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Studies on the brown alga Ectocarpus in culture : senescence, an ultrastructural study Oliveira, Luis Augusto Fernandes 1973

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STUDIES ON THE BROWN ALGA ECTOCARPUS IN CULTURE: SENESCENCE - AN ULTRASTRUCTURAL STUDY by LUIS AUGUSTO FERNANDES OLIVEIRA Licenciatura, University of Porto ( Portugal ), 19 65 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of BOTANY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1973 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f BOTANY The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 7 / 9 / 1 9 7 3 i i ABSTRACT A study of c e l l d i f f e r e n t i a t i o n and senescence on the brown alga Ectocarpus sp. was carried out using standard techniques of l i g h t - and electron microscopy as well as cytochemistry. The f i r s t 6-8 c e l l s i n each filament of Ectocarpus are characterized by a large round and well organized nucleus, a dense cytoplasm r i c h i n ribosomes, endoplasmic reticulum, dictyosomes, mitochondria, and chloroplasts with conspicuous stalked pyrenoids. Few small vacuoles are present i n the cytoplasm. New features not previously reported for the brown algae are also described. These include the presence of pore-like interruptions and bridge-like filaments i n the cisternae of dictyosomes and chloroplast thylakoids, as well as the formation of concen-t r i c bodies of chloroplast o r i g i n . Microbody-like organelles are also reported; cytochemical studies have shown catalase a c t i v i t y associated with them. Peroxidase a c t i v i t y i s repor-ted i n the c e l l wall, while acid phosphatase a c t i v i t y i s found i n association with both the endoplasmic reticulum and the Golgi elements, as well as inside the vacuoles. Adenosine triphosphatase i s present i n mitochondria, chloro-p l a s t thylakoids, and plasma membrane. These c e l l s , there-fore, can be described as meristematic or the immediate consequence of the d i f f e r e n t i a t i o n of the meristematic c e l l s . The processes of autophagy and vacuolation are studied i n d e t a i l and found to be p a r t i c u l a r l y s i g n i f i c a n t during the stages of c e l l d i f f e r e n t i a t i o n which lead to the aging of Ectocarpus sporophytic c e l l s . Increases i n vacuola-ti o n and autophagy are p a r a l l e l e d by an increase i n acid phosphatase a c t i v i t y . The ultimate r e s u l t s of these processes are: a reduction i n cytoplasmic matrix, a general deterioration of cytoplasmic organelles, and the formation of residual bodies which overcrowd the cytoplasm. Other i i i features are: the i r r e g u l a r i t y of the nuclear boundary, the disorganization of the E.R. system through v e s i c u l a t i o n , the increasing d i f f i c u l t y i n detecting mitochondria, the develop-ment of large stacks of chloroplast thylakoids as well as numerous patches of electron dense metabolites, d i s t i n c t from the p l a s t o g l o b u l i , and the formation of conspicuous c e l l wall ingrowths. Assays of enzyme systems other than acid phosphatase show t h e i r d i s t r i b u t i o n to be si m i l a r to those reported for young c e l l s . In the f i n a l stages of senescence the c e l l s become t y p i c a l l y necrotic; no enzymes can be l o c a l i z e d , except acid phosphatase whose reaction products are no longer compartmentalized but have become d i s t r i b u t e d a l l over the c e l l cavity. The nucleus and dictyosomes have disintegrated. Only small remnants of the E.R. system remain.y Mitochondria and chloroplasts have l o s t t h e i r i n t e r n a l organization, no cytoplasmic matrix can be detected. At the very end even the c e l l wall shows signs of disorganization. I t i s concluded that these c e l l s represent the f i n a l stage of autolysis. TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . . . .... . . . .... . 1 MATERIAL AND METHODS.......... . 6 OBSERVATIONS .... . . . . . . . . 11 PART I - LIGHT MICROSCOPE OBSERVATIONS 11 PART I I - ELECTRON MICROSCOPE OBSERVATIONS 13 A) ULTRASTRUCTURAL FEATURES OF YOUNG CELLS ( CELL TYPE #1 ) 13 B) ULTRASTRUCTURAL FEATURES OF THE TRANSITIONAL "1-2" CELLS 23 C) THE SENESCENT CELLS - ULTRASTRUCTURAL FEATURES OF THE "CELL TYPE #2" 26 D) THE SENESCENT CELLS - ULTRASTRUCTURAL FEATURES OF TRANSITIONAL "2-3" CELLS.. 30 E) THE SENESCENT CELLS - ULTRASTRUCTURAL FEATURES OF THE "CELL TYPE #3" 33 F) ENZYME LOCALIZATION 35 G) ULTRASTRUCTURAL IDENTIFICATION OF AGING PIGMENT 37 DISCUSSION 39 PART I - THE YOUNG CELLS ( CELL TYPE #1 ) 39 PART I I - TRANSITIONAL "1-2" CELLS 49 PART I I I - THE "CELL TYPE #2" 53 PART IV - TRANSITIONAL "2-3" CELLS 63 PART V - THE "CELL TYPE #3" 66 PART VI - ENZYME LOCALIZATION DURING DIFFEREN-TIATION AND SENESCENCE 70 CONCLUSION.. 76 LITERATURE CITED 78 KEY OF SYMBOLS AND PLATE EXPLANATION........ ......107 KEY OF SYMBOLS...... .. .. .. 108 PLATE EXPLANATION. . . . . . . . . 109 V LIST OF FIGURES FIGURE 1 Light microscope observation of an i n s i t u embedded filament ( prostrate system ) 2 Light microscope observation of an i n s i t u embedded filament ( erect system ) 3 Light microscope observation of an i n s i t u embedded filament ( prostrate system ) 4 Light microscope micrograph of a chloroplast 5 Light microscope micrograph of portion of a filament ( l i v i n g material ) 6 Chloroplast morphology ( l i v i n g material ) 7 Chloroplast morphology ( l i v i n g material ) 8 Pyrenoid d i v i s i o n ( l i v i n g material ) 9 Chloroplast and pyrenoid morphology ( l i v i n g material ) 10 C e l l wall ingrowths ( l i v i n g material ) 11 C e l l wall ingrowths ( l i v i n g material ) 12 Cytochemical i d e n t i f i c a t i o n of DNA containing structures ( l i g h t microscope micrograph ) 13 Cytochemical i d e n t i f i c a t i o n of DNA containing structures ( l i g h t microscope micrograph ) 14 Cytochemical i d e n t i f i c a t i o n of DNA containing structures ( l i g h t microscope micrograph ) 15 Cytochemical i d e n t i f i c a t i o n of RNA containing structures ( l i g h t microscope micrograph ) 16 Cytochemical i d e n t i f i c a t i o n of RNA containing structures ( l i g h t microscope micrograph ) 17 Cytochemical i d e n t i f i c a t i o n of RNA containing structures ( l i g h t microscope micrograph ) 18 Cytochemical. i d e n t i f i c a t i o n of proteins ( l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of proteins C l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of proteins ( l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of insoluble carbohydrates ( l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of insoluble carbohydrates ( l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of l i p i d s ( l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of l i p i d s ( l i g h t microscope micrograph ) Cytochemical i d e n t i f i c a t i o n of l i p i d s ( l i g h t microscope micrograph ) Schorml's reaction for the i d e n t i f i c a t i o n of l i p o f u s c i n ( l i g h t microscope micrograph ) Toluidine blue 0 staining ( l i g h t microscope micrograph ) Electron micrograph of a young c e l l ( prostrate system ) Plasmalemmasom.es Nuclear pores F i b r i l l a r aspect of chromatin Electron micrograph of a young c e l l ( erect system ) Electron micrograph of a young c e l l ( erect . system ) Electron micrograph of a young c e l l ( prostrate system ) Nuclear envelope- dictyosome association Endoplasmic reticulum - dictyosome association Freeze-etch r e p l i c a of the nuclear envelope Freeze-etch r e p l i c a of the- nuclear envelope v i i FIGURE 37a Plasmalemmasomes ( freeze-etch r e p l i c a ) 38 Portion of. the nucleus and perinuclear region of a young c e l l 39 Perinuclear space inclusions 40 Perinuclear space inclusions 41 Perinuclear space inclusions 42 Dictyosome morphology 43a Freeze-etch preparation of a chloroplast 43b D e t a i l of figure 43a 44 Endoplasmic reticulum morphology 45 Section passing through the perinuclear region of a young c e l l 46 Freeze-etch r e p l i c a of a chloroplast 47a Osmiophilic structured bodies ( o r i g i n ) 47b Osmiophilic structured bodies ( o r i g i n ) 48 Chloroplast-mitochondrion association 49 Tangential section through the region of the chloroplast E.R. 50 Chloroplast E.R. inclusions 51 Osmiophilic structured body ( morphology ) 52 Osmiophilic structured body ( morphology ) 53 Osmiophilic structured body-pyrenoid association 54 Discharge of osmiophilic structured bodies into the paramural space 55 Nuclear envelope-dictyosome association 56 Cross section through the periphery of a young c e l l ( concentration of mitochondria ) 57 Dictyosome morphology ( cisternae interruptions ) . 58 Dictyosome morphology ( cisternae interruptions ) 59 Dictyosome morphology ( bridge-like filaments ) 60 Dlctyosbme-pyrenoid association 61 Dictyosome-mitochondria association 62 Dictyosome-vacuole association v i i i FIGURE 63 Dictyosome-pyrenoid association 64 Dictyosome morphology ( lomasomes ) 65 Dictyosome morphology ( lomasbme o r i g i n ) 66 D e t a i l of a dictyosome derived v e s i c l e 6 7 Mitochondrion morphology 68 Mitochondrion morphology ( c r i s t a e inclusions ) 69 Nucleus-mitochondrion association 70 Chloroplast-mitochondrion association ( freeze-etch preparation ) 71 Mitochondrion-pyrenoid association 1 72 Mitochondrion-mitochondrion association 73 D e t a i l of the thylakoid system 74 Thylakoid architecture ( f r e e z e - e t c h preparation) 75 D e t a i l of a chloroplast 76 D e t a i l of a chloroplast 77 D e t a i l of the thylakoid system 7 8 Pore-like interruptions i n the thylakoids 79 Thylakoid architecture 80 Freeze-etch preparation of thylakoids 81 Freeze-etch r e p l i c a of a portion of a chloroplast 82 Chloroplast morphology ( pattern of thylakoid arrangement ) 83 Dictyosome morphology ( cisternae interruptions ) 84 Mitochondria morphology ( genophore ) 85 Whorled pattern of thylakoid arrangement 86 Concentric pattern of thylakoid arrangement 87 Abscission .of plastid-derived concentric bodies 88 Concentric body 89- Acid phosphatase a c t i v i t y associated with a concentric body 9 0 Acid phosphatase a c t i v i t y ( control preparation ) 91 D e t a i l of a concentric body 92 D e t a i l of a chloroplast ( bridge-like elements ) i x FIGURE 93 D e t a i l of a chloroplast and pyrenoid 94 Chloroplast morphology ( ribosomes ) 95 Freeze-etch r e p l i c a of a chloroplast ( general morphology ) 9 6 Thylakoid associated f i b r i l l a r elements 97 Plastoglobulus ( f i b r i l l a r composition ) 9 8 Plastoglobulus ( f i b r i l l a r composition ) 99 Plastoglobuli ( f i b r i l l a r composition ) 100 Endoplasmic reticulum-vacuole association 10.1 Chloroplast d i v i s i o n ( Laminaria type ). 102 Chloroplast d i v i s i o n ( Sphacelaria type ) 103 Chloroplast d i v i s i o n ( Sphacelaria type ) 104 P r o p l a s t i d - l i k e formation 105 Chloroplast d i v i s i o n ( p o t e n t i a l aspect ) 106 Chloroplast d i v i s i o n ( p o t e n t i a l aspect ) 107 Multiconstricted chloroplast 10 8 Chloroplast and pyrenoid morphology 109 Pyrenoid d i v i s i o n 110 Pyrenoid morphology 111 Plasmodesmata 112 Pyrenoid-vacuole association 113 C e l l wall morphology ( young c e l l ) 114 Pyrenoid d i v i s i o n 115 Cytoplasm morphology ( young c e l l ) 116 Vacuole development 117 C e l l wall ( freeze-etch preparation of a young c e l l ) 118 Provacuoles ( freeze-etch preparation ) 119 Autophagocytosis ( role of the E.R. ) 120 Autophagocytosis ( role of the E.R. ) 12.1 Autophagocytosis ( role, of the E.R. ) 1.22 Vacuole development 123 Vacuole development X FIGURE 1.24 Autophagocytosis ( r o l e of the tonoplast ) 125 Acid phosphatase (vacuole ) 1.26 Acid phosphatase ( vacuole ) 127 Vacuole development 12 8 Morphology of a aging c e l l 129 Acid phosphatase ( dictyosome ) 130 Acid phosphatase ( endoplasmic reticulum ) 131 Acid phosphatase ( control preparation ) 132 T r a n s i t i o n a l " 1-2 " c e l l s ; d e t a i l of a chloro-p l a s t 133 Permanganate f i x a t i o n ; d e t a i l of a chloroplast 134 " C e l l type #2 " ; permanganate f i x a t i o n 135 T r a n s i t i o n a l " 1-2. " c e l l ; general morphology 136 T r a n s i t i o n a l " 1-2 " c e l l ; general morphology 137 T r a n s i t i o n a l " 1-2 " c e l l ; cytoplasmic inclusions 138 T r a n s i t i o n a l " 1-2 " c e l l ; acid phosphatase a c t i v i t y 139 T r a n s i t i o n a l " 1-2 " c e l l ; acid phosphatase a c t i v i t y 140 T r a n s i t i o n a l " 1-2 " c e l l ; acid phosphatase a c t i v i t y ( control preparation ) 141 T r a n s i t i o n a l " 1-2 " c e l l ; d e t a i l of the dictyosome region 142 T r a n s i t i o n a l " 1-2 " c e l l ; c e l l wall morphology 143 " C e l l type #2 "; general ultr a s t r u c t u r e of a prostrate system c e l l 144 " C e l l type #2 "; c e l l wall morphology 145 T r a n s i t i o n a l . " 2-3 " c e l l ; general, morphology 146 Transition zone.between a " c e l l . t y p e #3 " and a " c e l l type J.2 " 147 T r a n s i t i o n a l " 2-3 " c e l l ; cytoplasm x i FIGURE 148 " C e l l type #2 "; general ul t r a s t r u c t u r e of an erect system c e l l 149 " C e l l type #2 "; d e t a i l of a chloroplast 150 " C e l l type #2 "; d e t a i l of the cytoplasm 151 " C e l l type #2 "; general ultrastructure of a prostrate system c e l l 152 11 C e l l type #.2 "; c e l l wall morphology 153 " C e l l type #2 "; nucleus and chloroplast morphology 154 " C e l l type #2 "; chloroplast morphology 155 " C e l l type #2 "; d e t a i l of a chloroplast 156 " C e l l type #2 "; d e t a i l of a chloroplast 157 " C e l l type #2 "; d e t a i l of a chloroplast 158 " C e l l type #2 "; c e l l wall morphology 159 " C e l l type #2 "; d e t a i l of a chloroplast ( freeze-etch preparation ) 160 " C e l l type #2 "; general morphology 161 " C e l l type #2 "; c e l l wall ingrowth 162 " C e l l type #2 "; c e l l wall ingrowths 163 T r a n s i t i o n a l " 1-2 " c e l l ; c e l l wall ingrowth 164 T r a n s i t i o n a l " 2-3 " c e l l ; cytoplasm morphology 165 T r a n s i t i o n a l " 2-3 " c e l l ; disruption of the tonoplast 166 T r a n s i t i o n a l " 2-3 " c e l l ; chloroplast 167 T r a n s i t i o n a l " 2-3 " c e l l ; chloroplast 168 T r a n s i t i o n a l " .2-3 " c e l l ; pyrenoid 169 T r a n s i t i o n a l " .2-3 " c e l l ; chloroplast 170 T r a n s i t i o n a l " 2-3 " c e l l ; nucleus 171 T r a n s i t i o n a l " 1-2 " c e l l ; c e l l wall, ingrowth 172 T r a n s i t i o n a l " 2-3 " c e l l ; chloroplast 173 T r a n s i t i o n a l " 2-3 " c e l l ; mitochondrion 174 T r a n s i t i o n a l " .2-3 " c e l l ; mitochondrion 175 T r a n s i t i o n a l 2-3 " c e l l ; mitochondrion x i i FIGURE 176 T r a n s i t i o n a l " 2-3 " c e l l ; concentric body 177 T r a n s i t i o n a l " .2-3 " c e l l ; chloroplast 178 T r a n s i t i o n a l " 2-3 " c e l l ; d e t a i l of a chloro-p l a s t .. . . 179 T r a n s i t i o n a l " 2-3 " c e l l ; d e t a i l of a chloroplast 180 T r a n s i t i o n a l " 2-3 " c e l l ; chloroplast 181 T r a n s i t i o n a l " 2-3 " c e l l ; d e t a i l of the cyto-plasm 182 T r a n s i t i o n a l " 2-3 " c e l l ; d e t a i l of a chloroplast 183 T r a n s i t i o n a l " 2-3 " c e l l ; pyrenoids 184 " C e l l type #3"; c e l l wall morphology 185 T r a n s i t i o n a l " 2-3 " c e l l ; chloroplast 186 T r a n s i t i o n a l " 2-3 " c e l l ; chloroplast 187 " C e l l type #3 "; general ultrastructure of a prostrate system c e l l 188 " C e l l type #3 "; general ultrastructure of a prostrate system c e l l 189 " C e l l type #3 "; general ultrastructure of an erect system c e l l 190 " C e l l type #3 "; chloroplast 191 " C e l l type #3 "; acid phosphatase a c t i v i t y 192 " C e l l type #3 "; d e t a i l of thylakoid arrangement 193 " C e l l type #3 "; thylakoid disorganization 194 " C e l l type #3 "; thylakoid disorganization 195 " C e l l type #3 "; thylakoid disorganization 196 " C e l l type #3 "; mitochondria and cytoplasm morphology 197 " C e l l type #3 11; mitochondria morphology 198 " C e l l type #3 "; thylakoid disorganization 199 " C e l l type #3 "; p l a s t o g l o b u l i accumulation x i i i FIGURE 200 " C e l l type #3 "; chloroplast morphology 201 " C e l l type #3 "; d e t a i l of the cytoplasm .202 " C e l l type #3 "; mitochondrion 203 " C e l l type #3 "; acid phosphatase a c t i v i t y ( control preparation ) .204 " C e l l type #3 "; acid phosphatase a c t i v i t y 205 " C e l l type #3 "; acid phosphatase a c t i v i t y ( control^preparation ) 206 " C e l l type #3 "; acid phosphatase a c t i v i t y 207 " C e l l type #3 "; c e l l wall disorganization 208 " C e l l type #3 "; c e l l wall disorganization 209 " C e l l type #1 "; catalase a c t i v i t y i n a microbody-like organelle 210 " C e l l type #1 "; catalase a c t i v i t y i n a microbody-like organelle 211 " C e l l type #1 "; catalase ( control preparation) 212 " C e l l type #1 "; catalase ( mitochondrial deposition of reaction product ) 213 " C e l l type #2 "; catalase a c t i v i t y 214 " C e l l type #1 "; peroxidase a c t i v i t y i n the c e l l wall 215 " C e l l type #1 "; microbody morphology 216 " C e l l type #1 "; peroxidase a c t i v i t y i n the c e l l wall 217 " C e l l type #3 "; acid phosphatase a c t i v i t y 218 " C e l l type #1 "; peroxidase a c t i v i t y ( control preparation ) 219 T r a n s i t i o n a l " 1-2 " c e l l ; ATPase a c t i v i t y i n association with the mitochondrion .220 " C e l l type. #3 acid phosphatase a c t i v i t y 221 " C e l l type #2 "; peroxidase a c t i v i t y 222 " C e l l type #2 "; peroxidase a c t i v i t y ( control preparation ) x i v C e l l type #1 "; ATPase.activity ( Na +-K +-Mg + +-+ + ++ C e l l type #1 "; ATPase a c t i v i t y ( Na -K -Mg -+ + ++ C e l l type #1 "; ATPase a c t i v i t y ( Na -K -Mg -C e l l type #1 "; ATPase a c t i v i t y ( Mg + +- a c t i -++ C e l l type #1 "; ATPase a c t i v i t y ( Mg - a c t i -activated system ) " C e l l type #1 activated system ) ".' C e l l type #1 "; activated system ) " vated system ) " C e l l type #J vated system ) ATPase a c t i v i t y ( Na +-K +-Mg + +- activated system ) ; control, preparation T r a n s i t i o n a l " 1-2 " c e l l ; ATPase a c t i v i t y + + ++ ( Na -K -Mg - activated system ) ATPase a c t i v i t y ( Na +-K +-Mg + +- activated system ); control preparation T r a n s i t i o n a l " 1-2 " c e l l ; l i p o f u s c i n ( Fontana's reaction ) ATPase a c t i v i t y ( Mg + +- activated system ); control preparation " C e l l type #2 "; l i p o f u s c i n ( Fontana 1s reaction ) Lipofuscin ( control preparation ) Lipofuscin ( Schorml's reaction ) X V ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. T. Bisalputra for his guidance and encouragement. Thanks are also extended to the other committee members: Drs. R.F. Scagel, R.E. Foreman, J. Berger, and I.E.P. Taylor, for th e i r h e l p f u l suggestions and. c r i t i c i s m . The.author also expresses his gratitude to Prof. Drs. A. Rozeira and R. Salema (Porto University) without whose i n t e r e s t my staying i n Canada would have been impossible. I am thankful to Dr. D.L. Brown for the reading and c r i t i c i s m of the f i r s t d r a f t of the thesis, and to my frie n d Miss S. Shinn for a l l her help. Special :thanks are due to the Calouste Gulben-kian Foundation for a 3 year study grant. Thanks are due to Dr. G. Hughes for his help during the period of sabatical leave of Dr. T. Bisalputra. 1 INTRODUCTION I. Morphology of Young Vegetative C e l l s The ultr a s t r u c t u r e of brown a l g a l c e l l s has been the subject of several studies ( von Wettstein,1954; Manton and Clark,1956; Manton,1957, 1959; Berkaloff,1961, 1963; Gibbs,1962a, 1962b; Giraud,1962; Greenwood,19 64; Ueda,1961; Bouck,1965; Bisalputra,1966; Evans,1966; Manton, 1966b; Bisalputra and Bisalputra,1967; Greenwood,19 67; Loiseaux,1967; Bourne and Cole,1968; Cole and Lin,1968; Cole et al.,1968; Evans,1968; McCully,1968; Neushul and Liddle,1968; Bailey and Bisalputra,19 69; Bisalputra and Bisalputra,1969; Bisalputra and Burton,1969; Bouck,1969; Cole,19 69; Liddle and Neushul,19 69; Bisalputra and Bisalputra,1970; Cole,1970; Cole and Lin,1970; Bisalputra et al.,1971; Chi,1971; Hori,1971; Neushul,1971; Neushul and Walker,1971; Hori,1972; Feldman and Guglielmi,1972; Neushul and Dahl,1972a, 1972b; Davies et al.,1973 ). From these studies the basic u l t r a s t r u c t u r a l features of brown a l g a l c e l l s were established, i t also became apparent that some c e l l u l a r c h a r a c t e r i s t i c s such as the p o s i t i o n of the Golgi bodies, the presence or absence of pyrenoids,the pyrenoid' morphology, and the arrangement of plasmodesmata could serve as useful c r i t e r i a i n establishing relationships among major groups of t h i s class of algae ( Evans,1966, 1968 Cole and Lin,1968; Cole,1970; Scagel,1966 ). Chloroplasts and pyrenoids have been more intensively studied than other organelles. The nucleus ( Neushul and Dahl,1972a; Leedale, 1970; Neushul and Walker,1971 ) and plasmalemmasomes ( Cole and Lin,1970 ) also have attracted the attention of phycologists. However,, only few representatives of the Phaeophyta have been studied i n d e t a i l ( Bouck,1965; Bourne and Cole,1968; Cole and Lin,1968; Cole,1969, 1970; McCully, 2 1968 ) . The Ectocarpales comprise plants with the simplest forms among the brown algae ( Fritsch,1945; Smith,1955 ); hence, they hold an important p o s i t i o n i n brown a l g a l phylogenetic schemes. Previous research on members of the Ectocarpales has included studies on : Ectocarpus mitchellae ( Longest,1946 ); P y l a i e l l a l i t t o r a l i s ( Gibbs,1962a, 1962b) Gif f ordia . sp. ( Bouck.,1965 ); Ectocarpus conf ervoides and PylaieTla l i t t o r a l i s ( Evans,1966 ) r e p r e s e n t a t i v e s of the Myrionemaceae ( Loiseaux,1967 ); Ectocarpus acutus ( Bailey and Bisalputra,1969 ); Eudesme virescens ( Cole,1969- the genus Eudesme i s considered by some investigators to belong to the order Chordariales, see Scagel,1966, for a review ); Ectocarpus b r e v i a r t i c u l a t u s , G i f f o r d i a i n d i c a ,  B o t r y t e l l a micromora ( Hori,1971 ); Ectocarpus crouanii ( Magne,19 71 ); G i f f o r d i a mitchellae,Hapterophycus sp., P y l a i e l l a l i t t o r a l i s ( Hori,1972 ). Studies on the genus "Ectocarpus were concerned with analysis of a r e s t r i c t e d number of features. Longest (1946) studied the external features of the f l a g e l l a of the zooids. Evans (1966) described the c h a r a c t e r i s t i c s of the pyrenoid i n zoospores and emphasized the absence of t h y l a -koids or thylakoid-derived structures from the pyrenoid matrix. Hori (1971) made sim i l a r observations using vegetative c e l l s . Bailey and Bisalputra (1969) used both freeze-etching and u l t r a t h i n sectioning techniques to study the c e l l wall morphology i n Ectocarpus acutus and concluded that the wall possesses a double layered organi-zation. Cytochemical techniques at the u l t r a s t r u c t u r a l l e v e l revealed the presence of polysaccharide-like substances i n association with l i p i d materials i n plasto-g l o b u l i ( Magne,1971 ). The aim of the f i r s t part of t h i s thesis i s to provide a more detailed description of the ultrastructure 3 of Ectocarpus and to compare i t s s a l i e n t features with those of other brown algae. II . C e l l u l a r Senescence In recent years, the biology of the brown algae has received increasing attention. Information on the mechanism of c e l l wall regeneration ( Fulcher and McCully, 1971 ), of f e r t i l i z a t i o n ( Pollock,1970 ), r h i z o i d formation ( Quatrano,1972 ), and c e l l u l a r p o l a r i t y ( Neushul and Dahl,19 72b ) have become available. Never-thless, so far I am aware, no u l t r a s t r u c t u r a l study has been c a r r i e d out on the aging of brown algae. Senescence has been defined i n many d i f f e r e n t ways. Strehler (1962) defined i t as changes which r e s u l t i n a-decreased s u r v i v a l capacity on the part of the i n d i v i d u a l organism. Leopold (1964) interpreted i t as the degenerative processes which terminated the functional l i f e of an organ or an organism. Osborne (1967) defined senescence as a general and increasing f a i l u r e of many synthetic reactions which are the normal forerunners of c e l l death. Woolhouse (1967) thought of senescence as a continuation of c e l l d i f f e r e n t i a t i o n ; while Rockstein (1967) that i t was the r e s u l t of reproducible time-related a l t e r a t i o n s i n structure and function i n an organism which r e s u l t i n the decreasing capacity of that organism to survive and thus would r e s u l t ultimately i n i t s death. McLean (1968) referred to senescence as those events which occur between maturity and death. Whatever the pos i t i o n taken, there i s general agreement that senescence ultimately leads to death of c e l l s or organisms. Aging and senescence have been used as synonymous terms ( Thung and Hollander,1967 ). Rockstein (1967), however, thinks of senescence as a more r e s t r i c t e d part 4 of a broader phenomenon c a l l e d aging. This, according to the author, includes: 1) the i n i t i a l period of development, 2) the middle l i f e stage of growth and maturation, and 3) the period of senescence. Carr and Pate (1967) also r e s t r i c t the term senescence to those changes which are c l e a r l y degenerative, while they think of aging as the sum of t o t a l changes i n the whole plant. In t h i s work senescence and aging w i l l be considered interchangeable terms. Senescence theories and mechanisms are as abundant as are d e f i n i t i o n s . The subject, however, was reviewed recently (Hahn, 1971; Medvedev, 1972). There i s now a considerable amount of information dealing with biochemical and ph y s i o l o g i c a l aspects of senescence (e.g. Varner, 1961; Wangerman, 1965; Woolhouse, 1967; Wareing and P h i l l i p s , 1970; Sax, 1962), but at the u l t r a s t r u c t u r a l l e v e l data are s t i l l sparse. Information dealing with higher plants was recently reviewed by Butler and Simon (1971). In comparison, there i s l i t t l e information on the algae. The aging phenomena of u n i c e l l u l a r algae has been studied i n Spongiochloris typica (McLean, 1968), Ochromonas (Schuster et a l . , 1968), and Euglena. .granulata (Walne et a l . , 1970; and Palisano and Walne, 1972). Fabbri and Palandri (1969) and Palandri (1972) studied aging i n the coenocytic filaments of Halimeda tuna. To contribute to our knowledge of the process of senescence, the u l t r a s t r u c t u r a l changes of Ectocarpus c e l l s during the o v e r a l l process of aging were studied. The selection of Ectocarpus for such a study has proved to have certa i n advantages. The simple filamentous form with prominent a p i c a l growth i s p a r t i c u l a r l y suitable i n that i t i s possible to follow a l l stages of c e l l u l a r d i f f e r e n t i a t i o n and disorganization i n a sequential order. The information obtained i n t h i s work w i l l provide background for future experimental work on the factors 5 governing senescence. Among those factors,the problem of co n t r o l l i n g senescence using growth regulators and related substances could be quite i n t e r e s t i n g to consider. Growth regulators were shown to influence senescence i n higher plants ( e.g. Butler and Simon,1971; Udvardy and Farkas, 1972 ). Recent reports have disclosed the existence of substances with s i m i l a r properties i n the brown algae ( e.g. Jennings,1968; Buggeln and Craigie,1971; Augier and Harada,1972; Hussain and Boney,1973 ). Therefore, the study of t h e i r e f f e c ts on the brown algae might provide interes-ti n g information on the aging mechanism i n general. This work also attempts to es t a b l i s h s i m i l a r i t i e s and differences that might e x i s t between the aging process i n organisms as d i f f e r e n t as a brown alga,a p r o t i s t , a higher plant or an animal. These attempts w i l l eventually help to judge assertions l i k e that of Palisano and Walne (1972), who defended the existence of a basic common mechanism of senescence from the lower to the higher forms of l i f e . 6 MATERIALS. AND METHODS Ectocarpus sp. was obtained from the Culture C o l l e c t i o n of Algae, Indiana University (culture LB 1433). Cultures were maintained i n Chihara's marine medium (Chihara, 1968) i n a growth chamber, at 14°C and illuminated by fluorescent lamps for 16 hours per day, at approximately 400 f t . c . The alga was grown i n "Nalgene" polypropylene p e t r i dishes (#5500). This f a c i l i t a t e d f i x a t i o n and embedding of the material (Bisalputra et a l . , 1971). To ensure optimum growth the medium was changed every 5 days. Stock cultures were handled i n a sim i l a r way except they were kept i n large pyrex culture j a r s , and the culture medium changed every 3 weeks. For dark treated material cultures were grown for 3 months under normal culture conditions, then transferred to t o t a l darkness for 1 to 5 days and 35 days. Light microscopy. L i v i n g filaments were studied d i r e c t l y on Nalgene p e t r i dishes or i n d i v i d u a l plants were placed i n a drop of culture medium on a microscope s l i d e . Fixed material was observed af t e r treatment i n one of the following procedures: a) 4% (v/v solution of glutaraldehyde i n cacodylate buffer (0.1 M Sodium Cacodylate, pH 1.0), to which 0.25 M of sucrose was added; b) 10% (v/v ac r o l e i n i n 0.025 M phosphate buffer (pH 6.8, 24 hours at 0°C). The material was post-fixed i n 1% HgCl 2 (24 hours, at 0°C; McCully, 1966); c) 4% (v/v phosphate buffered (0.05 M pH 7.0) paraformaldahyde. Except when s p e c i f i e d , a l l fixations, were car r i e d out at room temperature, from 3 to 28 hours. Fixations for dark treated material were car r i e d out i n t o t a l darkness. The fixed material was embedded i n Spurr 1s medium, which i s s i m i l a r to that described for electron microscopy and 7 sectioned at a thickness of approximately 1 micron. Sectioning was routinely done using an ultramicrotome equiped with glass knives. Ribbons of 5-6 sections were transferred with a platinum loop to glass s l i d e s and dried at 60 C on a e l e c t r i c hot plate. Slides bearing sections were either d i r e c t l y placed i n the staining solutions or deplasticized p r i o r to staining ( Rosenquist et al.,1971 ). The staining procedures were as summarized i n the following page. After staining the sections were washed, a i r dried, and mounted i n one drop of "Permount" mounting medium. Observations were made with a Zeiss photomicroscope using phase contrast and bright f i e l d i lluminations. Photographs were taken using I l f o r d FP4 f i l m . Electron microscopy. The material was fixed and processed according to one of the following methods: 1) glutaraldahyde-osmium f i x a t i o n . The material was fixed for 2 3 hours at room temperature, i n a f i x a t i v e solution containing 4% (v/v) glutaraldahyde and 0.25M sucrose, i n 0.1M sodium cacodylate buffer ( pH 7.0 ). The samples were transferred through a series of buffer solutions containing decreasing concentrations of sucrose, u n t i l f i n a l l y sucrose was completely eliminated from the r i n s i n g solutions. The material was post-fixed for a further 2h hours i n a 1.5% (v/v) Os04, s i m i l a r l y buffered, but without sucrose. I t was rinsed i n the buffer solution and dehydrated i n graded alchool s e r i e s . Spurr's low v i s c o s i t y embedding medium ( Spurr,1969 ) was used as the embedding vehicle according to the procedure described by Bisalputra et a l . (1971). This method was found to give the best r e s u l t s i n preserving the c e l l u l a r architecture of young c e l l s and c e l l s i n advanced stages of senescence. Sl i g h t modifications to the.above procedure were introduced by carrying the f i x a t i o n overnight, using a SUMMARY OF LIGHT MICROSCOPE STAIN METHODS STAINS REACTION OTHER DETAILS REFERENCES A. Prote i n s A n i l i n e blue- b r i g h t blue colour at Fisher,1968 black p r o t e i n s i t e s B. Insoluble P e r i o d i c a c i d - red colour at r e a c t i o n . DNPH blockage of Feder and carbohy- S c h i f f (P.A.S.) s i t e s aldehydes O'Brien,1968 drates r e a c t i o n C. N u c l e i c Methyl green- DNA-containing s t r u c - n u c l e i c acids Jensen,1962 ac i d s pyronin tures s t a i n blue-green e x t r a c t i o n s RNA-containing s t r u c - done by the tures s t a i n red P e r c h l o r i c a c i d method D. L i p i d s Sudan Black B black blue colour at Ap a r i c i o and r e a c t i o n s i t e s Mardsen,1969 procedure c a r r i e d Gomori,1952 out i n formaldehy-de f i x e d m a t e r i a l E. Polyphe- T o l u i d i n e blue green to turquoise m a t e r i a l f i x e d McCully,1966 nols 0 ' colour at r e a c t i o n according to s i t e s McCully (1966) F. Age Schorml r e a c t i o n deep blue colour Pigment at r e a c t i o n s i t e s Hendy,1971 8 5% (v/v) solution of glutaraldehyde. . The decrease - in. sucrose concentrations was done during the post-osmication steps. This v a r i a t i o n was found to give best r e s u l t s i n handling the " c e l l type #2" preservation, which has proved to be very d i f f i c u l t to preserve. 2) Permanganate f i x a t i o n . This was ca r r i e d out using 1% to 5% solutions of KMnO^ either i n d i s t i l l e d water or i n sodium cacodylate buffer (pH 7.0) without sucrose. A wide range of f i x a t i o n times were t r i e d but the images obtained were uniformly poor,-especially with respect to the older c e l l s . These data agree with those of Barton (19 66) who also found permanganate f i x a t i o n inadequate for studies of senescence. 3) Enzyme l o c a l i z a t i o n at the u l t r a s t r u c t u r a l l e v e l . The material used i n these experiments was b r i e f l y fixed (20 minutes, at room temperature) i n a 4% solution of p u r i f i e d glutaraldehyde (Electron Microscope Sciences, Washington) i n 0.1M cacodylate buffer (pH 7.0), to which sucrose was added to a f i n a l concentration of 7.5% (w/v). After b r i e f r i n s i n g , the material was incubated i n the appropriate incubation medium. The material was then quickly rinsed and fixed for a further 2 hours i n glutaraldehyde. Subsequent processing was as described before under 1. The composition of the incubation media were: a) Acid phosphatase. Tris-maleate, 60 mM (pH 5.0); lead n i t r a t e , 3 mM; Beta-glycerophosphate, 11.5 mM; sucrose, 220 mM (Gomori, 1952). The material was then incubated for periods of time ranging from 30 to 60 minutes, at a temperature of 37°C. Control material was incubated either i n the standard incubation medium with 10 mM of sodium flu o r i d e or from which Beta-glycerophosphate was removed. b) Catalase. 10 mg diaminobenzidine (DAB); 5 ml of 0.05M propanediol buffer; 1 ml of 3% (v/v) H 20 2. The f i n a l pH of the incubation medium was 9.0 (Frederick and Newcomb, 1969). 9 The incubation was c a r r i e d out at 37 C,for 60 minutes. Control material was incubated without I^C^, or i n the presence of 0.01M KCN. In another te s t the material was boiled for 5 minutes i n propanediol buffer p r i o r to incubation i n standard medium. c) Peroxidase. Media for incubation and controls were as indicated for catalase, but the f i n a l pH was adjusted to 7.6. The incubation was c a r r i e d out at room temperature for 60 minutes. d) Adenosine triphosphatase ( ATPase ).Two standard media were employed to study ATPase l o c a l i z a t i o n (adapted from Henrikson,19 71 ). Medium A.Tris-maleate, 80 mM ( pH 7.2 ); 10 mM MgS04; 3 mM ATP; 3 mM lead n i t r a t e ; 220 mM sucrose. The incubation was carried out for 1 hour at 37 C. Control material was incubated i n standard nedium without ATP. Medium B. Tris-maleate buffer, 80 mM ( pH 7.2 ); 10 mM Mg SC>4; 5 mM ATP; 100 mM NaCl; 30 mM KC1; 3 mM lead n i t r a t e ; 220 mM sucrose. The incubation was done for 1 hour, at 37 C. Control material was incubated i n standard medium without ATP or boiled p r i o r to p r e - f i x a t i o n i n standard medium. 4) U l t r a s t r u c t u r a l i d e n t i f i c a t i o n of the age pigment. These studies were done according to the modifications introduced by Hendy (1971) for the u l t r a s t r u c t u r a l i d e n t i f i c a t i o n of l i p o f u s c i n - l i k e materials. The material was fixed i n a 4% (v/v) solution of formaldahyde, i n 0.05M phosphate buffer ( pH 7.0 ), for 24 hours, 1 week or 2 weeks periods. After incubation i n either the Fontana's ( 25 ml of a 10% v/v aqueous s i l v e r n i t r a t e solution, ammonia ( s.g.= 0.880 ) added drop by drop, 25 ml d i s t i l l e d water ) or Schorml's ( 3% f e r r i c chloride, freshly prepared 1% potassium ferricyanide, 1:1, v/v ) solutions, some of the material was post-fixed i n buffered 1.5% (y/v) Os0 4. Incubation procedures were as described by Hendy (1971) and 10 subsequent handling of the material for electron microscopy was as described before. Material fixed according to the above procedure, but incubated i n neither the Fontana's nor the Schorml's solutions was used as control to determine the s i m i l a r i t i e s i n morphology and l o c a l i z a t i o n of the l a b e l l e d i n c l usions. 5) Freeze-etch. The material was pre-fixed for 1 hour i n a 4% (v/v) solution of glutaraldahyde, i n 0.1M cacodylate buffer ( pH 7.0 ), then transferred to 25 % (v/v) g l y c e r o l i n d i s t i l l e d water for periods of time ranging from 2 to 24 hours. The material was then quenched i n Freon 22, p r i o r to freezing i n l i q u i d nitrogen. The frozen material was fractured at -100 C and etched for 30 to 60 seconds using a Balzers BA 360M device. A l l preparations were viewed with a Zeiss EM 9A electron microscope. The u l t r a t h i n sectioning was done using either a duPont diamond knife or glass knives on a L.K.B. ultratome I or a Reichert OMU3 ultramicrotome. Both stained ( Reynolds,19 63 ) and unstained sections were used i n these studies. 11 OBSERVATIONS PART I—LIGHT MICROSCOPIC OBSERVATIONS Studies of l i v i n g filaments revealed features which were useful i n interpreting, the electron microscope r e s u l t s . The filaments were made up of a variable number of c e l l s . The c e l l s , rectangular i n shape, measured an average of - 3 - 3 2.5 x 10 X 2.0 x 10 cm i n the case of the erect system -3 -3 and 2.0 x 10 X 1.7 x 10 cm i n the case of the prostrate system. Other observations can be summarized as follows: 1) chloroplasts were found to be polymorphic (figures 4, 6, and 7); 2) d i f f e r e n t stages of pyrenoid d i v i s i o n were observed (figure 8); t h i s seems to occur by the f i s s i o n of the pyrenoid body; and 3) conspicuous wall ingrowths were apparent, both along the side walls as well as at the cross walls (figures 10 and 11). Observations of erect and prostrate filaments of the sporophytic generation of Ectocarpus sp., after glutaralde-hyde-osmium f i x a t i o n , indicate that the various c e l l s i n each filament do not respond equally to such treatment. Apical c e l l s are very l i g h t i n appearance, while the c e l l s below them become progressively darker (figure 1). Towards the basal portion of the filaments the c e l l s again appear to be less dense (figures 2 and 3). In the great majority of the cases studied, the t r a n s i t i o n between the darker c e l l s and the less dense basal ones was found to be abrupt (figure 3) . In a few cases, however., t r a n s i t i o n a l stages were detected. In the region where . l i g h t c e l l s followed dark ones, the cross walls between c e l l s always protruded into the l i g h t c e l l s (figure 3). Light microscope observations. of l i v i n g , filaments revealed ..similar, features, showing i n addition, that the i n t e r n a l organization of the c e l l s on either side of. the cross wall was d i f f e r e n t 12 ( figure 5 ). I t i s , therefore, apparent that at least. 3 major types of c e l l s may be recognized i n a filament of Ectocarpus l)the a p i c a l c e l l and i t s immediate derivatives; 2) the c e l l s which react intensely with OsO^; and 3) the c e l l s situated near the basal portion of the filament. I t i s also safe to assume the existence of t r a n s i t i o n a l stages within the filaments. In order to learn more about the cytoplasmic d i f f e -rences that might e x i s t i n these d i f f e r e n t types of c e l l s , several cytochemical tests were carried out. Results of these investigations are summarized i n the next page. To simplify the description, the following notations w i l l be adopted throughout t h i s work : 1) " c e l l type #1" refers to the group of c e l l s represented by the a p i c a l c e l l and i t s immediate neighbors; 2) " c e l l type #2" refers to the c e l l s which react intensely with OsO^; 3) " c e l l type #3" refers to those c e l l s which do not react so deeply with OsO. and are located near the basal end of the filaments; 4) intermediate stages between the 'type #1' and 'type #2' and between the 'type #2' and the 'type #3' w i l l be recogni-zed by the notations " t r a n s i t i o n a l 1-2" and " t r a n s i t i o n a l 2-3" respectively. To accurately r e l a t e the c e l l s to the proper stage of development; ribbons of 2-3 c e l l s were cut for E.M. examination and only aft e r was the l i g h t microscope study undertaken. SUMMARY OF THE LICHT MICROSCOPE RESOLTS C«ll Type DBA ( RNA extracted ) RNA ( DNA extracted ) Proteins L i p i d s Insoluble Carbohydrates Li p o f u s c i n Polyphenols 11 Nucleus smell, round i o Cytopleea intensely Intense reaction almost Few reacting spots. P.A.S. reaction v e i l Alnost undetectable Very fev r e a c t i o n s i t e s , p r o f i l e . Chromatin stained. Nuceolus throughout the cytoplesm. Figure 23. v i s i b l e i n the c e l l . r eaction p a r t i c u l a r l y with a c l e a r p o s i t i v e oore so i n pyrenoide. v a i l , riguxe 32. d i s t i n c t . Nucleolus r e a c t i o n { arrow ). Figure 18. unstained. Figure 12. Nucleus o u t l i n e v i s i b l e . Figure i». T r a n s i t i o - — - - - - - - - ~ — — : — P.A.S. reaction —---------------- — n a l "1-2" p a r t i c u l a r l y evident c e l l s ,' l n association v i t h 1 Golgi complex, i n the , paramural space ( arrow ), ^ and i n the c e l l w a l l . 1 f i g u r e 21. 12 Nucleus small and Irregu- 6talned area r e s t r i c t e d Staining r e s t r i c t e d to Reaction found P.A.S. reaction well' Conspicuous reaction Pattern of s t a i n i n g l a r l n p r o f i l e , but s t i l l to the perinuclear r e - •one cytoplasmic s p o t s i throughout the apparent l n the c e l l s i t e s throughout the complex. Sooe cytoplasmic conspicuously stained. ; gion. Nucleus unstained pyrenoids v e i l stained. cytoplasm. Figure w a l l . cytoplasm. Figure 26. inc l u s i o n s show a green Nucleolus unstained. end very i r r e g u l a r i n Figure 19. 24. to turquoise colour. Figure 13. • p r o f i l e . Figure 16. Figure 27. 11 Nuclei could not be Cytoplasm almost Very f e i n t or negative Reaction spots few P.A.S. reaction v e i l Reaction almost or L i t t l e or almost undetect-detected. Figure 14. unstained. Figure 17. s t a i n i n g throughout the end sparse. apparent i n the c e l l t o t a l l y absent. able r e a c t i o n . ; c e l l . Figure 20. Figure 25. v a i l . 13 PART I I . - ELECTRON MICROSCOPE. OBSERVATIONS Electron microscope observations.substantiate.evidence from l i g h t microscopy for the existence of 3 main types of c e l l s within the filaments of Ectocarpus, and whose charac-t e r i s t i c s are described In the following pages. A) ULTRASTRUCTURAL FEATURES OF YOUNG CELLS ( CELL TYPE # 1 ) The general morphology of these c e l l s i n both the erect and prostrate systems of Ectocarpus was found to be s i m i l a r ( figures 28a, 31, 32, and 33 ). Therefore, the following descriptions apply to c e l l s of both systems. Nucleus. The nucleus ( figures 28a, 31, 32, and 33 ) i s bound by a double membrane envelope interrupted by numerous pores. Freeze-etching studies of the nuclear envelope reveal the existence of surfaces with few p a r t i c l e s on them, while others are multiparticulate ( figure 36 ). Nuclear pores are e a s i l y i d e n t i f i e d i n freeze-etching preparations, but t h e i r morphology varies according to the exposed surface. This i s p a r t i c u l a r l y apparent i n figure 36 where the face 'A' pores have a c r a t e r - l i k e appearance and the ones on face 'B' appear as c i r c u l a r depressions. The pores,measuring approximately 80 nm i n diameter, are round i n appearance and reveal a defined substructure ( figures 29, and 36 ). They seem to possess a hollow central core from which spokes radiate toward the edge of the pore ( figure 29, arrowheads ). The pore rim appears to be made up of several globular units, whose number i s d i f f i c u l t to determine with accuracy.The nuclear pores i n figure 37 are not uniformly d i s t r i b u t e d throughout the nuclear membrane, but tend to occur i n certain areas and not i n others. Sections of the interphase nucleus of young 14 vegetative c e l l s of Ectocarpus sp. usually contain., one conspicuous nucleolus ( figures 31, 32, 33, and 38 ),which seems to consist of two d i s t i n c t components ( figures 32, and 38 ). One component i s f i b r i l l a r , measuring approxima-te l y 8nm i n diameter. The other component i s granular, with each unit measuring approximately 15 nm i n diameter. The nucleolus usually occupies a central p o s i t i o n i n the nucleoplasm, but i n some cases i t s association with the inner membrane of the nuclear envelope i s apparent ( figure 38 ). In t h i s case, the region of the nuclear envelope clos e l y associated with the nucleolus seems to be devoid of nuclear pores. Chromatin i s not conspicuous, although i t s d i s t r i b u -t i o n throughout the nucleoplasm as well as i t s r e l a t i o n s h i p to the inner membrane of the nuclear envelope i s obvious i n some micrographs.( figures 28a, and 32 ). At high magnification the f i b r i l l a r nature of the chromatin i s v i s i b l e ( figure 30 ). Each f i b r i l l a r unit measures approximately 10 nm i n diameter. The perinuclear space i s abundantly f i l l e d with g r a n u l a r - f i b r i l l a r material ( e.g. figures 30, 32, 38, 55, and 57 ). In addition, small v e s i c l e - l i k e structures . ( figures 38, arrow, 39, 40, 41, arrowheads ), whose relationship to the nuclear envelope membranes i s shown i n figures 41 ( arrowheads ) and 45 ( pns ), are also found inside the perinuclear space. More elaborated structures are found inco'close association with the outer membrane of the nuclear envelope (figures 38, empty arrowhead, and 42, arrowhead ). Besides the above mentioned features the association between the outer membrane of the nuclear enve-lope and the forming face, of the. dictyosomes.'is a consistent aspect of Ectocarpus c e l l s ( e.g. figure 34 ). Endoplasmic reticulum ( E.R. ). Most of the endoplasmic reticulum present i n these c e l l s seems to be of the 15 "rough", type (figures 28a, 32, and 44). C i s t e r n a l elements are fenestrated (figures 32, black arrow, 38, big arrow-heads /.and.44,.arrowhead). Fenestrae-llke formations, are also detectable i n freeze-etching preparations (figure 43a, arrowhead). .Some fenestrae display a -crater-like appearance (figure 43b, arrowhead). C r y s t a l l i n e inclusions are observed inside d i l a t e d portions of the.E.R. cisternae (figure 44, 1) . E.R. elements are d i s t r i b u t e d throughout the cytoplasm (figures 28a, 31, 32, and 33). In a few micrographs the E.R. elements are observed to display concentric arrangements (figure 44). In others, an orientation of the E.R. i n r e l a t i o n to the nuclear envelope i s apparent (figures 32, and 38). Continuity between the E.R. c i s t e r n a l space and the perinuclear space i s observed (figure 38). The E.R. c i s t e r n a l space i s occupied by g r a n u l a r - f i b r i l l a r . material (figures 32, 38, and 45) and vesicular structures (figure 45) s i m i l a r i n appearance to those found inside thei perinuclear space. In a few micrographs, multivesicular-l i k e structures are observed at the junction of the perinuclear and E.R. c i s t e r n a l spaces (figure 38, empty arrowhead). Endoplasmic reticulum membranes are also observed to establish s p a t i a l relationships and/or d i r e c t communication with several other organelles, i . e . associations between the endoplasmic . reticulum and the outer membrane of the mitochondrial, envelope (figures 35, arrowhead, and 72, arrow), and with the chloroplast (figures 44, 47a, b, 48), thereby forming a sp e c i a l section of the E.R. commonly known as the 'chloroplast. E.R..' (Bouck,. 1965). The chloroplast E.R.. follows the chloroplast envelope around the projecting.pyrenoid (figure 28a). Tubular-vesicular structures arise from the chloroplast E.R. membrane facing the chloroplast envelope (figures 32, 46, 16 and 48). These structures can be observed i n tangential view as a very complex network, of tubules (figure' 49). Intra-c i s t e r n a l structures s i m i l a r to those described, inside the E.R.. c i s t e r n a l space are found inside the cisternae of the chloroplast E.R. (figure 50) . Dark osmiophilic bodies with a complex i n t e r n a l structure, referred i n t h i s work as "osmiophilic structured bodies" (OSB), are also found i n association with the chloroplast E.R. (figures 45, 51, and 52). They can occupy the area shared by both the chloroplast E.R. and the nuclear envelope (figure 52), or they may occur i n close association with pyrenoids (figure 53). High magnification studies show these bodies to have two major components: 1) a very dark osmiophilic and unstructured material (figures 51, and 53); and 2) a myelin-like component (figures 51, 53, and 54). The arrangement of the two components i n r e l a t i o n to each other i s variable, and accounts for the heterogeneity of forms displayed by these bodies (figures 45, 51, 52, 53, 54, and 55). In some cases, the o r i g i n of the "OSB" can be traced d i r e c t l y from the chloroplast E.R. or from a complex system of membranes a r i s i n g by further elaboration of the chloroplast E.R. (figures 45, 47a, 47b). In other cases, the "OSB" are found i n the cytoplasm apart from the chloroplast E.R. (figure 56). In some instances they are apparently discharged into the paramural space (figure 54) . Golgi complex. The Golgi complex, an exclusively perinuclear organelle, i s represented by groups of 2, 3, or more dictyosomes (figures 28a, 31, and 33). Each dictyosome i s made up of 6 to 10 stacked cisternae, of which those closest to the nuclear envelope are conspicuously fenestrated (figures 57, and .58, arrowheads). The outer cisterna i s usually hypertrophied i n appearance (figures 28a, 33, 58, 59> and 61). Observations of dictyosomal cisternae at high magnification reveal the presence of two 17 sizes of interruptions or channels.. One type has an average diameter of 25 nm (figures 57, and 83, arrowheads lab e l l e d 'a'). The others average 5 hm i n diameter (figures 57, 83, arrowheads l a b e l l e d 'b'). Direct continuity between adjacent cisternae i s frequently observed (figures 33, 42, and 62), and the presence of i n t r a - as well as i n t e r c i s t e r n a l b ridge-like filaments i s also apparent (figure 59, arrowhead). As i n the case of other brown algae (Bouck, 19 65; Bourne and Cole, 1968; Cole, 1969, 1970; Bisalputra et a l . , 1971) a close association between the outer membrane of the nuclear envelope and the formative face of the dictyosomes i s evident i n Ectocarpus sp. This r e l a t i o n s h i p i s interpreted as being i n d i c a t i v e of the transfer of material from the perinuclear space to the dictyosomes by means of v e s i c l e s a r i s i n g from the outer membrane of the nuclear envelope (figures 61, and 83). Direct continuity between the perinuclear space and the innermost cisternae of the dictyosomes occurs i n some cases (figure 34, arrowhead). Besides the ubiquitous association of dictyosomes with the nuclear envelope, associations also occur between dictyosomes and other c e l l organelles; for instance, with mitochondria (figure 61), with pyrenoids (figures 60, and 63), and with large vacuoles. In t h i s case, incorporation of dictyosome-derived v e s i c l e s into vacuoles i s apparent (figure 62, big arrowhead). Evidence for the release of dictyosome-derived material into the paramural space i s obtained when comparing the inclusions (labelled 'L') inside the dictyosome cisterna (figure 64) with s i m i l a r inclusions inside the paramural space.(figure 49).. This type of i n c l u s i o n seems to a r i s e from invagination of the dictyosome c i s t e r n a l membrane.and often shows a high degree of morphological elaboration (figure 65). Occasionally, tube-l i k e inclusions are also found inside dictyosome cisternae (figure 66). A close association between osmiophilic 18 structured bodies . (OSB) and dictyosomes- i s sometimes observed (figure 55). Mitochondria. Mitochondria are ua.ually . found scattered throughout the cytoplasm (figures. 28a, 31, 32, and 33), although i n some cases they tend to accumulate close to the c e l i periphery., (figure 56) . Images suggesting mitochondrial d i v i s i o n are also detected (figure 62, small arrowhead). U l t r a s t r u c t u r a l l y these organelles (figures 38, 68, 84) do not appear to be d i f f e r e n t from those already described i n other brown a l g a l studies (Bouck, 1965; Bisalputra and Bisalputra, 1967; Bourne and Cole, 1968; Cole and L i n , 1968; McCully, 1968; Cole, 1969; Liddle and Neushul, 1969). Since they are polymorphic, t h e i r p r o f i l e may be quite elaborated (figure 67). D i s t i n c t genophore areas may be observed (figures 38, and 84, arrowheads). Inclusions are noticed inside the c r i s t a e (figures 68, and i n s e t ) . At high magnification, the inclusions appear to be c i r c u l a r with a hollow central core, with filaments radiating from the rims towards the c r i s t a e membranes. Mitochondria are found i n association with almost a l l the c e l l organelles. Mitochondria-dictyosome and mitochondria-endoplasmic reticulum associations have already been reported. Other associations are with the nucleus (figure 69), the chloroplast (figures 48, and .70) , the pyrenoid (figure 71), and with themselves (figure 72, arrowheads). Chloroplasts. .Chloroplasts.are bounded by a. double membrane envelope, outside of which l i e s , the chloroplast E.R. (figures 28a, .32, 33, 46, 4.7a,. 47b, and 48.) . Direct continuity between the c i s t e r n a l space of the.two above mentioned membrane, systems i s . detectable .(figures 47a, 48, arrowheads). Fenestra-llke interruptions can be observed along the chloroplast envelope membranes both In longitudinal as 19 well as i n tangential view (. figures 76, arrowhead 'c', and 84, arrowhead 'b'^respectively ). The photosynthetic lamellae consist of bands of 3 close l y associated thylakoids ( figures 76, and 79 ). Each thylakoid measures approximately 11 nm i r i width. The space between thylakoids of the same band as well as between neighboring bands has a high degree of uniformity, except for those regions where p l a s t o g l o b u l i intervened or where exchanges of thylakoids/;;between adjacent bands occur. Occasional exceptions to t h i s r e g u l a r i t y are observed ( figure 75 ). Bif u r c a t i o n of thylakoids i s observed ( figure 85, arrowhead ). Within the thylakoids interruptions are seen i n longitudinal ( figures 73, big arrowhead, and 76, arrowheads 'a' ) as well as i n tangential views ( figures 78, arrowheads, and inset; 84, arrowhead 'a' ). In tangential view the interruptions seem to be pore-like i n appearance. In some areas, thylakoid bands are interrupted by large portions of stroma material ( figures 46, and 92 'F' ). When freeze-etched thylakoids of Ectocarpus reveal two types of fracture surfaces. These ( figures 80, and 81 ) have either many p a r t i c l e s ( surfaces 'B' ) or only a few ( surfaces 'A' ). The peripheral band of thylakoids follows the contour of the inner membrane of the chloroplast envelope, enclosing the c e n t r a l l y located bands ( figures 28a, 31, 32, 33, and 46 ). In some cases, the peripheral band i s not s t r i c t l y peripheral, but i s continuous with the centa l l y placed ones ( figure 82, arrowheads ). The central bands normally traverse the chloroplast without interruption from t i p to t i p or terminate just short of the t i p s ( figures 28a, 32, 33, and 48 ). In some cases, thylakoids have a highly folded arrangement ( figure 85 ) or may be arranged concentrically ( figure 86 ). The whorled arrangement of thylakoids i s 20 apparently related to the abscission of c e r t a i n p l a s t i d portions ( f i g u r e s 87, and 88 ). Abscissed.portions seem to be incorporated into, vacuoles, . ( f i g u r e 91 )-. • AAcid phosphatase a c t i v i t y was found associated with these formations ( figure 89 ), but was absent from the control preparations ( figure 90 ). F i b r i l l a r bridge-like elements are observed inside and between thylakoids ( figures 73, 76, 79, small arrow-heads ). F i b r i l l a r elements were most noticeable i n sections cut tangentially to the thylakoids ( figure 96, white arrow-heads ). The f i b r i l l a r material i s not r e s t r i c t e d to the areas mentioned above, but i s also observed i n the space between the peripheral thylakoid band and the chloroplast envelope ( figure 92, arrowheads ). The presence of f i b r i l s inside the chloroplasts was further confirmed by freeze--etching studies ( figures 43a, 74, and 95, arrowheads ). The dimensions of the f i b r i l l a r elements were found to range from 3C to 5:nm i n diameter, with most of the measurements averaging 3.7 to 4.2 nm. In the stroma, ribosome-like p a r t i c l e s and plasto-g l o b u l i are detected ( figures 32, and 94 ). P l a s t o g l o b u l i possess a d e f i n i t e i n f r a s t r u c t u r e composed of f i b r i l s embedded i n an amorphous and less electron dense matrix ( figures 97, 98, and 99 ). The plastoglobular f i b r i l s show a clear r e l a t i o n s h i p to the thylakoid membranes ( figures 97, and 98, arrowheads ). In figure 99 d i r e c t continuity between the f i b r i l l a r phases of two adjacent pl a s t o g l o b u l i i s observed. DNA-containing regions at the t i p s of chloroplasts ( figures 47a, and 48, 'g' )are, as shown by Bisalputra and Bisalputra (1969), part of the genophore.of these organelles. In.Ectocarpus sp., as i n other algae. ( Evans,1966; Bisalputra and Bisalputra,1970 ), chloroplast d i v i s i o n i s not synchronous with c e l l d i v i s i o n . As a r e s u l t , various stages 21 of chloroplast m u l t i p l i c a t i o n are observed i n the. active c e l l s of the filaments. Processes of chloroplast m u l t i p l i -cation were. described i n the'..'brown algae ( e . g . von Wettstein,1954; Cole and Lin,1968; Cole,1970; Bisalputra and Bisalputra,1970 ).In the.case of Ectocarpus sp. more than one type of chloroplast division.seems-to take place i n the sporophytic vegetative c e l l s of both the erect and prostrate systems. Figures 102 and 103 depict two stages of a process of d i v i s i o n . This process of chloroplast d i v i s i o n resembles the chloroplast d i v i s i o n of Sphacelaria ( Bisalputra and Bisalputra,1970 ). In figure 103 the stage of d i v i s i o n seems to correspond to the onset of the formation of the peripheral lamellar bridge ( arrowhead). Figure 101 shows a d i v i s i o n by longitudinal c o n s t r i c t i o n . A s i m i l a r event has been recorded i n Laminaria gametophytes ( Bisalputra et al.,1971 ). Figures 105 and 106 are non-consecutive s e r i a l sections of another aspect resembling chloroplast d i v i s i o n . Apparently, th i s process involves the progressive c o n s t r i c t i o n of thylakoids without the formation of a peripheral lamellar bridge. The process cl o s e l y resembles that occuring i n Egregia chloroplasts ( Bisalputra, unpublished data ). Figure 107 suggests the p o s s i b i l i t y that a chloroplast i s undergoing d i v i s i o n at two c o n s t r i c t i o n s i t e s . Multicons-t r i c t i n g chloroplasts were described i n Fucus by von Wettstein (1954). In dark treated material conspicuous chloroplast blebbing somewhat resembling p r o p l a s t i d m u l t i p l i c a t i o n i s observed ( figure 104 ). The b l e b - l i k e structure contains only stroma and i s delimited by both the chloroplast envelope and the chloroplast E.R.. Since th i s phenomenon i s only found i n dark treated material, i t i s more l i k e l y to-be a reaction of.the organelles to dark treatment. Pyrenoid. In Ectocarpus, generally only one pyrenoid i s seen projecting from the chloroplast on the side facing the 22 nucleus ( figure 28a ). However, the presence of two or more pyrenoids per chloroplast i s not uncommon ( figure 108, see also 6, and 9 ).Most l i k e l y t h i s i s related to the phenomenon of pyrenoid d i v i s i o n ( figure 8 ). Gibbs (1962a) has shown that the.pyrenoid matrix of P y l a i e l l a consists of t i g h t l y packed f i b r i l s 6.5-7.0 nm i n diameter. Similar f i b r i l l a r elements are seen i n the matrix of Ectocarpus pyrenoids ( figure 109 ). A very close r e l a t i o n s h i p i s observed between pyrenoid filaments and thylakoids i n some micrographs ( figures 93, and 108 ), p a r t i c u l a r l y i n dark treated material ( figure 9 3 ). In addition to the chloroplast envelope and the chloroplast E.R., a t h i r d membrane system designated as the "pyrenoid sac" or "pyrenoid cap" i s found outside the chloroplast E.R.. This membrane system i s r e s t r i c t e d to the area around the pyrenoid body ( figure 28a ). The rel a t i o n s h i p existent among these three membrane systems i s better observed i n figure 110. Bouck (1965) and Cole (1969) reported that no connection exists between the outer membrane of the pyrenoid sac and any other cytoplasmic organelle. In Ectocarpus, however, d i r e c t communications seem to occur between the pyrenoid and dictyosomes ( figures 60, and 63 ), and the pyrenoid and the vacuole ( figure 112). Pyrenoid d i v i s i o n i s not synchronous with chloroplast d i v i s i o n ( figures 8, 9; see also Evans,1966 ). Images of pyrenoid d i v i s i o n are frequent ( figures 109, and 114 ); i n those situations the pyrenoid sac i s noticeably absent ( see also Manton,1966a ). The c e l l wall and associated structures. The c e l l wall i s composed of f i b r i l s arranged i n two d i s t i n c t zones separated by an abrupt boundary ( figure-142 ) . The inner layer ( i l ) i s made up of roughly p a r a l l e l m i c r o f i b r i l s and i s very compact. The outer layer (ol) i s formed by loosely associated m i c r o f i b r i l s arranged i n a r e t i c u l a t e pattern.The bilayeru. 23 organization of the c e l l . w a l l i s also evident i n freeze-etched preparations (figure. 117).. In t h i s picture inclusions of u nidentified o r i g i n are observed inside the inner layer of the c e l l wall. Osmiophilic material can be seen inside the paramural.space (figure 47). Other types of inclusions occupy the paramural space. The o r i g i n of some of. these can be traced from the plasmalemma i t s e l f (figure 28b, arrowhead). These structures can a t t a i n a high degree of morphological complexity (figures 28a, 28b, 102, pm). Following the c l a s s i f i c a t i o n of Marchant and Robards (1968), these structures ought to be designated as "plasmalemmasomes." They are e a s i l y recognizable i n freeze-etching preparations (figure 37a). However, as was previously considered, one has the impression that some of the paramural structures may have been derived from dictyosomal a c t i v i t y ( c f . figure 64—"L" with figure 49—"L"). These structures should, therefore, be designated "lomasomes" (Marchant and Robards, 1968). Plasmodesmata are observed i n the c e l l walls of adjacent c e l l s (figure 111), but they are not organized into p i t - l i k e areas. B) ULTRASTRUCTURAL FEATURES OF THE "TRANSITIONAL 1-2" CELLS Vacuole formation and processes of autophagy. One of the main processes for the transformation of the young c e l l ( c e l l type #1) architecture into that c h a r a c t e r i s t i c of the " c e l l type #2" i s related to vacuole formation and autophagy. In t h i s . s e c t i o n these events are analysed i n d e t a i l . 1) Vacuole o r i g i n . In Ectocarpus sp. sporophytic vegetative c e l l s , both endoplasmic reticulum and dictyosomes contribute to the development of the vacuolar system. This i s indicated by the existence of d i r e c t continuity and/or close 24 association between elements of the endoplasmic .reticulum and vacuoles (figure 100, arrowheads), as well as E.R.-derived provacuolar p r o f i l e s , which show a tendency, to fuse with each other and with f u l l y developed, vacuoles (figures .116, arrowhead, 14 8, arrows). The r o l e of.dictyosomes i n vacuole development i s depicted by the incorporation of dictyosomal v e s i c l e s into vacuolar structures (figure 62, big arrowhead). 2), Autophagic a c t i v i t y . An analysis at the u l t r a s t r u c t u r a l l e v e l of how cytoplasmic areas and/or organelles are i s o l a t e d from the remainder of the cytoplasm, become trans-formed, and gain access to vacuoles i s rendered d i f f i c u l t due to the fact that one i s studying s t a t i c pictures of a highly dynamic phenomenon. Bearing t h i s i n mind, and after examination of multiple micrographs, i t seems that autophagy i n Ectocarpus sp. can take place by more than one mechanism. One of the mechanisms seems to involve the i s o l a t i o n of cytoplasmic portions by the endoplasmic reticulum (figures 119, 120, 121). The i s o l a t e d area i s , hence, surrounded by a double membrane system. Continuity between these concentric E.R. and normal c i s t e r n a l elements i s apparent at an early stage of i s o l a t i o n (figures 120, 121). The is o l a t e d regions are gradually transformed into t y p i c a l vacuoles (figures 122, 123, and 127). In other cases, access to the vacuolar space i s gained by a d i f f e r e n t mechanism, which seems to involve the invagination o f the tonoplast i n the region where the material to be i s o l a t e d .contacts or approaches. „the vacuole boundary (figure 145, arrowheads.) . This process., appears to be responsible for the incorporation of. abscis.sed p l a s t i d parts into vacuoles (figures 145, upper arrowhead, see also figure 88). Invagination of tonoplast with a subsequent intake of cytoplasmic material i s also seen i n figure 124. 25 This mechanism d i f f e r s from the E.R.^dependent mechanism i n that the is o l a t e d material was not segregated from the cytoplasm p r i o r to i t s uptake by the invaginating tonoplast. 3) Autophagic a c t i v i t y — e v i d e n c e from cytochemistry of acid phosphatase. Although acid phosphatase a c t i v i t y was detected i n association with c i s t e r n a l E.R., cisternae of dictyosomes, and provacuole-llke structures (figures 129, and 130); recently i s o l a t e d cytoplasmic areas show no acid phosphatase a c t i v i t y . Control material f a i l s to show any signs of reaction products i n association with the above mentioned organelles (figure 131) . As the process of degradation of i s o l a t e d material proceeds, i t s appearance becomes more and more altered and membranous remains become v i s i b l e inside the vacuoles (figure 49). Acid phosphatase a c t i v i t y was found to be associated with these formations (figures 125, and 126), but absent from controls (figure 131). Further vacuplation and degradation of i s o l a t e d material leads to loss of r e c o g n i z a b i l i t y of organelles and cytoplasmic remnants inside the vacuoles. Instead, two types of inclusions are observed: 1) a homogeneous granular substance (figures 135, 136, 'a'), and 2) a polymorphic myelin-like figure (figures 135, 136, 'b'). The granular type of in c l u s i o n i s found alone or i n association with the myelin-like component (figures 135, 136, and 137, 'a'). The myelin-like form, however, does not occur by i t s e l f . With greater vacuolation and c e l l u l a r digestion, the cytoplasm of the c e l l s becomes more and more altered (figures 135, 136), approaching f i n a l l y (figure 128) the c e l l u l a r organization of " c e l l type #2" (figures 148, 151), which i s described below. The increase In acid phosphatase a c t i v i t y p a r a l l e l s vacuolation and degradation of cytoplasmic materials (figures 138, and 139). Reaction products are absent from control sections (figure 140). 26 4) Other cellular...alterations. Other, changes.in c e l l u l a r u l trastructure contribute to the establishment of the " c e l l type #2" morphology. These include : 1) a change i n thylakoid orientation into patches of large thylakoid stacks ( figure 132 ); 2) the occurrence of electron dense metabolites at the c e l l wall periphery and among the f i b r i l l a r elements of the c e l l wall ( figure 142 ); and 3) the appearance of l o c a l i z e d c e l l wall ingrowths ( figure 163, cwi ), a phenomenon that coincides with the observation of high a c t i v i t y at the Golgi complex l e v e l ( figure 141 ). This problem w i l l be dealt with i n d e t a i l l a terv C) THE SENESCENT CELLS - ULTRASTRUCTURAL FEATURES OF THE "CELL TYPE #2" Since a l l b i o l o g i c a l phenomena represent dynamic and continuous situations the determination of a boundary between the " t r a n s i t i o n a l 1-2" c e l l s and the " c e l l type #2" i s d i f f i c u l t . To solve t h i s d i f f i c u l t y i t i s established that whenever chloroplasts show increasing signs of produc-ti o n of highly electron dense metabolites together with altered thylakoid arrangements ( figures 143, 148, 149, 151, 152, 154, and 156 ), the c e l l i s considered as belonging to the " c e l l type #2". The u l t r a s t r u c t u r a l morphology of these c e l l s was found to be si m i l a r i n the erect and the prostrate systems ( figures 143, 148, and 151 ). Therefore the following descriptions apply to c e l l s of both systems. Nucleus. The nucleus remains c l e a r l y d i s c e r n i b l e at t h i s stage of c e l l u l a r development. The nuclear boundary has become i r r e g u l a r ( figures 143, and 148 ).Nuclear pores are d i f f i c u l t to detect. Chromatin-iike material can be seen i n some cases either throughout the nucleoplasm or i n associa-tion with the inner membrane of the nuclear envelope 27 (figure 143) . The nucleolus, when apparent, i s comprised of both f i b r i l l a r and granular elements (figure 153). The association between the outer membrane of the nuclear envelope and elements of the endoplasmic reticulum and dictyosomes are s t i l l maintained (figure 143). Endoplasmic reticulum. The E.R. system becomes less prominent i n these c e l l s than i n the " c e l l type #1" (figures 143, 148, 151). This i s due, at l e a s t i n part, to the disorganization of the E.R., which i s caused by the v e s i c u l a t i o n of i t s elements (figure 143, 151, regions la b e l l e d "E.R."). These ve s i c l e s then become incorporated into vacuoles (figure 151). In these areas, ribosomes of any form are d i f f i c u l t to detect. They are, however, observed i n less altered cytoplasmic regions (figure 153). Golgi complex. The Golgi complex i s s t i l l well represented at t h i s stage of c e l l u l a r d i f f e r e n t i a t i o n (figures 143, 148). Its functional association with the outer membrane of the nuclear envelope i s evident. However, the usual hypertrophied appearance of the outermost cisterna of the dictyosomes, so c h a r a c t e r i s t i c of the " c e l l type #1", i s no longer observed. Mitochondria. Mitochondria are very d i f f i c u l t to detect at th i s stage of aging. More favourable sections allow one to account for scarcely more than a double membrane envelope and c r i s t a e (figure 153) . Chloroplasts. Of a l l c e l l organelles, chloroplasts exhibit the most s t r i k i n g alterations which have been employed as a c r i t e r i o n for i d e n t i f i c a t i o n of the " c e l l type #2". The appearance of patches of highly electron dense metabolites i s v i s i b l e at many s i t e s Inside the chloroplasts (figures 143, 148, 151, 152, arrows). The i n t r a p l a s t i d metabolites, which s t a r t as small scattered formations (figure 143) , become bigger and more numerous as senescence proceeds (figures 148, 151). The regions where the 28 metabolite accumulates present conspicuous, patterns of thylakoid arrangement (figures 149, 154, 156, 157). The relationship between the metabolite and the special constant arrangement of thylakoids suggests involvement of thylakoids i n the production of the metabolite. This rel a t i o n s h i p i s also apparent i n freeze-etched material (figure 159, arrow-head) . Permanganate f i x a t i o n could preserve neither the i n t r a p l a s t i d regions where the metabolite i s found nor the p l a s t o g l o b u l i (figure 133, arrows). This implies a possible l i p i d nature to the i n t r a p l a s t i d metabolite; a p o s s i b i l i t y further confirmed by i t s a f f i n i t y for Sudan Black B. The i n t r a p l a s t i d metabolite seems to be released into the cytoplasm (figures 155, and 157), contributing to the osmiophilic appearance of the cytoplasm. Plastoglobuli are e a s i l y distinguished from the osmiophilic metabolite described above. There i s no rel a t i o n s h i p between the two materials, because: 1) both structures are present at the same time and are morphologically d i f f e r e n t (figures 143, 148, 151); 2) an increase i n the number of metabolite containing regions occurs with advancing senescence, while the number of p l a s t o g l o b u l i remains, at t h i s stage of aging, nearly constant; and 3) p l a s t o g l o b u l i bear a d i f f e r e n t type of relationship with thylakoids. Pyrenoids. Pyrenoids are e a s i l y detectable (figures 143, 148). The f i b r i l l a r nature of the pyrenoid matrix i s no longer apparent, nor are the boundary membranes. Cytoplasm morphology. Figures 143, 148, 150, 151, 160 depict the condition of the cytoplasm i n " c e l l type #2". The development and o r i g i n of cytoplasmic inclusions has already been presented. I t can be summarized that three main types of cyto-plasmic inclusions are ..present. F i r s t l y , myelin-like inclusions (figures 148, 150), at least p a r t i a l l y preserved afte r permanganate f i x a t i o n . The preserved portion represents 29 the non-myelinic component (figure 134, arrowhead '2'). The second i s a. granular type, of Inclusion. (figures 148, 150) which i s preserved by permanganate f i x a t i o n (figure 134, arrowhead '1'). Both types of inclusions have been shown to have lysosomal o r i g i n s . A t h i r d type of i n c l u s i o n i s expected to be present i n the cytoplasm of these c e l l s . This type of i n c l u s i o n would correspond to the i n t r a p l a s t i d metabolite, since i t s discharge into the cytoplasm has been demonstrated. Its presence i n the cytoplasm i s further substantiated by comparing the behaviour of the d i f f e r e n t types of inclusions after permanganate f i x a t i o n . Of the 3 types of inclusions reported, only one shows a response sim i l a r to that of.the i n t r a p l a s t i d metabolite, i . e . , non-preservation using the permanganate f i x a t i o n ( c f . figure 13 3, white arrow with figure 13 4, arrowhead '3'). C e l l w all. The c e l l wall as a whole seems to have increased i n thickness (figure 160). However, neither i t s thickness nor s t r u c t u r a l complexity are uniform, since conspicuous c e l l wall ingrowths are observed. The c e l l wall ingrowths are made up of metabolites trapped between the old layers of the c e l l wall and the newly formed ones underneath (figure 171). This phenomenon begins early i n the t r a n s i t i o n "1-2" period, and attains i t s highest degree of elaboration i n " c e l l type #2". Figures 56 and 63 (cwi) show a very early stage i n formation of the ingrowths. At t h i s stage, metabolites, probably of vacuolar o r i g i n (figure 153, arrows), begin to accumulate i n the paramural space. Figure 151 shows (at arrowheads) new c e l l wall material l a i d down i n the space between the paramural.metabolites and the plasma membrane. Deposition of new.cell wall material continues u n t i l a conspicuous w a l l . i s formed, i s o l a t i n g the metabo-l i t e s from the plasma membrane (figures 153, 158, '2'). New metabolites can now be discharged into the paramural space between the new c e l l wall material and the plasma membrane 30 giving to the conjunct a layered pattern (figures 152, 158). Deposition of new wall material alternately with discharge of metabolites can produce s t r i k i n g patterns of c e l l wall ingrowths (figure 161) . In the cross walls i t can be observed that ingrowths may match s i m i l a r structures i n adjacent c e l l s (figures 158, and 162). Plasmodesmata were seen to run through the newly l a i d wall material (figure 158, arrowheads"). These ingrowths are c h a r a c t e r i s t i c of t h i s stage of senescence and are e a s i l y detected at the l i g h t microscope l e v e l i n l i v i n g c e l l s (figures 10, 11, arrowheads), as well as i n fixed material (figure 26, arrowhead). Cytochemical tests carried out at the l i g h t microscope l e v e l have shown that the c e l l wall ingrowths s t a i n a greenish color after t o l u i d i n e blue 0 and deep blue aft e r the Schorml's reaction. C e l l wall ingrowths formation appears to.coincide with an increase i n a c t i v i t y of the Golgi apparatus, as indicated by the loaded appearance of the Golgi-derived v e s i c l e s (figure 141). Discharge of metabolites into the external medium also seems to occur (figure 144). D) THE SENESCENT CELLS—ULTRASTRUCTURAL FEATURES OF "TRANSITIONAL 2-3" CELLS The degree of difference between the " c e l l type #2" and " c e l l type #3" i s just as d i s t i n c t as that between " c e l l type #1" and " c e l l type #2". T r a n s i t i o n a l stages between types "2" and "3". c e l l s are, therefore, to be expected. These t r a n s i t i o n a l stages, however, must be very ephemeral, since they were d i f f i c u l t to f i n d and only on a few occasions were they detected i n electron microscopic, observations. For the most part, the t r a n s i t i o n between the type"#2" and type "#3" c e l l s was abrupt (figure 146). The general morphology of one of these t r a n s i t i o n a l 31 c e l l s i s shown i n figure 145. At high magnification, c e l l morphology does not seem to be s t r i k i n g l y d i f f e r e n t from that i n " c e l l type #2", but a closer examination shows that the cytoplasm has .a higher degree of autolysis (figures;; 147, 164). Disruption of the tonoplast i s common (figure 165, arrowheads). The E.R. system i s more and more d i f f i c u l t to detect, although small p r o f i l e s are s t i l l recognizable (figure 177) . Nucleus. The nucleus i s rarely observed i n c e l l s at thi s stage of senescence. When present, i t shows unmistakable signs of disorganization (figure 170). Disorganization of the nuclear envelope (arrows) seems to occur through the v e s i c u l a t i o n of i t s membranes. This process does not proceed uniformly throughout the nuclear boundary since, as can be observed i n figure 170, the nuclear envelope i s no longer detectable i n certai n places. I t should be noted that the portion of the nuclear envelope s t i l l i n t a c t i s that facing a dictyosome. Internally to the remains of the nuclear envelope, a disorganized g r a n u l a r - f i b r i l l a r material constitutes the remains of the nucleoplasm. No chromatin or nucleolus can be recognized. Golgi complex. Si m i l a r l y to the nucleus, dictyosomes are rarely detected. When present (figure 170, D), they are at y p i c a l . No steps i n the disorganization of these organelles could be followed. Nuclear-envelope-dictyosome associations cease to e x i s t at thi s stage of c e l l u l a r senescence; no transfer of material i s detectable between the two membrane systems. Mitochondria. These organelles seem to.be more numerous at th i s stage of c e l l u l a r senescence than i n " c e l l type #2". They are usually round i n p r o f i l e and small i n dimensions (figures 147, 164). Cristae are.conspicuous; some of them display a concentric arrangement (figures 174, 175). The matrix shows a rather homogeneous and dense appearance. 32 Chloroplasts. These organelles.possess a morphology reminiscent of that found i n " c e l l type #2".. Extensive stacks of thylakoids usually occupy most of the chloroplast body (figures 167, 169, and 172).. Occasionally,. patches of i n t r a p l a s t i d metabolites are seen In association with thylakoid stacks (figure 177, white arrow). An increase i n the number and size of p l a s t o g l o b u l i seems to take place (figures 166, 167, 169). The most conspicuous changes occurring i n the chloro-plasts are the budding o f f of portions of the p l a s t i d s . Different steps i n the formation and release of these plastid-derived bodies are shown i n figures 169, 172, 178, 179, 180, 182, 185, and 186. I t i s apparent from most of these pictures that thylakoids p a r t i c i p a t e i n the formation of such bodies. Release into the cytoplasm of plastid-derived bodies i s supported by studying figures 167, 169, 176, 181 (arrowheads), where formations s i m i l a r to those reported above are found either near p l a s t i d s or elsewhere i n the cytoplasm. Release of these formations seems to involve a process of budding (figures 169, 172, 178, 185). Figures 180 and 182 suggest that fusion of the outer membrane delimiting these bodies with the chloroplast envelope membrane may also be important i n achieving t h e i r release. The fate of these bodies i n the cytoplasm could not be followed, but the sequence of events and the morphological as well as enzymatic c h a r a c t e r i s t i c s of " c e l l type #3" suggest that they become degenerated along with a l l the other cytoplasmic inclusions. The s i g n i f i c a n t r e s u l t of t h i s phenomenon i s the reduction i n p l a s t l d . volume. The chloroplast boundary becomes extremely.variable. While some.micrographs s t i l l suggest the presence of both the envelope membranes and the chloroplast E.R. (figure 177), others show no indication.of the chloroplast E.R., outside the usual, double 33 membrane envelope (figures 185, 186). At other places, only one membrane of the chloroplast envelope remains (figure 182). There i s a large number of v e s i c l e s associated with such a membrane, suggesting, the v e s i c u l a t i o n of the outer membrane of the chloroplast envelope. Pyrenoid. Pyrenoids are observed at t h i s stage of senescence (figures 166, 167, 169). Although they were not found i n " c e l l type #3" (see next section), the steps involved i n pyrenoid disorganization could not be f u l l y elucidated at present, due to the s c a r c i t y with which t r a n s i t i o n a l stages were found. Certain micrographs (figure 168, arrowheads), however, suggested the p o s s i b i l i t y of pyrenoids becoming is.olated from chloroplasts. Other micrographs ( i . e . , figure 183) showed pyrenoidal structures inside vacuoles. Never-theless, i t i s d i f f i c u l t to ascertain i f such images represent true stages i n pyrenoid destruction or are simply the r e s u l t of peculiar planes of sectioning. C e l l w all. The c e l l wall, shows most of the features already mentioned i n " c e l l type #2". E) THE SENESCENT CELLS—ULTRASTRUCTURAL FEATURES OF THE "CELL TYPE #3" This c e l l type represents the l a s t step i n senescence and leads to t o t a l degeneration and c e l l death. Nuclei and dictyosomes are never detected. The E.R. i s reduced to remnants. Chloroplast and mitochondria are s t i l l recognizable, but show unmistakable signs of disorganization. The background cytoplasm i s also highly disorganized (figures 187, 188, 189, 196, 201, and 202). The presence of ribosomes cannot be determined with.accuracy. At very advanced stages the plasmalemma-seems to have vanished (figure 206), and the disappearance of the cytoplasmic matrix i s almost complete (figures 191, 206). Studies of acid phosphatase a c t i v i t y show a uniform d i s t r i b u t i o n 34 of reaction products throughout the c e l l cavity (figures 191, 206). Reaction products are absent from the control sections (figure 203). Mitochondria. Mitochondrial remnants are observed (figures 196, 197, 201, 202). These are round in'profile.and small i n s i z e , with none or very l i t t l e matrix. The mitochondrial envelope i s disrupted i n places (figures 201, 202). Vesicular structures, many s t i l l attached to the inner membrane, represent the remains of the c r i s t a e (figures 19 6, 197, 202). Chloroplasts. The morphology of the p l a s t i d s varies according to the plane of sectioning (figures 187, 188, 189), and/or the degree of degeneration (figures 191, 198). As senescence proceeds, these organelles show a tendency to round up (figure 191). The stroma has almost completely disappeared (figures 190, 191), except i n certain areas between thylakoids (figures 193, 194). The remains of the chloroplast envelope consist of a single membrane as described i n the l a s t section (figures 190, 198). In more advanced stages of degeneration t h i s envelope membrane breaks down i n places, leaving thylakoids i n d i r e c t contact with the remains of the cytoplasm (figure 191). The 3-thylakoid band pattern i s s t i l l recognizable, but there i s a tendency for the bands to clump and form stacks of up to 40 or more thylakoids (figures 192, 200). Subsequently, thylakoid breakdown begins (figures 193, 194, 195, 198). Conspicuous deposition of pl a s t o g l o b u l i i s also observed (figures 188, 189, 195, 199). In addition, a va r i a t i o n i n the electron opacity of p l a s t o g l o b u l i can be seen (figures 195, 199). Genophore-like regions are. i d e n t i f i a b l e i n a few degenerating p l a s t i d s (figure 200, g), but pyrenoids are t o t a l l y absent. C e l l wall. Plasmodesmata possess acid phosphatase a c t i v i t y , but enzyme a c t i v i t y i s absent from control sections (see 35 figures 204, and. .205, r e s p e c t i v e l y ) . Although the c e l l w a l l exhibits features, s i m i l a r to those reported i n " c e l l type #2" (figure 188) , there are signs of c e l l wall degeneration. In some c e l l s , a loosening of the packing of the wall f i b r i l l a r material leads to the appearance of.lacuna-like spaces (figure 184). In other c e l l s the disorganization of the outer layer of t h e . c e l l wall i s apparent (figure 207). Figure 208 i l l u s t r a t e s another step i n c e l l wall degeneration, where only the inner layer of the wall seems to remain. F) ENZYME LOCALIZATION In order to gain a better understanding of the processes of d i f f e r e n t i a t i o n and senescence, the l o c a l i z a t i o n of several enzyme systems was carried out' at the u l t r a s t r u c -t u r a l l e v e l i n the various c e l l types described. The enzyme systems studied included acid phosphatase, already reported elsewhere, catalase, peroxidase, and adenosine triphosphatase. The following are the res u l t s of the study. Catalase. I t appears that i n young c e l l s ( c e l l type #1), catalase reactions were primarily l o c a l i z e d i n microbody-l i k e structures (figures 209, 210), although mitochondria also showed some deposition (figure 212). Control sections of aminotriazole-treated material showed no deposition of reaction products i n microbodies, but mitochondria had some (figure 211). Microbody-like organelles are single membrane-bounded and possess a granular ..matrix . (figures 211, 215). In some cases,, a non-crystalline. core..is also apparent (figure 215, arrow). .. When, .the ..cor.e...is„,present,. reaction products seem.to be p r e f e r e n t i a l l y associated with i t as well as with the delimiting membrane.(figure 249). I t i s in t e r e s t i n g to note the mlcrobody-mitochondrion association (figures 209, 211, 213). Microbody-like structures also 36 seem t o b e p r e s e n t i n " c e l l t y p e #2". ( f i g u r e 2 1 3 , a r r o w -head) , b u t w e r e never, d e t e c t e d i n " c e l l t y p e #3". The membrane s u r r o u n d i n g t h e p y r e n o i d a l s o shows d e p o s i t i o n o f r e d u c e d p r o d u c t s ( f i g u r e 2 0 9 ) , b u t s i m i l a r r e s u l t s a r e o b t a i n e d i n c o n t r o l m a t e r i a l ( f i g u r e 2 1 1 ) , t h e r e f o r e i t i s l i k e l y t h a t t h i s i s n o t . t h e r e s u l t o f enzyme a c t i v i t y b u t due t o a r e a c t i o n b e t w e e n DAB and OsO^. P e r o x i d a s e . I n " c e l l t y p e #1" p e r o x i d a s e a c t i v i t y i s p r i m a r i l y a s s o c i a t e d w i t h t h e c e l l w a l l ( f i g u r e s 214, 2 1 6 ) . C o n t r o l e x p e r i m e n t s s u b s t a n t i a t e s u c h c o n c l u s i o n s . F i g u r e 218 shows t h e e f f e c t o f KCN i n h i b i t i o n on t h e m a t e r i a l . No r e a c t i o n i s f o u n d . D e p o s i t i o n o f r e a c t i o n p r o d u c t s a r e a l s o f o u n d i n t h e " t r a n s i t i o n a l 1-2" c e l l s . No d e p o s i t i o n i s d e t e c t e d i n " c e l l t y p e #3." The p r e s e n c e o f p e r o x i d a s e i n " c e l l t y p e #2" c a n n o t be a s c e r t a i n e d w i t h c o n f i d e n c e , due t o t h e e x i s t e n c e o f l a r g e amounts o f o s m i o p h i l i c m e t a b o l i t e s i n b e t w e e n t h e c e l l w a l l f i b r i l l a r m a t e r i a l ( f i g u r e 1 4 4 ) . F i g u r e s 2 2 1 , and 222 show p e r o x i d a s e l o c a l i z a t i o n a n d c o n t r o l e x p e r i m e n t s i n " c e l l t y p e #2", r e s p e c t i v e l y , t h u s f u r t h e r d e m o n s t r a t i n g t h e d i f f i c u l t i e s i n d e t e r m i n a t i o n o f t h e e x i s t e n c e o f p e r o x i d a s e a c t i v i t y . A d e n o s i n e t r i p h o s p h a t a s e . M a t e r i a l ( c e l l t y p e #1) i n c u b a t e d + + -++ i n a medium c o n t a i n i n g K -Na -Mg -ATP shows c o n s p i c u o u s d e p o s i t i o n s o f r e a c t i o n p r o d u c t s on t h e p l a s m a membrane ( f i g u r e s 2 2 3 , 2 2 5 ) , on m i t o c h o n d r i a l membranes ( f i g u r e 2 2 4 ) , and i n t h y l a k o i d s ( f i g u r e s 2 2 3 , 2 2 5 ) . F i g u r e 228 shows t h e c o n t r o l e x p e r i m e n t . . The. a b s e n c e o f r e a c t i o n p r o d u c t s i s a p p a r e n t . D e p o s i t i o n o f r e a c t i o n p r o d u c t s , i s o b s e r v e d i n " t r a n s i t i o n a l 1.-2" c e l l s ( f i g u r e 2 2 9 ) , b u t t h e enzyme a c t i v i t y i n " c e l l t y p e #2" c a n n o t b e d e t e r m i n e d : w i t h c e r t a i n t y due t o t h e c h a r a c t e r i s t i c s o f t h e s e c e l l s . F i g u r e 230 shows a c o n t r o l f o r " t r a n s i t i o n a l 1-2" c e l l s ; t h e 37 absence of r e a c t i o n , products., i s . e v i d e n t . . ATPase . a c t i v i t y was absent from " c e l l type #3". The Mg -ATP incubated m a t e r i a l shows ..in " c e l l , type #1" r e a c t i o n products .within the mi t o c h o n d r i a ( f i g u r e 226), on the plasmalemma and. i n t h y l a k o i d s ( f i g u r e . 227.) . However, the amount of d e p o s i t i o n does not seem to. be so i n t e n s e as i n the case of the K +-Na +-Mg + +-ATP-dependent system. D e p o s i t i o n of r e a c t i o n products seases when, the m a t e r i a l i s incubated i n a medium without ATP ( f i g u r e 232).. Reaction products are apparent i n the " t r a n s i t i o n a l 1-2" c e l l s ( f i g u r e 219), but again i t i s d i f f i c u l t t o c o n f i r m t h e i r presence i n " c e l l type #2". Reaction products are m i s s i n g i n the c e l l o r g a n e l l e s of " c e l l type #1". G) ULTRASTRUCTURAL IDENTIFICATION OF AGING PIGMENT Many i n c l u s i o n s found i n l y s o s o m e - l i k e s t r u c t u r e s p r e s e n t i n Ectocarpus resemble the s o - c a l l e d l i p o f u s c i n m a t e r i a l (aging pigment), which i s c o n s i d e r e d t o be one of the most important m o r p h o l o g i c a l f e a t u r e s of the aging process i n both protozoan and animal c e l l s (e.g., Rudzinska, 1961; S t r e h l e r , 1962). In order, t o t e s t f o r such m a t e r i a l i n aging c e l l s of Ectocarpus, two techniques adapted f o r u l t r a s t r u c t u r a l i d e n t i f i c a t i o n of the pigment (Hendy, 1971) were used i n the p r e s e n t study. L i g h t microscope .studies showed t h a t as the c e l l aged a s t r i k i n g i n c r e a s e i n li p o f u s c i n - r e a c t i n g . . . substances occurred, (figure.. 26) .. At the. e l e c t r o n microscope l e v e l the use of Fontana's modified,, procedure .showed, the l a b e l l i n g to take p l a c e p r e f e r e n t i a l l y i n .vacuolar, i n c l u s i o n s ( f i g u r e 231). Reactions i n c r e a s e s i g n i f i c a n t l y i n " c e l l type #2" ( f i g u r e 233). Furthermore, l a b e l l i n g i s r e s t r i c t e d 38 to vacuolar inclusions and does not.occur i n the. p l a s t i d -drived metabolites (figure 233, arrows). Schorml's modified prodedure increases, the electron opacity of. some, vacuolar inclusions (figure 235). Figure 234 shows vacuolar inclusions af t e r formaldehyde f i x a t i o n . 39 DISCUSSION PART I—THE YOUNG CELLS (CELL TYPE #1) C e l l s of Ectocarpus within a filament exhibit progressive increases i n the tendency to bind osmium from the apex downward, followed by sudden losses i n the a f f i n i t y for osmium towards the basal part of the filaments. Light and electron microscopic observations, supplemented by cytochemical and enzymatic studies, have revealed major and progressive differences i n the morphology and metabolic conditions of c e l l s i n each filament. Three main c e l l types are recognized. While " c e l l type #1" displays u l t r a s t r u c t u r a l features c h a r a c t e r i s t i c of non-senescent c e l l s ( c f . Bouck, 1965; Bourne and Cole, 1968; Cole and L i n , 1968; Cole, 1969, 1970; Bisalputra et a l . , 1971), " c e l l type #2" and " c e l l type #3" show unmistakable signs of senescence. The o v e r a l l morphology of the nucleus of Ectocarpus " c e l l type #1" i s s i m i l a r to that described i n other brown algae (Bouck, 1965; Bourne and Cole, 196 8; Cole and L i n , 1968; Liddle and Neushul, 1969; Cole, 1970; Bisalputra et a l . , 1971). In the resting stage, the nucleus i s t y p i f i e d by the inconspicuous appearance of chromatin. Chromatin becomes more apparent as c e l l s approach nuclear d i v i s i o n (Cole and Lin , 1968; Cole, 1969). Liddle and Neushul (1969) reported that ultracentrifugation.studies of Zonaria f a r l o w i i oogonium indicated that two granular phases, d i f f e r i n g i n size and density, could be recognized i n the nucleolus. McCully (1968) reported only one granular component i n Fucus n u c l e o l i . . Bourne and Cole (19 68) described Phaeostrophion irregulare n u c l e o l i as granular.structures, while the n u c l e o l i of Eudesme were described as i r r e g u l a r dense structures (Cole, 1969). .In Ectocarpus sp., n u c l e o l i have two d i s t i n c t components, which i n morphology and dimensions clo s e l y 40 resemble those described for n u c l e o l i of higher plants . (Lafontaine and Chaourdinard, 1963). A similar, organization has been reported i n n u c l e o l i of. Laminaria.meristodermatic c e l l s "(Davies et a l . , 1973). The association between chromatin and the inner membrane of the nuclear envelope i s observed at many s i t e s around the nucleus. This association has been found i n other material to occur with the annuli of the nuclear pores complex (Comings and Okada, 1970a; DuPraw, 1970). Neverthe-le s s , evidence also exists that such relationships could take place at s i t e s other than the annuli (Comings and Okada, 1970b). The f i b r i l l a r material detected i n the nucleoplasm i n close re l a t i o n s h i p with the inner membrane of the nuclear envelope could, according to i t s dimensions, be homologous to chromatin f i b e r s of interphase nuclei (Zirkin and Kim, 1972). The functional significance of chromatin-nuclear envelope associations has long been debatable. Several authors (e.g., Comings and.Okada, 1970a, 1970b; DuPraw, 1970) have postulated that such an association could be important i n determining arrangements of the chromatin fibers p r i o r to t h e i r condensation into chromosomes; thereby influencing nuclear d i v i s i o n . Other authors suggest that t h i s relationship could have an e f f e c t on organization and d i s t r i b u t i o n of nuclear pores (Maul et a l . , 1971; Thair and Wardrop, 1971; Teig l e r and Baerwald, 1972). The l a t t e r hypothesis could be important i n explaining nuclear pore arrangements i n Ectocarpus.. In Ectocarpus, as well as i n Dictyota (Neushul and Dahl, 1972a), freeze-etched preparations .of the nuclear envelope show.zones of uniformly arranged pores- alternating with pore-free.regions. Recent studies on brown a l g a l , n u c l e i (Neushul and Dahl, 1972a). have shown, that the staining properties of the peripheral nucleoplasm vary with changes i n nuclear 41 metabolism. I t i s possible, therefore,, that the i n t e r n a l architecture of the nucleus influences the.pattern of nuclear pores d i s t r i b u t i o n . The absence of nuclear pores from the region of the nuclear envelope associated with the nucleolus i s . a l s o known i n other material (LaCour and Wells, 1972). Although nuclear pores are ubiquitous structures, t h e i r f i n e organization has not yet been completely elucidated, and several s t r u c t u r a l models have been currently proposed (e.g., LaCour and Wells, 1972; Wunderlich and Speth, 1972). Furthermore, Franke (1970) has suggested that studies on nuclear pore structure should be extended to the 2 nm l e v e l before any conslusions can be drawn. Discussion of nuclear pore organization i n Ectocarpus i s therefore unwarranted at present. As i n the case of Dictyota (Neushul and Dahl, 1972a) , freeze-etch studies of the nuclear envelope reveal i n Ectocarpus two d i s t i n c t fracture faces, one representing the outer and the other the inner membrane of the envelope. The sc a r c i t y of data, however, makes any i n t e r p r e t a t i o n of the freeze-etch images d i f f i c u l t . The problem i s further complicated by c o n f l i c t i n g opinions on whether the fracture plane occurs along the organelle-cytoplasm interface or passes through the hydrophobic regions of the membranes. Recent evidence (DaSilva and Branton, 1970; T i l l a c k and Marchesi, 1970; T e i g l e r and Baerwald, 1972; Monneron et a l . , 1972) favors the l a t t e r hypothesis. However, the p o s s i b i l i t y that fracture planes are at the cytoplasm-membrane int e r f a c e , which has already been defended by Northcote and Lewis (1968), was revived by Thair and Wardrop (1971—see also Robinson and Preston, 1972). Production of membranous material by the nuclear envelope seems to be f a i r l y common i n brown a l g a l c e l l s (e.g., Cole and L i n , 1968; McCully, 1968; Bouck, 1969; Walker, 1971; Neushul and Dahl, 1972a). In the present 42 study e i t h e r v e s i c u l a r or more complex membranous...structures are observed, but t h e i r f u n c t i o n s arid f i n a l fates, are u n c e r t a i n . C o n t i n u i t y between the outer membrane of the n u c l e a r envelope and the p e r i n u c l e a r E.R. i s frequently, observed. P e r i n u c l e a r E.R. has.been d e t e c t e d i n other algae (e.g., M a s s a l s k i and Leedale, 1969; P a l a n d r i , 1972). P a l a n d r i (1972) has suggested t h a t the p e r i n u c l e a r E.R. arrangement might i n d i c a t e i n t e n s e m e t a b o l i c a c t i v i t i e s . T h i s might a l s o be the case f o r Ectocarpus, s i n c e s e c t i o n s w i t h such arrangements of the E.R. u s u a l l y c o n t a i n c r y s t a l l i n e d e p o s i t s i n s i d e d i l a t e d p o r t i o n s o f the E.R. C o n t i n u i t y between the E.R. and the outer membrane of the m i t o c h o n d r i a l envelope has been observed i n Ectocarpus as w e l l as i n hi g h e r p l a n t and animal c e l l s (Bracker and Grove, 1971; Morre e t a l . , 1971; Franke and Kartenbeck, 1971). Since d e t a i l s o f the f u n c t i o n a l s i g n i f i c a n c e o f E.R.-mito-c h o n d r i a l a s s o c i a t i o n s were f u l l y d i s c u s s e d by Bracker and Grove (1971) and Franke and Kartenbeck (1971) , no d e t a i l e d c o n s i d e r a t i o n s w i l l be necessary here. They p o i n t e d out t h a t the concept of a continuous c h a n n e l i n g system i n v o l v i n g a l l the c i s t e r n a l cytomembranes should be extended t o i n c l u d e the c i s t e r n a l compartment bordered by the outer and the i n n e r m i t o c h o n d r i a l membranes. I f such an assumption i s v a l i d , i t i s then l e g i t i m a t e t o f u r t h e r extend t h a t concept to i n c l u d e the space between the two membranes of the c h l o r o p l a s t envelope, s i n c e i t s c o n t i n u i t y w i t h the c h l o r o p l a s t E.R. c i s t e r n a l space i s obvious in.. E c t o c a r p u s . T h e . s t r u c t u r e s d e s i g n a t e d as o s m i o p h i l i c s t r u c t u r e d bodies are abundant.in c e r t a i n c e l l s . T h e i r o r i g i n . i s c l o s e l y a s s o c i a t e d w i t h the c h l o r o p l a s t . E.R. .... In some. s e c t i o n s i t i s p o s s i b l e t o f o l l o w t h e i r r e l e a s e i n t o the paramural space. However, t h e i r f u n c t i o n i s unknown. These s t r u c t u r e s c o u l d correspond t o the s o - c a l l e d physodes of other authors. 43 (FeId-man and Guglielmi, 19 72). Pore- and bridge-like formations similar, to those reported i n other plant material. (Franke.and Scheer, 1972; Franke et a l . , 1972). are found i n Ectocarpus dictyosomes. In a l l other aspects the Ectocarpus dictyosomes are morphogically s i m i l a r to those f©und i n other brown algae (Bouck, 1965; Bourne and Cole, 1968; Cole and L i n , 1968; McCully, 1968; Cole, 1969, 1970; Bisalputra et a l . , 1971). A d i s t i n c t association between dictyosomes and nuclei i s very much i n evidence. Direct continuity between the perinuclear space and dictyosomes can be seen, but i n general, the relationship between these two membrane systems i s indicated by the transfer of small vesi c l e s from the outer membrane of the nuclear envelope to the formative face of dictyosomes (Bouck, 1965; Bourne and Cole, 1968; Cole and Lin, 1968; Cole, 1969, 1970; Bisalputra et a l . , 1971). In addition dynamic interactions take place between dictyosomes and pyrenoids (figures 60, 63), mitochondria (figure 61) and vacuoles (figure 112). Of possible significance are the dictyosome-pyrenoid interactions, which could f a c i l i t a t e a d i r e c t and more e f f i c i e n t transfer of photosynthates to dictyosomes. I t i s possible to assume that such a d i r e c t transfer route could also a f f e c t metabolite composition i n r e l a t i o n to the photosynthates transferred v i a the chloroplast E.R.-perinuclear space-dictyosome pathway (Bouck, 1965) . Dictyosomes i n Ectocarpus are exclusively perinuclear i n p o s i t i o n , a feature that according to some authors (Bouck, 1965; .Cole and L i n , 1968; Cole, 19 69) may be of phylogenetic s i g n i f i c a n c e . The fine structure of mitochondria in. active c e l l s of Ectocarpus. i s similar, to that, reported in. other brown algae (Bouck, 1965; Bourne and Cole, 1968,; Cole and L i n , 1968; Cole, 1969; Liddle and Neushul, 1969). Ectocarpus chloroplast fine structure i s also 44 simi l a r to that reported for other brown algae (Bouck, 1965; Bourne and Cole, 1968; Cole and L i n , 1968; Evans, 1968; Bisalputra and Bisalputra, 1969; Cole, 1969, 1970; Liddle and Neushul, 1969; Bisalputra et a l . , 1971), except for the presence of filaments and pores i n the thylakoids. Filament-l i k e elements are seen jo i n i n g thylakoids of the same or neighboring thylakoid bands as well as i n an i n t r a l o c u l a r p o s i t i o n . So far as I am aware, th i s i s the f i r s t report of these structures i n brown a l g a l chloroplasts. Nevertheless, Staehelin (1966), applying freeze-etching techniques, was able to report i n C h l o r e l l a , the presence of f i b r i l s 4 nm i n diameter running from the plasmalemma through the ground cytoplasm into the chloroplast. In a l a t e r work, the same author was able to show that i n Cyanidium caldarium s i m i l a r f i b r i l s linked photosynthetic lamellae to one another. The filaments also traversed the l o c u l i of the thylakoids (Staehelin, 1967). In the present study, although dimensions of f i b r i l l a r elements were found to be variable, the mean of the averages closely approach those reported by Staehelin (1966, 1967). In Ectocarpus as well as i n other brown a l g a l chloroplasts (e.g., Bouck, 1965; Bourne and Cole, 1968; Cole and L i n , 1968; Cole, 1969) the space between thylakoids i n each band and between thylakoid bands as well as the relat i o n s h i p of the peripheral band to the chloroplast envelope show s t r i k i n g uniformity. I t i s , therefore, possible that the f i b r i l l a r elements described above could play an important r o l e i n maintaining space r e g u l a r i t i e s within the chloroplasts. Staehelin (1967) has expressed a sim i l a r point of view. This point of view would f i t into a broader concept, where establishment of st r u c t u r a l uniformity by membrane to membrane linkages may be p h y s i o l o g i c a l l y advantageous (e.g., Franke et a l . , 1971a, 1971b, 1972). Pore-like interruptions are observed In the 45 thylakoid membranes. These interruptions are probably not r e s t r i c t e d . t o Ectocarpus chloroplasts, but t h e i r presence has not yet been reported. I t i s not known i f . t h e y are permanent features .of the thylakoid membranes. Freeze-etch studies of the aspect and d i s t r i b u t i o n of p a r t i c l e s i n a l g a l thylakoid membranes were recently c a r r i e d out (Neushul, 1971) . Based on these studies, two types of fracture faces are recognized i n the brown alga Dictyota. Freeze-etch images of Ectocarpus thylakoids seem to be s i m i l a r to those i n Dictyota. The exposed faces, as shown i n e a r l i e r studies (Meyer and Winkelmann, 1969; Neushul, 1970), seem to r e s u l t from s p l i t t i n g of the thylakoid membranes. Further comments on the significance of these findings seem unwarranted here, since the subject was thoroughly considered by Neushul (1971). Concentric lamellar bodies of probable p l a s t i d o r i g i n were found i n the cytoplasm of the red algae (e.g. Brown and Weier, 1970) . Dawes and Rhamstine (1967) and Sabnis (1969) reported the presence of spherical lamellar bodies i n the cytoplasm of the green alga Caulerpa p r o l i f e r a , whose ch a r a c t e r i s t i c s also point towards a p l a s t i d o r i g i n . S t r i k i n g thylakoid patterns have been reported i n p l a s t i d s of Leathesia difformis sporelings (Cole and L i n , 1968; Cole et a l . , 1968), but t h i s was found to be related to a special process of p l a s t i d d i v i s i o n . In the present study, concentric lamellar bodies were shown to have a p l a s t i d o r i g i n . Incorporation of the concentric bodies into.vacuoles with associated acid phos-phatase a c t i v i t y suggests that t h i s process could f a c i l i t a t e massive transport of p l a s t i d material to the cytoplasm, where, a f t e r digestion i n lysosomal-like fashion, the products can be recovered and r e u t i l i z e d by the c e l l s . A l terations of the basic pattern of thylakoid arrangement (3 thylakoids/band) were recorded i n t 46 Chorda fHum ( Bouck,1965 ), Fucus serratus.and P e l v e t i a canaliculata ( Evans, 1968: ) , LeathesiaJdlf f ormls.. ( dole and Lin,1968 ).. In Ectocarpus, deviations from the.basic pattern also occur. These seem to be due either to the exchange of thylakoids between adjacent bands or to the b i f u r c a t i o n and merging of neighboring bands. The close r e l a t i o n s h i p between thylakoid membranes and p l a s t o g l o b u l i indicates the p o s s i b i l i t y of thylakoid involvement i n plastoglobular production, a phenomenon well known i n chromoplasts ( e. g. Harris,1970; Lichtenthaler, 1970 ) and p l a s t i d senescence ( e. g. Lichtenthaler,1968 ). In both cases, however, p l a s t o g l o b u l i formation seems to be the r e s u l t of thylakoid breakdown. In Ectocarpus " c e l l type #1" no signs of thylakoid breakdown are evident, so p l a s t o g l o b u l i accumulation i s most l i k e l y the r e s u l t of an excess of l i p i d production over the rate of thylakoid assembly. The l i p i d nature of p l a s t o g l o b u l i i s corroborated by t h e i r destruction a f t e r permanganate f i x a t i o n . However, Magne (1971) has recently shown that i n a wide range of plants, including Ectocarpus crouanii, insoluble poly-saccharides are present at the plastoglobular s i t e s . Therefore, the o r i g i n of p l a s t o g l o b u l i might be considerably more complex than simply related to thylakoid assembly and t h e i r role i n chloroplast metabolism more elaborated than previously supposed. Nowhere else has such a wide range of v a r i t i e s of chloroplast d i v i s i o n been observed i n the same plant as i n Ectocarpus . This could be the r e s u l t of an extensive study of a l l the c e l l s i n sequence along the filaments of both the erect and the protrate.systems of.Ectocarpus sp., rather than l i m i t i n g the studies to. certain, c e l l s . The. other and more l i k e l y explanation i s . t h a t images.suggesting p l a s t i d division. , i n most.cases r e f l e c t peculiar planes of sectioning through multilobed chloroplasts ( figure.4 ), 47 where several interpretations are possible.. However, figures 102 and 103 are without question v i s u a l i z a t i o n s of true stages of chloroplas.t division.. ..This conclusion i s based not only on the study, of.Ectocarpus •material i n s e r i a l sections, but a c t u a l l y these.images match chloroplast d i v i s i o n images.in the brown alga.Sphacelaria. (Bisalputra and Bisalputra, 1970) . I t seems apparent from the present studies that an i n t e r p r e t a t i o n of images suggesting chloroplast d i v i s i o n must be c a r e f u l l y considered and not taken to be accurate evidence without intensive studies. The general morphology of the pyrenoid agrees with that described i n other brown algae (see G r i f f i t h s , 1970, for a review). The pyrenoid body contains f i b r i l l a r material which i s highly reminiscent of that of P y l a i e i l a l i t t o r a l i s pyrenoids (Gibbs, 1962a). In the present material, pyrenoids have a c l e a r l y p o s i t i v e protein reaction (see also Rosowski, 19 70). No DNA or RNA reactions were detected i n the pyrenoids of Ectocarpus, although the presence of both substances have been reported i n Tetracystis excentrica (Brown and Arnott, 1970) and i n other a l g a l pyrenoids (Esser, 1967; Simon, 19 54) . Pyrenoids appear i n Ectocarpus as projecting stalked bodies, which usually arise from the side of the chloroplast facing the nucleus. This type of pyrenoid i s suggested to have phylogenetic implications (e.g., Evans, 1966, 1968; Bourne and Cole, 1968; Hori, 1971). .In.Ectocarpus sp., usually only one pyrenoid i s found per chloroplast. .Cases, of two or.more pyrenoids occurring i n the same chloroplast appear., to. be. .related, to. .the process of pyrenoid d i v i s i o n . I t i s apparent from. this.study, that sometimes a very close r e l a t i o n s h i p exists between the f i b r i l l a r , elements of the pyrenoid matrix and the thylakoid membranes. The meaning of t h i s r e l a t i o n s h i p requires 48 elucidation; the p o s s i b i l i t y exists that a ph y s i o l o g i c a l involvement of thylakoids i n pyrenoid development might occur. This rela t i o n s h i p i s p a r t i c u l a r l y apparent i n the dark treated material, a treatment which seems to stimulate pyrenoid d i v i s i o n ( Manton,1966a). Bailey and Bisalputra (19 69) have described the ultrastructure of the c e l l wall i n Ectocarpus acutus. The present findings support t h e i r description. Lomasomes and plasmalemmasome's ( Marchant and Robards, 1968 ) were found i n Ectocarpus sp. during sporeling develop-ment ( u n i l l u s t r a t e d data ) as well as i n sporophytes. Although these structures seem to be well represented i n algae ( see Cole and Lin,1970, for a review ), t h e i r presence i n the brown algae has only recently been reported i n sporelings of Petalonia d e b i l i s ( Cole and Lin,19 70 ). Bracker (1967) suggested 9 d i f f e r e n t functions for the lomasomes. As indicated by Cole and L i n (1970), evidence concerning the r o l e of these structures i s mostly circums-t a n t i a l , and therefore, d i f f i c u l t to sustain without further in v e s t i g a t i o n . Recent autoradiographic studies, however, implicate these structures i n the deposition of matrix materials i n the c e l l wall ( Cox and Juniper,1973 ). Plasmddesmatal continuity between neighboring c e l l s i n the brown algae was shown by Bisalputra (1966). Since the publication of t h i s work further evidence showing plasmodes-matal continuity i n these algae has rapidly accumulated. The organization of these structures i n the cross walls adjoining neighboring c e l l s has been suggested as phyloge-n e t i c a l l y s i g n i f i c a n t ( e. g. Cole and Lin,1968; Hori,1971 ). Plasmodesmata are also observed i n Ectocarpus sp. where the lack of organization.into pit.areas i s apparent. In conclusion, the ultr a s t r u c t u r e of young c e l l s of Ectocarpus i s not d i f f e r e n t from that reported i n other brown algae. The young c e l l s are t y p i c a l l y meristematic 49 or the immediate derivative of €hese. These c e l l s are confined to the f i r s t 6-8 c e l l s - i n each filament. They are characterized by a dense cytoplasm, with a well organized nucleus, mitochondria, chloroplasts, stalked pyrenoids, exclusive perinuclear location of the Golgi apparatus, and the.non-organization of plasmodesmata into p i t f i e l d s . Several of these features ( stalked pyrenoid morpho-logy, perinuclear location of dictyosomes, plasmodesmata non organized i n p i t f i e l d s ) support the hypothesis of e a r l i e r authors ( e.g. Fritsch,1945; Smith,1955 ) that Ectocarpus i s primitive among the brown algae. Neverthless, other authors ( e. g. Bisalputra et al.,1971; Chi,1971; Neushul and Dahl,1972a ) have warned that careful use of the above mentioned c h a r a c t e r i s t i c s as phylogenetic markers i s necessary u n t i l more information regarding t h e i r behaviour and occurrence i s obtained at a l l developmental stages. PART II - TRANSITIONAL "1-2" CELLS Vacuolation. I n i t i a l steps i n d i f f e r e n t i a t i o n of " c e l l type #2" involves the process of vacuolation. Vacuoles were reported to arise i n a variety of ways, including l o c a l d i l a t i o n s of the E.R., special a c t i v i t y of the Golgi apparatus or other structures such as mitochondria, or combinations of these ( e. g. Marinos,1963; Barton,1965; Ueda,1966; Mesquita,1969; Matile and Moor,1968; B e l i t s e r , 1972; Berjack,1972 ). The evidence from Ectocarpus indicates that vacuoles are the r e s u l t of E.R. a c t i v i t y , although dictyosomes are also involved. In addition to the.well known role of vacuoles as reservoirs of waste products i n plant c e l l s ( e. g. Robards, 19 70 ), the discovery of a wide range of associated lysosomal 50 enzymes ( e.g. Gahan,1969; Matile, 1968, 1969a, 1969b ) has led to the concept that vacuoles may function i n plant c e l l s as a lysosome equivalent ( e. g. Matile,1969a; H a l l and Davie,1971 ). In Ectocarpus remnants of cytoplasmic structu-res inside vacuoles as well as associated acid phosphatase a c t i v i t y are clear indicators of the role of vacuoles as lysosome equivalents. Autophagy. Several mechanisms have been described i n the l i t e r a t u r e to explain the o r i g i n of vacuolar inclusions; that i s , to explain how cytoplasmic material becomes trapped inside vacuoles ( see review by Fineran,1971 ). In Ectocarpus sp. more than one mechanism i s involved i n trapping cytoplasmic regions inside vacuoles. One of the mechanisms involves the i s o l a t i o n of pockets of cytoplasm by the E. R. The same process of c e l l u l a r autophagy was reported i n other plants ( e. g. Buvat,1968; Mesquita,1972; V i l l i e r s , 1972; Coulomb,1973; Marty,1973 ). The other mechanism of autophagy found i n Ectocarpus involves invagination of the tonoplast with subsequent incorporation of cytoplasmic material into the vacuoles. This process i s found to occur frequently i n other plant c e l l s ( Coulomb and Buvat,1968; Matile and Moor,1968; Wardrop,1968; Matile and Winkenbach, 1971; Belitser,1972; H a l l and Davie,1971; Coulomb,1973 ). There i s also evidence suggesting that the cytoplasm might take an active part i n t h i s process of autophagy. A s i m i l a r s i t u a t i o n was described i n Fraxinus excelsior ( V i l l i e r s , 1972 ). I t i s quite possible that i n any mechanism of incorporation of cytoplasmic material into vacuoles both the tonoplast and the cytoplasm may take an active part i n the process ( Fineran,1971 ). Subsequent to accumulation of materials inside vacuoles there i s . a marked reaction of acid phosphatase a c t i v i t y . Progressive a l t e r a t i o n s of the vacuolar inclusions are observed; leading to the appearance of two types of 51 remnant materials.. The f i r s t of these materials i s hetero-geneous and comprised of... a. granular component intermingled with a myelin-like component. The second i s simply granular. Morphologically, the."osmiophillc structured bodies" and the mixed vacuole remnants are much a l i k e , although t h e i r o r i g i n s are quite d i s t i n c t In both time (the "OSB" appears i n almost every young c e l l before autophagy becomes detectable) and space (the "OSB" originates from the chloroplast E.R. while the mixed remnants are the r e s u l t of autophagy and lysosomal a c t i v i t y ) . Nevertheless, the majority of electron-dense inclusions accumulated i n these t r a n s i -t i o n a l c e l l s can be broadly c l a s s i f i e d as "residual bodies", which clog the vacuoles. The reason for the clogging of the vacuoles i s not understood. Recent work has suggested some possible explanations. Tappel (1968) finds that proteins and membranes are less d i g e s t i b l e a f t e r being damaged by l i p i d peroxidation. He suggests that, i n d i r e c t l y , t h i s property contributes to lysosomal engorgement. Another l i n e of evidence shows that the hydrolase content of a c e l l varies due to the heterogeneity of the lysosomal enzyme population (e.g., Hayashi, 1967; Matile, 1968; Schultz and Jacques, 1971; Hanker et a l . , 1972). I t i s , therefore, quite understandable that lysosomal structures may be incapable of digesting the i n c l u s i o n material, e s p e c i a l l y when t h e i r enzyme complement i s not appropriate. This s i t u a t i o n can lead to a permanent or temporary engorgement of vacuoles. Comolli. et a l . (1972) have also suggested that lysosome engorgement could r e s u l t from changes i n the lysosomal enzymes brought about.by modifications i n t h e i r rates of synthesis and/or degradation. Acid phosphatase a c t i v i t y was. found ...in.. Ectocarpus to be associated with both the E.R. and Golgi apparatus. E.R.-associated acid phosphatase was demonstrated i n a number of plants (e.g., Camefort, 1966; Catesson and Czaninsky, 52 1968; Figier.,1968; Roland, 1969;' Robards and Kidway,1969; Zee',1.969; Poux,1970; Coulomb, and Coulomb., 1971.; Figier,1972; Gezelius, 19.72 ) , as was the. dlctyosome-assoclated acid phosphatase ( e. g. Poux,1962a, 1962b; Figier,1968; Coulomb, 1969; Halperin,1969; Roland,1969; Poux,1970; Coulomb and Coulon,1971.; Figier,1972; Micalef, 1972; Rougier,1972 ) . I t . i s not known, however, i f the E.R.-associated acid phospha-tase and that associated with the dictyosomes possess d i f f e r e n t hydrolytic p o t e n t i a l i t i e s ; a n d therefore, i f these findings bear any r e l a t i o n s h i p to the clogging of the vacuolar apparatus. Chloroplast. Also associated with the d i f f e r e n t i a t i o n of the " c e l l type #1" morphology into that c h a r a c t e r i s t i c of the " c e l l type #2" i s a tendency for chloroplast thylakoids to form large stacks. An increase i n thylakoid number with aging was also reported i n other plants ( e. g. McLean,1968, Dodge,1970; Messer and Ben-Shaul,1972 ). C e l l Wall. The existence of osmiophilic metabolites among the c e l l wall m i c r o f i b r i l s and at the c e l l wall periphery seems to support Fogg and Boalch (1958) and Armstrong and Boalch (1960) results for the release of metabolites into the culture medium. During these t r a n s i t i o n a l stages, formation of the so-called c e l l wall ingrowths (cwi) seems to reach a peak. Simultaneous with ingrowths formation, both electron and l i g h t microscope r e s u l t s ( l o c a l i z a t i o n of insoluble carbohydrates by the P.A.S. reaction) show intense dictyo-somal a c t i v i t y , further supporting other authors r e s u l t s that dictyosomes are involved i n c e l l wall development i n the brown algae ( Cole,19 69) as well as i n plant material i n general ( e. g. Pickett-Heaps,1967a, 1967b; Wooding,1968; Barton,1968 ). 53 PART III—THE "CELL TYPE # 2" Several authors consider i n t r a c e l l u l a r accumulation of l i p i d material to be symptomatic of senescence i n both, animal (e.g., Takah.ashi.et.al., 1970; Howse and Welford, 1972) and plant c e l l s (e.g., McLean, 1968; Schuster et a l . , 19 68; Palisano and Walne, 1972). As one studies the Ectocarpus c e l l s further away from the apical t i p , i t can be observed that simultaneously with an increasing'tendency to bind osmium there i s an increasing a f f i n i t y for Sudan black B. Electron microscope studies reveal a variety of inclusions crowding the cytoplasm. After permanganate f i x a t i o n some of these inclusions are not preserved, while others are only p a r t i a l l y preserved; further suggesting that l i p i d material i s present i n many of these inclusions. The designation of these c e l l s as senescent seems, therefore, j u s t i d i e d . This i s further corroborated by the morphology of t h e i r c e l l organelles. The nuclear boundary i s usually i r r e g u l a r , a common feature of senescing c e l l s i n both plants (Shaw and Manocha, 1965; Butler, 1967; Berjack and V i l l i e r s , 1970, 1972a, 1972b; Bria r t y et a l . , 1970; Fabbri and Palandri, 1970; V i l l i e r s , 1972) and animals (e.g., Sohal and A l l i s o n , 1971; Lipetz and C r i s t o f a l o , 1972). Nuclear pores are very d i f f i c u l t to detect. I t i s not known from the present data i f t h i s i s due to an increasing d i f f i c u l t y i n distinguishing the pores or i f t h i s i s the r e s u l t of a reduction i n the number of pores due to the low metabolic state of these c e l l s . Variations i n the number of nuclear pores due to the metabolic state of the c e l l s were reported i n other b i o l o g i c a l material (e.g... A f z e l i u s , 1955; Barnes and Davies, 1959; Grasso.et a l . , 1962; Moor.and Muhlethaler, 1963; Wiener et a l . , 1965;. Franke, 1967; Franke and Scheer, 1970; Wunderlich 54 Speth, 1972) . The reason for. the variat i o n s is. not under-stood. However, as suggested by some authors, (e.g. Thair and Wardrop, 1971; La Fountaine and La Fountaine, 1973), these variations may.reflect the synthetic a c t i v i t y of the nucleus and simultaneously indicate the degree of exchange that takes place between the nucleus and the cytoplasm. The evidence seems also to Indicate that c e l l s having a low metabolic a c t i v i t y have a lower number of nuclear pores (e.g. Barnes and Davies, 1959; Grasso et a l . , 1962; Moor and Muhlethaler, 19 63). P a r t i c u l a r l y pertinent are the re s u l t s obtained by Moor and Muhlethaler,(1963), who have shown decreases i n nuclear pore number to be associated with the aging process i n yeast c e l l s . The nuclear envelope ceases to produce v e s i c l e s or other membranous structures. N u c l e o l i , when observed, are s i m i l a r i n organization to those found i n " c e l l type #1". The presence of n u c l e o l i i s corroborated by l i g h t microscope cytochemistry. Increased nucleolar s i z e with rearrangement of the nucleolar components has been observed i n senescing nuclei i n other plants (Fowke and S e t t e r f i e l d , 1968; Chapman and Jordan, 1971; Jordan and Chapman, 1971; Jordan, 1972). No si m i l a r c o r r e l a t i o n could be done i n Ectocarpus. Some electron micrographs suggest the presence of chromatin. This i s confirmed by l i g h t microscope data. Therefore, even when other c e l l organelles (e.g. E.R., chloroplasts) already display a greatly altered morphology, nuclei s t i l l give a conspicuous DNA reaction. Nuclei have been considered very r e s i s t a n t to disruption during senescence. (Butler, 1,9 67; Roux and.McHale, 1968; Mittelheuser and van Steveninck,. 1971).. Shaw, and Manocha (.1965.) found that, even after, n u c l e i of. advanced senescing. c e l l s of wheat leaves have l o s t most of t h e i r RNA and,some p r o t e i n , DNA levels remained high. A decrease i n lev e l s of both RNA and protein i s 55 observed i n these c e l l s (see summary of l i g h t microscope cytochemical data). The present data, however, suggest that such decreases a f f e c t p r i m a r i l y the pattern of d i s t r i b u t i o n of "these substances. A reduction .in the size of RNA and protein staining-areas i s observed, without"any apparent e f f e c t on staining i n t e n s i t y . ( c f . figure 15 with figure 16, and figure 18 with figure 19). Reductions i n stained areas are e a s i l y understood.after electron microscope studies, which show the reduction to be mainly due to increasing vacuolation and autophagy. The s t a b i l i t y i n staining i n t e n s i t y i s i n t e r e s t i n g and indicates that i n the remaining cytoplasm RNA and protein contents are compara-t i v e l y high. Certain c h a r a c t e r i s t i c s of the nucleus, i . e . , nucleolar morphology, DNA content, together with high RNA and protein content indicate the capacity of these c e l l s for active protein synthesis. This does not imply that there i s no a l t e r a t i o n i n the proteins being synthesized. Protein synthesis i s a nucleic acid-directed phenomenon, although much less i s known about the mechanisms involved i n regulating t h e i r synthesis. There i s evidence to suggest that a v a r i a t i o n i n gene expression occurs with d i f f e r e n t metabolic conditions, that i s , some genes are expressed at one stage of the c e l l l i f e span but not at others (e.g. Carr and Pate, 1967). This would a f f e c t the type of proteins being produced and subsequently c e l l development, d i f f e r e n t i a t i o n and senescence (e.g. Heslop-Harrison, 1967; Shannon, 1968; Scandalios, 1969, Spencer and Titu s , 1972). .Such a p o s s i b i l i t y has. led Woolhouse (1967) to think of d i f f e r e n t i a t i o n and - senescence as two stages of the same process, which i s the r e s u l t of. a controlled gene expression (see also Carr and.Pate, 1967). Alterations i n the amount and. type of proteins being synthesized and disruption of normal c e l l functions can..be the r e s u l t of causes other than those mentioned above. "Pelc (1970) thinks of aging as the r e s u l t of an accumulation of damaged DNA 56 copies ( u n t i l no correct copies are l e f t ) , rather than the r e s u l t of a controlled expression.of genes.. The p o s s i b i l i t y also remains (Johnson and Strehler, 1972) that aging may r e s u l t from loss of genes coding for ribosomal RNA. Aging-dependent protein variations may also be the r e s u l t of mechanisms not d i r e c t l y related to protein synthesis (e.g. Simon, 19 67; Cherry, 1967) . Whatever the explanation, the fact remains that aging i s dependent upon the presence and production of certain s p e c i f i c proteins (e.g. Matile and Winkenbach, 19 71). An increase i n the amounts of l y t i c enzymes during aging i n both plant and animal c e l l s was reported by d i f f e r e n t authors (Klamer and Fennell, 1963; E l l i o t t and Bak, 1964; Blum, 1965; Sommer and Blum, 1965; Schuster et a l . , 1968; Grusky and Aaranson, 1969; Elens and Wattiaux, 1969; C r i s t o f a l o , 1970; Comolli, 1971; Matile and Winkenbach, 1971; Palisano and Walne, 1972). In Ectocarpus sp., as the c e l l ages, an increase i n acid phosphatase a c t i v i t y can be detected by cytochemical means. However, increases i n enzyme a c t i v i t y are not necessarily related to de novo synthesis of enzymes (Klamer and Fennell, 1963). The increase i n enzyme a c t i v i t y can be due to a c t i v a t i o n of latent enzymes (e.g. Simon, 1967). More recent evidence seems to suggest that the increase could, at l e a s t i n part, be the r e s u l t of an aging-dependent reduction i n the rate of enzyme degradation (e.g. Mainwaring, 1968, 1969; Srivastava, 1969; Comolli et a l . , 1972). Walne et a l . , (1970) and Palisano and Walne (19 72) have suggested that i n plant .as well as i n animal.cells, l i p o f u s c i n - l i k e pigments accumulate as the result, of aging. In Ectocarpus sp. " c e l l type #2" some of the cytoplasmic inclusions are morphologically, reminiscent, of the so-called l i p o f u s c i n inclusions of aging animal tissues (e.g. Takahashi et a l , 1970; Sohal and Sharma, 1972). Samorajski et a l . (1965) proposed that such lysosomal inclusions should be 57 considered as l i p o f u s c i n inclusions when myelin or laminated figures could be v i s u a l i z e d . However, as recently reviewed by Hasan and Glees (1972), morphological c h a r a c t e r i s t i c s of l i p o f u s c i n inclusions can be quite variable. Therefore, the use of f i n e s t r u c t u r a l c h a r a c t e r i s t i c s as a c r i t e r i o n i n i d e n t i f i c a t i o n of l i p o f u s c i n does not seem to be a v a l i d one ( see also Zeman,1971 ). Of the d i f f e r e n t hypothesis advanced to explain the o r i g i n of l i p o f u s c i n ( see Toth,1968, and Zeman,1971, for a review), the one r e l a t i n g them to lysosomes i s the most widely accepted ( e. g. Essner and Novikoff,1960; Strehler and Mildvan,1962; Koenig,1963; Samorajski et a l . , 1964, 1965; Goldfisher et al.,1966; Frank and Christensen,1968; Hirsch,1970; Brunk and Ericsson, 1972a ). Since most of " c e l l type #2" cytoplasmic i n c l u -sions seem to be of lysosomal o r i g i n , two techniques adapted by Hendy (1971) for the u l t r a s t r u c t u r a l i d e n t i f i c a -t i o n of l i p o f u s c i n were applied to the present material. Toluidine blue staining show an increase i n the amount of greenish-turquoise-stained inclusions with aging. These inclusions are, therefore, probably polyphenolic i n nature ( e. g. McCully,1966; Evans and Holligan,1972 ). Fontana's s i l v e r solution ( Hendy,19 71 ) r e l i e s on s i l v e r n i t r a t e impregnation to i d e n t i f y l i p o f u s c i n material. S i l v e r n i t r a t e , however, i s also known to impregnate phenolic compounds ( e. g. Chadefaud,1936 ). Schorml's solution ( Hendy,1971 ) uses a mixture of i r o n chloride and potassium ferricyanide to s p e c i f i c a l l y l a b e l l i p o f u s c i n inclusions. Phenolic vacuoles ( e. g. physodes ) respond p o s i t i v e l y to iron s a l t s ( e. g. Chadefaud,1936 ). However, F r i t s c h (1945) reported..that .no. response could..be recorded with i r o n chloride. C u l l i n g . (1963) considered. Schorml 1s method to be.quite s p e c i f i c for the i d e n t i f i c a t i o n of l i p o f u s c i n . In addition, an intense Sudan black B response characterizes " c e l l type #2". Physodes do not react to Sudan black B ( e. g. Chadefaud,1936; Evans and Holligan, 58 1972) , but l i p o f u s c i n - l i k e . material .gives, ...at.,least during certain phases of i t s formation,,a strong response to the st a i n (e.g. C u l l i n g , 1963; Pear.se, 1972) . ' I t seems, there-fore,: that the evidence points to the 'possibility -of some of the inclusions. found i n aging, c e l l s of Ectocarpus to be. l i p o f u s c i n related. G i f f o r d (1968) suggested that plastid-derived inclusions believed to contain both l i p i d s and phenolic compounds accumulated inside.vacuoles. Pearse (1972) also suggested the p o s s i b i l i t y of simultaneous occurrence of l i p o f u s c i n and materials r e s u l t i n g from transformation of phenolic compounds inside vacuoles. From the present studies, i t i s impossible to determine whether i n Ectocarpus phenolic compounds exist independently or are mixed with l i p o f u s c i n metabolites. The implications o f . l i p o f u s c i n accumulation and i t s r e l a t i o n to c e l l u l a r metabolism have remained largely obscure. The inclusions have been suggested to represent i n e r t waste products (e.g. Bjorkerud, 19 64), but most consider them to i n t e r f e r e with the physiology of the c e l l s , leading eventually to c e l l death (e.g. Samorajski et a l . , 1964, 1968; Raychaudhuri and Desai, 1971; Sohal and Sharma, 1972). Hers and van Hoof (cited i n Zeman, 1971) postulated that c e l l u l a r dysfunction was the r e s u l t of mechanical disturbances created by increasing accumulations of residual material i n the cytoplasm of the c e l l s . Accumulation of l i p o f u s c i n inclusions, known to contain reaction products of l i p i d peroxidation and other free r a d i c a l reactions (e.g. Tappel, 1965.; Bjorkerud, 1964), was shown to produce rapid.deterioration of the membrane systems of several organelles, including lysosomes. (Packer, et al.., 1967). Membrane deterioration, e s p e c i a l l y lysosomal, membranes, was suggested by.Hochschild (1971) to.lead to the leakage of hydrolytic enzymes into the cytoplasm; hence profoundly a f f e c t i n g c e l l metabolism. Recent evidence (Sullivan and 59 Debusk, 1973) seems to support t h i s hypothesis, and suggests that c e l l u l a r aging, may r e s u l t from membrane-damage and the consequent synthesis of altered proteins. Sohal and Sharma (1972) have proposed that the e f f e c t s of l i p o f u s c i n accumula-tion on the physiology of the c e l l could.be the r e s u l t of two i n t e r r e l a t e d phenomena: 1) as l i p o f u s c i n accumulates the amount of native cytoplasm i s reduced, disrupting normal c e l l u l a r functions, and 2) the accumulation of l i p o f u s c i n creates a new. environment around the nucleus that might influence genetic a c t i v i t y . This hypothesis i s p a r t i c u l a r l y a t t r a c t i v e i n the case of Ectocarpus. ' In Ectocarpus, most of the accumulation of inclusions coincides with v e s i c u l a t i o n of the E.R. Since there i s evidence to indicate that the nucleus might be Important i n maintaining the i n t e g r i t y of the E.R. (e.g. F l i c k i n g e r , 1968), a possible re l a t i o n s h i p might e x i s t i n Ectocarpus between in c l u s i o n accumulation and E.R. v e s i c u l a t i o n . The process of E.R. v e s i c u l a t i o n has often been noted i n other senescing plant material and has been interpreted as a sign of E.R. degenerency (Shaw arid Manocha, 1965; Bain and Mercer, 1966; Butler, 1967; T r e f f r y et a l . , 1967; V i l l i e r s , 1972; Potapov and Krishnamurthy, 1972). The disorganization of the E.R. i s also expected to a f f e c t the ribosomal population of these c e l l s . Shaw and Manocha (1965) reported that disappearence of the E.R. was simultaneous with that of ribosomes. Fukazawa and Higuchi (19 66) provided evidence for the existence of a c o r r e l a t i o n between a reduction i n the ribosomal. population, and a reduction i n the E.R. However, Mittelheuser and van Steveninck . (1971) have shown that ribosomes possess a greater s t a b i l i t y than other c e l l .organelles.. Berjack.and V i l l i e r s (1972b) reported that even.in stages.of extreme:disorganization monosomes could.be found randomly scattered throughout the cytoplasm (see also Gpik,. 1966; Berjack and V i l l i e r s , 1970; Mia, 1972).. On. the contrary, Palandri (1972) showed' that 60 although the E.R. i s s t i l l well represented, most of the ribosomal population has disappeared from seneseing c e l l s of Halimeda - tuna. In Ectocarpus,. small.clusters of p a r t i c l e s are s t i l l found here and there during early stages of " c e l l type #3". However, t h e i r ribosomal nature could not be determined and so the pattern of ribosomal behaviour i s d i f f i c u l t to e s t a b l i s h . The nucleus-dictyosome association i s s t i l l very apparent at t h i s stage of senescence and production of dictyosome-derived v e s i c l e s from the maturing face of dictyosomes is. also detectable. These ve s i c l e s seem to be loaded with metabolites, but t h e i r fate i s d i f f i c u l t to ascertain. This i s not only due to the c h a r a c t e r i s t i c s of the background cytoplasm, but also to the v e s i c u l a t i o n of the E.R. that makes i t d i f f i c u l t to d i s t i n g u i s h either type of v e s i c l e . C e l l wall thickening i s associated with aging i n d i f f e r e n t plant materials (e.g., James, 1966; Fabbri and Palandri, 1968, 1969; McLean, 1968; Palandri, 1972). In Ectocarpus senescence i s also accompanied by wall thickening with l o c a l i z e d formation of c e l l wall ingrowths. These c e l l wall ingrowths are made of two d i f f e r e n t components: 1) metabolites, probably of vacuolar o r i g i n , and 2) c e l l wall materials which i s o l a t e the metabolites from the plasma membrane. The metabolites cl o s e l y resemble some of the material accumulated i n the paramural space of seneseing root c e l l s of Glechoma hederacea (Bowes, 1972). This author has also shown, the metabolites to be vacuolar i n o r i g i n and possible.lomasomal i n nature. In Ectocarpus the vacuolar o r i g i n of the c e l l w a l l ingrowth metabolites i s at least p a r t i a l l y apparent, but-their morphological c h a r a c t e r i s t i c s hardly f i t into a lomasdme concept. One can say that t h i s process represents i n Ectocarpus a mechanism for the c e l l to get r i d of excess waste metabolites. However, t h i s hypothesis i s circumstantial 61 and f a i l s to account.for the reason(s) why the phenomenon does not occur uniformly a l l over, the c e l l wall,, but i t i s p a r t i c u l a r l y evident at places. C e l l wall ingrowths allow for.the easy recognition of t h i s c e l l type under the l i g h t microscope. Similar structures seem to e x i s t In other brown algae (Chadefaud, 19 36), but have never been described at the electron microscope l e v e l . Cytochemical tests carried out at the l i g h t microscope l e v e l showed these structures to give p o s i t i v e reactions for both phenolic compounds (greenish with toluidine blue 0--McCully, 1966) and l i p o f u s c i n (deep blue with Schorml's—Hendy, 1971). Their phenolic nature was also recorded by Chadefaud (1936). This double staining response could be rather s i g n i f i c a n t . Indeed, th i s could be interpreted as evidence for the occurrence i n the same incl u s i o n of both phenolic and l i p o f u s c i n - l i k e substances. I t i s i n t e r e s t i n g that as l o c a l i z e d ingrowths enlarge at the cross wall l e v e l , symplast continuity between c e l l s does not seem to be disrupted. This observation could be p a r t i c u l a r l y pertinent for the advance of senescence, since as proposed by Simon (1967), the rate of senescence could be dependent upon the existence of a sink (in Ectocarpus " c e l l type #1" could act as a sink) for materials exported from aging parts of the plants. An analysis of the mitochondrial population as well as mitochondrial features i n " c e l l type #2" seems unwarranted here, since these organelles are very d i f f i c u l t to detect. At f i r s t one gains the impression that.the mitochondrial population might have d r a s t i c a l l y decreased at t h i s stage of senescence. Two facts argue against.such.an hypothesis: 1) these c e l l s possess.a very crowded cytoplasm with a l l sorts of inclusions which make the mitochondria very d i f f i c u l t to d i s t i n g u i s h (see also.Schuster et a l . , 1968), and 2) more advanced stages of senescence have large mitochondrial 62 populations, which, .become, v i s i b l e when. the.. number of inclusions-in..the ground cytoplasm have decreased, revealing more e a s i l y -the remaining., structures. Conspicuous stacks of thylakoids.are v i s i b l e i n " c e l l type #2" chloroplasts.. A s i m i l a r phenomenon, was noticed i n other aging plant material (e.g. McLean, 1968; Dodge, 1970; Messer and Ben-Shaul, 1972), and has been considered as a sign of reduction of p l a s t i d a c t i v i t y (McLean, 1968). In Ectocarpus a very close r e l a t i o n s h i p exists between t h i s type of thylakoid arrangement and the formation of metabolites of high electron density, whose response to the osmium and permanganate fi x a t i o n s as well as to the Sudan Black B indicates a possible l i p i d content. The appearance of large amounts of l i p i d material i n p l a s t i d s was hypothesized by Harnischfeger (1972) as a sign of metabolic conditions which r e s u l t i n damage of the electron transport chain and therefore i n reduced p l a s t i d a c t i v i t y . Phenolic compounds (or phenolic compound precursors) were suggested to be p l a s t i d derived (e.g. G i f f o r d , 1968; Evans and Holligan, 1972; Davies et a l . , 1973). The p o s s i b i l i t y that the Ectocarpus p l a s t i d metabolite i s phenolic i n nature i s not supported by the s i l v e r n i t r a t e reaction (Chadefaud, 1936) , since i t f a i l s to l a b e l the i n t r a p l a s t i d metabolite (figure 233, arrows). I t i s also i n t e r e s t i n g to note that non-labelling of i n t r a p l a s t i d metabolites or s i m i l a r material found i n the cytoplasm support the concept that the l a b e l l i n g i s most, l i k e l y . r e s t r i c t e d to inclusions of lysosomal, origin... The. p o s s i b i l i t y , also exists that intraplastid.metabolites are the r e s u l t . o f f i x a t i o n a r t i f a c t s . Other.evidence seems to indicate, however, that i n t r a p l a s t i d inclusions are. s i g n i f i c a n t i n ..senescence. F i r s t -l y , these inclusions.are associated with a s p e c i a l arrange-ment of the thylakoid membranes. Secondly, i n permanganate fixed material as well as freeze-etching s i m i l a r preparations 63 bearing the same relat i o n s h i p to thylakoids are also detecta-ble. Discharge of p l a s t i d metabolites into the cytoplasm i s apparent and therefore contributes to the establishment of the ground cytoplasm c h a r a c t e r i s t i c s of " c e l l type #2". PART IV - "TRANSITIONAL 2-3" CELLS As senescence continues the cytoplasm becomes increa-singly a u t o l y t i c to the point where very few or almost no inclusions are present. At the l i g h t microscope l e v e l the transparency of the c e l l s becomes apparent. The s t r i k i n g differences e x i s t i n g between " c e l l type #2" and " c e l l type #3" make i t reasonable to assume the existence of intermediate stages. These t r a n s i t i o n a l stages were seldom detected and i n most cases the t r a n s i t i o n was abrupt. Loss of vacuolar compartmentation i s conspicous i n plant c e l l senescence ( e. g. Shaw and Manocha,1965; Butler, 1967; Roux and McHale,196 8; Halperin,1969; De Vecchi,1971; Habeshaw and Heyes,1971; Matile and Winkenbach,1971; Stearns and Wagenaar,1971; Berjack and Vi l l i e r s , 1 9 7 2 a , 1972b ). Gahan(1965) and Barton (1966), among others, have suggested that the rupture of the tonoplast marked the beginning of the sequence of events leading to the massive a u t o l y t i c condition of the c e l l s . The a u t o l y t i c condition i s most l i k e l y due to the massive release of toxic substances ( e. g. Shaw and Manocha,1965; Robards,1970 ) and l y t i c enzymes ( e. g. Matile,1969a ), which are known to be present i n the c e l l sap. Other authors ( e. g. Shaw and Manocha,1965; Butler, 1967 ) maintained that the vacuole content might dif f u s e through the tonoplast. The l a t t e r p o s s i b i l i t y seems.to be --. supported by recent cytochemical evidence ( Brunk and 64 Ericsson, 1972b). Di f f u s i o n through the tonoplast of c e l l sap substances, may thus, t r i g g e r an ..irreversible., chain of events ultimately leading to c e l l death; and the rupture of the tonoplast w i l l be a consequence rather than the cause. The causes of modification i n tonoplast permeability are as yet undetermined, although d i f f e r e n t p o s s i b i l i t i e s have been advanced i n the l i t e r a t u r e . An analysis of them i n r e l a t i o n to Ectocarpus re s u l t s w i l l be presented. Accumulation of l i p o f u s c i n inclusions such as those i n Ectocarpus has been suggested to cause damage to lysosome membranes, a f f e c t i n g permeability and r e s u l t i n g i n leakage of hydrolytic contents into the cytoplasm (Gabrielescu, 1970; Hochschild, 1971; Brunk and Brun, 1972). Berjack and V i l l i e r s (1970) thoroughly discussed the p o s s i b i l i t y that incorporation into vacuoles of dictyosome-derived v e s i c l e s carrying carbohydrate metabolites ultimately causes bursting of lysosomal membranes. The p o s s i b i l i t y also exists that l y t i c enzymes of extralysosomal o r i g i n may play a role i n " c e l l type #3" formation (Lin and Fishman, 1972). Whatever the true explanation, the evidence suggests that once a massive autolysis i s i n i t i a t e d , transformation of the c e l l content must proceed with extreme speed since i n most of the cases studied the t r a n s i t i o n between the " c e l l type #2" morphology and that c h a r a c t e r i s t i c of " c e l l type #3" i s sudden (see also Berjack and V i l l i e r s , 1970, 1972b; Woolhouse, 1967). A l l steps involved i n nuclear disorganization could not be followed, but available, information, shows many . s i m i l a r i t i e s with the.same s i t u a t i o n i n other senescing plants (e.g. Shaw and Manocha, 1965; Barton, 1966; Wooding, 1966) . The number of mitochondria.seemed to.have increased. One must, however, be cautious i n int e r p r e t i n g these 65 observations.. F i r s t l y because of the d i f f i c u l t y , i n studying " c e l l type #2" mitochondria, and secondly, m u l t i p l i c a t i o n of mitochondria at thi s stage of senescence would be d i f f i c u l t to explain. Despite t h i s , an increase i n the mitochondrial population was. reported in.aging c e l l s of Halimeda tuna (Palandri, 1972) and aging s l i c e s of potato tubers (Lee a'hd Chasson, 1966) . These observations are exceptions rather than the ru l e . Indeed, i n most of the cases studied, aging i n plant c e l l s has not been correlated with increases i n the mitochondrial population (see Butler and Simon, 1971). At t h i s stage of senescence, the mitochondrial envelope remains i n t a c t and the matrix i s darker. The cr i s t a e are less numerous than i n " c e l l type "1" and occasion-a l l y assume concentric arrangements, a configuration also detected i n aging wheat leaves (Shaw and Manocha, 1965). Similar features have been interpreted as an in d i c a t i o n of mitochondrial senescence i n other plant material (see Butler and Simon, 1971, for a review). I t i s known that a decline in.respiratory e f f i c i e n c y usually accompanies senescence (e.g. Drapper and Simon, 1971). Lund et al.,(1958) have reported that a l t e r a t i o n of the mitochondrial architecture r e f l e c t s decreases i n respiratory e f f i c i e n c y . Data for Ectocarpus are purely morphological and not backed up by experimental evidence. Moreover, a drop i n respiratory e f f i c i e n c y could have occurred long before i n i t i a t i o n of any a l t e r a t i o n i n mitochondria (see Drapper and Simon, 1971).. The problem, i s , nevertheless, in t e r e s t i n g to consider. Varner (1961) has suggested that changes i n respiratory metabolism,, p a r t i c u l a r l y i n oxida-t i v e phosphorylation, could be a major cause of senescence, due to the many c e l l u l a r a c t i v i t i e s , directly, or i n d i r e c t l y dependent upon i t . Of a l l the c e l l organelles, chloroplasts undergo the 66 most s t r i k i n g changes. In.some cases p l a s t i d . morphology s t i l l resembles that c h a r a c t e r i s t i c o f . " c e l l type #2". As production of i n t r a p l a s t i d metabolites cease,.changes i n thylakoid organization.become.more apparent. There i s also an apparent increase i n the number of p l a s t o g l o b u l i . Of int e r e s t i s the budding of stroma containing, structures from the p l a s t i d body. Morphologically si m i l a r phenomena were reported i n other plant material, but not i n connection with senescence (Gullvag, 1968; Schotz et a l . , 1971). Although the significance of.the p l a s t i d budding phenomenon might not be completely understood, i t seems reasonable to assume that i t plays a major role i n the reduction of p l a s t i d volume (see also Dodge, 1970; Fabbri and Palandri, 1970). Indeed, a decrease i n p l a s t i d size seems to occur i n Ectocarpus, p a r t i c u l a r l y i n " c e l l ..type #3" (see also F r i t s c h , 1945). A decrease i n p l a s t i d size i s rather common i n seneseing plant material (Ikeda and Ueda, 1964; Barton, 1966; Ljubesic, 1968; Dodge, 1970, De Vecchi, 1971; Stearhs and Wagenaar, 1971). Since pyrenoids were never found i n " c e l l type #3", i t seems safe to assume that they might have disappeared during these t r a n s i t i o n a l stages. However, as already pointed out, images suggesting pyrenoid breakdown were very d i f f i c u l t to interpret and the elucidation of the mechanism of pyrenoid disorganization must await further research. PART V—THE "CELL TYPE #3" It i s clear that t r a n s i t i o n a l "2-3" c e l l s possess a very altered c e l l u l a r architecture. ..The ground. cytoplasm shows extreme autolysis;. the. mitochondria and. p l a s t i d s are senescent; the tonoplast has. broken, .down i n places; the nucleus i s d i f f i c u l t to detect .and when present highly disorganized; the dictyosomes are r a r e l y detected and 67 abnormal i n apperance. A l l these features are unmistakable signs of c e l l necrosis. Therefore, " c e l l type #3" should be considered as a phase i n the f i n a l process of c e l l autolysis and not separated from the " t r a n s i t i o n a l 2-3" c e l l s . The a u t o l y t i c appearance of the background cytoplasm i n " c e l l type #3" correlates d i r e c t l y with the d i s t r i b u t i o n of acid phosphatase a c t i v i t y . Reaction product i s seen a l l over the cytoplasm, a sign that the compartmentation of lysosomes no longer exists and the c e l l cavity represents a single large lysosomal unit. A l l the other enzymes tested seem to have ceased to function and the l i g h t microscope data give further proof of c e l l degeneration. Nuclei and dictyosomes were never observed i n " c e l l type #3". This i s not surprising since the n u c l e i , as previously shown, begin to disorganize during the " t r a n s i -t i o n a l 2-3" stages. The E.R., i f present, i s reduced to very altered and almost u n i d e n t i f i a b l e membrane profiles.The plasmalemma i s discontinuous, the tonoplast no longer recognizable; a l l inclusions, with the exception of mitochondria and p l a s t i d s remains have disappeared. Mitochondrial p r o f i l e s are s t i l l numerous, but the mitochondrial envelope i s disrupted i n several places. The cr i s t a e and the matrix of the mitochondria are no longer detectable. These features are sim i l a r to those found i n degenerating mitochondria i n other senescent plants ( e. g. De Vecchi,1971; Butler and Simon,1971 ). Chloroplasts are most often delimited by a single membrane, a remnant of the chlo r o p l a s t envelope. They are almost devoid of stroma, and for the most part only large stacks of lamellae, remain. As disorganization.proceeds the lamellar system.becomes greatly..reduced and the envelope membrane i s also absent. The f i n a l steps i n disorganization of Ectocarpus p l a s t i d s are, therefore, si m i l a r to those reported for other plants ( e. g. Shaw and Manocha,1965; 68 De Vecchi, 1971; Messer and Ben-Shaul, 1972). In association with, thylakoid.breakdown conspicuous depositions, of plasto-p g l b u l i are detectable (e.g. Ikeda and Ueda, 1964; Barton, 1966; Fabbri and Palandri, 1970;. Dodge,\197.0: Butler and Simon, 1971; Stearns and. Wagenaar ,..1971) . . In some cases, one gains the impression that the plastoglobular population of degenera-t i n g chloroplasts i s heterogeneous i n t h e i r electron opacity. A similar s i t u a t i o n was reported by Fabbri and Palandri (1970) i n Ricinus communis senescing cotyledons. Stearns and Wagenaar (1971) also reported s i m i l a r phenomena i n senescing chloroplasts of autumn leaves and suggested that variations i n electron opacity of p l a s t o g l o b u l i were due to t h e i r role as waste baskets for breakdown products. Butler (1967) concluded that although p l a s t o g l o b u l i i n senescing chloroplasts were the r e s u l t of an accumulation of membrane breakdown products, they probably also acted as storage bodies for insoluble l i p i d materials not necessarily associated with membrane breakdown. Plastids seem to be the most r e s i s t a n t organelles i n senescing Ectocarpus c e l l s . I t i s i n t e r e s t i n g to note that the same s i t u a t i o n was found i n necrotic c e l l s of Ascophyllum  nodosum (Rawlence, 1972). Dissolution of the c e l l wall represents i n Ectocarpus the l a s t stage i n c e l l u l a r destruction. C e l l wall breakdown occurs i n other plants (e.g. Clowes and Juniper, 1968; Halperin, 1969; Bal and Payne, 1972). Bal and Payne (1972) found a c o r r e l a t i o n between c e l l wall breakdown and E.R. development (see also Clowes and Juniper, 1968). However,.when.wall breakdown.occurs i n Ectocarpus the c e l l i s reduced to.an.almost v i r t u a l l y , empty cavity. Acid phosphatase„is_..pr.esen.t...inside the c e l l s but deposition of reaction.products i s .not conspicuous i n the c e l l wall. De Jong (1966) reported that glutaraldehyde f i x a t i o n , although not a f f e c t i n g i n t r a c e l l u l a r acid phospha-tase a c t i v i t y , did block the c e l l wall reactions that were 69 otherwise evident i n ...unfixed c e l l s . Other reports (e.g. Halperin, 1969), show that acid, phosphatase, a c t i v i t y i s l o c a l i z e d i n c e l l walls, but even i n - these, cases.no function-a l r e l a t i o n s h i p between-its. presence and c e l l wall breakdown could be demonstrated. Horton.and Osborne (1967) have shown that as senescence proceeds an increase i n c e l l u l a s e a c t i v i t y i s also observed. C e l l wall breakdown was related to high l e v e l s of c e l l u l a s e a c t i v i t y i n cultured carrot c e l l s (Halperin, 1969). No attempts were made to study other enzyme a c t i v i t i e s in..Ectocarpus c e l l s , and so the enzymatic machinery behind c e l l wall disorganization remains unknown. The process of d i s i n t e g r a t i o n of the c e l l wall suggests the presence of.two d i f f e r e n t components: 1) a f i b r i l l a r component, and 2) a cement-like (matrix) material which seems to be the f i r s t to disorganize. Cronshaw et a l . (1958), and Myers and Preston (1959a, 1959b) reported that c e l l walls of marine algae were apparently composed of a network of f i b r i l s embedded i n an amorphous matrix. Recent evidence supports the occurrence of a s i m i l a r construction pattern for the c e l l wall of Fucus (Fulcher and McCully, 1971). The f i b r i l l a r component of the wall i s probably c e l l u l o s i c (Percival, 1968), but i t might also be a carboxylated polysaccharide, probably a l g i n i c acid, which i s known to assume a m i c r o f i b r i l l a r configuration (McCully, 196 8) . The nature of the cementing substance or amorphous matrix i s not e n t i r e l y known, but McCully. (19 66, 1968) has shown i t to be possibly a sulfated polysaccharide. Preston (1969), Lamport (1965), and.Thompson and.. Preston (1969) suggested that the orientation of the..microfibrils and the s t a b i l i z a t i o n . o f the c e l l wall structure could be on the r e s p o n s i b i l i t y of proteins. 70 PART VI—ENZYME LOCALIZATION DURING DIFFERENTIATION AND SENESCENCE The l a s t p a r t o f t h i s d i s c u s s i o n w i l l be r e s e r v e d f o r an a n a l y s i s o f e n z y m a t i c d a t a . S i n c e d e t a i l e d c o n s i d e r a -t i o n s r e g a r d i n g a c i d phosphatase a c t i v i t y and l i p o f u s c i n i d e n t i f i c a t i o n have a l r e a d y been d i s c u s s e d , the p r e s e n t c o n s i d e r a t i o n s w i l l be concerned w i t h t h e a n a l y s i s o f a denosine t r i p h o s p h a t a s e , c a t a l a s e , and p e r o x i d a s e r e s u l t s . A denosine t r i p h o s p h a t a s e (ATPase). R e c e n t l y a number o f papers have appeared w h i c h d e a l w i t h p l a n t ATPases (e.g. Coulomb and Coulomb, 1972; Cronshaw and G i l d e r , 1972; Hodges e t a l . , 1972; H a l l and D a v i e , 1971'; L a i and Thompson, 1972a, 1972b; M a i e r and M a i e r , 1972; Sundberg e t a l . , 1973). The r e p o r t s seem t o be c o n f l i c t i n g . Coulomb' and Coulomb (1972) and M a i e r and M a i e r (1972), f o r i n s t a n c e , r e p o r t e d Mg dependent ATPase a t t h e plasma membrane l e v e l . Other a u t h o r s r e p o r t e d t h a t ATPase a c t i v i t y i s s t i m u l a t e d by monovalent i o n s , b u t i n h i b i t e d (under c e r t a i n c o n d i t i o n s ) by d i v a l e n t i o n s ( A t k i n s o n and P o l y a , 1967). A pH-dependent K +-s t i m u l a t e d ATPase was d e t e c t e d i n t h e plasma membrane o f t h e green a l g a M o u g e o t i a (Sundberg e t a l . , 1973). F u r t h e r e v i d e n c e was p r e s e n t e d f o r the o c c u r r e n c e o f monovalent i o n s s t i m u l a t e d ATPases a t t h e l e v e l o f t h e plasmalemma, a l t h o u g h t h e system was s a i d t o be d i f f e r e n t from the K +-Na +-ATPase a c t i v a t e d systems (e.g. Hodges e t a l . , 1972; see a l s o R a t n e r and J a c o b y , 1973) . L a i and Thompson (1972a) p r o v i d e d e v i d e n c e f o r t h e e x i s t e n c e o f a K +-Na - s t i m u l a t e d ATPase a s s o c i a t e d w i t h t h e plasmalemma, as d i d B o w l i n g e t a l . (1972). B o w l i n g e t a l . (1972) c o n c l u d e d , however, t h a t t h e p l a n t ATPase t h e y s t u d i e d had d i f f e r e n t p r o p e r t i e s from the K +-Na +-ATPase system o f a n i m a l c e l l s . ATPases a r e a s s o c i a t e d w i t h some o f t h e most i m p o r t a n t f u n c t i o n s o f t h e c e l l (e.g. Heyden, 1969). A s t u d y 71 of ATPase l o c a l i z a t i o n i n - Ectocarpus,,, therefore, would allow a c o r r e l a t i o n between the architecture..of the : c e l l and i t s physiological status.. The results.. ;for:. Ectocarpus have shown that the pattern of reaction product, deposition..was the same for the Mg + +- and K +-Na +-Mg + +-ATPase systems. However, monovalent ions (Na +, K*) seem to have enhanced the deposi-t i o n of reaction products at both, the plasmalemma and the thylakoid l e v e l s . Involvement of monovalent ions dependent ATPases i n stimulation of ion .transport has been stressed i n plant c e l l s (e.g. H a l l and Davie., 19 71; Coulomb and Coulomb, 1972; Maier and Maier, 1972). Ratner and Jacoby (1973) found that i n t h e i r material monovalent s a l t effects on ATPase were not cation s p e c i f i c , and therefore not related to the c e l l p o t e n t i a l for cation absorption. Whether or not the enhancement of reaction product deposition i n Ectocarpus r e f l e c t s cation s p e c i f i c i t y remains to be determined. However, other authors have shown that Mg + + i s required i n order for plant ATPases to be stimulated by monovalent ions (Fisher and Hodges, 1969; Fisher et a l . , 1970; Leonard and Hanson, 1972). The reaction of the mitochondria i s probably due to the Mg + +-activated system only, without being affected by monovalent ions. Indeed, deposition of reaction products i s conspicuous i n both systems assayed. Novikoff.et.al.(1958) also reported that Mg + + ions p r e f e r e n t i a l l y stimulated the ATPase system of mitochondrial f r a c t i o n s . The above considerations hold for b o t h . " c e l l type #1" and " c e l l type #2". In " c e l l , type #3", however, none of the systems assayed led to. deposition...of....reaction products. The apparent lack, of.ATPases .in these.cells-leads one to conclude that they are no longer functional... The absence of ATPase at the plasma ..membrane., implies,, .deeply, altered physiological properties in. t h i s .membrane..system, although at the u l t r a s t r u c t u r a l l e v e l the membrane may s t i l l display 72 an i n t a c t appearance (figure 191). This i s i n agreement with reports showing that as senescence proceeds changes i n permeability, are observed i n cells,, (e.g. Eilam, 1965; Sacher, 1967; Fergusson and Simon, 1973). Catalase. Biochemical, cytochemical, and u l t r a s t r u c t u r a l studies have,during the .past few years, provided enough evidence for the i d e n t i f i c a t i o n In plants of a class of organelles usually described as."microbodies" (Molhenhauer et a l . , 1966; Frederick et a l . , 1968; Frederick and Newcomb, 1969a) or under more s p e c i f i c conditions as peroxysomes (Frederick and Newcomb, 1969b; Marty, 1969, 1970; Trelease et a l . , 1971) and glyoxysomes ( V i g i l , 1970; Beevers, 1970; Gerhardt and Beevers, 1970; Gruber et a l . , 1970; Trelease et a l . , 1971). Cytoplasmic organelles whose u l t r a s t r u c t u r a l features (single membrane boundary, granular matrix, occasional core-like structures ) resemble those, of the so-called microbodies are found i n a l l Ectocarpus sporophytic c e l l s , with the exception of " c e l l type #3". These microbody-l i k e structures are i d e n t i f i e d cytochemically by t h e i r response to diaminobenzidine (DAB) incubation. Under these conditions heavy deposition of reaction products i s associated with the microbodies. Control sections, mainly those incubated i n aminotriazole medium, show the reaction to be due to the presence of catalase (Margoliash and Novogrodsky, 1958; Margoliash et a l . , 1960) which i s the enzyme marker for microbodies (Tolbert, 1971).. This i s corroborated by the parameters of incubation, i . e . , optimum pH i n the alkaline, range.,, incubation, temperature (37°C) (e.g. Czaninski. and Catesson, 1970, 1971; Poux, 1972a, 1972b, 1972c). A consistent and close association was found i n . Ectocarpus between microbodies and mitochondria. Association with chloroplasts, although detectable, was not so evident. 73 The mitochondrion-microbody association i s not r e s t r i c t e d to the Ectocarpus material (e.g., H i l l i a r d et a l . , 1971; Poux, 1971, 1972b; Tourte, 1972). This association also occurs i n yeast and Tetrahymena c e l l s , where i t s occurrence i s related to the d i s t r i b u t i o n of glyoxylate cycle enzymes (Tolbert, 1971). I t i s impossible, however, without biochemical evidence,to make any conclusion about the significance of such an association i n Ectocarpus sp. Reaction products were also found i n mitochondria. The explanation for mitochondrial deposition of reaction products i s s t i l l debatable. Since reaction products are present even afte r incubation i n aminotriazole, one may rule out the p o s s i b i l i t y of catalase involvement, although catalase has been said to be present in. mitochondria (Herzog and Fahimi, 197.2) . Diaminobenzidine i s known to be oxidized by hemoproteins other than catalase, for instance, peroxidase and cytochrome oxidase (Seligman et a l . , 1968; Todd and V i g i l , 1972). Peroxidase i s indicated as a possible cause of mitochondrial reactions (Rothman, 196 8; Childs and M i l l e r , 1971; Gerhardt and Berger, 1971). In Ectocarpus, however, afte r incubation under conditions favouring optimum l o c a l i z a t i o n of peroxidase, no obvious mitochondrial reactions were found. The data of Plesnicar et a l . (1967) showed the absence of any kind of peroxidatic a c t i v i t y i n plant mitochondria. The p o s s i b i l i t y then remains that mitochondrial deposition of reaction products i s very l i k e l y due either to cytochrome oxidase a c t i v i t y (Seligman and Karnovsky, 1968; Gerhardt and Berger, 1971; Kataoka, 1971) or, as indicated by..Beard, and Novikoff .(1969) ,-Novikoff and Goldfisher (1969)., Novikoff (1970), Novikoff et a l . (1971), and Poux (1972a), to cytochrome c a c t i v i t y . Peroxidase. An increase i n peroxidase a c t i v i t y was reported to be c h a r a c t e r i s t i c of the ageing process (e.g., Galston and Davies, 1969). In Ectocarpus " c e l l type #3" peroxidase 74 a c t i v i t y i s . not detectable... Although i t i s possible that i n " c e l l type #2". there i s peroxidase . a c t i v i t y , .the o v e r a l l c h a r a c t e r i s t i c s of these cells.make i t very d i f f i c u l t to evaluate the pattern and i n t e n s i t y of the reaction. Therefore, any comparison with data av a i l a b l e . f o r " c e l l type #1" or the t r a n s i t i o n a l stages ..immediately following . i s . not possible. Peroxidase a c t i v i t i e s In plants have been l o c a l i z e d , using the DAB/I^O,, procedure, i n many parts of the c e l l s : c e l l wall (e.g. Ridge and Osborne, 1970; H a l l and Sexton, 1972; Poux, 1972a, 1972b, 1972c); dictyosomes (e.g. Poux 1969, 1972a; H a l l and Sexton, 1972); vacuoles (e.g. Coulomb, 1971; H a l l and Sexton, 1972; Hanzely and V i g i l , 1972; Poux, 1972a, 1972b, 1972c); ribosomes (e.g. Poux, 1972a, 1972b, 1972c); E.R., perinuclear spaces and plasma membranes (e.g. Poux, 1972a). In Ectocarpus, deposition of reaction products i s found i n the paramural space and i n the c e l l wall. That deposition of reaction products.is due to peroxi-dase a c t i v i t y can be i n f e r r e d from the experimental conditions (e.g. Poux, 1972a, 1972b, 1972c) as well as from the following control experiments: 1) aminotriazole incubation, which does not i n h i b i t deposition of reaction products, and 2) potassium cyanide incubation which i n h i b i t s deposition of reaction products (Strum and Karnovsky, 1970; Poux, 1972a). The absence of reaction products i n other i n t r a c e l l u l a r locations does not necessarily prove i t s absence. E x i s t i n g evidence shows that peroxidases, although ubiquitous i n d i s t r i b u t i o n , possess an unprecedented ..degree of heterogene-i t y (e.g. Shannon, 1968; Delincee and Radola., 1970; Radola and.Drawert, 1970).. Rucker and Radola (1971) have shown that, i n tobacco tissue cultures, a number ..of. peroxidase isoenzymes occur which could be separated ..into...3 groups on the basis of t h e i r i s o e l e c t r i c points. . H a l l and .Sexton (.1972) have studied the e f f e c t of pH on peroxidase.activity and.found a wide v a r i a t i o n i n pH-dependent responses. The evidence 75 shows, therefore, that d i f f e r e n t isoperoxidases might require i n d i v i d u a l incubation parameters to be. v i s u a l i z e d , as a recent study seems to indicate (Poux, 19 72a). The present data suggest: that peroxidase.activity i n Ectocarpus i s mainly a c e l l wall associated phenomenon. Although recent.years have seen Increasing i n t e r e s t i n the biochemical and p h y s i o l o g i c a l aspects of peroxidases, l i t t l e i s known about t h e i r r e a l functions. Ridge and Osborne (1970) have suggested that wall peroxidases might f a c i l i t a t e hydroxylation of proline i n the wall proteins, hence a f f e c t i n g c e l l wall e x t e n s i b i l i t y (Ridge and Osborne, 1971). Any role of peroxidases i n c e l l wall formation i n Ectocarpus must, however, wait for further knowledge i n wall composition i n t h i s alga. 76 CONCLUSION The present study has two objectives. The f i r s t intends to provide a more detailed knowledge of the c e l l u l a r morphology of non-senescing vegetative c e l l s of the sporo-phytic generation of Ectocarpus. Hopefully t h i s w i l l lead to a better understanding of the s i m i l a r i t i e s and the differences existent among the brown algae and between these and other organisms i n the plant kingdom. The second objective of the present work i s concerned with the study of the senescing process as i t occurs i n Ectocarpus under culture conditions. Culture conditions are known to a f f e c t the metabolism of the organisms ( e. g. Carol et al.,1972 ) and so the interpretation of the re s u l t s cannot be taken as duplication of f i e l d events. Neverthless, i t provide a useful and appropriate mean of understanding important changes i n development and senescence i n a reproducible way . In studying the aging process of any organism the i d e n t i f i c a t i o n of a single i n i t i a l change could provide a lead for a better understanding of the events of senescence. Neverthless, as pointed out by Butler (1967), i n i t i a l damage to the c e l l could be d i r e c t r e s u l t of a number of factors acting alone and i n concert, and therefore even a small change would have an accumulative e f f e c t . I d e n t i f i -cation of a single i n i t i a l a l t e r a t i o n at the u l t r a s t r u c t u r a l l e v e l i s a very d i f f i c u l t task. Therefore, any attempt to l o c a l i z e chronologically and s p a t i a l l y senescence-associa-ted phenomena must be regarded as an approximation to the r e a l sequence. Bearing t h i s i n mind, a summary of organelle c h a r a c t e r i s t i c s and modifications occurring in. Ectocarpus from " c e l l type #1" to c e l l death i s presented i n the following page. I 77 From the.data i t can be said that increases i n vacuolation and autophagy as well as chloroplast modifica-tions seem to be the f i r s t detectable signs of aging i n Ectocarpus. A f u l l cdmparision of the order of events i n r e l a t i o n to the data provided by senescence studies i n other plants i s unwarranted here; not only because of d i f f i c u l t i e s i n establishing with precision the actual order of events, but also because, as discussed by Roux and McHale (1968), variations i n the order of appearance of alterations might be species dependent. It i s apparent, however, from t h i s work that the pattern of c e l l u l a r disorganization i n Ectocarpus has many points i n common with the events reported during senescence i n other plants. The possible presence of l i p o f u s c i n metabolites during the aging of Ectocarpus also shows that a common denominator of aging might e x i s t from algae to man as suggested by Palisano and Walne (1972) during t h e i r study of senescence i n Euglena granulata. Other changes associated with aging i n other plant and animal c e l l s support such a suggestion : i . e. lobing of the nucleus ( L i p e t z and Cristofalo,1972; Munnell and Getty,1968 ), mitochondria with denser matrix and fewer c r i s t a e ( Lipetz and C r i s t o f a l o , 1972; Weinbach et al.,1967 ), increases i n c e l l u l a r autophagy ( Sohal and Allison,1971; Lipetz and Cristofalo,1972 ). In conclusion, i t can be said that no single technical approach w i l l elucidate such a complex phenomenon as i s the case of senescence. Each technical approach i s able to provide p a r t i a l information. This information, must then be compared and correlated with other data, obtained by di f f e r e n t means, and from a large number of tes t organisms. Only.afterwards can the complex process.of senescence as well as l i f e i n general be better understood. 78 LITERATURE CITED A f z e l i u s , B.A., 1955. "The ultrastructure of the nuclear membrane of the sea urchin oocyte as studied with the electron microscope." Exptl. C e l l Res. 8: 147-158. Aparicio, S.R., and Mardsen, P., 1969. "Application of standard micro-anatomical staining methods to epoxy r e s i n embedded sections." J . C l i n . Pathol. 2_2: 589-592. Armstrong, F.A.J., and Boalch, G.T., 1960. " V o l a t i l e organic matter i n a l g a l culture media and sea water." Nature 185: 761-762. Atkinson, M.R., and Polya, G.M., 1967. "Salt stimulated adenosine triphosphatase from carrot, beet and Chara  a u s t r a l i s . " Aust. J. B i o l . S c i . 20_: 1069-1086. Augier, H., and Harada, H., 1972. "Presence d'hormones de type cytokinine dans l e t h a l l e des algues marines." CR. Acad. Sc. (Paris) D 275: 1765-1768. Bailey, A., and Bisalputra, T., 1969. "Some u l t r a s t r u c t u r a l aspects of the c e l l wall of Ectocarpus acutus Setchell Garden and E l a c h i s t a f u e l c o l a (Velley) Areschong." Phycologia 8_: 57-63. Bain, J.M., and Mercer, F.V., 1966. "Subcellular organization of the cotyledons i n germinating seeds and seedlings of Pisum sativum L." Aust. J . B i o l . S c i . 19_: 69-84. Bal, A.K., and Payne, J.F., 1972. "Endoplasmic reticulum a c t i v i t y and c e l l wall breakdown i n quiescent root meristems of Allium cepa L."Z.pflanzenphysiol. 66: 265-272. Barnes, B.J., and Davies, J.M., 1959. "The structure of nuclear pores i n mammalian tissue." J . U l t r a s t r . Res. 3: 131-146. Barton, R., 1965. "Electron microscope studies on the o r i g i n and development of the vacuole i n root t i p c e l l s of Phaseolus." Cytologia 30: 266-273. Barton, R., 1966. "Fine structure of mesophyll c e l l s i n senescing leaves of Phaseolus." Planta 71: 314-325. Barton, R., 1968. "Autoradiographic studies on wall forma-ti o n i n Chara." Planta 82: 302-306. 79 Beard, M.E., and Novlkoff, A.B., 1969. "Reactions of mitochondria with diaminobenzidine." J . C e l l B i o l . 43: 12a. Beevers, H., 1970. "Glyoxysdmes of Castor bean endosperm and t h e i r r e l a t i o n to gluconeogenesis." Ann. N.Y. Acad. S c i . 168: 313-324. B e l i t s e r , N.V.j 1972. "The vacuolar system as the lysosomal apparatus of the plant c e l l . " Dok. Akad. Nauk. SSSR 203: 153-155 (English t r a n s l a t i o n ) . Berjack, P., 19 72. "Lysosomal compartmentation: u l t r a -s t r u c t u r a l aspects of the o r i g i n , development and function of vacuoles i n root c e l l s of Lepidium sativum." Ann. Bot. 36: 73-81. Berjack, P., and V i l l e r s , T.A., 1970. "Ageing i n plant embryos. I. The establishment of the sequence of development and senescence i n root cap during germina-t i o n . " New Phytol. 6_9: 929-938. Berjack, P., and V i l l i e r s , T.A., 1972a. "Ageing i n plant embryos. I I . Age-induced damage and i t s repair during early germination." New Phytol. 71: 135-144. Berjack, P., and V i l l i e r s , T.A., 1972b. "Ageing i n plant embroys. I I I . Acceleration of senescence following a r t i f i c i a l ageing treatment." New Phytol. 71_: 513-518. Berkaloff, C , 1961. "Etude au microscope electronique des plastes de Laminaria saccharina L." CR. Acad. Sc. (Paris)D 252: 2747-2749. Berkaloff, C , 1963. "Les c e l l u l e s meristematiques d' Himanthalia lorea (L.) S.F. Gray. Etude au microscope electronique." J . Microscopie 2_: 213-228. Bisalputra, T., 1966. "Electron microscopic study of the protoplasmic continuity i n c e r t a i n brown algae." Can. J. Bot. 44: 89-93. Bisalputra, T.., and Bisalputra, A..A. , 1967.. "Chloroplast and mitochondrial DNA i n a brown alga Egregia menziesii." J. C e l l Biol.. 33_: 511-520. Bisalputra, T., and Bisalputra, A.A., 1969. "The u l t r a -structure of the chloroplast of the .brown alga Sphacelaria sp. I. P l a s t i d DNA c o n f i g u r a t i o n — t h e chloroplast genophore." J. U l t r a s t r . Res. 2_9_: 151-170. 80 Bisalputra, T., and Bisalputra, A.A., 1970. "The u l t r a -structure of the. chloroplast of the brown alga Sphace1aria sp. I I I . The r e p l i c a t i o n and segregation of the chloroplast genophore." J . U l t r a s t r . Res. 32: 417-429. Bisalputra, T., and Burton, H., 1969. "The ultra s t r u c t u r e of the chloroplast of the brown alga Sphacelaria sp. I I . Association between the chloroplast DNA and the photo-synthetic lamellae." J . U l t r a s t r . Res. 29_: 224-235. Bisalputra, T., Shields, C , and Markham, J.W., 1971. "In s i t u observations of the fine structure of Laminaria gametophytes and embryos i n culture. I. Methods and the ultrastructure of the zygote." J . Microscopie 10_: 83-98. Bjorkerud, S., 1964. "Isolated l i p o f u s c i n g r a n u l e s — a survey of a new f i e l d . " In Advances i n Gerontological Research, v o l . 1, pp. 257-288. Blum, J. J . , 1965. "Observations on the acid phosphatases of Euglena g r a c i l i s . " J. C e l l B i o l . 24: 223-234. Bouck, G.B., 1965. "Fine structure and organelle association i n brown algae." J . C e l l B i o l . 2_6: 523-537. Bouck, G.B., 1969. "E x t r a c e l l u l a r microtubules. The o r i g i n , structure and attachment o f . f l a g e l l a r h a i r s . i n Fucus and As cophy Hum antherozoids. " J . C e l l B i o l . 4_0: -446-460. Bourne, V.L., and Cole, K., 1968. "Some observations on the fine structure of the marine brown alga Phaeostrophion irr e g u l a r e . " Can. J . Bot. ' 46: 1369-1375. Bowes, B.G., 1972. "Fine s t r u c t u r a l observations on necrotic adventitious root apices i n Glechoma hederacea L." Protoplasma 74: 41-52. Bowling, D.J.F., Turkina, M.V., Krasavina, M.S., and Kryucheshnikova, A.L., 1972. "Na +-K +-activated ATPase of conducting tissues." SOPPAA 19: 824-832 (English t r a n s l a t i o n ) . Bracker, C.E., 1967. "Ultrastructure of fungi." Ann. Rev. Phytopathol. _5: 343-374. Bracker, C.E., and Grove, S.N., 1971. "Continuity between cytoplasmic endomembranes and outer mitochondrial membranes i n fungi." Protoplasma 73: 15-34. B-riartry, L.G., Coult, D.A. , and Boulter, D. , 1970. "Protein bodies of germinating seeds of V i c i a faba. Changes i n 81 fine structure.and biochemistry." J..Experimental Botany 21: 513-524. Brown, R.M., and Arnott,.H.J., 19 70. "Structure and function of the a l g a l pyrenoid. I. Ultrastructure and cytochem-i s t r y during zoosporegenesis of Tetracystis excentrica." J. Phycol. 6: 14-22. Brown, D.L., and Weier, T.E., 1970. "Ultrastructure of the fresh water alga Batrachospermum. I. Thin section and freeze-etch analysis of juvenile and photosynthetic filament vegetative c e l l s . " Phycologia 9_: 217-235. Brunk, U., and Brun, A., 1972. "The e f f e c t of aging on lysosomal permeability i n nerve c e l l s of the central nervous system. An enzyme histochemical study i n r a t . " Histochemie 30_: 315-324. Brunk, U., and Ericsson, J.L.E., 1972a. "Electron micro-scopical study on r a t brain neurons. L o c a l i z a t i o n of acid phosphatase and mode of formation.of l i p o f u s c i n bodies." J . U l t r a s t r . Res. 38: 1-15. Brunk, U., and Ericsson, J.L.E., 1972b. "Cytochemical evidence for the leakage of acid phosphatase through u l t r a s t r u c t u r a l l y i n t a c t lysosomal membranes." Histochem. J. 4: 479-491. Buggeln, R.G. , and Craigie, J.S.', 1971. "Evaluation of evidence for the presence of Indole-3-acetic Acid i n marine algae." Planta 9J_: 7 3-17 8. Butler, R.D., 1967. "The f i n e structure of seneseing cotyledons of cucumber." J . Experimental Botany 18: 535-543. Butler, R.D., and Simon, E.W., 1971. " U l t r a s t r u c t u r a l aspects of senescence i n plants." In Advances i n Gerontological Research, G.L. Strehler (ed.), v o l . 3, pp. 73-129. Academic Press, New York and London. Buvat, R., 1968. "Diversite des vacuoles dans les c e l l u l e s de l a racine de 1'orge (Hordeum sativum)." C.R. Acad. Sc. (Paris)D 267: 296-298. Camefort, H., 1966. "Etude en microscopie electronique de l a degenerescence.du cytoplasme maternelle dans les oospheres embrionnees du Pinus l a r i c i o Poir. var. austriaca (P. nigra A.M.)." C.R. Acad. Sc. (Paris)D 263: 1443-1446. Carol, J . , Murray, S., Dixon, P.S., and Scherfig, J . , 1972. "Evaluation of material for use i n a l g a l culture systems." Hydrobiologia 40: 215-221. 82 Carr, D.J., and Pate, J.S., 1967. "Ageing i n the whole plant." In Aspects of the Biology of Ageing. Symp. Soc. Exp. B i o l . 21: 559-599. Catesson, A.M., et Czaninski, Y., 1968. "Localisation u l t r a s t r u c t u r a l e de l a phosphatase acide et cycle saissonier dans les tissues conducteurs de quelques arbres." B u l l . Soc. Franc. Physiol. Veget. X4: 165-173. Chadefaud, M., 1936. "Le cytoplasme des algues vertes et des algues brunes. Ses elements figures et ses inclusions." Rev. Algologique _8: 5-286. Chapman, J . , and Jordan, E.G., 1971. "Influence of g i b b e r e l l i c acid on nucleolar size changes i n storage tissue d i s c s . " J. Experimental Botany 22: 620-626. Cherry, J.H., 1967. "Nucleic acid metabolism i n ageing cotyledons." In Aspects of the Biology of Ageing. Symp. Soc. Exp. B i o l . 21/: 247-268. Chi, E.Y., 1971. "Brown a l g a l pyrenoids." Protoplasma 72: 101-104. Chihara, M., 1968. " F i e l d , culturing, and taxonomic studies of Ulva fenestrata P. and R. and Ulva s c a g e l i i sp. nov. (Chlorophyceae) i n B r i t i s h Columbia and Northern Washington." Syesis l_r 87-103. Childs, G.E., and M i l l e r , J.H., 1971. "Peroxidase l o c a l i z a t i o n i n HartmaneTla trophozoites." Proc. 29th Ann. E.M.S.A. Meeting. Claiton's Publishing D i v i s i o n — Baton Rouge. Clowes, F.A.L., and Juniper, B.E., 1968. Plant C e l l s , Blackwell S c i e n t i f i c Publications, Oxford and Edinburgh, pp. 293-294. Cole, K., 1969. "The cytology of Eudesme virescens (Carm.) J. Ag. I I . Ultrastructure of c o r t i c a l c e l l s . " Phycologia £: 101-108. Cole, K., 1970. "U l t r a s t r u c t u r a l c h a r a c t e r i s t i c s i n some species i n the order Scytosiphonales. " Phycologia 9_: 275-283. Cole, K., and L i n , S.C, 196 8. "The cytology of Leathesia difformis. I. Fine structure of vegetative c e l l s i n -f i e l d and cultured material." Syesis 1: 103-119. Cole, K., Bourne, V.L. , and L i n , S.C, 1968. "Electron microscope observations on chloroplasts of cultured Leathesia difformis (L.) Aresh." Can. J . Genet. Cytol. 83 10: 63-67. Cole, K., and L i n , S.C., 1970. "Plasmalemmasomes i n sporelings of the brown alga Petalonia d e b i l l s . " Can. J . Bot. 48: 265-268. Comings, D.E., and Okada, T.A., 1970a. "Association of chromatin f i b r e s with the annuli of the nuclear membrane." Exptl. C e l l Res. 6_2: 293-302. Comings, D.E., and Okada, T.A., 1970b. "Association of nuclear membrane fragments with metaphase. and anaphase chromosomes as observed by whole mount electron microscopy." Exptl. C e l l Res. 6_3: 62-68. Comolli, R., 1971. "Hydrolase a c t i v i t y and i n t r a c e l l u l a r pH i n l i v e r , heart, and diaphragm of ageing r a t s . " Exptl. Gerontol. 6_: 219-225. Comolli, R., F e r i o l i , M.E., and Azzola, S., 1972. "Protein turnover of the lysosomal and mitochondrial fractions of rat l i v e r during aging." Exptl. Gerontol. l_i 369-376. Coulomb, C , 1973. "Phenomenes d*autophagie l i e s a l a d i f f e r e n t i a t i o n c e l l u l a i r e s dans les jeunes racines de Scorsenere (Scorsonera hispanica)." C.R. Acad. Sc. (Paris)D 276: 1161-1164. Coulomb, C , and Buvat, R., 1968. "Processes de degeneres-cence cytoplasmique p a r t i a l l e dans les c e l l u l e s de jeunes racines de Cucurbita pepo." C.R. Acad. Sc. (Paris)D 267: 843-844. Coulomb, P., 1969. "Mise en evidence de structures analogues aux lysosomes dans le meristeme r a d i c u l a i r e de jeunes courges (Cucurbita pepo L. Cucurbitacee)." C.R. Acad. Sc. (Paris) D • 269: • 1400-1401. Coulomb, P., 1971. "Phytolysosomes dans l e meristeme r a d i c u l a i r e de l a courge (Cucurbita pepo L., Cucurbitacee) A c t i v i t e phosphatasique acide e:td a c t i v i t e peroxidasique." C.R. Acad. Sc. (Paris)D 272: 48-51. Coulomb, P., and Coulon, J . , 1971. "Functions de l ' a p p a r e i l de Golgi dans les meristemes.radiculaires de l a courge (Cucurbita pepo L., Cucurbitacee)." J . Microscopie 10: 203-214. Coulomb, P., and Coulomb, C., 1971. "Formation de phytolyso-somes, mise en evidence des r e l a t i o n s entre l e reticulum endoplasmique, les dictyosomes et les phytolysosomes dans l e meristeme r a d i c u l a i r e de l a courge (Cucurbita  pepo L., Cucurbitacee)." C.R. Acad. Sc. (Paris)D 272: 84 1634-1637. Coulomb, P., and Coulomb, C., 1972. " l o c a l i s a t i o n cytochimique u l t r a s t r u c t u r a l e d'une adenosine triphosphatase-Mg + + dependente dans les c e l l u l e s de meristeme r a d i c u l a i r e de l a courge (Cucurbita pe'po L., Cucurbitacee) ." CR. Acad. Sc. (Paris) D 275: 1035-1038. Cox, G.C, and Juniper, B.E., 1973. "Autoradiographic evidence for paramural-body function." Nature New Biology 243: 116-117. C r i s t o f a l o , V.J., 1970. Aging i n C e l l and Tissue Cultures, Plenum Press, New York and London, pp. 83. Cronshaw, J . , and Gilder, J . , 1972. "Cytochemical l o c a l i z a t i o n of ATPase a c t i v i t y i n d i f f e r e n t i a t i n g phloem c e l l s of Nicotiana and Cucurbita." J . C e l l B i o l . 55: 53a. Cronshaw, J . , Myers, A., and Preston, R.D., 1958. "A chemical and physical inv e s t i g a t i o n of the c e l l walls of some marine algae." Biochem. Biophys. Acta 27: 89-103. Cu l l i n g , C.F.A., 1963. Handbook of Histopathological Techniques, second e d i t i o n . Butterworths, London. Czaninski, Y., and Catesson, A.M., 1970. "Characterisation cytochimique de differentes a c t i v i t e s peroxydasiques dans les c e l l u l e s du Sycamore (Acer ps eudop1atanus L.)." 7e Congr. Intern. Microsc. Electron., Favard, P: ( e d i t . ) , Soc. Franc. Microsc. Electron. (Paris), v o l . 1, pp. 569-570. Czaninski, Y., and Catesson, A.M., 1971. " A c t i v i t e s peroxydasiques diverses dans les c e l l u l e s d'Acer  pseudop1atanus (tissus conducteurs et c e l l u l e s en culture)." J. Microscopie 9_i 1089-1102. Da S i l v a , P., and Branton, D., 1970. "Membrane s p l i t t i n g i n freeze-etching." J . C e l l B i o l . 4_5: 598-605. Davies, J.M., F e r r i e r , N.C, and Johnston, C.S., 1973. "The ultrastructure of the meristoderme c e l l s of the hapteron of Laminaria." J. Marine B i o l . Ass. U.K. 5_3: 237-246. Dawes, C.J., and Rhamstine, E.L., 1967. "An u l t r a s t r u c t u r a l study of the giant green alga coenocyte Caulerpa , ; - . p r o l i f e r a . " J . Phycol. 3: 117-126. De Jong, D.W., Olson, A.C, and Jansen, E.F., 1966. "Glutaraldehyde a c t i v a t i o n of nuclear acid phosphatase i n cultured plant c e l l s . " Science 155: 1672-1674. 85 De Vecchi, L. , 1971. "Fine structure of detached oat leaves senescing under d i f f e r e n t experimental conditions." I s r a e l J. Bot. 20: 169-183. Delincee, H., and Radola, B.J., 1970. "Thin layer i s o e l e c t r i c focussing on Sephadex layers of horseradish peroxidase." Biochem. Biophys. Acta 200: 404-407. Dodge, J.D., 1970. "changes i n chloroplast fine structure during the autumnal senescence of Betula leaves." Ann. Bot. 34: 817-824. Draper, S.R., and Simon, E.W., 1971. "changes i n free f a t t y acid content and respiratory a c t i v i t y during the senescence of cotyledons of Cucumber." J . Experimental Botany 2_2: 481-486. Du Praw, E.J., 1970. DNA and Chromosomes, Molecular and c e l l u l a r biology series, Holt, Rhinehart, and Wilston, Inc. Eilam, Y., 1965. "Permeability changes i n senescing t i s s u e s . " J. Experimental Botany 16: 614-627. Elens, A., and Wattiaux, R., 1969. "Age-correlated changes i n lysosomal enzyme,'-; a c t i v i t i e s : an index of ageing?" Exptl. Gerontol. 4: 131-135. E l l i o t , A.M., and Bak, I.J.,1964. "The fate of mitochondria during aging i n Tetrahymena pyriformis." J . C e l l B i o l . 20: 113-130. Esser, K., 1967. "Electronenmikrospichen nachweis von DNA en den pyrenoiden von Strepthotheea thamesis." Z. Naturforsch 22b: 993. Essner, E., and Novikoff, A.B., 1960. "Human hepatocellular pigments and lysosomes." J . U l t r a s t r . Res. 3_: 374-391. Evans, L.V., 1966. "Distri b u t i o n of pyrenoids among some brown algae." J. C e l l S c i . 1: 449-454. Evans, L.V., 1968. "Chloroplast morphology and fine structure i n B r i t i s h fucoids." New Phytol. 6_7: 173-178. Evans, L.V., and Holligan, M.S., 1972. "Correlated l i g h t and electron microscope studies on brown algae. I I . Physode production i n Dictyota." New Phytol. 71: 1173-1180. Fabbri, F., and Palandri, M., 1968. "Ihdagini a l microscopio o t t i c o ed el l e t r o n i c o . s u appici v e g e t a t i v i quiescenti i n talee d i Psiloturn nudum (L.) Beauv." Giorn. Bot. I t a l . 102: 33-53/ 86 FBbbri, F., and Palandri, M., 19 69. " F i r s t observations on the ultrastructure of young and old filaments of Halimeda  tuna." Giorn. Bot. I t a l . 103: 610. Fabbri, F., and Palandri,. M., 1970. " U l t r a s t r u c t u r a l modifications i n cotyledonous leaves of Ricihus communis L. during aging." Caryologia 23_: 677-714; -Feder, N., and O'Brien, T.P., 1968. "Plant microtechnique: some p r i n c i p l e s and new methods." Amer. J. Bot. 5_5: 123-142. Feldmann, G., and Guglielmi, G., 1972. "Les physodes et les corps i r i s a n t s du Dictyota dichotoma (Hudson) Lamouroux." C.R. Acad. Sc. (Paris)D 275: 751-754. Fergusson, C.H.R., and Simon, E.W., 1973. "Membrane l i p i d s i n seneseing green tissues." J. Experimental Botany 24: 307-316. F i g i e r , J., 1968. "Localisation i n f r a s t r u c t u r a l e de l a phosphomonosterase acide dans l a s t i p u l e de V i c i a faba L. au niveau du nectaire. Roles possible de cet enzyme dans les mecanismes de l a secretion." Planta 8_3: 60-79. F i g i e r , J. , 1972.- "Localisation i n f rastructurale de l a phosphatase acide dans les glandes p e t i o l a i r e s d 1  Impatiens h o l s t i i . " Planta 108: 215-226. Fineran, B.A., 1971. "Ultrastructure of vacuolar inclusions i n root t i p s . " Protoplasma 72: 1-18. Fisher, D.B., 1968. "Protein staining of ribboned epon sections for l i g h t microscopy." Histochemie 16_: 92-96. Fisher, J.D., and Hodges, T.K., 1969. "Monovalent ion stimulated adenosine triphosphatase from oat roots." Plant Physiol. 44: 385-395. Fisher, J.D. , Hansen, D. , and Hodges, ,T.K. , 1970. "Correlation between ion fluxes and ion stimulated adenosine triphosphatase a c t i v i t y of plant roots." Plant Physiol. 46_: 812-814.-Fl i c k i n g e r , C.J., 1968. "The e f f e c t s of enucleation on the cytoplasm membranes of Amoeba proteus." J . C e l l B i o l . 37: 300-315. Fogg, G.E., and Boalch, G.T., 1958. " E x t r a c e l l u l a r products i n a pure culture of a brown alga." Nature 181: 789-790. 87 Fowke, L.C., and S e t t e r f i e l d , G., 1968. "Cytological responses i n Jerusalem aritchoke tuber s l i c e s during ageing and subsequent auxin treatment." In Physiology and Biochemistry of Plant Growth Substances, F. Wightman and G. S e t t e r f i e l d ( e d i t . ) , Range Press, Ottawa, pp. 581-602. Frank, A.L., and Christensen, A.K., 1968. "Localization of acid phohphatase i n l i p o f u s c i n granules and possible H autophagic vacuoles i n i n t e r s i t i t a l c e l l s of the guinea pig t e s t i s . " J . C e l l B i o l . 36_: 1-13. Franke, W.W., 1967. "Zur frei n s t r u k t u r i s o l i e r t e r Kernmem-branen aus t i e r i s c h e n z e l l e n . " Z. Z e l l f o r s c h . 80: 585-593. Franke, W.W., 1970. "On the u n i v e r s a l i t y of nuclear pore complex structures." Z. Z e l l f o r s c h . TO5: 405-429. Franke, W.W., and Kartenbeck, J . , 1971. "Outer mitochondrial membrane continuous with the endoplasmic reticulum." Protoplasma 7_3: 35-41. Franke, W.W., and Scheer, U., 1970. "The ultrastructure of the nuclear envelope of the amphibian oocyte: a reinvestigation. I I . The immature oocyte and dynamic aspects." J. U l t r a s t r . Res. 30: 317-327. Franke, W.W., and Scheer, U., 1972. "Structural d e t a i l s of dictyosomal pores." J. U l t r a s t r . Res. 4_0: 132-144. Franke, W.W., Eckert, W., and Krien, S., 1971a. "Cytomembrane d i f f e r e n t i a t i o n i n a c i l i a t e , Tetrahymena pyriformis. I. Endoplasmic reticulum and dictyosomal equivalents." Z. Z e l l f o r s c h . 119: 577-604. Franke, W.W., Kartenbeck, J . , Zentgraf, H., Scheer, U., and Falk, H., 1971b. "Membrane-to-membrane cross bridges, a means to orientation and in t e r a c t i o n of membrane faces." J. C e l l B i o l . 51: 881-888. Franke, W.W., Kartenbeck, J . , Krien, S., Vanderwoude, W.J., Scheer, U., and Morre, D.J., 1972. "Inter- and i n t r a -c i s t e r n a l .elements of the Golgi apparatus. A system of membrane-to-membrane cross l i n k s . " Z. Z e l l f o r s c h . 132: 365-380. Frederick, S.E., and Newcomb, E.H., 1969a. "Microbody-like organelles i n leaf c e l l s . " Science 163: 1353-1355. Frederick, S.E., and Newcomb, E.H., 19 69b. "Cytochemical l o c a l i z a t i o n of catalase i n leaf microbodies (peroxi-somes)." J . C e l l B i o l . 43: 343-353. 88 F r e d e r i c k , S.E., Newcomb, E.H., V i g i l , E . F . , a n d W e r g i n , W.P., 1968. " F i n e s t r u c t u r a l c h a r a c t e r i z a t i o n o f p l a n t m i c r o b o d i e s . " P l a n t a 81: 2 2 9 - 2 5 2 . F r i t s c h , F.E., 1 9 4 5 . The S t r u c t u r e a n d t h e R e p r o d u c t i o n o f t h e A l g a e , v o l . . 2 , C a m b r i d g e U n i v e r s i t y P r e s s . F u k a z a w a , K., and H l g u c h i , T., 1966. " S t u d i e s o n t h e m e c h a n i s m o f h e a r t w o o d f o r m a t i o n . V I . RNA c o n t e n t i n r a y p a r e n c h y m a c e l l s . " J . J a p . Wood R e s . S o c . 1 2 : 221-2 2 6 . F u l c h e r , R.G., and M c C u l l y , M.E., 1 9 7 1 . " H i s t o l o g i c a l s t u d i e s on t h e genus F u c u s . V. An a u t o r a d i o g r a p h i c a n d e l e c t r o n m i c r o s c o p i c s t u d y o f t h e e a r l y s t a g e s o f r e g e n e r a t i o n . " C a n . J . B o t . 49_: 1 6 1 - 1 6 5 . G a b r i e l s c u , E., 1 9 7 0 . "The l a b i l i t y o f l y s o s o m e s d u r i n g r e s p o n s e o f n e u r o n s t o s t r e s s . " H i s t o c h e m . J . 2: 1 2 3 -130. G a han, P.B., 1 9 6 5 . " H i s t o c h e m i c a l e v i d e n c e f o r t h e p r e s e n c e o f l y s o s o m e - l i k e p a r t i c l e s i n r o o t m e r i s t e m c e l l s o f V i c i a f aba'." J . E x p e r i m e n t a l B o t a n y 16_: 35 0 - 3 5 5 . Gahan, P.B., 1 9 6 9 . " S u b c e l l u l a r l o c a l i z a t i o n a n d f u n c t i o n o f h y d r o l a s e s i n d i f f e r e n t i a t i n g c e l l s . " B i o c h e m . J . I l l : 27p. G a l s t o n , A.W., and D a v i e s , P . J . , 1 969. " H o r m o n a l r e g u l a t i o n i n h i g h e r p l a n t s . " S c i e n c e 1 6 3 : 1 2 8 8 - 1 2 9 7 . G e r h a r d t , B.P., a n d B e e v e r s , H., 1970. " D e v e l o p m e n t a l s t u d i e s on g l y o x y s o m e s i n R i c i n u s e n d o s p e r m . " J . C e l l B i o l . 44_: 94 - 1 0 2 . G e r h a r d t , B.P., and B e r g e r , C , 1 9 7 1 . " M i c r o b o d i e s u n d d i a m i n o b e n z i d i n - r e a k t i o n i n d e n a c e t a t f l a g e l l a t e n P o l y t o m e l l a c a e c a und C h l o r o g o n i u m e l o n g a t u m . " P l a n t a 100: 1 5 5 - 1 6 6 . G e z e l i u s , K., 1972. " A c i d p h o s p h a t a s e l o c a l i z a t i o n d u r i n g d i f f e r e n t i a t i o n i n t h e c e l l u l a r s l i m e m o l d D i c t y o s t e l i u m  d i s c o i d e u m . " A r c h . M i k r o b i o l . 85_: 51-76. G i b b s , S.P., 1 9 6 2 a . "The u l t r a s t r u c t u r e o f t h e p y r e n o i d s o f a l g a e , e x c l u s i v e o f t h e g r e e n a l g a e . " J . U l t r a s t r . R e s . 7: 2 4 7 - 2 6 1 . G i b b s , S.P., 1962b. "The u l t r a s t r u c t u r e o f t h e c h l o r o p l a s t s o f a l g a e . " J . U l t r a s t r . R e s . 7_: 41 8 - 4 3 5 . 89 Grf-ford, E.M., 1968. In Plant C e l l s , Clowes, *F.A.L. and Juniper, B.E., eds., Blackwell S c i e n t i f i c Press, Oxford and Edinburgh, pp. 146-147. Giraud, G., 1962. "Les infrastructures de quelques algues et leur physiologie." J . Microscopie 1_: 251-274. Goldfisher, S., Vi l l a v e r d e , H., and Forschirm, R., 1966. "The demonstration of acid hydrolase, thermostable diphosphopyridine nucleotide tetrazolium reductase and peroxidase a c t i v i t i e s i n human l i p o f u s c i n pigment granules." J. Histoehem. Cytochem. 14: 641-652. Gomori, G., 1952. 'Microscopic Histochemistry, P r i n c i p l e s and P r a c t i c e . 1 Univ. of Chicago Press. Grasso, J.A., Swift, H., and Ackerman, G.A., 1962. "Observations on the development of erythrocytes i n mammalian f e t a l l i v e r . " J . C e l l B i o l . 14: 235-254. Greenwood, A.D., 1964. nThe Structure of Chloroplasts i n Lower Plants." 10th Intern. Congr. Bot. (Edinburgh), pp. 212-213. Greenwood, A.D., 1967. In The P l a s t i d s , Kirk, J.T.O. and Tilney-Bassett, R.A.E., eds., WHH. Freeman, San Francisco. G r i f f i t h s , D.J., 1970. "The pyrenoid." Bot. Rev. 36: 29-58. Gruber, P.J., Trelease, •R.N.. , Becker, W.M. , and Newcomb, E.H., 1970. "A co r r e l a t i v e u l t r a s t r u c t u r a l and enzymatic study of cotyledonary microbodies following germination of f a t storing seeds." Planta 93: 269-288. Grusky, G.E., and Aaranson, S., 1969. "Cytochemical changes i n aging Ochromonas; evidence for an alk a l i n e phospha-tase." J . Protozool. 16: 686-689. Gullvag, B.M., 1968. "Fine structure of the p l a s t i d s and possible ways of d i s t r i b u t i o n of chloroplast products i n some spores of archegoniatae." Phytomorphology 18: 520-535. Habeshaw, D., and Heyes, J.K., 1971. "Metabolic changes and senescence i n cultured pea root tissue." New Phytol. 70_: 149-162. H a l l , J.L., and Davie, C.A.M., 1971. "Localization of acid hydrolase a c t i v i t y i n Zea mays L. root t i p s . " Ann. Bot. 35: 849-855. 90 H a i l , J . L . , and S e x t o n , R., 1972. " C y t o c h e m i c a l l o c a l i z a t i o n o f p e r o x i d a s e a c t i v i t y i n r o o t c e l l s . " P l a n t a 108: 103-120. H a l p e r i n , W. , 1969. " U l t r a s t r u c t u r a l l o c a l i z a t i o n o f a c i d phosphatase i n c u l t u r e d c e l l s o f Daucus c a r o t a . " P l a n t a 88: 91-102. Hanker, J.S., Y a t e s , P.E., C l a p p , D.H., and Anderson, W.A., 1972. "New methods f o r the d e m o n s t r a t i o n o f l y s o s o m a l h y d r o l a s e s by t h e f o r m a t i o n o f osmium b l a c k s . " H i s t o c h e m i e 30: 201-214. H a n z e l y , L., and V i g i l , E.L., 1972. "Comparative c y t o c h e m i c a l a n a l y s i s o f m i c r o b o d i e s d u r i n g c y t o k i n e s i s i n u n t r e a t e d and d i g i t o n i n - t r e a t e d A l l i u m r o o t t i p c e l l s . " J . C e l l B i o l . 55: 10 6a. H a r n i s c h f e g e r , G., 1972. " P h o t o s e n s i t i z e d i n h i b i t o r f o r m a t i o n i n i s o l a t e d a g i n g c h l o r o p l a s t s . " P l a n t a 104: 316-328. H a r r i s , W.M., 1970. "Chromoplasts o f tomato f r u i t s . I I . The h i g h d e l t a tomato." B o t . Gaz. 131: 163-166. Hasan, M., and Glees, P., 1972. "Electron microscopical appearance of neuronal l i p o f u s c i n using d i f f e r e n t preparative techniques, including freeze-etching." E x p t l i Gerontol. 7: 345-351. Hayashi, H., 1967. "Comparative histochemical l o c a l i z a t i o n of lysosomal enzymes i n rat tissue." J . Histochem. Cytochem. X5: 83-92. Hendy, R., 1971. "Electron microscopy of l i p o f u s c i n pigment stained by the Schorml and Fontana techniques." Histochemie '26: 311-318. Henrikson, R.S., 1971. "Mechanism of sodium transport across ruminal epithelium and histochemical l o c a l i z a t i o n of ATPase." Exptl. C e l l Res. 6_8: 456-458. Herzog, V., and Fahimi, H.D., 1972. "Glutaraldehyde f i x a t i o n : a prerequisite for demonstration of the peroxidatic a c t i v i t y of catalase i n i s o l a t e d r a t l i v e r peroxisomes and c r y s t a l l i n e beef l i v e r catalase." J . C e l l B i o l . 55: 113a. Heslop-Harrison, J . , 1967. " D i f f e r e n t i a t i o n . " Ann. Rev. Plant Physiol. 18: 325-348. Heyden, G., 1969. "Adenosinetriphosphatases. A l i t e r a t u r e survey (I) and some methodological r e s u l t s ( I I ) . " Acta Histochem. 34: 273-286. 91 H i l l i a r d , J.H., Gracen, V.E., and West, S.H., 1971. "Leaf microbodies (peroxisomes) and catalase l o c a l i z a t i o n i n plants d i f f e r i n g i n t h e i r photosynthetic carbon pathway." Planta 97: 93-105. Hirsch, H.I., 1970. "Enzyme le v e l s of i n d i v i d u a l neurons i n r e l a t i o n to l i p o f u s c i n content." J.•Histochem. Cytochem. 18: 268-270. Hochschild, R., 1971/ "Lysosomes, membranes, and aging." Exptl. Gerontol. 6_: 153-166. Hodges, T.K., Leonard, R.T., Bracker, C.E., and Keenan, T.W., 19 72. " P u r i f i c a t i o n of an ion stimulated adenosine triphosphatase from plant roots: association with plasma membrane." Proc. Nat. Acad. S c i . U.S.A. 69: 3307-3311. Hori, T., 1971. "Survey of pyrenoid d i s t r i b u t i o n i n brown algae." Bot. Mag. (Tokyo) 134: 231-242. Hori, T., 1972. "Further survey of pyrenoid d i s t r i b u t i o n i n Japanese brown, algae." Bot. Mag. (Tokyo) 8_5: 125-134. Horton, R.F., and Osborne, D.J., 1967. "Senescence, abcission, and c e l l u l a s e a c t i v i t y i n Phaseolus vul g a r i s . " Nature 214: 1086-1088. Howse, H.D., and Welford, RlfF. , 1972. "A histochemical and u l t r a s t r u c t u r a l study, e s p e c i a l l y of the l i p o f u s c i n i n the endothelium, of the toadfish endocardium." Trans. Amer. Microsc. Soc. 93.: 24-35. Hussain, A., and Boney, A.D., 1973. "Hydrophilic growth i n h i b i t o r s from Lamiriaria and AscophyHum." New Phytol. 7_2: 403-410. Ikeda, T., and Ueda, R.R., 1964. "Light and electron microscopic studies on the senescence of chloroplasts of Elodea leaf c e l l s . " Bot. Mag. (Tokyo) " T7: 336-341. James, F.E.L., 1966. "M.Sc. Thesis." Univ. of Wales. Cited by Butler and Simon (1971). Jennings, R.C., 1968. "Gibberellins as endogenous growth regulators i n green and brown algae." Planta 80_: 34-42. Jensen, W.A., 1962. Botanical H i s t o c h e m i s t r y — p r i n c i p l e s and practice." Freeman and Co., San Francisco and London. Johnson, R., and Strehler, B.L., 19 72. "Loss of genes coding for ribosomal RNA i n ageing brain c e l l s . " Nature 240: 412-414. 92 JoTrdan, E.G., and Chapman, J.M., 1971. " U l t r a s t r u c t u r a l changes i n the n u c l e o l i of Jerusalem artichoke (Helianthus tuberosus) tuber discs." J . Experimental Botany 22: 627-634. Kataoka, K., 1971. "Fine s t r u c t u r a l l o c a l i z a t i o n of peroxidase a c t i v i t y i n the epithelium and the gland of the r at larynx." Histochemie 26: 319-326. Klamer, B., and Fennell, R.A., 1963. "Acid phosphatase a c t i v i t y during growth and synchronous d i v i s i o n of Tetrahymena pyriformis." Exptl. C e l l Res. 29: 166-175. Koenig, H., 1963. "The autofluorescence of lysosomes. Its value for the i d e n t i f i c a t i o n of lysosomal constituents." J. Histochem. Cytochem. XI: 556-557. Kordan, H.A., 1972. "Nucleolar s i z e and morphology i n lemon f r u i t s and plants exposed to d i f f e r e n t osmotic environ-ments." Z. Pflanzenphysiol. 6_7: 311-317. La Cour, L.F., and Wells, B., 1972. "The nuclear pores of early meiotic prophase, of plants." Z. Z e l l f o r s c h . 123: 178-194. La Fountaine, J.G., and Chaourdinard, L.A., 1963. "A correlated l i g h t and electron microscopic study of the nucleolar material during mitosis i n V i c i a faba." J . C e l l B i o l . 17: 167-201. La Fountaine, J.R., and La Fountaine, K.L., 1973. "Comparison of density of nuclear pores on vegetative and generative nuclei i n pollen of Tradescantia." Exptl. C e l l Res. 78: 472-476. L a i , Y.F., and Thompson,.J.E., 1972a. "Effects of germination on Na +-K +-stimulated adenosine-5'-triphosphatase and ATP-dependent ion transport of i s o l a t e d membranes from cotyledons." Plant Physiol. 50_: 452-457. L a i , Y.F., and Thompson,. J.E., 1972b. "Distinguishable ATPases a c t i v i t i e s of c e l l wall and plasma membrane." Phytochemistry 11: 2747-2749. Lamport, D.T.A., 19 65. "The protein component of primary c e l l walls." Advances i n Botanical Research, v o l . 2, Academic Press, New York and London, pp. 151-218. Lee, S.G., and Chasson, R.M. , 1966. "Aging and mitochondrial development i n potato tuber t i s s u e . " Physiol. Plantarum 19.: 199-206. Leedale, G.F., 1970. "Phylogenetic aspects of cytology i n 93 the algae." Ann. New York Acad. S c i . 175: 429-453. Leonard, R.T., and Hanson, J.B., 1972. "Increased membrane bounded adenosine triphosphatase a c t i v i t y accompanying development of enhanced solute uptake i n washed corn root tissue." Plant Physiol. 49_: 436-440. Leopold, A.C., 1964. Plant Growth and Development, Academic Press, New York and London. Lichtenthaler, H.K., 1968. "Plastoglobuli and the fine structure of p l a s t i d s . " Endeavour 2_7: 144-149. Lichtenthaler, H.K. , 1970. "Die feinstruktur der chromo-plasten i n plasmochromen perigon blaettern von Tulipa." Planta 9_3: 143-151. Liddle, L.B., and Neushul, M., 1969. "Reproduction i n Zonaria f a r l o w i i . I I . Cytology and ul t r a s t r u c t u r e . " J . Phycol. 5: 4-12. Li n , C.W., and Fishman, W.H. , 1972. "Microsomal and lysosomal acid phosphatase isoenzymes of the mouse kidney: characterization and separation." J . Histochem. Cytochem. 20_: 487-498. Lipetz, J. , and C r i s t o f a l o , V.J'., 1972. " U l t r a s t r u c t u r a l changes accompanying the aging of human d i p l o i d c e l l s i n culture." J . U l t r a s t r . Res. 39: 43-56. Ljubesic, N., 1968. "Feinbau der chloroplasten der wahrend der vergilbund und wiederergrunung der b l a t t e r . " Protoplasma 66^ 369-379. Loiseaux, S., 1967. "Morphologie et cytologie des Myrior-nemacees. Criteres taxonomiques." Rev. Gen. Bot. 74: 329-347. Longest, P., 1946. "Structure of the c i l i a i n Ectocarpus mitchellae and Codium decortleaturn." J . E l i s h a M i t c h e l l Soc. £2: 249-252. Lund, H.A., Watter, A.E., and Hanson, J.B., 1958. "Biochemical and c y t o l o g i c a l changes accompanying growth and d i f f e r e n t i a t i o n i n the roots of Zea mays." J . Biophysic. Biochem. Cytol. £: 87-98. Magne, F., 1971. "Sur l a presence vraisemblable de polysaccharides dans les globules.osmiophiles des plastes." CR. Acad. Sc. (Paris) D 273: 340-343. Maier, K., and Maier, U., 1972. "Localization of Beta-glycerophosphatase and Mg+*—activated adenosine 94 triphosphatase i n a moss haustorium, and the r e l a t i o n of these enzymes to the c e l l wall labyrinth." Protoplasma 75: 91-112. Mainwaring, W.I.P., 1968. "Changes i n the ribonucleic acid metabolism of ageing mouse tissues with p a r t i c u l a r reference to the prostrate gland." Biochem. J. 110: 79-86. Mainwaring, W.I.P., .1969. "The e f f e c t of age on protein synthesis i n mouse l i v e r . " Biochem. J. 113: 869-879. Manton, I., 1957. "Observations with the electron microscope on the i n t e r n a l structures, of .the zoospores of a brown alga." J. Experimental Botany '' 8_: 294-303. Manton, I., 1959. "Observations on the i n t e r n a l structure of the spermatozoid of Dictyota." J . Experimental Botany 10_: 448-461. Manton, I., 1966a. "Further observations on the fine structure of ChrysOchromuTina chiton, with sp e c i a l reference to the pyrenoid." J . C e l l B i o l . 1: 187-192. Manton, I., 1966b. "Some possible s i g n i f i c a n t s t r u c t u r a l relations between chloroplasts and other c e l l components." Proc. NATO Advanced Study I n s t i t u t e , v o l . 1, Goodwin, T.W., ed., Academic Press, New York and London, pp. 23-47. Manton, I., and Clarke, B., 1956. "Observations with the electron microscope on the i n t e r n a l structure of the spermatozoid of Fucus. " J. Experimental Botany 7^: 416-432. Marchant, R., and Robards, A.W., 1968. "Membrane systems associated with the plasmalemma of plant c e l l s . " Ann. Bot. 32: 457-471. Margoliash, E., and Novogrodsky, A., 1958. "A study of the i n h i b i t i o n of catalase by 3-amino-^l: 2 :4-triazole. " Biochem. J. 6_8: 468-475. Margoliash, E., Novogrodsky, A., and Schejter, A., 1960. "Irrevers i b l e reaction of 3-amino-l:2:4-triazole and. related i n h i b i t o r s with the protein catalase." Biochem. J. 7_4: 339-348. Marinos, N.G., 1963. "Vacuolation i n plant c e l l s . " J . U l t r a s t r . Res. !9: 177-185. Marty, F., 1969. "Caracterisation cytochemique u l t r a s t r u c -turale de peroxysomes (microbodies sensu s t r i c t o ) chez 95 Euphorbia characias. " CR. Acad. Sc. (Paris)D 268 : 1388-1391. Marty, F., 1970. "Les peroxysomes (microbodies) des l a c t i c i f e r e s d'Euphorbia characias." J . Microscopie 9_: 923-948. Marty, M., 1973. "Mise en evidence d'un appareil pro-vacuolaire et de son r o l e dans l'autophagie c e l l u l a i r e et l ' o r i g i n e des vacuoles." CR. Acad. Sc. (Paris)D 276: 1549-1552. Massalski, A., and Leedale, G.F., 1969. "Cytology and ultrastructure of the Xanthophyceae. I. Comparative morphology of the zoospores of BumiTier1a s i c u l a Borzi and Tribonema vulgare Pascher." Br. Phycol. J. 4_: 159-180. Matile, P.H., 19 68. "Lysosomes of root t i p c e l l s i n corn seedlings." Planta 79: 181-196. Matile, P.H., 19 69a. "Plant lysosomes." In Lysosomes i n Biology and Pathology, Dingle and F e l l , eds., North Holland Publishing Co., Amsterdam, and London, pp. 406. Matile, P.H., 19 69b. "Vacuoles as lysosomes of plant c e l l s . " Biochem:;. J. I l l : 26p. Matile, P.H., and Moor, H., 1968. "Vacuolation: o r i g i n and development of the lysosomal apparatus i n root t i p c e l l s . " Planta 8Ck 159-175. Matile, P.H., and Winkenbach, F., 1971. "Function of lysosomes and lysosomal enzymes i n the senescing c o r o l l a o f f the morning glory (Ipomoea purpurea)." J. Experimental Botany 2_2: 759-771. Maul, G.G., Price, J.W., and Lieberman, A.W., 1971. "Forma-tion and d i s t r i b u t i o n of nuclear pores i n interphase." J. C e l l B i o l . 51: 405-418. McCully, M.E., 1966. " H i s t o l o g i c a l studies on the genus Fucus. I. Light microscopy of the mature vegetative plant." Protoplasma 62,: 287-305. McCully, M.E., 1968. " H i s t o l o g i c a l studies on the genus Fucus. I I I . Fine structure: and possible functions of the epidermal c e l l s of the vegetative t h a l l u s . " J . C e l l S c i . 3: 1-16. McLean, R.J., 1968. "Ultrastructure of SporigiochTor1s typica during senescence." J . Phycol.- 4_: 277-283. 96 Medvedev, Z .H.A. , .1972 . " R e p e t i t i o n of molecular g e n e t i c i n f o r m a t i o n as a p o s s i b l e f a c t o r i n evolutionary-changes i n l i f e span." E x p t l . G e r o n t o l . 7: 227-238. Mesquita, J.F., 1969. " E l e c t r o n , microscope study of the o r i g i n and development of v a c u o l e s i n r o o t t i p c e l l s of Lupinus albus L." J . U l t r a s t r . Res. 26: 242-250. Mesquita, J.F., 1972. " U l t r a s t r u c t u r e des formations comparables aux vacuoles autophagiques dans l e s c e l l u l e s des r a c i n e s d ' A l l i u m cepa L. e t du Lupinus albus L." C y t o l o g i a 3_7: 95-110. Messer, G., and Ben-Shaul, ¥., 1972. "Changes i n c h l o r o p l a s t s t r u c t u r e d u r i n g c u l t u r e growth of P e r i d i n i u m cihctum Fa. W e s t i i (Dinophyceae)." P h y c o l o g i a 11: 291-299. Meyer, H.W., and Winkelmann, H., 1969. "Die g e f r i e r a t z u n g und d i e s t r u k t u r b i o l o g i s c h e r membranen." Protoplasma 6_8: 253-270. Mia, A.J., 1972. "Fine s t r u c t u r e of the ray parenchyma c e l l s i n Populus tremuloides i n r e l a t i o n t o senescence and seasonal changes." Texas J . S c i . 24: 245-260. M i c a l e f , H., 1972. "Mise en evidence de phosphatase a c i d e dans l ' a p p a r e i l de G o l g i des c e l l u l e s v e g e t a t i v e s d 1  Ulva l a c t u c a L. ( C h l o r o p h y c e a e — U l v a l e s ) . " C.R. Acad. Sc. ( P a r i s ) D 275: 2481-2484. M i t t e l h e u s e r , C.J., and van Steveninck, R.F.M., 1971. "The u l t r a s t r u c t u r e of wheat l e a v e s . " I. Changes due to n a t u r a l senescence and the e f f e c t s of K i n e t i n and ABA on detached leaves incubated i n the dark." Protoplasma 73_: 239-252. Mollenhauer, H.H., Morre, D.J., and K e l l e y , A.G., 1966. "The widespread occurrence of p l a n t cytosomes resembling animal microbodies." Protoplasma 6_2: 44-52. Monneron, A., B l o b e l , G., and Palade, G.E., 1972. " F r a c t i o n a t i o n of the nucleus by d i v a l e n t c a t i o n s . I s o l a t i o n of n u c l e a r membranes." J . C e l l B i o l . 55: 104-125. Moor, H., and M u h l e t h a l e r , K., 1963. "Fine s t r u c t u r e i n f r o z e n - e t c h e d y e a s t c e l l s . " J . C e l l B i o l . 17: 609-628. Morre, D.J., M e r r i t t , W.D., and Lembi, C.Y., 1971. "Connection between m i t o c h o n d r i a and endoplasmic r e t i c u l u m i n r a t l i v e r and onion stem." Protoplasma 73: 43-49. 97 Munnell, J.F., and Getty, R. , 1968. "Nuclear lobulation and amitotic d i v i s i o n associated with increasing c e l l size i n the aging.canine myocardium." J . Gerontol. 23: 363-369. Myers, A., and Preston, R.D., 1959a. "Fine structure i n the red algae. I I . The structure of the c e l l wall of Rhodymenia palmata." Proc. Roy. Soc. London B 150: 447-455. Myers, A., and Preston, R.D., 1959b. "Fine structure i n the red algae. I I I . A general study of the c e l l wall struc-ture i n the red algae." Proc. Roy. Soc. London B 150: 456-559. Neushul, M., 1970. "A freeze-etching study of the red-alga Porphyridlum." Amer. J . Bot. 57_: 1231-1239. Neushul, M., 1971. "Uniformity of thylakoid structure i n a red, a brown, and two blue-green algae." U l t r a s t r . Res. 3J7: 532-543. Neushul, M., and Liddle, L.B., 1968. "A l i g h t and electron microscope study of primary heterogeneity i n the eggs of two brown algae." Amer. J. Bot. 5_5: 1068-1073. Neushul, M., and Walker, D., 1971. "Ultrastructure of the dictyotalean nucleus." J . Phycol. (suppl.) 7_: 3. Neushul, M., and Dahl, A.L., 1972a. " U l t r a s t r u c t u r a l studies of brown a l g a l n u c l e i . " Amer. J. Bot. 59: 401-410. Neushul, M., and Dahl, A.L., 1972b. "Zonation i n the ap i c a l c e l l of Zonarla." Amer. J. Bot. 59^ : 393-400. Northcote, D.H., and Lewis, D.R., 1968. "Freeze-etched surfaces of membranes and organelles i n the c e l l s of the pea root t i p s . " J. C e l l S c i . 3: 199-206. Novikoff, A.B., 1970. "V i s u a l i z a t i o n of c e l l organelles by diaminobenzidine reactions." 7e Congr. Intern. Micro-scop. E l e c t r . (P. Favard, e d i t . ) , v o l . 1, Soc. Franc. Microscop. E l e c t r . , pp. 565-566. Novikoff, A.B., and Goldfischer, S., 1969. "V i s u a l i z a t i o n of peroxisomes (microbodies) and mitochondria with diaminobenzidine." J . Histochem. Cytochem. 17: 675-680. Novikoff, A.B., Hausman, D.H., and Podber, E., 1958. "The l o c a l i z a t i o n of adenosine triphosphatase i n l i v e r : i n s i t u staining and c e l l f r a c t i o n a t i o n studies." J. 98 Histochem. Cytochem. 6_: 61-71. Novikoff, A.B., Beard, M.E., Albala, A., Sheid, B., Quintana, N., and Biempica, L., 1971. "Localization of endogenous peroxidase i n animal.tissues." J. Microscopie 12: 381-404. Opik, H., 1966. "Changes i n c e l l f i n e structure i n the cotyledons of Phaseolus vulgaris during germination." J. Experimental Botany 17: 427-439. Osborne, D.J., 1967. "Hormonal regulation of leaf senescence." In Aspects of the Biology of Ageing. Symp. Soc. Exptl. B i o l . 21: 305-321. Packer, L., Deamer, D.W., and Heath, R.L., 1967. "Regulation and deterioration of structures i n membranes." In Advances i n Gerontological Research, B.L. Strehler, ed., v o l . 2, Acad. Press., New York and London, pp. 77. Palandri, M., 1972. "Aspetti u l t r a s t r u t t u r a l i d e l l ' invecchiamento dei filamenti c e n o c i t i c i i n Halimeda tuna ( E l l . et Sol.) Lamour." Caryologia 25: 211-235. Palisano, J.R., and Walne, P.L., 1972. "Acid phosphatase a c t i v i t y and ultra s t r u c t u r e of aged c e l l s of Euglena  granulata." J. Phycol. 8: 81-88. Pelc, S.R., 1970. "Metabolic DNA and the problem of ageing." Exptl:: o Gerontol. 5_: 217-226. Pearse, A.G.E., 1972. "Histochemistry,Theoretical and Applied.' v o l . 2, C h u r c h i l l and Livingstone, London, pp. 1076-1093. Pe r c i v a l , E., 1968. "Marine a l g a l carbohydrates." Oceanogr. Marine B i o l . Annu. Rev. 6_: 137-161. Pickett-Heaps, J.D., 1967a. "Ultrastructure and d i f f e r e n t i a -t i o n i n Chara sp. I. Vegetative c e l l s . " Aust. J. B i o l . S c i . 20.: 539-551. Pickett-Heaps, J.D., 1967b. "Further observations on the Golgi apparatus and i t s functions i n c e l l s of wheat seedlings." J. U l t r a s t r . Res. 18: 287-303. Plesnicar, M., Bonner, W.D., and Storey, B.T., 1967. "Peroxidase associated with higher plant mitochondria." Plant Physiol. 42^ 366-370. Pollock, E.G., 1970. " F e r t i l i z a t i o n i n Fucus." Planta 92: 85-99. Po'tapov, N.G., and Krishnamurthy, K.V. , 1972. "Excised root 99 cultures of Lupine (Lupinus angustifolius L.). IV. U l t r a s t r u c t u r a l changes i n parenchymatous', c e l l s with age." Acta B i o l . Acad. S c i . Hung. 23.: 217-236. Poux, N., 1963a. "Localisation de l a phosphatase acide dans les c e l l u l e s meristematiques du ble (Tr1ticum vulgare V i l l . ) . " J . Microscopie 2: 485-489. Poux, N., 1963b. "Localisation des phosphates et de l a phosphatase acide dans les c e l l u l e s des embryons de ble (Triticum vulgare) l o r s de l a germination." J. Micro-scopie 2: 557-568. Poux, N., 1969. "Localisation d ' a c t i v i t e s enzymatiques dans les c e l l u l e s du meristeme r a d i c u l a i r e de Cucumis  sativus L." J . Microscopie £: 855-866. Poux, N. , 1970. "Localisation d ' a c t i v i t e s enzymatiques dans le meristeme r a d i c u l a i r e de Cucumis' sativus L. I I I . A c t i v i t e phosphatasique acide. " 3T Microscopie 9_: 407-434. Poux, N., 1971. "Reactions avec le DAB dans l e limbe f o l i a i r e de Dacty11s.glomerata L. .(Graminees). Mise en evidence de peroxisomes." J. Microscopie 10: 87-88. Poux, N. , 1972a. "Localisation d'activites:?enzymatiques dans le meristeme. r a d i c u l a i r e de Cucumis sativus L. IV. Reactions avec l a diaminobenzidine mise en evidence de peroxisomes." J . Microscopie 14: 183-218. Poux, N., 1972b. "Observations sur les a c t i v i t e s peroxida-siques l i e e s aux peroxisomes f o l i a i r e s du Dactylis glomerata L. (Graminees)." C.R. Acad. Sc. (Paris)D 274: 2647-2650. Poux, N., 1972c. "Nouvelles observations sur les a c t i v i t e s peroxidasiques du mesophylle de Dactylis glomerata L. (Graminees)." J. Microscopie 14_: 82a. Preston, R.D., 1961. "Cellulose-protein complexes i n plant c e l l walls." In.Maeromolecular Complexes, M.V. Edds, ed,. , Ronald Press, pp. 229-253. Quatrano, R.S., 1972. "An u l t r a s t r u c t u r a l study of the determined s i t e of r h i z o i d formation i n Fucus zygotes." Exptol. C e l l Res. 70: 1-12. Radola, B.J., and Drawert, F., 1970. "Isoelektrische fokussierung wasserloslicher proteine aus gerste und malz." Brauwiss 2_3: 449-458. Ratner, A., and Jacoby, B., 1973. "Non-specificity of s a l t e f f e c t s on Mg + +-dependent ATPase from grass roots." J . 100 Experimental. Botany 24: 231-238. Rawlence, D.J.,1972. "An u l t r a s t r u c t u r a l study of the relationship between rhizoids of Polysiphonia 1 alios a (L.) Tandy . (Rhodophyceae) and.tissue of Ascophyllum nodosum (L.) Le J o l i s (Phaeophyceae)." Phycologia 11: 279-280. Raychaudhuri, C , and Desai, I.D., 1971. "Ceroid pigment formation and i r r e v e r s i b l e s t e r i l i t y i n vitamin E deficiency." Science 173: 1028-1029. Reynolds, E.S., 1963.. "The use of lead c i t r a t e at high pH as an electron opaque st a i n i n electron microscopy." J. C e l l B i o l . 17: 208-212. Ridge, I., and Osborne, D.J., 1970. "Hydroxyproline and peroxydases i n c e l l walls of Pisum sativum: regulation by ethylene." J. Experimental Botany 21: 843-856.-Ridge, I., and Osborne, D.J., 1971. "Role of peroxidase when hydroxyproline-rich protein i n plant c e l l walls i s increased by ethylene." Nature 229: 205-208. Robards, A.W., 1970. "Electron Microscopy and Plant ultrastructure. McGraw-Hill, London. Robards, A.W., and Kidway, P., 1969. "Cytochemical l o c a l i z a t i o n of phosphatase i n d i f f e r e n t i a t i n g secondary vascular c e l l s . " Planta 87_: 227-238. Robinson, D.J., and Preston, R.D., 1972. "Plasmalemma structure i n r e l a t i o n to m i c r o f i b r i l biosynthesis i n Oocystis." Planta 104: 234-246. Rockstein, M., 1967. " C e l l u l a r ager changes i n insects." In Aspects of the.Biology of Ageing. Symp. Soc. Exptl. B i o l . 21: 337-349, Roland, J . C , 1969. "Localisation d ' a c t i v i t e s enzymatiques au niveau des ultrastructures des collocytes en croissance de Balota nigra." CR. Acad. Sc. (Paris)D 268: 2052-2055. Rosenquist, T.H., and Slavin, B.G., and Bernick, S., 1971. "The Pearson s i l v e r - g e l a t i n method for l i g h t microscopy of 0.5-2.0M p l a s t i c sections." Stain Technol. '46_: 253-257. Rosowski, J.R., 1970. "Staining a l g a l pyrenoids with carmine afte r f i x a t i o n i n an a c i d i f i e d hypochlorite solution." Stain Technol. 45: 293-298. 101 Rothman, A.H.,' 1968. "Peroxidase, a c t i v i t y i n Platyhelminte c u t i c u l a r mitochondria." Exptl. P a r a s i t o l . .' 23: 51-55. Rougier, M., 1972. "Etude cytochimiques des squamules d 1 Elodea canadensis. Mise en evidence de leur secretion polysaccharidique et de leur a c t i v i t e phosphatasique acide." Protoplasma 74_: 113-131. Roux, S.J., and McHale, J.T., 1968. "U l t r a s t r u c t u r a l changes during the senescence of detached tomato leaves." Phyton 25: 113-122. Rucker, W., and Radola, B.J., 1971. " I s o e l e c t r i c patterns of peroxidase isoenzymes from tobacco tissue cultures." Planta 99: 192-198. Rudzinska, M.A., 1961. "The use of a Protozoan for studies on aging. I. Differences between young and old organisms of Tokophora Infusorium as revealed by l i g h t and electron microscopy." J. Gerontol. 16: 213-224. Sabnis, D.D., 19 69. "Observations on the ultrastructure of the coenocytic marine alga CaUlerpa p r o l i f e r a , with special reference to some unusual cytoplasmic components." Phycologia 7: 24-32. Sacher, J.A., 1967. "Studies of permeability, RNA, and protein turnover during ageing of f r u i t and leaf tissue." In Aspects of the: Biology of Ageing. Symp. Soc. Exptl. B i o l . 21: 269-303. Samorajski, T., and Ordy, J.M., 1967. "The histochemistry and ultrastructure of lipid.pigment i n the adrenal i f . ' gland of aging mice." J . Gerontol. 2J2: 253-267. Samorajski, T., Keefe, J.R., and Ordy, J.M., 1964. " I n t r a c e l l u l a r l o c a l i z a t i o n of l i p o f u s c i n age pigment i n the nervous system." J . Gerontol. 19: 262-276. Samorajski, T., Ordy, J.M., and Keefe, J.R., 1965. "The fine structure of l i p o f u s c i n age pigment i n the nervous system of aged mice." J. C e l l B i o l . 26: 779-795. Samorajski, T., Ordy, J.M., and Rady-Reimer, P., 1968. "Lipofuscin pigment accumulation i n the nervous system of aging mice." Anat. Rec. 160: 555-574. Sax, K., 1962. "Aspects of aging i n plants." Annu. Rev. Plant Physiol. 13: 489-506. Scagel, R.F., 1966. "Phaeophyceae i n perspective." Annu. Rev. Oceanogr. & Mar. B i o l . 4: 123-194. 102 Scandalios, J . G., 1969. "Genetic control of multiple forms of enzymes i n plants: a review." Biochem. Genet. 3_: 37-79. Schotz, F., Diers, L., und Ruffer, P., 1971. "Abgabe von pl a s t i d e n t e i l e n i n das cytoplasma. Eine vergleichend lichtmikroskopische und elektronenmikroskopische unter-schung." Ber. Dtsch.Bot. Ges. 84_: 41-51. Schultz, R.L., and Jacques, P.J., 1971. "Characteristics of lysosomes i n rat placental c e l l s . " Arch. Biochem. 144: 293-303. Schuster, F.L., Hershenov, B., and Aaranson, S., 1968. "Ul t r a s t r u c t u r a l observations on aging of stationary cultures and feeding i n Ochromohas." J . Protozool. 15: 335-346. Seligman, A.M., and Karnovsky, M.J., 1968. "Non droplet u l t r a s t r u c t u r a l demonstration of cytochrome oxidase a c t i v i t y with a polymerizing osmiophylic reagent diaminobenzidine (DAB)." J. C e l l B i o l . 38: 1-14. Shannon, L.M., 1968. "Plant isoenzymes." Annu. Rev. Plant Physiol. 19: 187-204. Shaw, M., and Manocha, M.S., 1965. "Fine structure i n detached senescing wheat leaves." Can. J . Bot. 43: 747-755. Simon, M.F., 1954. "Recherches sur les pyrenoides des Phaeophycees." Rev. Cytol. B i o l . 15_: 73-105. Simon, E.W., 1967. "Types of le a f senescence." In Aspects of the Biology of Ageing. Symp. Soc. Exptol. B i o l . 21: 215-230. Smith, G.M., 1955. Cryptogamic Botany, v o l . 1, McGraw-Hill Book Co., Inc., New York and London. Sohal, R.S., and A l l i s o n , V.F., 1971. "Senescent changes i n the cardiac myofiber of the house f l y Musea domestica. An electron microscopic study." J. Gerontol. 26: 490-496. Sohal, R.S., and Sharma, S.P., 1972. "Age-related changes i n the fine structure and number of neurons i n the brain of the house f l y Musca domestica.". Exptl. Gerontol. 7: 243-249. Sommer, J.R., and Blum, J.J.,'. 19 65. "Cytochemical l o c a l i z a t i o n s of acid phosphatases i n Euglena g r a c i l i s . " J . C e l l B i o l . 24: 235-251. 103 Spencer, P.W., and T i t u s , J.S., 1972. "Biochemical and enzymatic changes i n apple l e a f tissue during autumnal senescence." Plant Physiol. 49: 746-750. Spurr, A.R., 1969. "Low v i s c o s i t y epoxy r e s i n embedding medium for electron microscopy." J . U l t r a s t r . Res. 26 31-43. Srivastava, U., 1969. "Polyribosome concentration of mouse ske l e t a l muscle, as a function of age." Arch. Biochem. Biophys. 130: 129-139. Staehelin, A., 1966. "Die u l t r a s t r u k t u r der zellwand und de chloroplasten von C h l o r e l l a . " Z. Z e l l f o r s c h . 74_: 325 350. Staehelin, A., 1967. "Chloroplast f i b r i l s l i n k i n g the photosynthetic lamellae." Nature 214: 1158. Stearns, M.E., and Wagenaar, E.B., 1971. " U l t r a s t r u c t u r a l changes i n chloroplasts of autumn leaves." Can. J . Genet. Cytol. 13: 550-560. Strehler, B.L., 1962. Time, C e l l s , and Aging. Academic Press, New York and London. Strehler, B.L., and Mildvan, A.S., 1962. "Studies on the chemical properties of l i p o f u s c i n age pigment." In B i o l o g i c a l Aspects of Aging, Columbia Univ. Press, New York, pp. 174-181. Strum, J.M., and Karnovsky, M.J., 1970. " U l t r a s t r u c t u r a l l o c a l i z a t i o n of peroxidase i n submaxillary acinar c e l l s J. U l t r a s t r . Res. 31: 323-336. Sul l i v a n , J.L., and Debusk, A.G., 1973. " I n o s i t o l - l e s s death i n Neurospora and c e l l u l a r ageing." Nature New Biology 243: 72-74. Sundberg, I., Doskocil, M.J., and Lembi, CA., 1973. "Studies on the i s o l a t i o n of plasma membranes from filamentous green algae." J. Phycol. SKsuppl.): 21. Takahashi, A., P h i l p o t t , D.E., andMiquel, J . , 1970. "Electron microscope studies on aging Drosophila me1anogaster. I. Dense bodies." J. Gerontol. 25: 210-217. Tappel, A.L., 1965. "Free r a d i c a l l i p i d peroxidation damage and i t s i n h i b i t i o n by vitamin E and Selenium." Fed. Proc. 24_: 73-78. Tappel, A.L., 1968. "Will antioxydant nutrients, slow aging 104 processes?" G e r i a t r i c s 23 (10); 97-105. Teigler, D.J., and Baerwald, R.J., 1972. "A freeze-etched study of clustered nuclear pores." Tissue and C e l l 4_: 447-456. Thair, B.W., and Wardrop, A.B., 1971. "The structure and arrangement of nuclear pores in. plant c e l l s . " Planta TOO: 1-17. Thompson, E.W., and Preston, R.D., 1968. "Evidence for a st r u c t u r a l role of proteins i n a l g a l c e l l walls." J . Experimental Botany 19_: 690-697. Thung, P.J., and Hollander, C.R., 1967. "Regenerative growth and accelerated ageing." In Aspects of the Biology of Ageing, Symp. Soc. Exptl. B i o l . 21_: 455-462. T i l l a c k , T.W., and Marchesi, V;T., 1970. "Demonstration of the outer surface of freeze-etched red blood c e l l membranes." J . C e l l B i o l . £5: 649-653. Todd, M.M. , and V i g i l , E.i.L. , 1972. "Cytochemical l o c a l i z a t i o n of peroxidase a c t i v i t y i n Saccharomyces cerevisiae." J. Histochem. Cytochem. 20: 344-349. Tolbert, N.E., 1971. "Microbodies, peroxisomes and glyoxysomes." Annu. Rev. Plant Physiol. 2_2: 45-74. Toth, S.E., 1968. "The o r i g i n of l i p o f u s c i n age pigments." Exptl. Gerontol. _3_: 19-30. Tourte, M., 1972. "Mise en evidence d'une a c t i v i t e catalasique dans les peroxisomes de Micrasterias fimbriata." Planta 105: 50-59. T r e f f r y , T., Kl e i n , S., and Abrahemsen, M., 1967. "Studies of fine s t r u c t u r a l and biochemical changes i n cotyledons of germinating soybeans." Aust. J . B i o l . S c i . 20: 859-868. Trelease, R.W., Becker, W.M., Gruber, P.J., and Newcomb, E.H., 1971. "Microbodies (peroxisomes and glyoxysomes) i n Cucumber cotyledons." Plant Physiol. 48: 461-475. Udvardy, J . , and Farkas, G.L., 1972. "Abscisic acid stimulation of the formation of an ageing s p e c i f i c nuclease i n Avena leaves." J. Experimental Botany 23: 914-920. Ueda, K., 1961. "Structures of plant c e l l s with sp e c i a l reference to lower plants." Cytologia 26_: 34-35. 105 "Ue'da, K. , 1966. "Fine structure of Ch 1 orogohlum elongatum with special reference to vacuole development." Cytologia 31: 461-472. Varner, J.E. , 1961. "Biochemistry, of.senescence." Annu. Rev. Plant Physiol. 12: 245-264. V i g i l , E.L., 1970. "Cytochemical and developmental changes i n microbodies (glyoxysomes) and related organelles of castor bean endosperm." J. C e l l B i o l . 4_6: 435-454. V i l l i e r s , T.A., 1972. "Cytological studies i n dormancy. I I . Pathological ageing changes during prolonged dormancy and recovery upon dormancy release." New Phytol 71: 145-152. von Hahn, H.P., 1971. "Failures of regulation mechanisms as causes of c e l l u l a r aging." In Advances i n Gerontological Research, v o l . 3, B.L. Strehler, ed., Academic Press, New York and London, pp. 1-38. von Wettstein, D., 1954. "Formwechsel und t e i l u n g der chromotaphoren von Fucus vesiculosus." Z. Naturf. 9b: 476-481. Walker, D., 1971. "Experimental studies of cytoplasmic organization i n Zonaria apical c e l l s . " M.Sc. Thesis. Univ. of C a l i f o r n i a , Davis. Walne, P.L., Palisano, J.R., and Gomez, M.P., 1970. " C e l l u l a r changes associated with aging i n Euglena  granulata (Klebs) Lemm." J. Phycol. 6_ (suppl.): 11. Wangermann, E., 1965. "Longevity and ageing i n plants and plant organs." In Encyclopedia of Plant Physiology, W. Ruhland, ed., v o l . 15, Springer, B e r l i n , pp. 1026-1057. Wardrop, A.B., 1968. "Occurrence of structures with lysosome-l i k e functions i n plant c e l l s . " Nature 218: 978-9 80. Wareing, P.F., and P h i l l i p s , I.D.J., 1970. The Control of Growth and D i f f e r e n t i a t i o n i n Plants, MacMillan (Pergamon), New York. Weinbach, E.C., Garbus, J . , and S h e f f i e l d , H.G., 1967. "Morphology of mitochondria i n the. coupled, uncoupled and recoupled stages." Exptl. C e l l Res. 46: 129-143. Weiner, J . , Spiro, D., and Loewenstein, W.R., 1965. "Ultrastructure and permeability of nuclear membranes." J. C e l l B i o l . 27: 107-117. 106 Wooding, F.P.B., 1966. "The development of sieve elements of Pinus piriea." Planta 69: 230-243. Wooding, F.P.B., 1968. "Radioautographic and chemical investigation of incorporation into Sycamore vascular tissue walls." J . C e l l S c i . 3.: 71-81. Woolhouse, H.W., 1967. "The nature of senescence i n plants. In Aspects of the Biology of Ageing. Symp. Soc. Exptl. B i o l . 21: 179-214. Wunderlich, F., and Speth, V., 1972. "The macronuclear envelope of Tetrahymeha pyriformis GL i n d i f f e r e n t ph y s i o l o g i c a l stages. IV. Structural and functional aspects of nuclear pore complexes." J . Microscopie 13: 361-382. Zee, S.Y., 1969. "The l o c a l i z a t i o n of acid phosphatase i n the sieve elements of Pisum." Aust. J . B i o l . S c i . 22: 1051-1054. Zeman, W., 1971. "The neuronal ceroid-lipofuscinoses-Batten-Vogt Syndrome: a model for human ageing?" In Advances i n Gerontological Research, v o l . 3, B.L. Strehler, ed., Academic Press; New York and London, pp. 147-170. Z i r k i n , B.R., and Kim, S.K., 1972. "Fine structure of f i b e r s i n t h i n sections of condensed chromatin." E x p t l . C e l l Res. 75: 490-496. KEY OF SYMBOLS AND PLATE EXPLANATION 108 KEY. OF SYMBOLS ac api c a l c e l l ce chloroplast envelope cer chloroplast endoplasmic reticulum Ch chloroplast cw c e l l wall cwi c e l l wall ingrowth D dictyosome Dv dictyosome v e s i c l e ER endoplasmic reticulum er endoplasmic reticulum g genophore i l inner layer of the c e l l wall L lomasome M mitochondrion m microbody N nucleus n l nucleolus o l outer layer of the c e l l wall OSB osmiophilic structured body Pg pl a s t o g l o b u l i pm paramural space pns perinuclear space Py pyrenoid Pys pyrenoid sac r ribosomes V vacuole 10 9 Figure 1 Phase contrast observation of an i n s i t u embedded filament ( prostrate system ). A progressive increase i n the of a f f i n i t y for osmium i s observed from the a p i c a l c e l l (a.e.) downwards. X 750 Figure 2 Phase contrast observation.of an i n s i t u embedded filament ( erect system ). A sudden loss i n a f f i n i f y for osmium occurs i n c e l l s of the older portion of filaments, X 1,000 Figure 3 In s i t u embedded filament ( prostrate system ).The loss of a f f i n i t y for osmium i s sudden ( c e l l s located at the older end of the filament ). Notice that the c e l l wall between the dark and the l i g h t c e l l i s convex. X 1,000 Figure 4 Multilobed chloroplast. The pyrenoid i s i d e n t i f i e d by an arrowhead. Phase contrast observation of l i v i n g material. X 1,500 Figure 5 Bright f i e l d observation of l i v i n g material, The t r a n s i t i o n between two d i f f e r e n t c e l l types ( older portion of a filament ) i s i d e n t i f i e d by the c e l l wall convexity. X 1,000 Figure 6 Phase contrast observation of l i v i n g ... material.Possible phases of chloroplast d i v i s i o n ( arrowheads ). X 1,500 110 Figure 7 Phase contrast observation of l i v i n g material. Chloroplast morphology.. X 800 Figure 8 Phase contrast observation of l i v i n g material. Two d i f f e r e n t stages of pyrenoid d i v i s i o n are observed ( arrowheads ). X 900 Figure 9 Phase contrast observation of l i v i n g material. A chloroplast with 3 pyrenoids i s depicted ( arrowheads ). X 1,500 Figure 10 Phase contrast observation of l i v i n g material. At arrowheads l o c a l i z e d ingrowths of the c e l l wall are shown. X 1,000 Figure 11 Phase contrast observation of l i v i n g material. A c e l l wall ingrowth i s seen at arrowhead. X 750 Figures 12, 13, & 14 Cytochemical i d e n t i f i c a t i o n of DNA-containing structures, a f t e r RNA extraction. Figure 12 " C e l l type #1". Intense staining of the nucleus, e s p e c i a l l y apparent at arrowheads. X 1,200 Figure 13 " C e l l type #2". Nucleus perfe-c t l y stained. Nucleolus un-stained ( arrowhead ). X 1,200 Figure 14 " C e l l type #3". Absence of DNA staining characterizes t h i s c e l l type. X 1,200 I l l Figures 15, 16, & 17 Cytochemical i d e n t i f i c a t i o n of RNA-containing structures, a f t e r DNA extraction. Figure 15 " C e l l type #1". Nucleus un-stained (N). Intense staining i s found a l l over the cyto-plasm. X 1,200 Figure 16 " C e l l type #2". Nucleus un-stained (N). Staining r e s t r i -cted to the perinuclear region. X 1,200 Figure 17 " C e l l type #3". The almost absence of staining characte-r i z e s t h i s c e l l type. X 1,000 Figure 18 Cytochemical i d e n t i f i c a t i o n of protein staining regions. " C e l l type #1". Intense staining i s seen almost a l l over the cytoplasm, p a r t i c u l a r l y at the chloroplasts (Ch) and pyrenoids (Py) l e v e l s . X 800 112 Figures 19 .& 20 Cytochemical i d e n t i f i c a t i o n of protein staining regions. Figure 19 " C e l l type #2". Chloroplasts (Ch) and pyrenoids (Py) show intense staining. Cytoplasmic staining i s r e s t r i c t e d to certain areas. X 800 Figure 20 " C e l l type #3". The almost absence of staining from the cytoplasm of these c e l l s i s c h a r a c t e r i s t i c . X 1,200 Figures 21 & 22 Cytochemical i d e n t i f i c a t i o n of insoluble carbohydrates. Figure 21 C e l l belonging to the " t r a n s i t i o n a l 1-2" stages. Staining i s observed i n the perinuclear region (Golgi area), paramural space (arrowhead), and c e l l wall (cw). X 1,200 Figure 22 " C e l l type #1". Section through the c e l l wall showing intense staining properties. X, 1,000 Figures. 23, 24 & 25 Cytochemical study of l i p i d d i s t r i b u t i o n (Sudan Black B). Figure 23 " C e l l type #1". Very few 113 s i t e s with a f f i n i t y for Sudan. X 1,000 Figure 24 " C e l l type #2.Conspicuous staining a l l over the cyto-plasm. X 1,000 Figure 25 " C e l l type #3". An almost absence of staining characte-r i z e s t h i s c e l l type. X 1,000 Figure 26 " C e l l type #2". L i p o f u s c i n - l i k e pigment d i s t r i b u t i o n a f t e r treatment with the Schorml's reagent i s shown i n thi s picture. X 750 Figure 27 " C e l l type #2". Toluidine blue 0. Some of the cytoplasmic inclusions show a green to turquoise colour i n d i c a t i v e of polyphenolic content. X 750 114 Figure 28a General ul t r a s t r u c t u r e of a young c e l l ( c e l l type #1) belonging to the prostrate system of Ectocarpus sp. X 18,000 Figure 28b Plasmalemmasomes. The relat i o n s h i p of one of these structures to the plasmalemma i s shown at arrowhead. X 22,000 Figure 29 Section tangential to the nuclear envelope showing nuclear pores. Observe that from a hollow central core (arrowheads) spokes radiate toward the outer rim. X 100,000 Figure 30 F i b r i l l a r aspect of chromatin. X 90,000 Note: To best detect the intranuclear f i b r i l l a r system t h i s picture must be observed i n the d i r e c t i o n indicated by the arrowhead. 1 115 Figures 31 & 32 General u l t r a s t r u c t u r a l morphology of erect system c e l l s . " C e l l type #1". X 18,000 116 Figure 33 General u l t r a s t r u c t u r a l morphology of a young prostrate system c e l l . X 18,000 Figure 34 Direct continuity between the nuclear envelope and the dictyosome i s depicted at arrowhead. X 16,000 Figure 35 Association between the endoplasmic reticulum (E.R. ) and the mitochondrion (M) i s shown at arrowhead. X 30,000 117 Figure 36 Freeze-etch. r e p l i c a of the nuclear envelope A many-particulate (A) and a sparsely p a r t i c u l a t e (B) surfaces are recognized. X 60,000 Figure 37 Freeze-etch r e p l i c a of the nuclear envelope Pore containing areas are permeated by pore free regions. X 30,000 Figure 37a Freeze-etch r e p l i c a of the periphery of a young c e l l . Plasmalemmasomes are observed i n the paramural space (pm). X 26,000 118 Figure 38 Portion of. the nucleus and perinuclear region of a young c e l l . The close associa-t i o n between the' nucleolus (nl) and the inner membrane of the nuclear envelope i s apparent. Direct continuity of the p e r i -nuclear space with the E.R. i s observed. At other place ( empty arrowhead ) a multivesicular structure occupies a posi t i o n shared by both the E.R. and the perinuclear space. Other features are E.R. fenestrae ( arrowheads ) and mitochondria (M). The small arrowhead points to a mitochondrion DNA-containing area. X 55,000 Figures 39 & 40 V e s i c u l a r - l i k e inclusions are seen inside the perinuclear space (arrowheads ). X 18,000 Figure 41 The rel a t i o n s h i p of the perinuclear inclusions to the nuclear envelope mem-branes i s shown at arrowheads. X 40,000 Figure 42 A vesicul a r structure ( arrowhead ) i s observed i n association with the outer membrane of the nuclear envelope. A dictyosome (D) i s also depicted. X 25,000 119 Figure 43a Freeze-etch. preparation of a chloroplast, "g" indicates the pos i t i o n of the genophore.-Thylakoid associated f i b r i l s are depicted at small arrowheads and at nearby positions. A fenestra-like structure i s shown i n the chloroplast E.R. ( big arrowhead ). X 40,000 Figure 43b D e t a i l of the area l a b e l l e d "A" i n figure 43a. Both the chloroplast envelope (ce) and ( probably ) the chloroplast E.R. (cer) are shown. A pore-like structure i s observed at arrowhead. X 30,000 Figure 44 Endoplasmic reticulum morphology. A c r y s t a l l i n e - l i k e i n c l u s i o n i s seen inside. the E.R. ( i ) . The arrowhead points to a fenestra-like structure. X 60,000 120 Figure 45 S i m i l a r i t y i n the o r i g i n and morphology of the perinuclear space ( pns ) and endoplasmic reticulum ( er ) inclusions i s shown i n t h i s picture. A " Osmiophilic Structured Body " (OSB) i s seen i n association with the chloroplast (Ch). X 90,000 Figure 46 Freeze-etch preparation of a chloroplast. The pattern of thylakoid arrangement and the p o s i t i o n of the chloroplast envelope (ce) i n r e l a t i o n to the chloroplast E.R. (cer) are shown. Other features include the presence of a thylakoid free area (F), and of vesicula r structures i n the narrow cytoplasmic region found i n between the "ce" and the "cer" ( big arrowhead ). X 36,000 121 Figure 47a A portion of a chloroplast i s depicted; "g" indicates the pos i t i o n of the geno-phore. The existence of d i r e c t continuity between the chloroplast E.R. (cer) c i s t e r -nal space and the chloroplast envelope (ce) i s shown at arrowhead. Also apparent i s an i n i t i a l step i n the formation of a " Os-miophilic Structured Body " (OSB). Meta-b o l i t e s of high electron opacity are seen to be discharged i n the paramural space (pm). X 42,000 Figure 47b A step i n the process of formation of a " OSB " i s depicted. The relat i o n s h i p of the " OSB " to the chloroplast E.R. (cer) i s apparent. X 40,000 Figure 48 Portion of a chloroplast showing a geno-phore-like region (g). Direct luminal continuity between the chloroplast E.R. (cer) c i s t e r n a l space and the chloroplast envelope (ce) i s apparent ( arrowhead ). The close association between the chlo-roplast and the mitochondrion (M) i s also shown. X 42,000 Figure 49 An almost tangential section through the region of the chloroplast E.R. (cer) shows the i n t r i c a t e pattern of arrangement of the "cer" derived tubular structures. Lomasome-like structures (L) are present i n the paramural space. Membrane residues are seen inside the vacuoles (V). X 24,000 Figure 50 V e s i c u l a r - l i k e inclusions are present inside the chloroplast E.R; c i s t e r n a l space (cer). X 36,000 Figure 51 Osmiophilic structured body morphology. Notice that a non structured component i s intermingled with a myelin-like one. X 60,000 Figure 52 An osmiophilic structured body (osb) i s observed i n the space shared by the chlo-roplast E.R. (cer) and the perinuclear space (pns). X 30,000 Figure 53 The association between a osmiophilic structured body (osb) and the pyrenoid (Py) i s shown. A portion of the pyrenoid sac (Pys) i s also depicted. X 36,000 Figure 54 The discharge of osmiophilic structured bodies (osb) into the paramural space (pm) i s shown. X 36,000 Figure 55 The nuclear envelope-dictyosome relation-ship i s shown. Also depicted i s the association of the dictyosome (D) with a osmiophilic structured body (osb). X 50,000 123 Figure 56 Portion of a young c e l l ( c e l l type #1 ) showing peripheral l o c a l i z a t i o n of mitochondria. Osmiophilic structured bodies ( osb) are found either i n associa-tion with the chloroplast (Ch) or they l i e i n the cytoplasm without any apparent rela t i o n s h i p to these organelles. An early stage i n the process of formation of a c e l l wall ingrowth (cwi) i s also depicted. X 30,000 124 Figure 57 Dictyosome.morphology. Interruptions can be recognized i n the cisternae of the dictyosome. These seem to be of two types: 1) a narrow tu b u l a r - l i k e interruption ( arrowheads l a b e l l e d b ), and 2) a larger fenestra-like interruption ( arrowheads l a b e l l e d a ). X 80,000 Figure 5 8 Dictyosome morphology. Interrutpions are recognizable i n the cisternae of the dictyosome ( arrowheads ). These i n t e r r u -ptions seem to be more;abundant i n the innermost cisternae. The outermost c i s -terna displays a hypertrophied appearance (D). X 70,000 125 Figure 59 Dictyosome morphology. Bridge-like elements are observed i n between the cisternae of the dictyosome as well as i n an i n t r a c i s t e r n a l p o s i t i o n ( arrowheads ). X 90,000 Figure 60 The existence of a very close association between the dictyosome (D) and pyrenoid (Py) i s observed. X 30,000 Figure 61 Dictyosome (D)-mitochondria (M) associa-t i o n i s depicted. X 30,000 126 Figure 62 Incorporation of dictyosbme-derived v e s i -cles ( big arrowheads ) into vacuoles (V). Three possible cases of mitochondrial d i v i s i o n are shown at small arrowheads. X 36,000 Figure 63 Dictyosome (D)-pyrenoid (Py) association. X 20,000 Figure 64 A stage i n the formation of a lomasome-l i k e structure (L) i s seen inside an hypertrophied dictyosome cisterna ( c. f. with figure 49, showing a p o t e n t i a l lomasome-like structure 'L' inside the paramural space ). X 24,000 Figure 65 Multimembranous i n c l u s i o n occupies the outermost cisterna of a dictyosome. The mode of formation of t h i s structure through the invagination of the cisterna peripheral membrane i s depicted at the arrowhead. X 80,000 Figure 66 Tubular-like inclusions are seen inside a dictyosome-derived v e s i c l e (Dv). X 80,000 127 Figure 67 Mitochondrion.morphology. X 20,000 Figure 68 D e t a i l of the mitochondrion c r i s t a e Inclusions are shwon at arrowhead and i n the inset. X 50,000 Inset X 120,000 Figure 69 Mitochondrion (M)-nucleus (N) association, X 24,000 Figure 70 Freeze-etch preparation showing the asso-c i a t i o n of a mitochondrion (M) with a chloroplast (Ch). X 22,000 Figure 71 Mitochondrion (M)-pyrenoid (Py) associa-t i o n . X 24,000 Figure 72 Mitochondrion-mitochondrion association ( arrowheads ). At arrow the mitochon-drion- E.R. association i s depicted. X 30,000 Figure 73 High magnification observation of thylakoids. Big arrowhead points to an interruption. Small arrowheads indicate the presence of bridge-like elements. X 120,000 Figure 74 Freeze-etch preparation of a portion of the thylakoid system of a chloroplast. Arrowheads indicate the presence of b r i -dge-like elements. X 120,000 128 Figure 75 Deviations from the normal pattern of thylakoid arrangement are shown. X 60,000 Figure 76 Thylakoid interruptions are seen at arrowheads "a". Arrowheads "b" point to bridge elements. Arrowheads . "c" indicate pore-like interruptions i n the chloroplast envelope. X 80,000 Figure 77 High magnification observation of the arrowhead l a b e l l e d "a" i n figure 78. X 100,000 Figure 78 Section tangential to the thylakoid membranes showing the presence of pore-l i k e interruptions ( arrowheads ). X 50,000 Figure 79 High magnification observation of the thylakoid architecture. Arrowheads point to bridge-like elements. X 90,000 Figure 80 Freeze-etch preparation of thylakoids showing the p a r t i c u l a t e nature of the exposed surfaces. A many p a r t i c u l a t e (B) and a sparsely p a r t i c u l a t e (A) surfaces are recognized. X 60,000 129 Figure 81 Freeze-etch preparation of a chloroplast. The p a r t i c u l a t e nature of exposed surfaces i s depicted. A many p a r t i c u l a t e (B) and a sparsely p a r t i c u l a t e (A) surfaces are observed. X 60,000 Figure 82 Chloroplast architecture. The pattern of bi f u r c a t i o n and merging of neighboring thylakoid bands i s depicted. Notice that the peripheral band of thylakoids i s not continuous, instead i s made up of portions of the c e n t r a l l y placed bands ( arrow-heads ). X 40,000 130 F i g u r e 83 T a n g e n t i a l s e c t i o n t h r o u g h a d i c t y o s o m e . I n t e r r u p t i o n s : 1) l a r g e ( arrowheads "a" ) , 2) narrow ( arrowheads "b" ) a r e o b s e r v e d i n one o f t h e c i s t e r n a e . X 80,000 F i g u r e 84 M i t o c h o n d r i a l a r c h i t e c t u r e . : The m i t o -c h o n d r i o n genophore i s i n d i c a t e d by arrowheads. I n t h e c h l o r o p l a s t (Ch), p o r e - l i k e i n t e r r u p t i o n s a r e d e t e c t e d i n the t h y l a k o i d s ( arrowheads "a" ) and i n th e c h l o r o p l a s t envelope ( arrowhead "b" ),. X 75,000 131 Figures 85, 86, 87, and 88 Different stages In the formation and-detachment from, chloroplasts of concentric bodies. Figure 85 the arrowhead points to the place where b i f u r c a t i o n of one thylakoid has occurred. X 45,000 Figure 8 6 Concentric arrangement of thylakoids i s observed inside the chloroplast. X 40,000 Figure 87 Concentric body becomes deta-ched from the p l a s t i d (arrow-head) . X 36,000 Figure 88 Concentric body l i e s free i n the cytoplasm. X 26,000 Figure 89 Acid phosphatase a c t i v i t y i s found associated with concentric bodies once they are present inside vacuoles. X 30,000 Figure 90 Acid phosphatase a c t i v i t y (control preparation). Notice the absence of. reaction product. X. 30,000 Figure 91 Concentric body X 30,000 Inside vacuole. Portion of a chloroplast showing a th y l a -koid free region (F). Bridge-Tike elements are found between the outermost thylakoid of the peripheral band and the chloro-p l a s t envelope. X 80,000 The close relationship between the pyrenoid matrix f i b r i l s and the surroun-ding thylakoid membranes i s observed ( material held for 5 days i n t o t a l darkness ). X 60,000 133 Figure 94 Section tangential to the thylakoid membranes. Ribosome-like p a r t i c l e s (r) are seen i n close association with thylakoids. X 50,000 Figure 9 5 Freeze-etch preparation of a chloroplast. F i b r i l l a r elements are seen i n associa-t i o n with the thylakoids ( arrowheads ). A plastoglobulus (Pg) i s also depicted. X 36,000 Figure 9 6 Section p a r a l l e l to the thylakoid membra-nes. F i b r i l l a r elements are seen to run i n between thylakoids ( white arrows ). X 90,000 134 Figures.97, 98, and 99 Plastoglobuli architecture. Notice the f i b r i l l a r organization of these structures. At places the f i b r i l l a r elements that composed these bodies seem to be linked to the thylakoid membranes ( arrowheads ). One of the micrographs ( figure 99 ) shows the f i b r i l l a r phase of one plastoglobulus to be continuous with the f i b r i l l a r phase of another. Figures 97, and 98 X 100,000 Figure 99 X 80,000 Figure 100 E.R.- vacuole continuity i s depicted at arrowheads. X 25,000 Figure l o i Laminaria type of chloroplast d i v i s i o n . The c o n s t r i c t i n g region of the chloroplast i s indicated by an arrowhead. X 20,000 Figures 102, and 103 Sphacelaria type of chloroplast d i v i s i o n . Figure 102 Plasmalemmasome-like (pm) structures are also depicted. X 12,000 Figure 103 Arrowhead points to the place of the peripheral lamellar bridge formation. X 18,000 135 Figure 10 4 Bleb-like structure produced by a chloroplast of dark treated material ( 5 days i n t o t a l darkness ). X 60,000 136 Figures 105, and 106. Non consecutive s e r i a l sections showing a c o n s t r i c t i n g chloroplast. Figure 105 X 16,000 Figure 106 X 26,000 Figure 107 Chloroplast with two d i s t i n c t c o n s t r i -ction s i t e s ( big arrowheads ). Geno-phore i s indicated by small arrowheads. X 24,000 Figure 108 Chloroplast with two pyrenoids. The pyrenoid on the r i g h t side of the micrograph shows a close r e l a t i o n s h i p to thylakoids. X 24,000 Figure 109 This figure depicts a stage i n the process of pyrenoid d i v i s i o n ; notice both the chloroplast envelope (ce) and the chloroplast E.R. (cer). X 100,000 Figure 110 Cross section of a pyrenoid depicting the rel a t i o n s h i p existent among the d i f f e r e n t membrane systems which surround the pyrenoid. X 40,000 Figure 111 Cross wall adjoining two consecutive c e l l s . Notice that the plasmadesmata are not organized into a p i t - l i k e area. X 30,000 Figure 112 Direct continuity between the pyrenoid sac space (Pys) and the vacuole (v). X 22,000 Figure 113 C e l l wall organization. Two regions are recognized . i n the c e l l , w a ll: 1) an Inner layer (II) showing a p a r a l l e l arrangement of i t s f i b r i l l a r elements, 2) an outer layer (ol) r e t i c u l a t e i n appearance. X 60,000 Figure 114 Longitudinal section passing through the region of d i v i s i o n of a pyrenoid. X 50,000 Figure 115 At arrowheads, one process of autopha-gocytosis, involving the invagination of the tonoplast, i s depicted. Provacuole-l i k e structures are indicated by arrows. Their o r i g i n from the E.R. i s apparent. X 36,000 Figure 116 D e t a i l of a portion of the cytoplasm showing provacuolar structures and t h e i r tendency to merge with themselves and with vacuoles (arrowheads). X 40,000 Figure 117 Freeze-etch preparation of the c e l l w all, showing the bilayered organization. X 30,000 Figure 118 Freeze-etch preparation of a region of the cytoplasm showing provacuolar structures, and t h e i r tendency to fuse with each other ( arrows ). X 40,000 Figure 119 The involvement of E.R. elements i n the i s o l a t i o n of cytoplasmic t e r r i t o r i e s i s shown. Notice at arrow the b i f u r c a t i o n of the E.R. X 40,000 Figures 120, and 121 Aspects of the i s o l a t i o n of cytoplasmic t e r r i t o r i e s involving the p a r t i c i p a t i o n of the E.R. X 60,000 Figures 122, and 123 Two d i f f e r e n t stages i n the process of transformation of E.R. membranes and i s o l a t e d materials into t y p i c a l vacuoles are shown. X 60,000 Figure 124 The traping of cytoplasmic material by tonoplast invagination i s shown. X 45,000 140 Figures 125, and 126 Acid phosphatase a c t i v i t y i s associated with vacuoles. X 30,0.00 Figure 127 Vacuole showing remnants of disorgani-zed membrane inclusions. X 30,000 Figure 128 Inclusions crowding the cytoplasm of a t r a n s i t i o n a l " 1-2 " c e l l . X 6,000 Figures 129, and 130 Acid phosphatase a c t i v i t y i n dictyosomes ( figure 129 ) and E.R. ( figure 130 ). Figure 129 X 10,000 Figure 130 X 20,000 Figure 131 Acid phosphatase a c t i v i t y ( control preparation ). Notice the absence of reaction product. X 12,000 Figure 132 T r a n s i t i o n a l " 1-2 " c e l l . Notice the pattern of thylakoid arrangement. X 25,000 Figure 133 Notice that neither the pl a s t o g l o b u l i (Pg) nor the region of i n t r a p l a s t i d meta-b o l i t e ( big arrowhead ) are preserved by permanganate f i x a t i o n . X 20,000 Figure 134 Permanganate e f f e c t s on the preservation of cytoplasmic inclusions. Notice that while some inclusions are preserved "1"; others are only p a r t i a l l y "2", and s t i l l others are not preserved at a l l "3". X 17,500 Figures 135, and 136 T r a n s i t i o n a l " 1-2 " c e l l s . Two types of vacuolar. Inclusions seem to be present i n these c e l l s . One type (a) i s granular i n appearance, the other i s myelin-like (b). X 36,000 142 Figure 137 High: magnification observation of . vacuolar inclusions found i n t r a n s i t i o n a l " 1-2 " c e l l s . A mixed, myelin-like, i n c l u s i o n (B) and granular Inclusions (A) are depicted. X 80,000 Figures 138, and 139 T r a n s i t i o n a l " 1-2 " c e l l s . D i s t r i b u t i o n of acid phosphatase a c t i v i t y X 12,000 Figure 140 T r a n s i t i o n a l "1-2 " c e l l . Acid phospha-tase a c t i v i t y ( control preparation ). Notice the absence of reaction product. X 10,000 Figure 141 T r a n s i t i o n a l " 1-2 " c e l l . Aspect of the Golgi region. Dictyosome-derived v e s i c l e s (Dv) are f i l l e d with metabolites. X 20,000 Figure 142 T r a n s i t i o n a l " 1.-2 " c e l l . C e l l wall. Notice the presence of electron dense metabolites among the c e l l wall micro-f i b r i l s and at the wall periphery. X 36,000 Figure 143 " C e l l type #2". General ultrastructure of a prostrate system c e l l . At black arrows several chloroplast regions involved i n metabolite production are indicated. White arrows point to a highly vesiculated E.R. region. X 18,000 Figure 144 " C e l l type #2". C e l l w all. Notice the presence of conspicuous patches of metabolites among the c e l l wall micro-f i b r i l s and at the wall periphery. X 6,000 Figure 145 T r a n s i t i o n a l " 2-3 " c e l l . General morphology of an erect system c e l l . X 8,000 Figure 146 This picture displays the abrupt t r a n s i -t i o n between " c e l l type #2" and " c e l l type #3" morphologies. X 8,000 Figure 147 Transition " 2-3 " c e l l s . General aspect of the background cytoplasm. X 26,000 144 Figure 148 " C e l l type #2". General u l t r a s t r u c t u r e of an erect system c e l l . At arrows several chloroplast regions involved i n metabolite production are Indicated. X 14,000 Figure 149 High magnification observation of a chloroplast region involved i n metabolite production. X 60,000 Figure 150 High magnification observation of the background cytoplasm of a " c e l l type #2". X 60,000 145 Figure 151 " C e l l type #2". General ultrastructure of a prostrate system c e l l . Several chloroplast regions engaged i n metabolite production are indicated ( arrows ). A region of E.R. disorganization i s also depicted. X 20,000 Figure 152 " C e l l type #2". C e l l wall-ingrowths are observed. The alternate disposition, of ~ metabolite ( !, 3, 5 ) and wall material ( 2, 4 ) are indicated. X 40,000 146 Figure 153 " C e l l type #2". The nucleus (N) and the nucleolus (nl) are depicted. Mitochondria (M) are also" observed. A. c e l l , wall ingrowth i s seen and i t s double c o n s t i -tution indicated ( labels 1, and 2 ). Black arrows indicate the s i m i l a r i t y existent between the c e l l wall i n c l u s i o n material and the vacuole i n c l u s i o n material. X 50,000 Figures 154, and 156 High magnification observa-t i o n of chloroplast zones of metabolite production. Figure 154 X 70,000 Figure 156 X 36,000 Figure 155 Release of i n t r a p l a s t i d metabolite into the cytoplasm. X 18,000 Figure 157 ) Release of i n t r a p l a s t i d metabolites into the cytoplasm. X 32,000 Figure 158 Portion of a cross wall separating two " c e l l type #2" neighboring c e l l s . The layering constitution of one of the c e l l wall ingrowths i s indicated. At arrow-heads plasmodesmata are indicated. X 40,000 Figure 159 Freeze-etch preparation of portion of a chloroplast depicting a region of metabolite production ( arrowhead ). X 34,000 Figure 160 " C e l l type #2". General ultrastructure of an erect system c e l l . The morphology of the c e l l wall i s also depicted. X 10,000 Figures 161, and 162 C e l l wall ingrowths morphology. Figure 161 X 12,000 Figure 162 X 20,000 Figure 163 T r a n s i t i o n a l " 1-2 " c e l l . An early stage i n the formation of a c e l l wall ingrowth i s shown. X 30,000 Figure 164 Tr a n s i t i o n a l " 2-3 " c e l l . General aspect, of the background cytoplasm. X 12,000 Figure 165 T r a n s i t i o n a l " 2-3 " cell.Tonoplast i s -absent at certain places ( arrowheads ). X 50,000 Figure 166 T r a n s i t i o n a l " 2-3 " c e l l . Chloroplast morphology. Notice at arrowhead the protrusion from the chloroplast body of a stroma containing structure. .Notice also that t h i s structure i s d i s t i n c t from the pyrenoid (Py), but s i m i l a r to other structures seen i n the cytoplasm of other c e l l s ( c. f; figures 167, 169, arrowheads ). X 30,000 Figures 167, and 169 T r a n s i t i o n a l " 2-3 " c e l l s . Chloroplast and pyrenoid morphology. Notice the lamellar nature of the region l a b e l l e d "A". Plastid-derived bodies are detected i n the cytoplasm ( arrowheads ). X 22,000 Figure 16 8 T r a n s i t i o n a l " 2-3 " c e l l . The chloro-p l a s t envelope (ce) i s c l e a r l y defined around the pyrenoid body (Py). At the arrowhead the abscission of the pyrenoid from the chloroplast seems to be occurring. X 36,000 149 Figure 170 T r a n s i t i o n a l " 2-3 " c e l l . Notice that the nucleus i s i n the process of disorganization. The nuclear envelope i s no longer recognizable at places, while i n others seems to be.degenerating ( arrows ). The portion of the nuclear envelope longer remaining i n t a c t seems to be the one facing the dictyosome (D). X 50,000 Figure 171 T r a n s i t i o n a l " 1-2 " c e l l . A phase i n the formation of a c e l l wall ingrowth ( cwi ) i s depicted. Deposition of new wall material i s seen at arrowheads. X 38,000 Figure 172 T r a n s i t i o n a l " 2-3 " c e l l . Chloroplast morphology. Notice the lamellar nature of the area l a b e l l e d "A" and the pheno-menon of abscission of p l a s t i d derived structures "B". X 50,000 Figures 173, 174, and 175 Tr a n s i t i o n a l " 2-3 " c e l l s . Mitochondrial morphology. Notice the c i r c u l a r arrangement of the mitochondrial c r i s t a e ( figures 174, and 175 ). X 30,000 Figure 176 Plastid-derived body. X 36,000 Figure 177 T r a n s i t i o n a l " 2-3 " c e l l . The r e l a t i o n -ship of metabolite to the chloroplast lamellae i s s t i l l apparent i n t h i s picture ( white arrowhead ). X 36,000 Figure 178 T r a n s i t i o n a l " 2-3 " c e l l . Abscission of chloroplast-derived structures from the p l a s t i d body. X 42,000 Figure 179 T r a n s i t i o n a l " 2-3 " c e l l . The involve-ment of thylakoids i n the i s o l a t i o n of stroma regions i s depicted ( arrowheads ) X 50,000 Figure 180 T r a n s i t i o n a l " 2-3 " c e l l . Chloroplast morphology. The i s o l a t i o n of stroma, regions from the main p l a s t i d body i s shown. X 20,000 Figure 181 Tr a n s i t i o n a l " 2-3 " c e l l . Chloroplast derived bodies ( arrowheads ) are seen i n the cytoplasm. Mitochondria are seen at "A". X 16,000 Figure 182 T r a n s i t i o n a l " 2-3 " c e l l . A portion of a p l a s t i d showing an area of i s o l a t i o n of stroma material i s depicted. At arrowhead the membrane of one of the i s o l a t e d areas has fused with the chloroplast envelope membrane,:which i s single membrane i n cons t i t u t i o n . X 40,000 Figure 183 T r a n s i t i o n a l " 2-3 " c e l l . Pyrenoid-like structures (Py) are observed inside vacuoles. X 24,000 Figure 184 " C e l l type #3". Aspect of the c e l l wall. X 60,000 152 Figures 185, and 186 T r a n s i t i o n a l " 2-3 " c e l l s . S e r i a l sections showing the release of p l a s t i d derived bodies into the cytoplasm. X 36,000 Figures 187, 188, and 189 " C e l l type #3". General u l t r a s t r u c t u r a l morphology. Figures 187, and 188 Prostrate system c e l l s . X 6,000 Figure 189 Erect system c e l l . X 6,000 Figure 190 " C e l l type #3". Chloroplast morphology. X 30,000 Figure 191 " C e l l type #3". Acid phosphatase a c t i v i t y ( erect system c e l l ). Notice that reac-t i o n product i s di s t r i b u t e d throughout the c e l l cavity. X 20,000 Figure 192 " C e l l type. #3". De t a i l of a thylakoid stack. X 70,000 Figures 193, and 194 " C e l l type #3". Portions of chloroplasts showing signs of thylakoid disorganization. X 30,000 Figure 195 " C e l l type #3". Portion of a.chloroplast showing extensive disorganization of the thylakoid system. X 24,000 Figure 196 " C e l l type #3". Aspect of the disorganiz t i o n of the cytoplasm and mitochondria (M). X 36,000 Figure 197 " C e l l type #3". Mitochondrial (M) remnants showing d i f f e r e n t stages of disorganization. X 36,000 Figure 198 " C e l l type;'#3". Chloroplast showing widespread signs of thylakoid disorgani-t i o n . X 36,000 Figure 199 " C e l l type #3". Accumulation of plastoglo-b u l i inside the chloroplast. X 26,000 Figure 200 " C e l l type #3". De t a i l of thylakoid arrangement. A genophore-like region (g) i s apparent. X 30,000 Figure 201 " C e l l type #3". Aspect of disorganization of cytoplasm and mitochondria (M). X 28,000 Figure 202 " C e l l type #3". Mitochondrial remnant. X 42,000 Figure 203 " C e l l type #3". Acid phosphatase a c t i v i t y ( control preparation ) . Notice the. absence of reaction product. X 12,000 " C e l l type #3". Acid phosphatase a c t i v i t y . Reaction product i s apparent i n plasmo-desmata and cytoplasm. X 36,000 " C e l l type #3". Acid-phosphatase a c t i v i t y (control preparation ). Notice that no reaction product i s found associated with plasmodesmata. X 30,000 " C e l l type #3". Acid phosphatase a c t i v i t y . Reaction product i s d i s t r i b u t e d throughout the c e l l c avity. X 12,000 Figures 207, and 208 " C e l l type #3". Aspects of disorganization of the c e l l wall. Figure 207 X 30,000 Figure 208 X 45,000 Figure 204 Figure 205 Figure 206 Figures 209, and 210 " C e l l type #1". Material incub ted for determination., of - catalase a c t i v i t y Microbody-like organelles (m) show deposi-t i o n of reaction product. Figure 209 X 30,000 Figure 210 Arrow points to core-like structure. X 36,000 Figure 211 " C e l l type #1". Aminotriazole incubation (catalase c o n t r o l ) . No reaction product i s observed i n the microbodies (m). X 22,000 Figure 212 " C e l l type #1". Material incubated for determination of catalase a c t i v i t y . Reac-tio n product i s observed i n mitochondria (M). X 30,000 Figure 213 " C e l l type #2". Material incubated for determination of catalase a c t i v i t y . Arrow head points to zone of deposition of reaction products. X 30,000 Figure 214 " C e l l type #1". Peroxidase a c t i v i t y i s observed i n the c e l l wall. X 16,000 Figure 215 " C e l l type #1". A microbody showing a core-like structure (arrow) i s depicted. X 36,000 Figure 216 " C e l l t y p e # l " . Peroxidase a c t i v i t y i s apparent i n the c e l l wall (cw) and para-mural space. X 13,000 Figures 217, and 220 . " C e l l type #3". Acid phospha-tase a c t i v i t y . Reaction product i s di s t r i b u t e d throughout the c e l l cavity. X 26,000 Figure 218 " C e l l type #1". Peroxidase a c t i v i t y ; con-t r o l preparation ( KCN ) . Notice, the absence of reaction product. X 12,000 Figure 219 T r a n s i t i o n a l " 1-2 " c e l l . ATPase a c t i v i t y ( Mg + +-dependent system ). Notice deposition of reaction product i n the mitochondrion ( arrow ). X 18,000 Figure 221 " C e l l type #2". Material incubated for l o c a l i z a t i o n of peroxidase a c t i v i t y . X 30,000 Figure 222 " C e l l type #2". Peroxidase a c t i v i t y ( control preparation ). X 30,000 159 Figures 223, and 225 " C e l l type #1". ATPase a c t i v i t y + + ++ ( Na -K -Mg. - dependent system ). Deposition of reaction product i s cons-picuous at the plasmalemma and i n associa-t i o n with thylakoids. Figure 223 X 16,000 Figure 225 X 36,000 Figure 224 " C e l l type #1". ATPase a c t i v i t y ( Na -K -Mg - dependent system ). Deposition of reaction product i s observed i n the mitochondrion (M). X 32,000 Figure 226 " C e l l type #1". ATPase a c t i v i t y ( Mg + +-dependent system ). Deposition of reaction product i s apparent i n the mitochondrion. X 40,000 Figure 227 " C e l l type #1". ATPase a c t i v i t y ( Mg + +-dependent system ). Reaction product i s observed i n association with the plasma-lemma and thylakoids. The reaction, however, does not seem so intense as i n the case of the I system. X 12,000 Na +-K +-Mg + +- dependent Figure 228 ATPase a c t i v i t y ( Na +-K +-Mg + +- activated system control preparation ) . Notice the absence of reaction product. X 14,000 160 Figures 229, and 230 ATPase a c t i v i t y ( Na +-K +-Mg + +-activated system ). Figure 229 Deposition of reaction product i s observed i n asso-c i a t i o n with the plasmalemma and thylakoids. X 14,000 Figure 230 Control preparation ( incubation medium minus ATP ). Notice the absence of reaction product. X 12,000 161 Figures 231, and 233 I d e n t i f i c a t i o n of l i p o f u s c i n -l i k e material ( Hendy,1971, modification of the Fontana' s technique ')'••* Notice that l a b e l l i n g i s primarily found i n vacuolar inclusions. Figure 231 X 20,000 Figure 233 Notice that zones of i n t r a -p l a s t i d metabolite formation are free of l a b e l l i n g ( arrows ), and so are simi l a r inclusions present i n the cytoplasm. X 20,000 Figure 232 ++ ATPase a c t i v i t y ( Mg - activated system; control preparation ). Notice the absence of reaction product. X 18,000 Figure 234 Lipofuscin ( control preparation ). X 18,000 Figure 235 Schorml's modification ( Hendy,1971 ) for the u l t r a s t r u c t u r a l i d e n t i f i c a t i o n of l i p o f u s c i n . X 18,000 

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