the  mother c e l l , even p r i o r to the production i t i s probable that t h e i r production v i s i b l e growth of the i n i t i a l .  tetrasporangial  of the tetrasporangial i n i t i a l ,  commences before the onset of  any  Whether this indicates that the process i s  continuous i n a l l of the mature c e l l s , or perhaps only the p o t e n t i a l mother c e l l s , i s impossible apparently  to say from our present knowledge.  Proplastids  also produced by the mature chloroplasts within the  are  tetrasporangial  i n i t i a l at l e a s t i n stage C, and i t i s quite l i k e l y that t h i s occurs i n t e r mittently throughout the development up to and including the very early s i n g l e - c e l l e d tetrasporangium.  Immature chloroplasts, some of which appear  to be undergoing d i v i s i o n , have been observed i n , as well as c l o s e l y associated with,-the stage C i n i t i a l .  The study also shows that during the l a s t  part of stage C (which occurs j u s t before the developing i n i t i a l i s cut o f f from the mother c e l l ) and during the young s i n g l e - c e l l e d stage, there i s a d i v i s i o n of at l e a s t some of the large mature chloroplasts which have been forced into the i n i t i a l during i t s growth. chloroplasts may  be multiple, with as many as f i v e c o n s t r i c t i o n s having been  seen per chloroplast. may  As shown, the d i v i s i o n s of these  This indicates that at l e a s t s i x small d i s c o i d p l a s t i d s  be derived from one mature chloroplast.  This type of d i v i s i o n has been  reported e a r l i e r by Nichols, et a l (1966) i n 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 i n Antithamnion. The results thus f a r obtained show that proplastids and forming proplastids occur much more frequently i n the t e t r a sporangial mother c e l l than i n the tetrasporangial i n i t i a l .  This does not  necessarily mean that d i v i s i o n of the mature chloroplasts i s the major c o n t r i butor to the increase i n chloroplast numbers which i s seen i n the 2-celled and 4-celled  tetrasporangium.  The r e s u l t s of previous works on the growth and d i f f e r e n t i a t i o n of proplastids indicate that this process i s probably quite rapid (3, 9,  29)  and that d i v i s i o n s of the immature chloroplasts i n the a p i c a l c e l l s are probably both rapid and frequent (29).  Further i n v e s t i g a t i o n of a f i n e l y  graded series of stages between stage B and the formation of the t e t r a sporangium might reveal the r e l a t i v e importance of these two methods of chloroplast formation i n the continuity of p l a s t i d s through  tetraspore  formation. One fact that has been shown by this study i s that the pale  colour-^  ation of the i n i t i a l and colourless condition of the young tetrasporangium i s 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 chloroplasts.  The r e s u l t s indicate the presence of phycobilisomes on the  of the chloroplasts.  thylakoids  The r e s u l t s indicate the presence of phycobilisomes  on the thylakoids of the chloroplasts within the stage C i n i t i a l and even i n the e a r l i e s t s i n g l e - c e l l e d stage.  However, i n the rapidly d i v i d i n g chloro-  plasts of the nearly mature tetrasporangium phycobilisomes are nearly  absent.  31 Investigation of subsequent stages ( i . e . the 2-celled and 4-celled t e t r a sporangia) with the l i g h t microscope reveals a considerable pigmentation.  These observations  increase i n  are borne out u l t r a s t r u c t u r a l l y by  the  reappearance of the phycobilisomes j u s t p r i o r to the 2-celled stage, and t h e i r increased numbers on the surface of the thylakoids i n the 4-celled stage.  Peyriere  (35) has shown that there i s an increase of s i z e i n the  chloroplasts and i n the number of i n t e r n a l thylakoids per chloroplast  during  the growth of the tetrasporangium of G r i f f i t h s i a from the j u v e n i l e to the mature stage.  However, from the observations  made i n 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 s i g n i f i c a n c e of thylakoid number i n the chloroplasts of the developing tetrasporangium.  The  reason for this i s that a f a i r l y high percentage of  these small d i s c o i d p l a s t i d s found i n the tetrasporangium are derived from the d i v i s i o n of the mature chloroplasts which were included i n the  initial;  t h i s means that the derived p l a s t i d s have, i n most cases, a number of i n t e r n a l thylakoids which i s very close to that of the parent p l a s t i d s . described  As  previously  (10, 11, 12) t h i s number i s i t s e l f v a r i a b l e , since the chloroplasts  of the tetrasporangial mother c e l l s found lowest on the t h a l l u s have the highest number of i n t e r n a l thylakoids.  Therefore,  observations  on numbers  of thylakoids are only s i g n i f i c a n t i f chloroplasts of the tetrasporangium are derived through the d i f f e r e n t i a t i o n of a r e l a t i v e l y pure population proplastids, or i f tetrasporangia  of  from comparable regions of the t h a l l u s are  observed.  Peyriere concentration  (35) also reports the presence of phycobilisomes i n low  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 d i v i s i o n figures among the chloroplast population within  the  32  tetrasporangium.  The tetrasporangia of G_. flosculosa and A. subulatum  are  both s e s s i l e upon the mother c e l l s , and i n comparable stages the u l t r a s t r u c t u r a l aspects of t h e i r development seems to be p r a c t i c a l l y i d e n t i c a l . The exception i s the presence of phycobilisomes i n the chloroplasts of G r i f f i t h s i a i n both the j u v e n i l e and the nearly mature tetrasporangium. However, since the period of lowest pigmentation during the development of the tetrasporangium i s of short duration (probably only a matter of a few hours), i t i s possible that the stages examined i n G r i f f i t h s i a did not  fall  within t h i s time range, though i t i s also possible that the method of p l a s t i d continuity i s not the same.  The question i s what i s the cause of the disappearance of the phycobilisomes evident i n the developing tetrasporangium of Antithamnion? explanations seem f e a s i b l e :  Two  the f i r s t i s 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 d i v i s i o n of t h i s stage.  The  second p o s s i b i l i t y i s that the same rate of production i s maintained, but that this production i s at a low enough rate i n comparison  to the rate of  growth of' the membranes that a considerable d i l u t i o n occurs, r e s u l t i n g i n a low phycobilisome d i s t r i b u t i o n per unit area of membrane.  This imbalance  would be gradually n u l l i f i e d as the d i v i s i o n and i n t e r d i v i s i o n a l growth rate of the chloroplasts slows down i n the 2-celled and 4-celled stages. There i s some i n d i c a t i o n within this study that the l a t t e r p o s s i b i l i t y i s the more probable.  The immature chloroplasts described e a r l i e r 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 t h e i r higher frequency of d i v i s i o n and rate of membrane growth.  One further observation chloroplast ontogeny.  has been made which i s highly relevant to  A considerable  increase i n the number of genophores  per chloroplast and i n the s i z e of the i n d i v i d u a l genophores has been noted i n the d i v i d i n g chloroplasts of the nearly mature tetrasporangium.  On the  other hand, sections of material taken through the 2-celled stage show very few genophores per p l a s t i d , although the s i z e remains f a i r l y constant.  There  appears to be nothing remarkable about the genophore i n the 4-celled stage. It has been shown that b a c t e r i a l chromosomes may be r e p l i c a t e d at a higher rate i n an a c t i v e l y growing culture with the r e s u l t that a s i n g l e c e l l may contain several genomes, and that a balance between number of genomes and c e l l s i s restored as the d i v i s i o n rate of the c e l l s f a l l s o f f (25).  The  occurrence of genophores i n greater numbers and of increased s i z e within these chloroplasts of the nearly mature s i n g l e - c e l l e d stage i s considered by t h i s author to be the v i s i b l e r e s u l t of an increased rate of DNA r e p l i c a t i o n i n response to the increased d i v i s i o n rate.  Presumably the DNA r e p l i c -  ation rate increases to i t s highest point p r i o r to the onset of the rapid chloroplast d i v i s i o n , and then drops o f f quite quickly, so that during the subsequent d i v i s i o n s there i s a segregation to the daughter chloroplasts.  of the newly formed genophores  This would result i n the lowest number of  genophores to the daughter chloroplasts.  This would r e s u l t i n the lowest  number of genophores being found i n the chloroplasts j u s t a f t e r the decline i n chloroplast d i v i s i o n s .  This stage corresponds to the 2-celled stage.  the 4-celled stage the chloroplast density i s reaching  i t s peak, with the  By  34  r e s u l t that the d i v i s i o n rate f a l l s o f f toward zero, and the genophores then a t t a i n t h e i r c h a r a c t e r i s t i c number r e l a t i v e to the s i z e of the  may  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 i n , and during the outgrowth of the i n i t i a l , and the mature chloroplasts which undergo a multiple d i v i s i o n provide the chloroplast c o n t i nuity between the tetrasporophytic and gametophytic generations. of these events i s presented i n F i g . 7 3 .  A summary  From present observations,  the  mature chloroplasts seem to be most important i n t h i s r o l e , but further work i s required to c l a r i f y  the r e l a t i v e values of each of these methods of chloro-  p l a s t formation during tetraspore development.  I t may  be added here as an  incentive to further study that the s i g n i f i c a n c e of p r o p l a s t i d formation may  not be i n tetraspore formation, but rather i n i t s germination.  function may  be much more s i g n i f i c a n t during the production  ductive structures as carpogonia and spermatia.  This  of such repro-  A number of the questions  which have been raised by t h i s 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  osmophilic granule  ax  =  axial c e l l  p  proplastid  c  =  mature chloroplast  pe  p l a s t i d envelope  dc  =  d i v i d i n g mature chloroplast  pg  plastoglobuli  DNA  =  DNA f i b r i l s  ph  phycobilisome  fp  =  forming p r o p l a s t i d  pis  p r o p l a s t i d - l i k e structure  ge  =  genophore  pt  peripheral thylakoid  ic  =  immature chloroplast  s  starch grains  it  =  i n t e r n a l thylakoid  sp  septation  lb  =  l a t e r a l branchlet  t  tetrasporangium  n  =  nucleus  ne  =  nuclear envelope  nu  =  nucleolus  ti tmc  tetrasporangial i n i t i a l tetrasporangial mother cell  w  wall  36 Plate I  Figure 1.  Diagrammatic representation of the thallus of Antithamnion subulatum. showing sequential production of tetrasporangia.  37  Plate I I Figure 2.  Light micrograph of stage A t e t r a s p o r a n g i a l i n i t i a l ( t i ) x  Figure 3.  1200  Light micrograph of stage B tetrasporangial i n i t i a l ( t i , top c e l l ) and stage C ( t i , lower c e l l ) . a p i c a l cytoplasm of the i n i t i a l s  Note that the  i s denser and more homo-  genous than that of the mother c e l l . x Figure 4.  1200  Light micrograph of three early stages i n the development of the tetrasporangium.  Septation i s incomplete i n the  upper c e l l , and the nucleus has not yet become evident. x Figure 5.  520  Light micrograph of nearly mature one--celled tetrasporangium. Nucleus with conspicuous nucleolus (jm) i s evident.  Apical  cytoplasm remains homogeneous. x Figure 6.  1300  Light micrograph of mature tetrasporangium and 2-celled tetrasporangium.  Note even d i s t r i b u t i o n of very granular  cytoplasm i n the mature tetrasporangium. x Figure 7.  530  Light micrograph of three, 4-celled tetrasporangia. Lower right i s the youngest of the three. granular cytoplasm.  Note retention of  38  Plate I I I  Figure 8.  Phase contrast micrograph showing the morphology of the mature chloroplast (c) i n the tetrasporangial mother c e l l . Numerous s p h e r i c a l 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 Figure 9.  1500  Phase contrast micrograph showing s i m i l a r chloroplast morphology i n c e l l of a l a t e r a l branchlet.  Several pro-  p l a s t i d s are evident, as are c o n s t r i c t i o n s of the mature chloroplasts. x Figure 10.  1500  Low^ power electron micrograph of median l o n g i t u d i n a l section of mother c e l l . grains  (s).  A x i a l cytoplasm i s f i l l e d with starch  Portions of the larger mature chloroplasts  (c) are seen i n 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 t i g h t packing of the chloroplasts ( c ) . are c l e a r l y  Numerous scattered genophores  (ge)  evident. x  16,400  Tangential section of mother c e l l showing a portions of two mature chloroplasts (c) and one immature chloroplast (ic).  P l a s t o g l o b u l i may  p l a s t (pg). ing  be seen i n both types of chloro-  A lobe free of i n t e r n a l thylakoids and  a genophore may  contain-  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 i n t e r n a l ( i t ) 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 and i n t e r n a l thylakoids koids i s evident  (it).  (pt)  Branching of i n t e r n a l t h y l a -  (white arrows).  Genophores (ge) are seen  between the i n t e r n a l thylakoids, interrupting the continuity of i n t e r n a l thylakoids  ( s o l i d arrows) and crossing an i n -  t e r n a l thylakoid (dotted arrows). x Figure 15.  24,000  A small portion of the tetrasporangial mother c e l l showing a p r o p l a s t i d (p) c l o s e l y associated with a large a l l y oriented chloroplast ( c ) . terminating  peripher-  Further examples of genophores  i n t e r n a l thylakoids may  be seen  (arrows).  Al Plate VI  Figure 16.  Part of mature chloroplast (c) showing genophores (ge) c l o s e l y associated with the peripheral thylakoid i n the l a t e r a l aspect of the chloroplast. x  Figure 17.  31,600  Portions of two chloroplasts showing genophores (ge) associated with the peripheral thylakoid (pt) both l a t e r a l l y and terminally. x  Figure 18.  31,000  A genophore i n the apex of a lobe of a mature chloroplast. Association between genophore (ge) and peripheral thylakoid (pt) i s again evident. x  Figure 19.  18,600  Higher power micrograph showing one complete genophore (ge) c l o s e l y associated  (arrow) with the peripheral thylakoid  (pt) as well as i n t e r n a l thylakoids ( i t ) . x  51,000  42  High power electron micrograph showing genophore (ge) i n terminal portion of mature chloroplast. 25-30 A. thylakoids  Some of the f i n e s t  DNA f i b r i l s may be seen attached to the i n t e r n a l ( i t ) , seen here i n oblique section, and to the  peripheral thylakoid (lower arrows). x  72,200  A l o n g i t u d i n a l section of an immature chloroplast ( i c ) containing a large genophore (ge) and showing reorientation of i n t e r n a l thylakoids associated with subterminal swelling. A connection between the i n t e r n a l and peripheral may also be seen (white arrow).  thylakoids  The mature chloroplast  above (c) also shows a subterminal swelling containing a genophore (black arrow). x  55,400  43 Plate VIII  Figure 22.  A p r o p l a s t i d (p) c l o s e l y 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 Figure 23.  22,300  High power view of same p r o p l a s t i d shoving r e l a t i o n s h i p of peripheral thylakoid to p l a s t i d envelope, and s l i g h t l y eccentric genophore containing DNA f i b r i l s .  Phycobilisomes  may be seen on the surfaces of the peripheral thylakoid. x Figure 24.  50,000  A s i n g l e p r o p l a s t i d containing a s i n g l e i n t e r n a l thylakoid ( i t ) and genophore (ge) which i s c l o s e l y associated with the peripheral thylakoid ( p t ) .  Phycobilisomes (ph) are  again evident. x Figures 25, 26, 27.  49,500  Adjacent sections showing a p r o p l a s t i d (p) l y i n g close to the nucleus (n). Figure  25  Figure 26, 27  x  49,000  x  52,000  44  Plate IX  Figure 28.  P r o p l a s t i d (p) containing  single i n t e r n a l thylakoid and  genophore (ge) i s shown to be closely appressed to the mature chloroplast  (c) i n top h a l f of micrograph. x  Figure 29.  50,700  High power micrograph of the connection between a prop l a s t i d - l i k e - s t r u c t u r e (pis) and a mature chloroplast (c). The  structure contains a peripheral thylakoid  bilisomes  (ph) and a genophore (ge).  (pt), phyco-  Continuity between  the p r o p l a s t i d - l i k e - s t r u c t u r e and the mature chloroplast may be seen (arrows). x Figure 30.  53,200  S e r i a l section of connection shown i n Figure 29, showing continuity of peripheral thylakoid with that of mature chloroplast  (arrows). x  52,500  45  Plate X  Figures 36-36  S e r i a l sections taken through the connection between a p r o p l a s t i d - l i k e structure (pis) and a mature chloroplast (c), showing that four sections are required to pass through the genophore (ge) of the p r o p l a s t i d - l i k e - s t r u c t u r e and mature chloroplast.  Note phycobilisomes i n 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 chloroplast  mature  (c) and single p r o p l a s t i d - l i k e - s t r u c t u r e , i n  a x i a l cytoplasm, ( p i s ) , containing a single i n t e r n a l t h y l a koid and genophore. x Figure 38.  15,600  Adjacent section of a stage C i n i t i a l , showing the attachment (arrow) of the p r o p l a s t i d - l i k e structure  (pis) to the  l a t e r a l aspect of the mature chloroplast ( c ) . x  15,600  47 Plate XII  Figure 39-42.  Sequence of sections of the p r o p l a s t i d - l i k e - s t r u c t u r e (pis) i n a stage C i n i t i a l showing the connection with the mature chloroplast.  The connection i s s l i g h t l y oblique through  the point of connection; hence the membrane continuity i s not c l e a r i n the connecting piece (arrows, Figs. 41 and 42.  Fine f i b r i l s of DNA i n the genophore are c l e a r l y e v i -  dent i n Figures 41 and 42.  Inserts, Figures 39 and 40.  Figure 39  x  42,700  Figure 40  x  43,200  Figure 41  x  44,600  Figure 42  x  44,600  High power micrograph showing  s e 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 p r o p l a s t i d - l i k e - s t r u c t u r e (pis) connected to a mature chloroplast ( c ) . envelope (arrows).  Note continuity of p l a s t i d  P r o p l a s t i d - l i k e - s t r u c t u r e contains  two i n t e r n a l thylakoids. x Figure 44.  S e r i a l section of Figure 43.  53,000  Note proximity of mature  chloroplast genophore to connection, and i n c l u s i o n of genophore i n p r o p l a s t i d - l i k e structure. x  54,400  49 Plate XIV  Figure 45.  Diagrammatic representation of p r o p l a s t i d formation.  Figure 46.  Diagrammatic representation of p r o p l a s t i d formation, showing how i n t e r n a l thylakoid may be included i n the p r o p l a s t i d .  50 Plate XV  Figure 47,  Electron micrograph of s l i g h t l y oblique, median l o n g i t u d i n a l section of stage A tetrasporangial i n i t i a l . (c) follow contours of outgrowth of i n i t i a l grains  (ti).  Starch  (s) are present i n the mother c e l l cytoplasm.  small portion of the nucleus (n) i s also  A  evident. x  Figure 48.  Chloroplasts  5,700  Stage B i n i t i a l , f i l l e d with mature chloroplasts ( c ) . (s) at t h i s stage seems to be confined to the mother cytoplasm.  Starch cell  Note the large nucleus (n) and conspicuous  nucleolus (nu). x Figure 49.  4,900  Apex of the stage B i n i t i a l shown above, showing s i m i l a r i t y of mature chloroplasts to those found i n mother c e l l .  Note  the dense cytoplasm of the apex. x 15,300 Figure 50.  Lobe of mature chloroplast which i s being cut o f f i n stage B initial.  Note c o n s t r i c t i o n , indicated by arrows. x 16,000  Figure 51.  P r o p l a s t i d observed i n stage B i n i t i a l . x 27,500  51  Plate XVI  Figure 52.  Longitudinal section of l a t e stage C i n i t i a l .  Note the  continuing high concentration of chloroplasts (c) i n apex. Starch (s) i s found i n the a x i a l cytoplasm. x  5,100  Insert (a). Enlargement of boxed area i n Figure 52 showing d e t a i l s of mature chloroplasts i n l a t e stage C i n i t i a l . Phycobilisomes (ph) are evident on outer surface of t h y l a koids . x  51,800  Insert (b). Beginning of septation of l a t e stage C i n i t i a l evident i n f i r s t  few tangential cuts of this  initial. x  Figures 53 and 54.  13,800  S e r i a l sections of a d i v i d i n g immature chloroplast ( i c ) observed i n the apex of a stage C i n i t i a l .  Note the small  number of thylakoids and t h e i r reorganization. genophores are present (ge).  Numerous  52 Plate XVII  Figures 55 and 56.  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 ( i c ) are evident, one of which i s d i v i d i n g .  A pro-  p l a s t i d (p) i s also evident.  Figure 57.  Figure 55  x  16,200  Figure 56  x . 26,000  Very young single c e l l e d stage showing high density of chlorop l a s t s (c) and c e n t r a l l y oriented starch grains ( s ) . Prop l a s t i d v i s i b l e i n boxed area.  x Figure 58.  5,900  High power of boxed area showing p r o p l a s t i d (p).  x  41,000  53 Plate XVIII  Figure 59.  Young tetrasporangium ( t ) . Multiple d i v i s i o n of mature chloroplast i s evident on the l e f t hand side of the c e l l (see arrows i n d i c a t i n g c o n s t r i c t i o n s ) . A p i c a l cytoplasm of the tetrasporangium remains dense and i s f i l l e d with chloroplasts ( c ) . Numerous starch grains  (s) surround the  conspicuous, c e n t r a l l y located nucleus (n). x Figure 60.  S l i g h t l y older tetrasporangium.  3,600  Most of the p l a s t i d s are  d i s c o i d and several of these appear to be undergoing  divi-  sions (see arrows at c o n s t r i c t i o n s ) . x Figures 61 and 62.  5,200  Chloroplasts of nearly mature tetrasporangium undergoing division.  D i v i s i o n 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 and 6 4 .  Higher magnification of d i v i d i n g chloroplasts from nearly mature tetrasporangium,  showing d i v i s i o n planes, reorienta-  t i o n of i n t e r n a l thylakoids and numerous genophores. that phycobilisomes  are scarce and i n d i s t i n c t ,  Note  (ph).  Figure 63  x  40,000  Figure 64  x  38,800  55 Plate XX  Figure 65.  Low  power electron micrograph of l o n g i t u d i n a l section of  2-celled tetrasporangium. (w).  Note wall separating  the 2 spores  Almost a l l chloroplasts are discoid and several within  the plane of sectioning, marked (c) are undergoing d i v i s i o n . x Figure 66.  4,500  Higher power micrograph of boxed area i n Figure 65., d e t a i l s of the d i v i d i n g chloroplasts.  showing  D i v i s i o n i s unequal  i n the chloroplast upper r i g h t . x Figure 67.  A s i n g l e chloroplast from a 2-celled tetrasporangium. nections between the i n t e r n a l thylakoids and thylakoid are c l e a r l y discernable  Con-  peripheral  (arrows). x  Figure 68.  13,900  27,600  A portion of a chloroplast from a 2-celled tetrasporangium. The  section i s tangential to the surface of an i n t e r n a l  thylakoid ( i t ) upon which phycobilisomes (ph) may  be seen.  A genophore (ge) i s evident, associated with the peripheral thylakoid (pt) . A possible connection also e x i s t s between the outer membrane of the peripheral thylakoid and the membrane of the chloroplast envelope.  (arrow) x  40,900  inner  56 Plate XXI  Figure 69.  Portion of a four c e l l e d tetrasporangium.  Note the r e l a t i v e l y  thick w a l l (w) which separates the four tetraspores.  Phyco-  bilisomes are obvious on the thylakoids of a l l micrographs of this stage ( i e . Figs. 69-72). x Figure 70.  Longitudinal and cross sections of chloroplasts are present i n this micrograph. thylakoids i s evident  Continuity of i n t e r n a l and peripheral (arrow) i n cross s e c t i o n a l view. x  Figure 71.  15,000  M u l t i p l e d i v i s i o n of chloroplast.  35,800  A portion of another chloro-  plast undergoing d i v i s i o n may be seen i n the lower right of t h i s micrograph. x Figure 72.  24,000  Longitudinal section of chloroplasts of four c e l l e d sporanium.  tetra-  Branching of an i n t e r n a l 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 ation of tetrasporangia. are (a)  Two types of chloroplast  the formcontinuity  illustrated: Through formation of proplastids, occurring i n a l l stages up to and including the s i n g l e - c e l l e d stage,  (b)  through d i v i s i o n of mature chloroplasts, occuring stage C.  after  58  BIBLIOGRAPHY  1.  B e l l , P.R. and K. 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