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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 L i c e n c i a t u r a , U n i v e r s i t y o f Porto  ( P o r t u g a l ), 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 t h e s i s as conforming required  THE  standard  UNIVERSITY OF BRITISH COLUMBIA August, 1973  t o the  In p r e s e n t i n g an  this  thesis i n partial  advanced degree a t t h e U n i v e r s i t y  the  Library  s h a l l make i t f r e e l y  f u l f i l m e n t o f the requirements f o r o f B r i t i s h Columbia, I agree  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 extensive for  copying o f t h i s  thesis  s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t 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 understood that copying or p u b l i c a t i o n  of t h i s  thesis f o r financial  written  permission.  Department o f  7  /  9  /  gain  BOTANY  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  Date  that  1  9  7  3  Columbia  s h a l l n o t be a l l o w e d w i t h o u t my  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 a l g a Ectocarpus techniques  sp. was c a r r i e d out u s i n g  of l i g h t - and e l e c t r o n microscopy  c y t o c h e m i s t r y . The f i r s t Ectocarpus  standard  as w e l l as  6-8 c e l l s i n each f i l a m e n t o f  are c h a r a c t e r i z e d by a l a r g e round and w e l l  organized nucleus, a dense cytoplasm endoplasmic r e t i c u l u m , dictyosomes, c h l o r o p l a s t s w i t h conspicuous  r i c h i n ribosomes, m i t o c h o n d r i a , and  s t a l k e d p y r e n o i d s . Few s m a l l  vacuoles are p r e s e n t i n the cytoplasm. New  f e a t u r e s not  p r e v i o u s l y r e p o r t e d f o r the brown algae are a l s o d e s c r i b e d . These i n c l u d e the presence  o f p o r e - l i k e i n t e r r u p t i o n s and  b r i d g e - l i k e f i l a m e n t s i n the c i s t e r n a e of dictyosomes and c h l o r o p l a s t t h y l a k o i d s , as w e l l as the formation of concent r i c bodies of c h l o r o p l a s t o r i g i n . M i c r o b o d y - l i k e o r g a n e l l e s are a l s o r e p o r t e d ; c y t o c h e m i c a l s t u d i e s have shown c a t a l a s e a c t i v i t y a s s o c i a t e d w i t h them. Peroxidase  a c t i v i t y i s repor-  ted i n the c e l l w a l l , w h i l e a c i d phosphatase a c t i v i t y i s found i n a s s o c i a t i o n with both the endoplasmic r e t i c u l u m and the G o l g i elements, as w e l l as i n s i d e the v a c u o l e s . Adenosine t r i p h o s p h a t a s e i s p r e s e n t i n m i t o c h o n d r i a , c h l o r o p l a s t t h y l a k o i d s , and plasma membrane. These c e l l s , f o r e , can be d e s c r i b e d as m e r i s t e m a t i c  there-  o r the immediate  consequence of the d i f f e r e n t i a t i o n o f the m e r i s t e m a t i c  cells.  The processes o f autophagy and v a c u o l a t i o n are s t u d i e d i n d e t a i l and found t o be p a r t i c u l a r l y  significant  d u r i n g the stages of c e l l d i f f e r e n t i a t i o n which l e a d t o the aging o f Ectocarpus  s p o r o p h y t i c c e l l s . Increases i n v a c u o l a -  t i o n and autophagy are p a r a l l e l e d by an i n c r e a s e i n a c i d phosphatase a c t i v i t y . The u l t i m a t e r e s u l t s of these processes a r e : a r e d u c t i o n i n c y t o p l a s m i c m a t r i x , a g e n e r a l d e t e r i o r a t i o n o f c y t o p l a s m i c o r g a n e l l e s , and the formation of r e s i d u a l bodies which overcrowd the cytoplasm.  Other  iii f e a t u r e s a r e : the i r r e g u l a r i t y of the n u c l e a r boundary, the d i s o r g a n i z a t i o n o f the E.R. system through v e s i c u l a t i o n , the i n c r e a s i n g d i f f i c u l t y i n d e t e c t i n g m i t o c h o n d r i a , the development o f l a r g e s t a c k s o f c h l o r o p l a s t t h y l a k o i d s as w e l l as numerous  patches o f e l e c t r o n 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 o f conspicuous c e l l w a l l ingrowths. Assays o f enzyme systems o t h e r than a c i d phosphatase  show t h e i r d i s t r i b u t i o n t o be s i m i l a r t o  those r e p o r t e d f o r young c e l l s . In the f i n a l stages o f senescence  the c e l l s become  t y p i c a l l y n e c r o t i c ; no enzymes can be l o c a l i z e d , a c i d phosphatase compartmentalized  except  whose r e a c t i o n products are no l o n g e r but have become d i s t r i b u t e d a l l over the  c e l l c a v i t y . The nucleus and dictyosomes  have d i s i n t e g r a t e d .  Only s m a l l remnants o f the E.R. system remain.y M i t o c h o n d r i a and c h l o r o p l a s t s have l o s t t h e i r i n t e r n a l o r g a n i z a t i o n , no c y t o p l a s m i c m a t r i x can be d e t e c t e d . At the very end even the c e l l w a l l shows s i g n s of d i s o r g a n i z a t i o n . I t i s concluded t h a t these c e l l s r e p r e s e n t the f i n a l stage o f autolysis.  TABLE OF  CONTENTS  INTRODUCTION. . . . .  . . . . . . . .  MATERIAL AND METHODS..........  .... . . . .... .  .  OBSERVATIONS  6  .... . . . . . . . .  PART I -  LIGHT MICROSCOPE  11  OBSERVATIONS  PART I I - ELECTRON MICROSCOPE  11  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" C E L L S . . 30 E) THE SENESCENT CELLS - ULTRASTRUCTURAL FEATURES OF THE "CELL TYPE #3"  33  F) ENZYME LOCALIZATION G) ULTRASTRUCTURAL  35  IDENTIFICATION OF  AGING PIGMENT  37  DISCUSSION PART I  39 - 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 I V - TRANSITIONAL "2-3" CELLS  63  PART V  66  - THE "CELL TYPE #3"  PART V I - ENZYME LOCALIZATION DURING TIATION AND  DIFFEREN-  SENESCENCE  70  CONCLUSION..  76  LITERATURE CITED  78  KEY OF SYMBOLS AND PLATE EXPLANATION........ KEY OF SYMBOLS...... PLATE EXPLANATION. . . .  .. ..  ......107 .. . . . . .  108 109  1  V  LIST OF FIGURES FIGURE 1  L i g h t microscope o b s e r v a t i o n embedded f i l a m e n t  2 3  ( p r o s t r a t e system )  L i g h t microscope o b s e r v a t i o n embedded f i l a m e n t  o f an i n s i t u  ( e r e c t system )  L i g h t microscope o b s e r v a t i o n embedded f i l a m e n t  of an i n s i t u  o f an i n s i t u  ( p r o s t r a t e system )  4  L i g h t microscope micrograph of a c h l o r o p l a s t  5  L i g h t microscope micrograph of p o r t i o n o f a filament  ( l i v i n g material )  6  C h l o r o p l a s t morphology  ( l i v i n g material )  7  C h l o r o p l a s t morphology  ( l i v i n g material )  8  Pyrenoid  9  C h l o r o p l a s t and p y r e n o i d  division  ( l i v i n g material ) morphology  ( l i v i n g material ) 10  C e l l w a l l ingrowths  ( l i v i n g material )  11  C e l l w a l l ingrowths  ( l i v i n g material )  12  Cytochemical i d e n t i f i c a t i o n structures  13 14 15 16 17 18  containing  o f RNA  containing  o f RNA  containing  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n structures  of DNA  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n structures  containing  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n structures  of DNA  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n structures  containing  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n structures  of DNA  o f RNA  containing  ( 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 o f p r o t e i n s 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 o f i n s o l u b l e carbohydrates  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n o f i n s o l u b l e carbohydrates  ( l i g h t microscope micrograph )  Cytochemical i d e n t i f i c a t i o n o f 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 o f l i p i d s ( l i g h t microscope micrograph ) Schorml's r e a c t i o n f o r the i d e n t i f i c a t i o n o f lipofuscin  ( l i g h t microscope micrograph )  Toluidine blue 0 s t a i n i n g  ( l i g h t microscope  micrograph ) E l e c t r o n micrograph o f a young c e l l  ( prostrate  system ) Plasmalemmasom.es Nuclear pores Fibrillar  aspect o f chromatin  E l e c t r o n micrograph o f a young c e l l  ( erect  system ) E l e c t r o n micrograph o f a young c e l l  ( erect  .  system ) E l e c t r o n micrograph o f a young c e l l  ( prostrate  system ) Nuclear envelope- dictyosome Endoplasmic r e t i c u l u m  association  - dictyosome  association  F r e e z e - e t c h r e p l i c a of the n u c l e a r envelope F r e e z e - e t c h r e p l i c a of the- n u c l e a r envelope  vii FIGURE 37a 38  Plasmalemmasomes  ( freeze-etch r e p l i c a )  P o r t i o n of. the nucleus and p e r i n u c l e a r of a young  cell  39  Perinuclear  40  P e r i n u c l e a r space i n c l u s i o n s  41  P e r i n u c l e a r space i n c l u s i o n s  42  Dictyosome morphology  space i n c l u s i o n s  43a  Freeze-etch  43b  D e t a i l o f f i g u r e 43a  preparation  of a c h l o r o p l a s t  44  Endoplasmic r e t i c u l u m morphology  45  Section passing of a young  46  region  through the p e r i n u c l e a r  region  cell  Freeze-etch  r e p l i c a of a c h l o r o p l a s t  47a  Osmiophilic  s t r u c t u r e d bodies  ( origin )  47b  Osmiophilic  s t r u c t u r e d bodies  ( origin )  48  Chloroplast-mitochondrion  49  Tangential  association  s e c t i o n through the r e g i o n o f the  c h l o r o p l a s t E.R. 50  C h l o r o p l a s t E.R. i n c l u s i o n s  51  Osmiophilic  s t r u c t u r e d body  ( morphology )  52  Osmiophilic  s t r u c t u r e d body  ( morphology )  53  Osmiophilic  s t r u c t u r e d body-pyrenoid a s s o c i a t i o n  54  Discharge o f o s m i o p h i l i c s t r u c t u r e d bodies i n t o the paramural space  55  Nuclear envelope-dictyosome a s s o c i a t i o n  56  Cross s e c t i o n through the p e r i p h e r y cell  ( concentration  o f a young  of mitochondria )  57  Dictyosome morphology  ( cisternae interruptions )  . 58  Dictyosome morphology  ( cisternae interruptions )  59  Dictyosome morphology  ( b r i d g e - l i k e filaments )  60  Dlctyosbme-pyrenoid a s s o c i a t i o n  61  Dictyosome-mitochondria a s s o c i a t i o n  62  Dictyosome-vacuole a s s o c i a t i o n  viii FIGURE 63  Dictyosome-pyrenoid a s s o c i a t i o n  64  Dictyosome morphology  ( lomasomes )  65  Dictyosome morphology  ( lomasbme o r i g i n )  66  D e t a i l of a dictyosome d e r i v e d v e s i c l e  67  Mitochondrion morphology  68  Mitochondrion morphology  69  Nucleus-mitochondrion a s s o c i a t i o n  70  Chloroplast-mitochondrion etch p r e p a r a t i o n  1  ( cristae inclusions ) association  ( freeze-  )  71  Mitochondrion-pyrenoid  association  72  Mitochondrion-mitochondrion a s s o c i a t i o n  73  D e t a i l of the t h y l a k o i d  74  Thylakoid  75  D e t a i l of a c h l o r o p l a s t  76  D e t a i l of a c h l o r o p l a s t  77  D e t a i l of the t h y l a k o i d  78  P o r e - l i k e i n t e r r u p t i o n s i n the  79  Thylakoid  80  Freeze-etch preparation  81  F r e e z e - e t c h r e p l i c a of a p o r t i o n of a c h l o r o p l a s t  82  C h l o r o p l a s t morphology  architecture  system (freeze-etch  preparation)  system thylakoids  architecture of  thylakoids  ( pattern  of  thylakoid  arrangement ) 83  Dictyosome morphology  84  M i t o c h o n d r i a morphology  85  Whorled p a t t e r n  86  Concentric  87  A b s c i s s i o n .of p l a s t i d - d e r i v e d c o n c e n t r i c  88  Concentric  89-  A c i d phosphatase a c t i v i t y a s s o c i a t e d w i t h a concentric  ( cisternae interruptions ) ( genophore )  of t h y l a k o i d arrangement  p a t t e r n of t h y l a k o i d arrangement bodies  body body  90  A c i d phosphatase a c t i v i t y  91  D e t a i l of a c o n c e n t r i c  92  D e t a i l of a c h l o r o p l a s t  ( control preparation  body ( b r i d g e - l i k e elements )  )  ix FIGURE 93  D e t a i l of a c h l o r o p l a s t and  94  C h l o r o p l a s t morphology  95  Freeze-etch  pyrenoid  ( ribosomes )  r e p l i c a of a c h l o r o p l a s t ( g e n e r a l  morphology ) 96  T h y l a k o i d a s s o c i a t e d f i b r i l l a r elements  97  Plastoglobulus  ( f i b r i l l a r composition  )  98  Plastoglobulus  ( f i b r i l l a r composition  )  99  P l a s t o g l o b u l i ( f i b r i l l a r composition  )  100  Endoplasmic r e t i c u l u m - v a c u o l e  association  10.1  Chloroplast division  ( Laminaria  102  Chloroplast division  ( S p h a c e l a r i a type )  103  Chloroplast d i v i s i o n  ( S p h a c e l a r i a type )  104  Proplastid-like  105  Chloroplast division  ( p o t e n t i a l aspect  106  Chloroplast division  ( p o t e n t i a l aspect )  107  Multiconstricted chloroplast  10 8  C h l o r o p l a s t and p y r e n o i d  109  Pyrenoid  division  110  Pyrenoid  morphology  111  Plasmodesmata  112  Pyrenoid-vacuole  113  C e l l w a l l morphology  114  Pyrenoid  115  Cytoplasm morphology  116  Vacuole development  117  C e l l wall  type ).  formation  morphology  association ( young c e l l  )  ( young c e l l  )  division  ( f r e e z e - e t c h p r e p a r a t i o n of a  young c e l l ) 118  Provacuoles  ( freeze-etch preparation )  119  Autophagocytosis  ( r o l e of the E.R.  )  120  Autophagocytosis  ( r o l e of the E.R.  )  12.1  Autophagocytosis  ( r o l e , of the E.R.  )  1.22  Vacuole development  123  Vacuole  development  )  X  FIGURE 1.24  Autophagocytosis  ( r o l e of the  tonoplast  )  125  A c i d phosphatase  (vacuole )  1.26  A c i d phosphatase  ( vacuole )  127  Vacuole development  12 8  Morphology of a aging  129  A c i d phosphatase  ( dictyosome )  130  A c i d phosphatase  ( endoplasmic r e t i c u l u m  )  131  A c i d phosphatase  ( control preparation )  132  T r a n s i t i o n a l " 1-2  cell  " c e l l s ; d e t a i l of a  chloro-  plast 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  135  T r a n s i t i o n a l " 1-2. " c e l l ;  136  T r a n s i t i o n a l " 1-2  " cell;  g e n e r a l morphology  137  T r a n s i t i o n a l " 1-2  " cell;  cytoplasmic  " cell;  a c i d phosphatase  " cell;  a c i d phosphatase  " cell;  a c i d phosphatase  " ; permanganate f i x a t i o n g e n e r a l morphology  inclusions 138  T r a n s i t i o n a l " 1-2 activity  139  T r a n s i t i o n a l " 1-2 activity  140  T r a n s i t i o n a l " 1-2 activity  141  (  control preparation )  T r a n s i t i o n a l " 1-2 dictyosome  T r a n s i t i o n a l " 1-2  143  " C e l l type #2  system  " C e l l type #2  145  T r a n s i t i o n a l . " 2-3  146  Transition  147  the  " cell;  c e l l w a l l morphology  "; g e n e r a l u l t r a s t r u c t u r e of a  144  a " cell  d e t a i l of  region  142  prostrate  " cell;  cell  "; c e l l w a l l morphology "cell;  general, morphology  zone.between a " c e l l . t y p e #3 type J.2 "  T r a n s i t i o n a l " 2-3  " cell;  cytoplasm  "  and  xi FIGURE 148  " C e l l type #2 "; g e n e r a l u l t r a s t r u c t u r e of an erect  system  cell  149  "Cell  type #2 "; d e t a i l  150  " C e l l type #2 "; d e t a i l  151  "Cell  type  prostrate 152 153  11  #2  of a  chloroplast  of the cytoplasm  "; g e n e r a l u l t r a s t r u c t u r e of a  system  cell  C e l l type #.2 "; c e l l w a l l morphology  " C e l l type #2  "; nucleus and  chloroplast  morphology 154  " C e l l type #2  "; c h l o r o p l a s t  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 o f a  chloroplast  158  " C e l l type #2  "; c e l l w a l l morphology  159  " C e l l type #2  "; d e t a i l  of a  morphology  chloroplast  ( freeze-etch preparation ) 160  " C e l l type #2  "; g e n e r a l morphology  161  " C e l l type #2  "; c e l l w a l l  ingrowth  162  " C e l l type #2  "; c e l l w a l l  ingrowths  163  T r a n s i t i o n a l " 1-2  " cell;  c e l l wall  164  T r a n s i t i o n a l " 2-3  " cell;  cytoplasm morphology  165  T r a n s i t i o n a l " 2-3  " cell;  d i s r u p t i o n of the  ingrowth  tonoplast 166  T r a n s i t i o n a l " 2-3  " cell;  chloroplast  167  T r a n s i t i o n a l " 2-3  " cell;  chloroplast  168  T r a n s i t i o n a l " .2-3 " c e l l ; p y r e n o i d  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  " cell;  nucleus  171  T r a n s i t i o n a l " 1-2  " cell;  c e l l wall, ingrowth  172  T r a n s i t i o n a l " 2-3  " cell;  chloroplast  173  T r a n s i t i o n a l " 2-3  " cell;  mitochondrion  174  T r a n s i t i o n a l " .2-3 " c e l l ;  mitochondrion  175  Transitional  mitochondrion  2-3  " cell;  xii FIGURE 176  T r a n s i t i o n a l " 2-3  177  T r a n s i t i o n a l " .2-3 " c e l l ;  178  T r a n s i t i o n a l " 2-3 plast  179  " cell; " cell;  concentric  body  chloroplast d e t a i l of a  chloro-  .. . .  T r a n s i t i o n a l " 2-3  " cell;  d e t a i l of a chloroplast  chloroplast 180  T r a n s i t i o n a l " 2-3  " cell;  181  T r a n s i t i o n a l " 2-3  " cell;  d e t a i l of the c y t o -  " cell;  d e t a i l of a  " cell;  pyrenoids  plasm 182  T r a n s i t i o n a l " 2-3 chloroplast  183  T r a n s i t i o n a l " 2-3  184  " C e l l type #3";  185  T r a n s i t i o n a l " 2-3  " cell;  chloroplast  186  T r a n s i t i o n a l " 2-3  " cell;  chloroplast  187  " C e l l type #3 prostrate  188 189  "; g e n e r a l u l t r a s t r u c t u r e of a  system  " C e l l type #3 prostrate  c e l l w a l l morphology  "; g e n e r a l u l t r a s t r u c t u r e of a  system  " C e l l type #3 e r e c t system  cell cell  "; g e n e r a l u l t r a s t r u c t u r e of an  cell  190  " C e l l type #3  ";  chloroplast  191  " C e l l type #3  "; a c i d phosphatase  192  " C e l l type #3  "; d e t a i l of  thylakoid  activity  arrangement 193  " C e l l type #3  "; t h y l a k o i d  disorganization  194  " C e l l type #3  "; t h y l a k o i d  disorganization  195  " C e l l type #3  "; t h y l a k o i d  disorganization  196  " C e l l type #3  "; m i t o c h o n d r i a and  cytoplasm  morphology 197  " C e l l type #3  198  " C e l l type #3  199  " C e l l type #3 "; p l a s t o g l o b u l i  11  ; m i t o c h o n d r i a morphology  "; t h y l a k o i d  disorganization accumulation  xiii FIGURE 200  " C e l l type #3  "; c h l o r o p l a s t morphology  201  " C e l l type #3  "; d e t a i l of the  .202  " C e l l type #3 "; mitochondrion  203  " C e l l type #3  cytoplasm  "; a c i d phosphatase  ( control preparation  activity  )  .204  " C e l l type #3  "; a c i d phosphatase  activity  205  " C e l l type #3 "; a c i d phosphatase  activity  ( control^preparation type #3  )  206  "Cell  "; a c i d phosphatase  activity  207  " C e l l type #3  "; c e l l w a l l d i s o r g a n i z a t i o n  208  " C e l l type #3  "; c e l l w a l l d i s o r g a n i z a t i o n  209  " C e l l type #1 "; c a t a l a s e a c t i v i t y i n a microbody-like organelle  210  " C e l l type #1  "; c a t a l a s e a c t i v i t y i n  a microbody-like  organelle  211  " C e l l type #1  "; c a t a l a s e  ( control preparation)  212  " C e l l type #1  "; c a t a l a s e  ( mitochondrial  d e p o s i t i o n of r e a c t i o n product ) 213  " C e l l type #2  "; c a t a l a s e  214  " C e l l type #1  "; peroxidase a c t i v i t y i n the  cell  activity  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  cell  wall  217  " C e l l type #3  "; a c i d phosphatase  218  " C e l l type #1  "; peroxidase a c t i v i t y  preparation 219  activity ( control  )  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 a s s o c i a t i o n w i t h the mitochondrion .220  " C e l l type. #3  221  " C e l l type #2  222  " C e l l type #2 "; p e r o x i d a s e a c t i v i t y preparation  )  a c i d phosphatase "; p e r o x i d a s e  activity  activity ( control  xiv  C e l l type #1 "; A T P a s e . a c t i v i t y  ( Na -K -Mg +  +  ++  a c t i v a t e d system ) " C e l l type #1 C e l l type #1 "; ATPase a c t i v i t y a c t i v a t e d system )  + + ++ ( Na -K -Mg -  ".' C e l l type #1 "; C e l l type #1 "; ATPase a c t i v i t y a c t i v a t e d system )  + + ++ ( Na -K -Mg -  " CC ee ll ll type type #1 "; ATPase a c t i v i t y  ( Mg ++  acti-  vated system ) " C e l l type #J C e l l type #1 "; ATPase a c t i v i t y vated system ) ATPase a c t i v i t y  ++ ( Mg  - acti-  ( Na -K -Mg - activated +  +  ++  system ) ; control, p r e p a r a t i o n 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 - a c t i v a t e d system ) ATPase a c t i v i t y  ( Na -K -Mg - activated +  +  ++  system ); c o n t r o l p r e p a r a t i o n T r a n s i t i o n a l " 1-2 " c e l l ;  lipofuscin  ( Fontana's r e a c t i o n ) ATPase a c t i v i t y  ( M g - a c t i v a t e d system ); ++  control preparation " C e l l type #2 "; l i p o f u s c i n  ( Fontana s  reaction ) Lipofuscin  ( control preparation )  Lipofuscin  ( Schorml's r e a c t i o n )  1  XV  ACKNOWLEDGEMENTS The  author wishes t o express h i s g r a t i t u d e t o Dr. T.  B i s a l p u t r a f o r h i s guidance and encouragement. Thanks are a l s o extended t o the other committee members: Drs. R.F. S c a g e l , R.E. Foreman, J . Berger, and I.E.P. T a y l o r , f o r t h e i r h e l p f u l suggestions expresses  and. c r i t i c i s m . The.author a l s o  h i s g r a t i t u d e t o P r o f . Drs. A. R o z e i r a and R.  Salema (Porto U n i v e r s i t y ) without  whose i n t e r e s t my s t a y i n g  i n Canada would have been i m p o s s i b l e . I am t h a n k f u l t o Dr. D.L. Brown f o r the r e a d i n g and c r i t i c i s m o f the f i r s t d r a f t o f the t h e s i s , and t o my f r i e n d Miss S. Shinn f o r a l l her h e l p . S p e c i a l t h a n k s :  are due t o the Calouste  k i a n Foundation f o r a 3 year study  Gulben-  grant.  Thanks are due t o Dr. G. Hughes f o r h i s h e l p the p e r i o d of s a b a t i c a l leave o f Dr. T. B i s a l p u t r a .  during  1 INTRODUCTION I. Morphology of Young V e g e t a t i v e C e l l s The u l t r a s t r u c t u r e o f brown a l g a l c e l l s has been the s u b j e c t of s e v e r a l s t u d i e s  ( 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;  B i s a l p u t r a , 1 9 6 6 ; Evans,1966; Manton,  1966b; B i s a l p u t r a and B i s a l p u t r a , 1 9 6 7 ; Greenwood,19 67; Loiseaux,1967; Bourne and Cole,1968; Cole and Lin,1968; Cole e t al.,1968; Evans,1968; McCully,1968; Neushul and Liddle,1968; B a i l e y and B i s a l p u t r a , 1 9 69; B i s a l p u t r a and B i s a l p u t r a , 1 9 6 9 ; B i s a l p u t r a and Burton,1969;  Bouck,1969;  Cole,19 69; L i d d l e and Neushul,19 69; B i s a l p u t r a and B i s a l p u t r a , 1 9 7 0 ; Cole,1970; Cole and Lin,1970; B i s a l p u t r a e t al.,1971; Chi,1971; Hori,1971; Neushul,1971; Neushul and Walker,1971; Hori,1972; Feldman and Guglielmi,1972; Neushul and Dahl,1972a, 1972b; Davies e t al.,1973 ). From these s t u d i e s the b a s i c u l t r a s t r u c t u r a l f e a t u r e s of brown a l g a l c e l l s were e s t a b l i s h e d , i t a l s o became apparent t h a t 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 G o l g i b o d i e s , the presence or absence o f p y r e n o i d s , t h e pyrenoid' morphology, and the arrangement of plasmodesmata c o u l d serve as u s e f u l c r i t e r i a i n e s t a b l i s h i n g among major groups of t h i s c l a s s o f algae  relationships  ( Evans,1966,  1968  Cole and Lin,1968; Cole,1970; Scagel,1966 ). C h l o r o p l a s t s and pyrenoids have been more i n t e n s i v e l y s t u d i e d than o t h e r o r g a n e l l e s . The nucleus  ( Neushul and Dahl,1972a; L e e d a l e ,  1970; Neushul and Walker,1971  ) and plasmalemmasomes  ( Cole and Lin,1970 ) a l s o have a t t r a c t e d the a t t e n t i o n o f p h y c o l o g i s t s . However,, only few r e p r e s e n t a t i v e s o f the Phaeophyta have been s t u d i e d 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 p l a n t s w i t h the  forms among the brown algae  ( Fritsch,1945;  simplest  Smith,1955 );  hence, they h o l d an important p o s i t i o n i n brown a l g a l phylogenetic  schemes. Previous  Ectocarpales  has  r e s e a r c h on members of  i n c l u d e d s t u d i e s on  : Ectocarpus m i t c h e l l a e  ( Longest,1946 ); P y l a i e l l a l i t t o r a l i s G i f f o r d i a . sp.  ( Gibbs,1962a, 1962b)  ( Bouck.,1965 ); Ectocarpus conf e r v o i d e s  PylaieTla l i t t o r a l i s Myrionemaceae  the  ( Evans,1966 ) r e p r e s e n t a t i v e s of  ( Loiseaux,1967 ); Ectocarpus acutus  and Bisalputra,1969  and  ); Eudesme v i r e s c e n s  genus Eudesme i s c o n s i d e r e d  the  ( Bailey  ( Cole,1969- the  by some i n v e s t i g a t o r s to  belong to the order C h o r d a r i a l e s , see Scagel,1966, f o r 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 c r o u a n i i  ( Magne,19 71 ); G i f f o r d i a mitchellae,Hapterophycus Pylaiella littoralis  sp.,  ( Hori,1972 ).  Studies on the genus "Ectocarpus were concerned with a n a l y s i s of a r e s t r i c t e d number of f e a t u r e s . Longest s t u d i e d the e x t e r n a l f e a t u r e s of the f l a g e l l a of zooids. Evans pyrenoid  (1966) d e s c r i b e d  i n zoospores and Hori  vegetative  the  the c h a r a c t e r i s t i c s of  the  emphasized the absence of t h y l a -  koids or t h y l a k o i d - d e r i v e d s t r u c t u r e s from the matrix.  (1946)  (1971) made s i m i l a r o b s e r v a t i o n s  pyrenoid using  c e l l s . B a i l e y and B i s a l p u t r a (1969) used both  f r e e z e - e t c h i n g and u l t r a t h i n s e c t i o n i n g techniques study the c e l l w a l l morphology i n Ectocarpus acutus concluded t h a t the w a l l possesses a double l a y e r e d z a t i o n . Cytochemical techniques  at the  to and organi-  ultrastructural  l e v e l r e v e a l e d the presence of p o l y s a c c h a r i d e - l i k e substances i n a s s o c i a t i o n w i t h globuli  ( Magne,1971 ). The  provide  l i p i d materials i n plasto-  aim of the f i r s t p a r t of t h i s t h e s i s i s to  a more d e t a i l e d d e s c r i p t i o n of the u l t r a s t r u c t u r e  3 of Ectocarpus and t o compare i t s s a l i e n t f e a t u r e s  with  those o f other brown algae. I I . C e l l u l a r Senescence In r e c e n t y e a r s , the b i o l o g y o f the brown algae has  r e c e i v e d i n c r e a s i n g a t t e n t i o n . Information  mechanism o f c e l l w a l l r e g e n e r a t i o n 1971  ), o f f e r t i l i z a t i o n  formation  on the  ( F u l c h e r and McCully,  ( Pollock,1970 ), r h i z o i d  ( Quatrano,1972 ), and c e l l u l a r  ( Neushul and Dahl,19 72b  polarity  ) have become a v a i l a b l e . Never-  t h l e s s , so f a r 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 o f brown a l g a e . Senescence has been d e f i n e d i n many d i f f e r e n t ways. Strehler  (1962) d e f i n e d i t as changes which r e s u l t i n a-  decreased s u r v i v a l c a p a c i t y on the p a r t o f the i n d i v i d u a l organism. Leopold processes  (1964) i n t e r p r e t e d i t as the degenerative  which terminated  or an organism. Osborne  the f u n c t i o n a l l i f e o f an organ  (1967) d e f i n e d senescence as a  g e n e r a l and i n c r e a s i n g f a i l u r e o f many s y n t h e t i c r e a c t i o n s which are the normal forerunners  o f c e l l death. Woolhouse  (1967) thought o f senescence as a c o n t i n u a t i o n o f c e l l differentiation  ; while  Rockstein  (1967) t h a t 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 s t r u c t u r e and f u n c t i o n i n an organism which r e s u l t i n the decreasing  c a p a c i t y o f t h a t organism t o s u r v i v e and thus  would r e s u l t u l t i m a t e l y i n i t s death. McLean (1968) r e f e r r e d t o senescence as those events which occur between maturity  and death. Whatever the p o s i t i o n taken, there i s  g e n e r a l agreement t h a t senescence u l t i m a t e l y leads t o death of c e l l s o r organisms. Aging and senescence have been used as synonymous terms  ( Thung and Hollander,1967 ) . Rockstein  (1967),  however, t h i n k s o f senescence as a more r e s t r i c t e d p a r t  4 of  a broader phenomenon c a l l e d aging.  the author, i n c l u d e s : 2) the middle  life  T h i s , according to  1) the i n i t i a l p e r i o d of development,  stage of growth and maturation, and  the p e r i o d of senescence. the term senescence  C a r r and Pate  (1967) a l s o  3)  restrict  t o those changes which are c l e a r l y  d e g e n e r a t i v e , w h i l e they t h i n k of aging as the sum changes i n the whole p l a n t .  of t o t a l  In t h i s work senescence  and  aging w i l l be c o n s i d e r e d i n t e r c h a n g e a b l e terms. Senescence t h e o r i e s and mechanisms are as abundant as are d e f i n i t i o n s . recently  The  (Hahn, 1971;  There i s now  s u b j e c t , however, was  reviewed  Medvedev, 1972).  a c o n s i d e r a b l e amount of i n f o r m a t i o n  d e a l i n g w i t h b i o c h e m i c a l and p h y s i o l o g i c a l aspects of senescence 1967;  (e.g. Varner,  1961;  Wareing and P h i l l i p s ,  Wangerman, 1965;  1970;  Sax,  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 d e a l i n g w i t h h i g h e r p l a n t s was and Simon (1971). on the a l g a e .  1962), but a t the  sparse.  Information  r e c e n t l y reviewed by B u t l e r  In comparison,  there i s l i t t l e i n f o r m a t i o n  The aging phenomena of u n i c e l l u l a r algae  been s t u d i e d i n S p o n g i o c h l o r i s t y p i c a Ochromonas (Schuster e t a l . , (Walne e t a l . ,  Woolhouse,  1970;  has  (McLean, 1968),  1968), and Euglena. .granulata  and P a l i s a n o and Walne, 1972).  Fabbri  and P a l a n d r i (1969) and P a l a n d r i (1972) s t u d i e d aging i n the c o e n o c y t i c f i l a m e n t s of Halimeda tuna. To c o n t r i b u t e t o 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  cells  d u r i n g the o v e r a l l process of aging were s t u d i e d . The proved  s e l e c t i o n of Ectocarpus f o r such a study  t o have c e r t a i n advantages.  The  simple  filamentous  form w i t h prominent a p i c a l growth i s p a r t i c u l a r l y in  has suitable  t h a t i t i s p o s s i b l e t o f o l l o w a l l stages o f c e l l u l a r  d i f f e r e n t i a t i o n and d i s o r g a n i z a t i o n i n a s e q u e n t i a l o r d e r . The i n f o r m a t i o n obtained i n t h i s work w i l l p r o v i d e background f o r f u t u r e experimental work on the  factors  5 governing senescence. Among those f a c t o r s , t h e problem o f c o n t r o l l i n g senescence u s i n g growth r e g u l a t o r s and r e l a t e d substances c o u l d be q u i t e i n t e r e s t i n g t o c o n s i d e r .  Growth  r e g u l a t o r s were shown t o i n f l u e n c e senescence i n h i g h e r plants 1972  ( e.g. B u t l e r and Simon,1971; Udvardy and Farkas,  ). Recent r e p o r t s have d i s c l o s e d the e x i s t e n c e o f  substances w i t h s i m i l a r p r o p e r t i e s i n the brown  algae  ( e.g. Jennings,1968; Buggeln and Craigie,1971;  A u g i e r and  Harada,1972; Hussain and Boney,1973 ) . T h e r e f o r e ,  the study  of t h e i r e f f e c t s on the brown algae might p r o v i d e  interes-  t i n g information  on the aging mechanism i n g e n e r a l .  T h i s work a l s o attempts t o e s t a b l i s h s i m i l a r i t i e s and  d i f f e r e n c e s t h a t 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 a l g a , a p r o t i s t , a h i g h e r p l a n t o r an animal. These attempts w i l l  eventually  help t o judge a s s e r t i o n s l i k e t h a t o f P a l i s a n o  and Walne  (1972), who  common  defended the e x i s t e n c e  of a basic  mechanism o f senescence from the lower t o the h i g h e r of  life.  forms  6  MATERIALS. AND METHODS Ectocarpus sp. was obtained from the C u l t u r e C o l l e c t i o n of Algae, Indiana U n i v e r s i t y Cultures (Chihara,  ( c u l t u r e LB  1433).  were maintained i n Chihara's marine medium  1968) i n a growth chamber, a t 14°C and i l l u m i n a t e d  by f l u o r e s c e n t lamps f o r 16 hours per day, a t approximately 400 f t . c .  The a l g a was grown i n "Nalgene"  p e t r i d i s h e s (#5500).  This f a c i l i t a t e d  embedding o f the m a t e r i a l  polypropylene  f i x a t i o n and  ( B i s a l p u t r a e t a l . , 1971).  To  ensure optimum growth the medium was changed every 5 days. Stock c u l t u r e s were handled i n a s i m i l a r way except they were kept i n l a r g e pyrex c u l t u r e j a r s , and the c u l t u r e medium changed every 3 weeks.  F o r dark t r e a t e d  c u l t u r e s were grown f o r 3 months under normal conditions,  material  culture  then t r a n s f e r r e d t o t o t a l darkness f o r 1 t o 5  days and 35 days. L i g h t microscopy.  L i v i n g f i l a m e n t s were s t u d i e d  d i r e c t l y on Nalgene p e t r i d i s h e s o r i n d i v i d u a l p l a n t s were placed  i n a drop of c u l t u r e medium on a microscope  slide.  F i x e d m a t e r i a l was observed a f t e r treatment i n one of the f o l l o w i n g procedures: i n cacodylate buffer  a) 4% (v/v s o l u t i o n of g l u t a r a l d e h y d e (0.1 M Sodium Cacodylate, pH 1.0),  to which 0.25 M o f sucrose was added; b) 10% (v/v a c r o l e i n i n 0.025 M phosphate  buffer  (pH 6.8, 24 hours a t 0 ° C ) .  was p o s t - f i x e d i n 1% H g C l  2  The m a t e r i a l  (24 hours, a t 0°C; McCully,  1966); c) 4% (v/v phosphate pH 7.0)  buffered  (0.05 M  paraformaldahyde.  Except when s p e c i f i e d , a l l f i x a t i o n s , were c a r r i e d out a t room temperature,  from 3 t o 28 hours.  Fixations  f o r dark t r e a t e d m a t e r i a l were c a r r i e d out i n t o t a l  darkness.  The f i x e d m a t e r i a l was embedded i n S p u r r s medium, which 1  i s s i m i l a r to that described  f o r e l e c t r o n microscopy and  7 s e c t i o n e d a t a t h i c k n e s s of approximately  1 micron.  S e c t i o n i n g was r o u t i n e l y done u s i n g an ultramicrotome equiped w i t h g l a s s k n i v e s . Ribbons of 5-6 s e c t i o n s were t r a n s f e r r e d with  a platinum  loop t o g l a s s s l i d e s and d r i e d  at 60 C on a e l e c t r i c hot p l a t e . S l i d e s b e a r i n g  sections  were e i t h e r d i r e c t l y p l a c e d i n the s t a i n i n g s o l u t i o n s o r d e p l a s t i c i z e d p r i o r t o s t a i n i n g ( Rosenquist e t al.,1971 ). The  s t a i n i n g procedures were as summarized i n the f o l l o w i n g  page. A f t e r s t a i n i n g the s e c t i o n s were washed, a i r d r i e d , and mounted i n one drop of "Permount" mounting medium. Observations were made with  a Z e i s s photomicroscope  u s i n g phase c o n t r a s t and b r i g h t f i e l d i l l u m i n a t i o n s . Photographs were taken u s i n g I l f o r d FP4 f i l m . E l e c t r o n microscopy. The m a t e r i a l was f i x e d and processed  according  t o one of the f o l l o w i n g methods:  1) glutaraldahyde-osmium f i x a t i o n . The m a t e r i a l was for 2  fixed  3 hours a t room temperature, i n a f i x a t i v e s o l u t i o n  c o n t a i n i n g 4% (v/v) g l u t a r a l d a h y d e 0.1M sodium c a c o d y l a t e  buffer  and 0.25M sucrose, i n  ( pH 7.0 ). The samples were  t r a n s f e r r e d through a s e r i e s of b u f f e r s o l u t i o n s c o n t a i n i n g decreasing  concentrations  was completely  of sucrose,  until finally  sucrose  e l i m i n a t e d from the r i n s i n g s o l u t i o n s . The  m a t e r i a l was p o s t - f i x e d f o r a f u r t h e r 2h hours i n a 1.5% (v/v) Os04, s i m i l a r l y b u f f e r e d , but without sucrose.  I t was  r i n s e d i n the b u f f e r s o l u t i o n and dehydrated i n graded a l c h o o l 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 v e h i c l e  according  to the procedure d e s c r i b e d by B i s a l p u t r a e t a l . (1971). T h i s method was found t o g i v e the best r e s u l t s i n p r e s e r v i n g the c e l l u l a r a r c h i t e c t u r e of young c e l l s and c e l l s i n advanced stages  of senescence. S l i g h t m o d i f i c a t i o n s t o the.above procedure were  i n t r o d u c e d by c a r r y i n g the f i x a t i o n o v e r n i g h t ,  using a  SUMMARY  REACTION  STAINS A. P r o t e i n s  OF LIGHT MICROSCOPE STAIN METHODS  Aniline  blue-  black  OTHER DETAILS  b r i g h t blue protein  colour at  REFERENCES Fisher,1968  sites  Periodic acid-  red colour a t reaction .  DNPH b l o c k a g e o f  F e d e r and  carbohy-  Schiff  sites  aldehydes  O'Brien,1968  drates  reaction Jensen,1962  B. I n s o l u b l e  C. N u c l e i c acids  (P.A.S.)  Methyl green-  DNA-containing s t r u c -  nucleic  pyronin  tures s t a i n  extractions  blue-green  RNA-containing s t r u c tures  stain red  acids  done by t h e Perchloric acid method  D. L i p i d s  Sudan B l a c k B  black  blue  reaction  A p a r i c i o and  colour at  sites  Mardsen,1969 procedure c a r r i e d  Gomori,1952  out i n f o r m a l d e h y -  E. P o l y p h e nols  Toluidine 0  '  blue  de f i x e d  material  green t o turquoise  material  fixed  colour  according to  at reaction  McCully  sites F. Age Pigment  Schorml r e a c t i o n deep b l u e  colour  at reaction  sites  McCully,1966  (1966) Hendy,1971  8 5%  (v/v) s o l u t i o n of g l u t a r a l d e h y d e . . The  decrease - in. sucrose  c o n c e n t r a t i o n s was  done d u r i n g the p o s t - o s m i c a t i o n  steps.  T h i s v a r i a t i o n was  found to g i v e b e s t r e s u l t s i n h a n d l i n g  the " c e l l type #2"  p r e s e r v a t i o n , which has proved to be  v e r y d i f f i c u l t to p r e s e r v e . 2) Permanganate f i x a t i o n .  T h i s was  c a r r i e d out u s i n g 1% to  5% s o l u t i o n s of KMnO^ e i t h e r i n d i s t i l l e d water or i n sodium c a c o d y l a t e b u f f e r (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 u n i f o r m l y p o o r , - e s p e c i a l l y with r e s p e c t to the o l d e r cells.  These data agree with those of Barton  found permanganate f i x a t i o n inadequate  (19 66) who  also  f o r s t u d i e s of  senescence. 3) Enzyme l o c a l i z a t i o n a t the u l t r a s t r u c t u r a l l e v e l .  The  m a t e r i a l used i n these experiments was  (20  b r i e f l y fixed  minutes, a t room temperature) i n a 4% s o l u t i o n of p u r i f i e d glutaraldehyde i n 0.1M  ( E l e c t r o n Microscope S c i e n c e s , Washington)  c a c o d y l a t e b u f f e r (pH 7.0),  to which sucrose  added to a f i n a l c o n c e n t r a t i o n of 7.5% r i n s i n g , the m a t e r i a l was i n c u b a t i o n medium. and  (w/v).  incubated i n the  The m a t e r i a l was  was  After brief  appropriate  then q u i c k l y r i n s e d  f i x e d f o r a f u r t h e r 2 hours i n g l u t a r a l d e h y d e .  Subsequent p r o c e s s i n g was The  composition  of the i n c u b a t i o n media were:  a) A c i d phosphatase. n i t r a t e , 3 mM; 220 mM  as d e s c r i b e d before under 1.  T r i s - m a l e a t e , 60 mM  Beta-glycerophosphate,  (Gomori, 1952).  (pH 5.0);  11.5  The m a t e r i a l was  mM;  lead  sucrose,  then incubated f o r  p e r i o d s of time ranging from 30 to 60 minutes, at a temperature of 37°C.  C o n t r o l m a t e r i a l was  incubated  either  i n the standard i n c u b a t i o n medium w i t h 10 mM  of sodium  f l u o r i d e or from which Beta-glycerophosphate  was  b) C a t a l a s e .  10 mg  diaminobenzidine  p r o p a n e d i o l b u f f e r ; 1 ml of 3% the i n c u b a t i o n medium was  9.0  removed.  (DAB); 5 ml of 0.05M  (v/v) H 0 . 2  2  The  f i n a l pH  of  ( F r e d e r i c k and Newcomb, 1969).  9 The  i n c u b a t i o n was  c a r r i e d out a t 37 C , f o r 60 minutes.  C o n t r o l m a t e r i a l was presence of 0.01M  incubated  KCN.  without I^C^,  In another t e s t the m a t e r i a l  b o i l e d f o r 5 minutes i n p r o p a n e d i o l i n c u b a t i o n i n standard c) Peroxidase.  or i n the  b u f f e r p r i o r to  medium.  Media f o r i n c u b a t i o n and  c o n t r o l s were as  i n d i c a t e d f o r c a t a l a s e , but the f i n a l pH was 7.6.  The  was  i n c u b a t i o n was  adjusted  to  c a r r i e d out a t room temperature f o r  60 minutes. d) Adenosine t r i p h o s p h a t a s e  ( ATPase ).Two  standard  were employed to study ATPase l o c a l i z a t i o n Henrikson,19 71 ). Medium A . T r i s - m a l e a t e , 10 mM The  MgS0 ; 3 mM 4  ATP;  i n c u b a t i o n was  m a t e r i a l was  3 mM  80 mM  l e a d n i t r a t e ; 220  i n standard  ( pH  7.2  3 mM  l e a d n i t r a t e ; 220  Mg  SC> ; 5 mM 4  mM  ATP;  sucrose.  medium without ATP  i n standard  mM  100 The  7.2  mM  );  sucrose. Control ATP.  b u f f e r , 80  mM  NaCl; 30 mM  i n c u b a t i o n was  f o r 1 hour, at 37 C. C o n t r o l m a t e r i a l was standard  ( pH  nedium without  Medium B. T r i s - m a l e a t e ); 10 mM  (adapted from  c a r r i e d out f o r 1 hour at 37 C.  incubated  media  incubated  KC1; done  in  or b o i l e d p r i o r to p r e - f i x a t i o n  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 s t u d i e s were done a c c o r d i n g by Hendy  to the m o d i f i c a t i o n s  (1971) f o r 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  l i p o f u s c i n - l i k e m a t e r i a l s . The 4%  (v/v)  ( pH  7.0  introduced  m a t e r i a l was  of  fixed i n a  s o l u t i o n of formaldahyde, i n 0.05M phosphate b u f f e r ), f o r 24 hours, 1 week or 2 weeks p e r i o d s . A f t e r  i n c u b a t i o n i n e i t h e r the Fontana's  ( 25 ml of a 10%  aqueous s i l v e r n i t r a t e s o l u t i o n , ammonia ( s.g.=  v/v  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 c h l o r i d e , f r e s h l y prepared 1% potassium f e r r i c y a n i d e , 1:1, was  v/v  ) s o l u t i o n s , some of the  p o s t - f i x e d i n b u f f e r e d 1.5%  (y/v) O s 0 .  procedures were as d e s c r i b e d by Hendy  4  material  Incubation  (1971) and  10 subsequent h a n d l i n g of the m a t e r i a l f o r e l e c t r o n microscopy was  as d e s c r i b e d b e f o r e . M a t e r i a l f i x e d a c c o r d i n g  above procedure, but incubated the Schorml's s o l u t i o n s  was  to  the  i n n e i t h e r the Fontana's  nor  used as c o n t r o l 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  labelled inclusions. 5) F r e e z e - e t c h . a 4%  (v/v)  buffer  The m a t e r i a l was  p r e - f i x e d f o r 1 hour i n  s o l u t i o n of g l u t a r a l d a h y d e ,  ( pH  7.0  ), then t r a n s f e r r e d to 25 %  i n d i s t i l l e d water f o r p e r i o d s 24 hours. The  i n 0.1M  m a t e r i a l was  f r a c t u r e d at -100  u s i n g a B a l z e r s BA  C and  (v/v) g l y c e r o l  of time ranging  etched  e l e c t r o n microscope. The  frozen material  f o r 30 to 60 seconds  were viewed w i t h  a Z e i s s EM  u l t r a t h i n s e c t i o n i n g was  e i t h e r a duPont diamond k n i f e or g l a s s knives stained  22,  360M d e v i c e .  A l l preparations  ultratome  from 2 to  then quenched i n Freon  p r i o r to f r e e z i n g i n l i q u i d n i t r o g e n . The was  cacodylate  9A  done u s i n g  on a L.K.B.  I or a R e i c h e r t OMU3 ultramicrotome. Both ( Reynolds,19 63 ) and  i n these s t u d i e s .  unstained  s e c t i o n s were used  11 OBSERVATIONS PART I — L I G H T MICROSCOPIC OBSERVATIONS S t u d i e s of l i v i n g f i l a m e n t s r e v e a l e d f e a t u r e s which were u s e f u l i n i n t e r p r e t i n g , the e l e c t r o n microscope r e s u l t s . The  f i l a m e n t s were made up of a v a r i a b l e number of  cells.  The  c e l l s , r e c t a n g u l a r 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 e r e c t system -3 -3 and 2.0 x 10 X 1.7 x 10 cm i n the case of the p r o s t r a t e system.  Other o b s e r v a t i o n s  can be summarized as f o l l o w s :  1) c h l o r o p l a s t s were found to be polymorphic and  ( f i g u r e s 4,  6,  7); 2) d i f f e r e n t stages of p y r e n o i d d i v i s i o n were  observed  ( f i g u r e 8); t h i s seems to occur by the  of the p y r e n o i d body; and  fission  3) conspicuous w a l l ingrowths were  apparent, both along the s i d e w a l l s as w e l l as at the walls  ( f i g u r e s 10 and Observations  cross  11).  of e r e c t and p r o s t r a t e f i l a m e n t s of the  s p o r o p h y t i c g e n e r a t i o n of Ectocarpus  sp., a f t e r g l u t a r a l d e -  hyde-osmium f i x a t i o n , i n d i c a t e t h a t the v a r i o u s c e l l s i n each f i l a m e n t do not respond e q u a l l y to such treatment. A p i c a l c e l l s are very l i g h t i n appearance, w h i l e the below them become p r o g r e s s i v e l y darker  ( f i g u r e 1).  Towards the b a s a l p o r t i o n of the f i l a m e n t s the c e l l s appear to be l e s s dense m a j o r i t y of the cases darker  c e l l s and  abrupt  ( f i g u r e 3) .  ( f i g u r e s 2 and  3).  In the  great  In a few  found t o be  cases, however., t r a n s i t i o n a l  In the r e g i o n where . l i g h t  f o l l o w e d dark ones, the c r o s s w a l l s between c e l l s protruded  again  s t u d i e d , the t r a n s i t i o n between the  the l e s s dense b a s a l ones was  stages were d e t e c t e d .  cells  i n t o the l i g h t c e l l s  (figure 3).  cells always  L i g h t microscope  o b s e r v a t i o n s . of l i v i n g , f i l a m e n t s r e v e a l e d ..similar, f e a t u r e s , showing i n a d d i t i o n , t h a t the i n t e r n a l o r g a n i z a t i o n of c e l l s on e i t h e r s i d e of. the c r o s s w a l l was  different  the  12 ( f i g u r e 5 ). I t i s , t h e r e f o r e , apparent t h a t a t l e a s t . 3 major types of c e l l s may  be r e c o g n i z e d i n a f i l a m e n t of Ectocarpus  l ) t h e a p i c a l c e l l and i t s immediate d e r i v a t i v e s ; 2) the c e l l s which r e a c t i n t e n s e l y w i t h OsO^;  and 3) the  cells  s i t u a t e d near the b a s a l p o r t i o n of the f i l a m e n t . I t i s a l s o safe to assume the e x i s t e n c e of t r a n s i t i o n a l stages w i t h i n the f i l a m e n t s . In order t o l e a r n more about the c y t o p l a s m i c d i f f e rences t h a t 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 , s e v e r a l c y t o c h e m i c a l t e s t s were c a r r i e d out. R e s u l t s of these i n v e s t i g a t i o n s are summarized i n the next page. To s i m p l i f y the d e s c r i p t i o n , the f o l l o w i n g n o t a t i o n s w i l l adopted  throughout  t h i s work : 1) " c e l l type #1"  be  refers  to the group of c e l l s r e p r e s e n t e d 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" c e l l s which r e a c t i n t e n s e l y w i t h OsO^;  r e f e r s t o the  3) " c e l l type  #3"  r e f e r s t o those c e l l s which do not r e a c t so deeply w i t h OsO.  and are l o c a t e d near the b a s a l end of the f i l a m e n t s ;  4) i n t e r m e d i a t e stages between the and between the  'type #2'  and the  'type #1' and  #2'  "transitional  r e s p e c t i v e l y . To a c c u r a t e l y r e l a t e the c e l l s t o the  proper stage of development; r i b b o n s of 2-3 f o r E.M. study  'type  'type #3' w i l l be r e c o g n i -  zed by the n o t a t i o n s " t r a n s i t i o n a l 1-2" 2-3"  and  examination  undertaken.  and o n l y a f t e r was  c e l l s were cut  the l i g h t  microscope  SUMMARY OF THE LICHT MICROSCOPE RESOLTS C « l l Type 11  DBA ( RNA e x t r a c t e d )  RNA  N u c l e u s s m e l l , round i o  Cytopleea  p r o f i l e . Chromatin  ( DNA e x t r a c t e d  )  intensely  s t a i n e d . Nuceolus  . reaction particularly  with  a clear positive  d i s t i n c t . Nucleolus  r e a c t i o n { arrow ) .  u n s t a i n e d . F i g u r e 12.  Nucleus o u t l i n e v i s i b l e .  Proteins Intense  r e a c t i o n almost  throughout the cytoplesm.  Lipids  I n s o l u b l e Carbohydrates  Lipofuscin  Few r e a c t i n g s p o t s .  P.A.S. r e a c t i o n v e i l  Alnost undetectable  Very f e v r e a c t i o n s i t e s ,  F i g u r e 23.  v i s i b l e i n the c e l l  —----------------  —  o o r e so i n p y r e n o i d e .  vail,  Polyphenols  r i g u x e 32.  F i g u r e 18.  F i g u r e i». Transitional  —-------~  —  —  :  —  "1-2"  l n association vith  1  G o l g i complex, i n t h e  ,  paramural space  6 t a l n e d area  lar  to  l n p r o f i l e , but s t i l l  conspicuously Nucleolus  stained.  g i o n . Nucleus  ;  unstained.  end  F i g u r e 13. •  11  restricted  the perinuclear r e unstained  very i r r e g u l a r i n  Staining r e s t r i c t e d to  ( arrow ) ,  and i n the c e l l w a l l . f i g u r e 21.  1  Nucleus s m a l l and I r r e g u -  evident  ,'  ^  12  P.A.S. r e a c t i o n particularly  cells  P.A.S. r e a c t i o n w e l l '  Conspicuous r e a c t i o n  Pattern of s t a i n i n g  •one c y t o p l a s m i c 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  pyrenoids  cytoplasm. Figure  wall.  cytoplasm. F i g u r e 26.  i n c l u s i o n s show a green  veil  stained.  F i g u r e 19.  Reaction  found  24.  to  p r o f i l e . F i g u r e 16.  N u c l e i c o u l d n o t be d e t e c t e d . F i g u r e 14. ;  turquoise  cytoplasmic colour.  F i g u r e 27.  C y t o p l a s m almost  Very f e i n t o r n e g a t i v e  u n s t a i n e d . F i g u r e 17.  s t a i n i n g t h r o u g h o u t the  end  c e l l . F i g u r e 20.  F i g u r e 25.  Reaction  spots few  sparse.  P.A.S. r e a c t i o n v e i l  R e a c t i o n almost o r  L i t t l e o r almost  a p p a r e n t i n the c e l l  t o t a l l y absent.  able r e a c t i o n .  vail.  undetect-  13 PART I I . - ELECTRON MICROSCOPE. OBSERVATIONS E l e c t r o n microscope  observations.substantiate.evidence  from l i g h t microscopy f o r the e x i s t e n c e of 3 main types of c e l l s w i t h i n the f i l a m e n t s of E c t o c a r p u s ,  and whose charac-  t e r i s t i c s are d e s c r i b e d In the f o l l o w i n g pages. A) ULTRASTRUCTURAL FEATURES OF YOUNG CELLS  ( CELL  TYPE # 1 ) The  g e n e r a l morphology of these c e l l s i n both  e r e c t and p r o s t r a t e systems of Ectocarpus was similar  ( f i g u r e s 28a,  31, 32,  and  33 ). T h e r e f o r e ,  f o l l o w i n g d e s c r i p t i o n s apply to c e l l s of both Nucleus. The nucleus  ( f i g u r e s 28a,  found  31,  the to be the  systems.  32, and  33 ) i s  bound by a double membrane envelope i n t e r r u p t e d by numerous pores. F r e e z e - e t c h i n g  s t u d i e s of the n u c l e a r envelope r e v e a l  the e x i s t e n c e of s u r f a c e s with few p a r t i c l e s on them, w h i l e others are m u l t i p a r t i c u l a t e ( f i g u r e 36  ). Nuclear pores are  e a s i l y i d e n t i f i e d i n f r e e z e - e t c h i n g p r e p a r a t i o n s , but  their  morphology v a r i e s a c c o r d i n g to the exposed s u r f a c e . T h i s i s p a r t i c u l a r l y apparent i n f i g u r e 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' as c i r c u l a r d e p r e s s i o n s . The pores,measuring 80 nm  i n diameter, a hollow  approximately  are round i n appearance and r e v e a l a  defined substructure possess  appear  ( f i g u r e s 29, and  36 ). They seem to  c e n t r a l core from which spokes r a d i a t e  toward the edge of the pore  ( f i g u r e 29, arrowheads ). The  pore rim appears to be made up of s e v e r a l g l o b u l a r u n i t s , whose number i s d i f f i c u l t to determine w i t h n u c l e a r pores i n f i g u r e 37 are not u n i f o r m l y  accuracy.The distributed  throughout the n u c l e a r membrane, but tend to occur i n c e r t a i n areas and not i n o t h e r s . S e c t i o n s of the i n t e r p h a s e nucleus  of young  14 v e g e t a t i v e c e l l s of E c t o c a r p u s sp. u s u a l l y contain., one conspicuous n u c l e o l u s ( f i g u r e s 31, 32, 33, and 38 seems t o c o n s i s t of two d i s t i n c t and 38 ). One tely  8nm  components  ),which  ( figures  component i s f i b r i l l a r , measuring  32,  approxima-  i n diameter. The o t h e r component i s g r a n u l a r , w i t h  each u n i t measuring  approximately 15 nm i n diameter.  The  n u c l e o l u s u s u a l l y occupies a c e n t r a l p o s i t i o n i n the nucleoplasm, but i n some cases i t s a s s o c i a t i o n w i t h the i n n e r membrane of the n u c l e a r envelope i s apparent 38 ). In t h i s  ( figure  case, the r e g i o n of the n u c l e a r envelope  c l o s e l y a s s o c i a t e d w i t h the n u c l e o l u s seems t o be devoid of n u c l e a r pores. Chromatin tion  i s not conspicuous, although i t s d i s t r i b u -  throughout the nucleoplasm as w e l l as i t s r e l a t i o n s h i p  to the i n n e r membrane of the n u c l e a r envelope i s obvious in  some micrographs.(  f i g u r e s 28a, and 32 ). At h i g h  m a g n i f i c a t i o n the f i b r i l l a r nature of the chromatin i s visible  ( f i g u r e 30 ). Each f i b r i l l a r  u n i t measures  approximately 10 nm i n diameter. The p e r i n u c l e a r space i s abundantly f i l l e d w i t h granular-fibrillar  material  ( e.g. f i g u r e s 30, 32, 38,  and 57 ). In a d d i t i o n , s m a l l v e s i c l e - l i k e  55,  structures .  ( f i g u r e s 38, arrow, 39, 40, 41, arrowheads ), whose r e l a t i o n s h i p t o the n u c l e a r envelope membranes i s shown i n f i g u r e s 41  ( arrowheads ) and 45  ( pns  ), are a l s o found  i n s i d e the p e r i n u c l e a r space. More e l a b o r a t e d s t r u c t u r e s are  found inco'close a s s o c i a t i o n w i t h the outer membrane of  the n u c l e a r envelope  ( f i g u r e s 38, empty arrowhead, and  arrowhead ). Besides the above mentioned  42,  f e a t u r e s the  a s s o c i a t i o n between the outer membrane of the n u c l e a r envelope and the forming face, of the. dictyosomes.'is a c o n s i s t e n t aspect of E c t o c a r p u s c e l l s Endoplasmic  reticulum  ( E.R.  ( e.g. f i g u r e 34 ). ). Most of the  endoplasmic  r e t i c u l u m p r e s e n t i n these c e l l s seems t o be of the  15 "rough", type are  ( f i g u r e s 28a, 32, and 44).  fenestrated  arrowhead). ( f i g u r e 43b,  elements  ( f i g u r e s 32, b l a c k arrow, 38, b i g arrow-  heads /.and.44,.arrowhead). also detectable  Cisternal  Fenestrae-llke  i n freeze-etching  .Some f e n e s t r a e  f o r m a t i o n s , are  preparations  (figure  43a,  d i s p l a y a - c r a t e r - l i k e appearance  arrowhead).  C r y s t a l l i n e i n c l u s i o n s are observed i n s i d e d i l a t e d portions  of the.E.R. c i s t e r n a e  ( f i g u r e 44, 1) .  E.R. elements are d i s t r i b u t e d throughout the cytoplasm ( f i g u r e s 28a, 31, 32, and 33).  In a few micrographs the  E.R. elements are observed to d i s p l a y c o n c e n t r i c ( f i g u r e 44).  In o t h e r s ,  an o r i e n t a t i o n o f the E.R. i n  r e l a t i o n t o the n u c l e a r envelope i s apparent and 38).  Continuity  the p e r i n u c l e a r  arrangements  (figures  32,  between the E.R. c i s t e r n a l space and  space i s observed  ( f i g u r e 38).  The  E.R.  c i s t e r n a l space i s o c c u p i e d by g r a n u l a r - f i b r i l l a r . m a t e r i a l ( f i g u r e s 32, 38, and 45) and v e s i c u l a r s t r u c t u r e s  (figure  45) s i m i l a r i n appearance t o those found i n s i d e t h e i perinuclear  space.  l i k e structures perinuclear  In a few micrographs,  multivesicular-  are observed a t the j u n c t i o n of the  and E.R. c i s t e r n a l spaces  ( f i g u r e 38, empty  arrowhead). Endoplasmic r e t i c u l u m  membranes are a l s o observed t o  e s t a b l i s h s p a t i a l r e l a t i o n s h i p s and/or d i r e c t with s e v e r a l o t h e r o r g a n e l l e s , the endoplasmic . r e t i c u l u m m i t o c h o n d r i a l , envelope  communication  i . e . associations  between  and the o u t e r membrane o f the  ( f i g u r e s 35, arrowhead, and  arrow), and w i t h the c h l o r o p l a s t  72,  ( f i g u r e s 44, 47a, b, 48),  thereby forming a s p e c i a l s e c t i o n of the E.R.  commonly  known as the ' c h l o r o p l a s t . E.R..' (Bouck,. 1965). The c h l o r o p l a s t E.R.. f o l l o w s around the p r o j e c t i n g . p y r e n o i d vesicular structures  the c h l o r o p l a s t  ( f i g u r e 28a).  Tubular-  a r i s e from the c h l o r o p l a s t  membrane f a c i n g the c h l o r o p l a s t  envelope  envelope  E.R.  ( f i g u r e s 32,  46,  16 and  48). These s t r u c t u r e s  can be observed i n t a n g e n t i a l  view as a very complex network, o f t u b u l e s c i s t e r n a l structures  (figure' 49).  Intra-  s i m i l a r t o those described, i n s i d e the  E.R.. c i s t e r n a l space are found i n s i d e the c i s t e r n a e o f the c h l o r o p l a s t E.R.  ( f i g u r e 50) .  Dark o s m i o p h i l i c  bodies w i t h a complex i n t e r n a l  s t r u c t u r e , r e f e r r e d i n t h i s work as " o s m i o p h i l i c bodies"  structured  (OSB), are a l s o found i n a s s o c i a t i o n w i t h the  c h l o r o p l a s t E.R.  ( f i g u r e s 45, 51, and 52). They can occupy  the area shared by both the c h l o r o p l a s t E.R. and the n u c l e a r envelope  ( f i g u r e 52), o r they may occur i n c l o s e  a s s o c i a t i o n with pyrenoids studies  ( f i g u r e 53). High  magnification  show these bodies t o have two major components:  a very dark o s m i o p h i l i c  and u n s t r u c t u r e d m a t e r i a l component  1)  (figures  51,  and 53); and 2) a m y e l i n - l i k e  ( f i g u r e s 51,  53,  and 54). The arrangement o f the two components i n  r e l a t i o n t o each other i s v a r i a b l e , and accounts f o r the h e t e r o g e n e i t y of forms d i s p l a y e d  by these bodies  (figures  45,  51, 52, 53, 54, and 55).  In some cases, the o r i g i n o f  the  "OSB" can be t r a c e d d i r e c t l y from the c h l o r o p l a s t E.R.  or from a complex system o f membranes a r i s i n g by f u r t h e r elaboration  o f the c h l o r o p l a s t E.R.  ( f i g u r e s 45, 47a, 47b).  In other cases, the "OSB" are found i n the cytoplasm from the c h l o r o p l a s t E.R. ( f i g u r e 56).  In some  apart  instances  they are a p p a r e n t l y d i s c h a r g e d i n t o the paramural space ( f i g u r e 54) . G o l g i complex. organelle, dictyosomes  The G o l g i complex, an e x c l u s i v e l y  perinuclear  i s r e p r e s e n t e d by groups o f 2, 3, o r more ( f i g u r e s 28a, 31, and 33). Each dictyosome  i s made up o f 6 t o 10 stacked c i s t e r n a e , o f which those c l o s e s t t o the n u c l e a r envelope are c o n s p i c u o u s l y ( f i g u r e s 57, and .58,  arrowheads).  fenestrated  The outer c i s t e r n a i s  u s u a l l y h y p e r t r o p h i e d i n appearance ( f i g u r e s 28a, 33, 58, 59> and 61). Observations o f d i c t y o s o m a l c i s t e r n a e a t high magnification  r e v e a l the presence o f two  17 s i z e s of i n t e r r u p t i o n s or channels.. One average diameter of 25 nm labelled 57,  83,  'a').  The  ( f i g u r e s 57,  type has  and  others average 5 hm  83,  an  arrowheads  i n diameter  (figures  arrowheads l a b e l l e d 'b').  D i r e c t c o n t i n u i t y between adjacent f r e q u e n t l y observed  ( f i g u r e s 33,  42,  cisternae i s  and  62),  and  the  presence of i n t r a - as w e l l as i n t e r c i s t e r n a l b r i d g e - l i k e f i l a m e n t s i s a l s o apparent As and  ( f i g u r e 59,  arrowhead).  i n the case of other brown algae  Cole,  1968;  Cole,  1969,  1970;  (Bouck, 19 65;  B i s a l p u t r a et a l . ,  a c l o s e a s s o c i a t i o n between the outer membrane of nuclear  envelope and  evident  i n Ectocarpus sp.  as being  the formative  Bourne 1971)  the  face of the dictyosomes i s  This r e l a t i o n s h i p i s interpreted  i n d i c a t i v e of the t r a n s f e r of m a t e r i a l from  the  p e r i n u c l e a r space to the dictyosomes by means o f v e s i c l e s a r i s i n g from the outer membrane of the n u c l e a r ( f i g u r e s 61,  and  83).  p e r i n u c l e a r space and  D i r e c t c o n t i n u i t y between the the innermost c i s t e r n a e of  dictyosomes occurs i n some cases Besides the u b i q u i t o u s with the n u c l e a r dictyosomes and mitochondria  envelope  ( f i g u r e 34,  the  arrowhead).  a s s o c i a t i o n of dictyosomes  envelope, a s s o c i a t i o n s a l s o occur between other  c e l l organelles;  for instance,  ( f i g u r e 61), with p y r e n o i d s  63), and w i t h l a r g e v a c u o l e s .  with  ( f i g u r e s 60,  In t h i s case,  and  incorporation  of dictyosome-derived v e s i c l e s i n t o vacuoles i s apparent ( f i g u r e 62, b i g arrowhead).  Evidence f o r the r e l e a s e  of  dictyosome-derived m a t e r i a l i n t o the paramural space i s obtained  when comparing the i n c l u s i o n s ( l a b e l l e d  'L')  i n s i d e the dictyosome c i s t e r n a ( f i g u r e 64) w i t h s i m i l a r i n c l u s i o n s i n s i d e the paramural s p a c e . ( f i g u r e  49)..  This  type of i n c l u s i o n seems t o a r i s e from i n v a g i n a t i o n o f  the  dictyosome c i s t e r n a l membrane.and o f t e n shows a h i g h degree of m o r p h o l o g i c a l e l a b o r a t i o n  ( f i g u r e 65).  Occasionally,  tube-  l i k e i n c l u s i o n s are a l s o found i n s i d e dictyosome c i s t e r n a e ( f i g u r e 66).  A c l o s e a s s o c i a t i o n between o s m i o p h i l i c  18 s t r u c t u r e d bodies . (OSB) observed  and dictyosomes- i s sometimes  ( f i g u r e 55).  Mitochondria.  M i t o c h o n d r i a are ua.ually . found  throughout the cytoplasm  scattered  ( f i g u r e s . 28a, 31, 32, and  although i n some cases they tend t o accumulate c e l i periphery., ( f i g u r e 56) .  33),  c l o s e t o the  Images suggesting m i t o c h o n d r i a l  d i v i s i o n are a l s o d e t e c t e d ( f i g u r e 62, s m a l l  arrowhead).  U l t r a s t r u c t u r a l l y these o r g a n e l l e s ( f i g u r e s 38, 68, 84)  do  not appear t o be d i f f e r e n t from those a l r e a d y d e s c r i b e d i n other brown a l g a l s t u d i e s B i s a l p u t r a , 1967; McCully, 1968;  (Bouck,  1965;  Bourne and C o l e , 1968;  C o l e , 1969;  i n s i d e the c r i s t a e  1969).  Since  be q u i t e e l a b o r a t e d  D i s t i n c t genophore areas may  ( f i g u r e s 38, and 84, arrowheads).  and  Cole and L i n , 1968;  L i d d l e and Neushul,  they are polymorphic, t h e i r p r o f i l e may ( f i g u r e 67).  Bisalputra  be  observed  I n c l u s i o n s are n o t i c e d  ( f i g u r e s 68, and i n s e t ) .  At h i g h  m a g n i f i c a t i o n , the i n c l u s i o n s appear t o be c i r c u l a r w i t h a hollow c e n t r a l c o r e , w i t h f i l a m e n t s r a d i a t i n g from the rims towards the c r i s t a e membranes. M i t o c h o n d r i a are found i n a s s o c i a t i o n w i t h almost a l l the c e l l o r g a n e l l e s . mitochondria-endoplasmic been r e p o r t e d .  Mitochondria-dictyosome  r e t i c u l u m a s s o c i a t i o n s have a l r e a d y  Other a s s o c i a t i o n s are w i t h the nucleus  ( f i g u r e 69), the c h l o r o p l a s t pyrenoid  and  ( f i g u r e s 48, and .70) , the  ( f i g u r e 71), and w i t h themselves  (figure  72,  arrowheads). Chloroplasts.  . C h l o r o p l a s t s . a r e bounded by a. double membrane  envelope, o u t s i d e of which l i e s , the c h l o r o p l a s t ( f i g u r e s 28a, .32, 33, 46, 4.7a,. 47b,  and 48.) .  E.R.  Direct  c o n t i n u i t y between the c i s t e r n a l space o f the.two above mentioned  membrane, systems i s . d e t e c t a b l e .(figures 47a,  48,  arrowheads). F e n e s t r a - l l k e i n t e r r u p t i o n s can be observed along the c h l o r o p l a s t envelope membranes both In l o n g i t u d i n a l as  19 w e l l as i n t a n g e n t i a l view (. f i g u r e s 76, arrowhead 'c', and  84, arrowhead ' b ' ^ r e s p e c t i v e l y ) . The  photosynthetic  closely associated  lamellae  thylakoids  c o n s i s t o f bands o f 3  ( f i g u r e s 76, and 79 ) . Each  t h y l a k o i d measures approximately 11 nm i r i width. The space between t h y l a k o i d s  o f the same band as w e l l as between  n e i g h b o r i n g bands has a high degree o f u n i f o r m i t y , f o r those regions  where p l a s t o g l o b u l i i n t e r v e n e d  except  o r where  exchanges o f thylakoids/;;between adjacent bands occur. Occasional  exceptions t o t h i s r e g u l a r i t y are observed  ( f i g u r e 75 ). B i f u r c a t i o n o f t h y l a k o i d s  is  ( f i g u r e 85, arrowhead ) . W i t h i n the t h y l a k o i d s  observed interruptions  are seen i n l o n g i t u d i n a l ( f i g u r e s 73, b i g arrowhead, and 76,  arrowheads 'a' ) as w e l l as i n t a n g e n t i a l views  78,  arrowheads, and i n s e t ; 84, arrowhead  ( figures  'a' ) . In t a n g e n t i a l  view the i n t e r r u p t i o n s seem t o be p o r e - l i k e i n appearance. In some areas, t h y l a k o i d bands are i n t e r r u p t e d by l a r g e portions  o f stroma m a t e r i a l  When f r e e z e - e t c h e d two  ( f i g u r e s 46, and 92 'F' ).  thylakoids  types o f f r a c t u r e s u r f a c e s . These  have e i t h e r many p a r t i c l e s ( s u r f a c e s ( surfaces The  o f Ectocarpus r e v e a l ( f i g u r e s 80, and 81 ) 'B' ) o r only  'A' ). p e r i p h e r a l band o f t h y l a k o i d s  f o l l o w s the contour  of the i n n e r membrane o f the c h l o r o p l a s t envelope, the c e n t r a l l y l o c a t e d bands 46  a few  enclosing  ( f i g u r e s 28a, 31, 32, 33, and  ). In some cases, the p e r i p h e r a l band i s n o t s t r i c t l y  p e r i p h e r a l , but i s continuous w i t h the c e n t a l l y p l a c e d  ones  ( f i g u r e 82, arrowheads ) . The c e n t r a l bands normally t r a v e r s e the c h l o r o p l a s t without i n t e r r u p t i o n from t i p t o tip 33,  o r terminate j u s t s h o r t o f the t i p s  ( f i g u r e s 28a, 32,  and 48 ) . In some cases, t h y l a k o i d s have a h i g h l y  arrangement  folded  ( f i g u r e 85 ) o r may be arranged c o n c e n t r i c a l l y  ( f i g u r e 86 ) . The whorled arrangement o f t h y l a k o i d s i s  20  a p p a r e n t l y r e l a t e d to the a b s c i s s i o n of c e r t a i n p l a s t i d portions to be  ( f i g u r e s 87,  incorporated  and  ( f i g u r e 89  preparations  ). A b s c i s s e d . p o r t i o n s seem  i n t o , vacuoles, . ( f i g u r e 91  phosphatase a c t i v i t y was formations  88  found a s s o c i a t e d ), but was  ( f i g u r e 90  )-. • AAcid  w i t h these  absent from the  control  ).  F i b r i l l a r b r i d g e - l i k e elements are observed i n s i d e and  between t h y l a k o i d s  ( f i g u r e s 73,  76,  79,  small  heads ). F i b r i l l a r elements were most n o t i c e a b l e cut t a n g e n t i a l l y t o the t h y l a k o i d s heads ). The  f i b r i l l a r material  areas mentioned above, but between the p e r i p h e r a l envelope  ( f i g u r e 92,  i n s i d e the -etching 3C to 5:nm  i s not r e s t r i c t e d to  arrowheads ). The  fibrillar  sections  ( f i g u r e 96, white arrow-  t h y l a k o i d band and  the  74,  and  space  presence of 95,  the  chloroplast  f u r t h e r confirmed by  ( f i g u r e s 43a,  dimensions of the  in  i s a l s o observed i n the  c h l o r o p l a s t s was  studies  arrow-  fibrils  freeze-  arrowheads ).  The  elements were found to range from  i n diameter, w i t h most of the measurements  averaging 3.7 In the  to 4.2  nm.  stroma, r i b o s o m e - l i k e p a r t i c l e s and  g l o b u l i are d e t e c t e d  ( f i g u r e s 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 embedded i n an amorphous and ( f i g u r e s 97,  98,  and  99  and  98,  c o n t i n u i t y between the  fibrils  l e s s e l e c t r o n dense m a t r i x  ). The  show a c l e a r r e l a t i o n s h i p t o the ( f i g u r e s 97,  plasto-  plastoglobular  fibrils  t h y l a k o i d membranes  arrowheads ). In f i g u r e 99 d i r e c t f i b r i l l a r phases of two  adjacent  p l a s t o g l o b u l i i s observed. DNA-containing r e g i o n s at the t i p s of ( f i g u r e s 47a, Bisalputra  and  48,  'g'  chloroplasts  ) a r e , as shown by B i s a l p u t r a  (1969), p a r t of the genophore.of these  In.Ectocarpus sp., B i s a l p u t r a and  and  organelles.  as i n o t h e r algae. ( Evans,1966;  B i s a l p u t r a , 1 9 7 0 ), c h l o r o p l a s t d i v i s i o n i s not  synchronous w i t h c e l l  d i v i s i o n . As  a r e s u l t , various  stages  21 of c h l o r o p l a s t m u l t i p l i c a t i o n are observed i n the. a c t i v e c e l l s o f the f i l a m e n t s . Processes o f c h l o r o p l a s t c a t i o n were. d e s c r i b e d  i n the'..'brown algae  multipli-  ( e . g . von  Wettstein,1954; Cole and Lin,1968; Cole,1970; B i s a l p u t r a and B i s a l p u t r a , 1 9 7 0 ).In the.case o f Ectocarpus sp. more than one  type o f c h l o r o p l a s t d i v i s i o n . s e e m s - t o  sporophytic  vegetative  systems. F i g u r e s  take p l a c e  i n the  c e l l s o f both the e r e c t and p r o s t r a t e  102 and 103 d e p i c t two stages o f a process  of d i v i s i o n . T h i s process o f c h l o r o p l a s t d i v i s i o n the c h l o r o p l a s t d i v i s i o n o f S p h a c e l a r i a  resembles  ( B i s a l p u t r a and  B i s a l p u t r a , 1 9 7 0 ). In f i g u r e 103 the stage o f d i v i s i o n seems to correspond t o the onset o f the formation o f the p e r i p h e r a l lamellar bridge  ( arrowhead). F i g u r e  101 shows a d i v i s i o n by  l o n g i t u d i n a l c o n s t r i c t i o n . A s i m i l a r event has been r e c o r d e d i n L a m i n a r i a gametophytes Figures  ( B i s a l p u t r a e t al.,1971 ).  105 and 106 are non-consecutive s e r i a l s e c t i o n s o f  another aspect resembling c h l o r o p l a s t d i v i s i o n . A p p a r e n t l y , t h i s p r o c e s s i n v o l v e s the p r o g r e s s i v e  c o n s t r i c t i o n of  t h y l a k o i d s without the formation o f a p e r i p h e r a l  lamellar  bridge.  The p r o c e s s c l o s e l y resembles t h a t o c c u r i n g i n  Egregia  chloroplasts  Figure  ( B i s a l p u t r a , unpublished data ).  107 suggests the p o s s i b i l i t y t h a t a c h l o r o p l a s t i s  undergoing d i v i s i o n a t two c o n s t r i c t i o n s i t e s . t r i c t i n g c h l o r o p l a s t s were d e s c r i b e d Wettstein  i n Fucus by von  ( 1 9 5 4 ) . In dark t r e a t e d m a t e r i a l  chloroplast blebbing  conspicuous  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 structure contains  Multicons-  ( f i g u r e 104 ). The b l e b - l i k e  only stroma and i s d e l i m i t e d by both  the c h l o r o p l a s t envelope and the c h l o r o p l a s t E.R.. Since t h i s phenomenon i s only it  found i n dark t r e a t e d  material,  i s more l i k e l y to-be a r e a c t i o n o f . t h e o r g a n e l l e s t o  dark treatment. P y r e n o i d . In E c t o c a r p u s , g e n e r a l l y only one pyrenoid  i s seen  p r o j e c t i n g from the c h l o r o p l a s t on the s i d e f a c i n g the  22 nucleus  ( f i g u r e 28a ) . However, the presence o f two or  more pyrenoids p e r c h l o r o p l a s t i s n o t uncommon 108,  ( figure  see a l s o 6, and 9 ) . M o s t l i k e l y t h i s i s r e l a t e d t o the  phenomenon of p y r e n o i d d i v i s i o n ( f i g u r e 8 ) . Gibbs has  (1962a)  shown t h a t t h e . p y r e n o i d m a t r i x o f P y l a i e l l a c o n s i s t s o f  t i g h t l y packed f i b r i l s  6.5-7.0 nm i n diameter. S i m i l a r  fibrillar  elements are seen i n the m a t r i x o f Ectocarpus  pyrenoids  ( f i g u r e 109 ). A very c l o s e r e l a t i o n s h i p i s  observed between p y r e n o i d f i l a m e n t s micrographs  and t h y l a k o i d s  i n some  ( f i g u r e s 93, and 108 ), p a r t i c u l a r l y i n  dark t r e a t e d m a t e r i a l  ( f i g u r e 9 3 ).  In a d d i t i o n t o the c h l o r o p l a s t envelope and the c h l o r o p l a s t E.R., a t h i r d membrane system designated as the "pyrenoid sac" o r "pyrenoid cap" i s found o u t s i d e the c h l o r o p l a s t E.R.. T h i s membrane system i s r e s t r i c t e d t o the area around the p y r e n o i d body  ( f i g u r e 28a ) . The r e l a t i o n s h i p  e x i s t e n t among these three membrane systems i s b e t t e r observed i n f i g u r e 110. Bouck reported  (1965) and Cole  (1969)  t h a t no connection e x i s t s between the o u t e r  membrane o f the p y r e n o i d sac and any o t h e r c y t o p l a s m i c organelle.  In E c t o c a r p u s , however, d i r e c t communications  seem t o occur between the p y r e n o i d and dictyosomes 60,  and 63 ), and the p y r e n o i d and the vacuole  ( f i g u r e 112).  P y r e n o i d d i v i s i o n i s not synchronous w i t h division  chloroplast  ( f i g u r e s 8, 9; see a l s o Evans,1966 ) . Images o f  p y r e n o i d d i v i s i o n are frequent in  ( figures  ( f i g u r e s 109, and 114 );  those s i t u a t i o n s the p y r e n o i d sac i s n o t i c e a b l y  absent  ( see a l s o Manton,1966a ). The  c e l l w a l l and a s s o c i a t e d  composed o f f i b r i l s  s t r u c t u r e s . The c e l l w a l l i s  arranged i n two d i s t i n c t zones separated  by an abrupt boundary  ( figure-142 ) . The i n n e r  layer ( i l )  i s made up o f 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 l a y e r  (ol) i s formed by l o o s e l y  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 b i l a y e r u .  23 o r g a n i z a t i o n of the c e l l . w a l l i s a l s o e v i d e n t i n f r e e z e etched p r e p a r a t i o n s (figure. 117)..  In t h i s p i c t u r e  of  u n i d e n t i f i e d o r i g i n are observed  of  the c e l l w a l l .  i n s i d e the i n n e r l a y e r  O s m i o p h i l i c m a t e r i a l can be seen  the paramural.space  ( f i g u r e 47).  occupy the paramural  space.  inside  Other types of i n c l u s i o n s  The o r i g i n of some of. these  can be t r a c e d from the plasmalemma i t s e l f arrowhead).  inclusions  (figure  28b,  These s t r u c t u r e s can a t t a i n a h i g h degree of  m o r p h o l o g i c a l complexity  ( f i g u r e s 28a,  28b,  102,  pm).  F o l l o w i n g the c l a s s i f i c a t i o n of Marchant and Robards  (1968),  these s t r u c t u r e s ought t o be designated as "plasmalemmasomes." They are e a s i l y r e c o g n i z a b l e i n f r e e z e - e t c h i n g p r e p a r a t i o n s ( f i g u r e 37a).  However, as was  p r e v i o u s l y considered,  one  has the impression t h a t some of the paramural s t r u c t u r e s may  have been d e r i v e d from dictyosomal a c t i v i t y  64—"L" with f i g u r e 4 9 — " L " ) .  These s t r u c t u r e s should,  t h e r e f o r e , be designated "lomasomes" (Marchant 1968).  Plasmodesmata are observed  adjacent c e l l s into p i t - l i k e B)  Vacuole  and Robards,  i n the c e l l w a l l s of  ( f i g u r e 111), but they are not o r g a n i z e d areas.  ULTRASTRUCTURAL FEATURES OF THE 1-2"  ( c f . figure  "TRANSITIONAL  CELLS formation and processes of autophagy.  One  the main processes f o r the t r a n s f o r m a t i o n of the young ( c e l l type #1)  cell  a r c h i t e c t u r e i n t o t h a t c h a r a c t e r i s t i c of  the " c e l l type #2" autophagy.  of  i s r e l a t e d t o vacuole formation  and  In t h i s . s e c t i o n these events are analysed i n  detail. 1) Vacuole o r i g i n .  In Ectocarpus sp. s p o r o p h y t i c v e g e t a t i v e  c e l l s , both endoplasmic to  r e t i c u l u m and dictyosomes c o n t r i b u t e  the development of the v a c u o l a r system.  This i s indicated  by the e x i s t e n c e of d i r e c t c o n t i n u i t y and/or c l o s e  24 a s s o c i a t i o n between elements of the endoplasmic .reticulum and v a c u o l e s  ( f i g u r e 100,  arrowheads),  as w e l l as  E.R.-  d e r i v e d p r o v a c u o l a r p r o f i l e s , which show a tendency, to fuse w i t h each other and w i t h f u l l y developed, vacuoles .116,  arrowhead, 14 8, arrows).  (figures  The r o l e of.dictyosomes  in  vacuole development i s d e p i c t e d by the i n c o r p o r a t i o n of dictyosomal v e s i c l e s i n t o v a c u o l a r s t r u c t u r e s ( f i g u r e big  62,  arrowhead).  2), Autophagic l e v e l of how  activity.  An a n a l y s i s a t the u l t r a s t r u c t u r a l  c y t o p l a s m i c areas and/or o r g a n e l l e s are  i s o l a t e d from the remainder  of the cytoplasm, become t r a n s -  formed, and g a i n access t o vacuoles i s rendered due  difficult  to the f a c t t h a t one i s s t u d y i n g s t a t i c p i c t u r e s of a  h i g h l y dynamic phenomenon. examination  B e a r i n g t h i s i n mind, and  of m u l t i p l e micrographs,  after  i t seems t h a t  autophagy i n Ectocarpus sp. can take p l a c e by more than  one  mechanism. One of  of the mechanisms seems t o i n v o l v e the  c y t o p l a s m i c p o r t i o n s by the endoplasmic  119,  120,  121).  c o n c e n t r i c E.R.  reticulum (figures  The i s o l a t e d area i s , hence,  by a double membrane system.  surrounded  C o n t i n u i t y between these  and normal c i s t e r n a l elements i s apparent  at an e a r l y stage of i s o l a t i o n  ( f i g u r e s 120,  121).  i s o l a t e d r e g i o n s are g r a d u a l l y transformed i n t o vacuoles In  isolation  ( f i g u r e s 122,  123,  and  The  typical  127).  o t h e r cases, access to the v a c u o l a r space i s  gained by a d i f f e r e n t mechanism, which seems t o i n v o l v e the i n v a g i n a t i o n o f the t o n o p l a s t i n the r e g i o n where the m a t e r i a l t o be i s o l a t e d .contacts or approaches. „the vacuole boundary  ( f i g u r e 145,  arrowheads.) .  T h i s process., appears  to  be r e s p o n s i b l e f o r the i n c o r p o r a t i o n of. abscis.sed p l a s t i d p a r t s i n t o vacuoles f i g u r e 88).  ( f i g u r e s 145,  upper arrowhead, see  also  I n v a g i n a t i o n of t o n o p l a s t w i t h a subsequent  i n t a k e of c y t o p l a s m i c m a t e r i a l i s a l s o seen i n f i g u r e  124.  25 T h i s mechanism d i f f e r s from the E.R.^dependent mechanism i n t h a t the i s o l a t e d m a t e r i a l was not segregated from the cytoplasm p r i o r t o i t s uptake by the i n v a g i n a t i n g t o n o p l a s t . 3) Autophagic  a c t i v i t y — e v i d e n c e from c y t o c h e m i s t r y o f a c i d  phosphatase.  Although a c i d phosphatase  a c t i v i t y was  d e t e c t e d i n a s s o c i a t i o n w i t h c i s t e r n a l E.R., c i s t e r n a e o f dictyosomes,  and p r o v a c u o l e - l l k e s t r u c t u r e s  ( f i g u r e s 129,  and 130); r e c e n t l y i s o l a t e d c y t o p l a s m i c areas show no a c i d phosphatase  activity.  Control material f a i l s  t o show any  s i g n s o f r e a c t i o n products i n a s s o c i a t i o n w i t h the above mentioned o r g a n e l l e s ( f i g u r e 131) .  As the process of  degradation o f i s o l a t e d m a t e r i a l proceeds, i t s appearance becomes more and more a l t e r e d and membranous remains become v i s i b l e i n s i d e the vacuoles  ( f i g u r e 49). A c i d  phosphatase  a c t i v i t y was found t o be a s s o c i a t e d w i t h these formations ( f i g u r e s 125, and 126), b u t absent from c o n t r o l s 131).  (figure  F u r t h e r v a c u p l a t i o n and degradation o f i s o l a t e d  m a t e r i a l leads t o l o s s o f r e c o g n i z a b i l i t y o f o r g a n e l l e s and c y t o p l a s m i c remnants i n s i d e the v a c u o l e s . types o f i n c l u s i o n s are observed: g r a n u l a r substance  Instead, two  1) a homogeneous  ( f i g u r e s 135, 136, ' a ' ) , and 2) a  polymorphic m y e l i n - l i k e f i g u r e  ( f i g u r e s 135, 136,  'b').  The g r a n u l a r type o f i n c l u s i o n i s found alone o r i n a s s o c i a t i o n w i t h the m y e l i n - l i k e component 136,  and 137, ' a ' ) .  ( f i g u r e s 135,  The m y e l i n - l i k e form, however, does not  occur by i t s e l f . With g r e a t e r v a c u o l a t i o n and c e l l u l a r d i g e s t i o n , the cytoplasm of the c e l l s becomes more and more a l t e r e d 135,  136), approaching f i n a l l y  (figures  ( f i g u r e 128) the c e l l u l a r  o r g a n i z a t i o n o f " c e l l type #2" ( f i g u r e s 148, 151), which i s d e s c r i b e d below.  The i n c r e a s e In a c i d phosphatase  activity  p a r a l l e l s v a c u o l a t i o n and d e g r a d a t i o n o f c y t o p l a s m i c materials  ( f i g u r e s 138, and 139). Reaction products are  absent from c o n t r o l s e c t i o n s ( f i g u r e 140).  26 4) Other c e l l u l a r . . . a l t e r a t i o n s . Other, changes.in ultrastructure contribute  cellular  t o the e s t a b l i s h m e n t o f the " c e l l  type #2" morphology. These i n c l u d e  : 1) a change i n t h y l a k o i d  o r i e n t a t i o n i n t o patches o f l a r g e t h y l a k o i d stacks 132  ( figure  ); 2) the occurrence of e l e c t r o n dense m e t a b o l i t e s a t  the c e l l w a l l p e r i p h e r y of the c e l l w a l l  and among the f i b r i l l a r elements  ( f i g u r e 142 ); and 3) the appearance o f  l o c a l i z e d c e l l w a l l ingrowths  ( f i g u r e 163, cwi ), a  phenomenon t h a t c o i n c i d e s w i t h the o b s e r v a t i o n a c t i v i t y a t the G o l g i complex l e v e l  o f high  ( f i g u r e 141 ) . T h i s  problem w i l l be d e a l t w i t h i n d e t a i l l a t e r v 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 r e p r e s e n t  dynamic and  continuous s i t u a t i o n s the d e t e r m i n a t i o n o f 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 #2" i s d i f f i c u l t .  type  To s o l v e t h i s d i f f i c u l t y i t i s e s t a b l i s h e d  t h a t whenever c h l o r o p l a s t s show i n c r e a s i n g signs of product i o n o f h i g h l y e l e c t r o n dense m e t a b o l i t e s t o g e t h e r w i t h a l t e r e d t h y l a k o i d arrangements 152,  ( f i g u r e s 143, 148, 149, 151,  154, and 156 ), the c e l l i s considered  as b e l o n g i n g  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 o f these c e l l s was  found t o be s i m i l a r i n the e r e c t and the p r o s t r a t e  systems  ( f i g u r e s 143, 148, and 151 ) . T h e r e f o r e the f o l l o w i n g d e s c r i p t i o n s apply t o c e l l s o f both systems. Nucleus. The nucleus remains c l e a r l y d i s c e r n i b l e a t t h i s stage o f c e l l u l a r development. The n u c l e a r  boundary has  become i r r e g u l a r ( f i g u r e s 143, and 148 ).Nuclear pores are difficult  to detect. Chromatin-iike material  can be seen i n  some cases e i t h e r throughout the nucleoplasm o r i n a s s o c i a t i o n w i t h the i n n e r membrane o f the n u c l e a r  envelope  27 ( f i g u r e 143) . of  The n u c l e o l u s , when apparent,  both f i b r i l l a r and g r a n u l a r elements  is  comprised  ( f i g u r e 153).  The  a s s o c i a t i o n between the outer membrane of the n u c l e a r envelope  and elements of the endoplasmic r e t i c u l u m and  dictyosomes are s t i l l maintained  (figure  Endoplasmic r e t i c u l u m .  system becomes l e s s  The E.R.  143).  prominent i n these c e l l s than i n the " c e l l type ( f i g u r e s 143, to  148,  151).  T h i s i s due,  the d i s o r g a n i z a t i o n of the E.R.,  v e s i c u l a t i o n of i t s elements labelled  "E.R.").  i n t o vacuoles  any form are d i f f i c u l t  which i s caused  ( f i g u r e 143,  to d e t e c t .  the  regions  They are, however, (figure  observed  153).  The G o l g i complex i s s t i l l w e l l represented  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  148).  151,  by  In these areas, ribosomes of  i n less a l t e r e d cytoplasmic regions at  at l e a s t i n p a r t ,  These v e s i c l e s then become i n c o r p o r a t e d  ( f i g u r e 151).  G o l g i complex.  #1"  (figures  143,  I t s f u n c t i o n a l a s s o c i a t i o n w i t h the outer membrane of  the n u c l e a r envelope  i s evident.  However, the u s u a l  h y p e r t r o p h i e d appearance of the outermost c i s t e r n a of the dictyosomes, longer  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  observed.  Mitochondria.  M i t o c h o n d r i a are very d i f f i c u l t  t h i s stage of aging. account  to detect at  More f a v o u r a b l e s e c t i o n s allow one  f o r s c a r c e l y more than a double membrane  and c r i s t a e  to  envelope  ( f i g u r e 153) .  Chloroplasts.  Of a l l c e l l o r g a n e l l e s , c h l o r o p l a s t s e x h i b i t  the most s t r i k i n g a l t e r a t i o n s which have been employed as a c r i t e r i o n f o r i d e n t i f i c a t i o n of the " c e l l type The appearance of patches  #2".  of h i g h l y e l e c t r o n dense  m e t a b o l i t e s i s v i s i b l e a t many s i t e s I n s i d e the c h l o r o p l a s t s ( f i g u r e s 143,  148,  151,  152,  arrows).  The  intraplastid  m e t a b o l i t e s , which s t a r t as s m a l l s c a t t e r e d formations ( f i g u r e 143) , become b i g g e r and more numerous as senescence proceeds  ( f i g u r e s 148,  151).  The r e g i o n s where the  28 m e t a b o l i t e accumulates p r e s e n t conspicuous, p a t t e r n s of t h y l a k o i d arrangement  ( f i g u r e s 149,  154,  156,  157).  The  r e l a t i o n s h i p between the m e t a b o l i t e and the s p e c i a l arrangement of t h y l a k o i d s suggests  involvement  i n the p r o d u c t i o n of the m e t a b o l i t e . a l s o apparent head) .  constant  of t h y l a k o i d s  This relationship i s  i n f r e e z e - e t c h e d m a t e r i a l ( f i g u r e 159,  arrow-  Permanganate f i x a t i o n c o u l d p r e s e r v e n e i t h e r the  i n t r a p l a s t i d r e g i o n s where the m e t a b o l i t e i s found nor plastoglobuli  ( f i g u r e 133,  arrows).  the  This implies a possible  l i p i d nature to the i n t r a p l a s t i d m e t a b o l i t e ; a p o s s i b i l i t y f u r t h e r confirmed by i t s a f f i n i t y f o r Sudan Black B.  The  i n t r a p l a s t i d m e t a b o l i t e seems to be r e l e a s e d i n t o the cytoplasm  ( f i g u r e s 155,  and 157),  c o n t r i b u t i n g t o the  o s m i o p h i l i c appearance of the cytoplasm.  Plastoglobuli  are  e a s i l y d i s t i n g u i s h e d from the o s m i o p h i l i c m e t a b o l i t e d e s c r i b e d above.  There i s no r e l a t i o n s h i p between the two m a t e r i a l s ,  because:  1) both s t r u c t u r e s are p r e s e n t a t the same time  and are m o r p h o l o g i c a l l y d i f f e r e n t  ( f i g u r e s 143,  148,  151);  2) an i n c r e a s e i n the number of m e t a b o l i t e c o n t a i n i n g r e g i o n s occurs with advancing  senescence,  w h i l e the number  of p l a s t o g l o b u l i remains, a t t h i s stage of aging, n e a r l y 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  r e l a t i o n s h i p with t h y l a k o i d s . Pyrenoids. The  Pyrenoids  are e a s i l y d e t e c t a b l e  ( f i g u r e s 143,  f i b r i l l a r nature of the p y r e n o i d matrix i s no  apparent,  148).  longer  nor are the boundary membranes.  Cytoplasm morphology.  F i g u r e s 143,  148,  d e p i c t the c o n d i t i o n of the cytoplasm  150,  151,  160  i n " c e l l type  #2".  The development and o r i g i n of c y t o p l a s m i c i n c l u s i o n s  has  a l r e a d y been presented. I t can be summarized t h a t t h r e e main types of c y t o plasmic i n c l u s i o n s are ..present. inclusions  ( f i g u r e s 148,  150),  a f t e r permanganate f i x a t i o n .  F i r s t l y , myelin-like at least p a r t i a l l y  preserved  The preserved p o r t i o n r e p r e s e n t s  29 the non-myelinic component The second  ( f i g u r e 134,  arrowhead '2').  i s a. g r a n u l a r type, o f I n c l u s i o n . ( f i g u r e s  150)  which i s p r e s e r v e d by permanganate f i x a t i o n  134,  arrowhead  '1').  148,  (figure  Both types of i n c l u s i o n s have been  shown t o 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 t o be p r e s e n t i n the cytoplasm of these  cells.  T h i s type of i n c l u s i o n would correspond t o the i n t r a p l a s t i d m e t a b o l i t e , s i n c e i t s d i s c h a r g e i n t o the cytoplasm has been demonstrated.  I t s presence i n the cytoplasm i s f u r t h e r  s u b s t a n t i a t e d by comparing the behaviour of the d i f f e r e n t types of i n c l u s i o n s a f t e r permanganate f i x a t i o n .  Of the 3  types of i n c l u s i o n s r e p o r t e d , only one shows a response s i m i l a r to t h a t o f . t h e i n t r a p l a s t i d m e t a b o l i t e , i . e . , n o n - p r e s e r v a t i o n u s i n g the permanganate f i x a t i o n ( c f . f i g u r e 13 3, white arrow w i t h f i g u r e 13 4, arrowhead '3'). Cell wall.  The c e l l w a l l as a whole seems t o have i n c r e a s e d  i n t h i c k n e s s ( f i g u r e 160).  However, n e i t h e r i t s t h i c k n e s s  nor s t r u c t u r a l complexity are uniform, s i n c e conspicuous w a l l ingrowths  are observed.  The c e l l w a l l ingrowths  cell  are  made up of m e t a b o l i t e s trapped between the o l d l a y e r s of the c e l l w a l l and the newly formed ones underneath 171).  (figure  T h i s phenomenon begins e a r l y i n the t r a n s i t i o n  "1-2"  p e r i o d , and a t t a i n s i t s h i g h e s t degree of e l a b o r a t i o n i n " c e l l type #2".  F i g u r e s 56 and 63  (cwi) show a very e a r l y  stage i n formation of the ingrowths.  At t h i s stage,  m e t a b o l i t e s , probably of v a c u o l a r o r i g i n begin t o accumulate  i n the paramural  shows (at arrowheads) new  space.  Figure  c e l l wall material laid  space between the paramural.metabolites membrane.  ( f i g u r e 153,  arrows),  151  down i n the  and the plasma  D e p o s i t i o n of n e w . c e l l w a l l m a t e r i a l 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 metabolites  from the plasma membrane ( f i g u r e s 153,  m e t a b o l i t e s can now between the new  158,  '2').  be d i s c h a r g e d i n t o the paramural  New  space  c e l l w a l l m a t e r i a l and the plasma membrane  30 g i v i n g to the conjunct a l a y e r e d p a t t e r n ( f i g u r e s 158).  D e p o s i t i o n of new  152,  wall material alternately  with  d i s c h a r g e of m e t a b o l i t e s can produce s t r i k i n g p a t t e r n s of c e l l w a l l ingrowths can be observed  ( f i g u r e 161) .  t h a t ingrowths  i n adjacent c e l l s  ( f i g u r e s 158,  were seen t o run through ( f i g u r e 158, of  may  In the c r o s s w a l l s i t match s i m i l a r s t r u c t u r e s  and  162).  Plasmodesmata  the newly l a i d w a l l m a t e r i a l  arrowheads").  These ingrowths  are  characteristic  t h i s stage of senescence and are e a s i l y d e t e c t e d a t 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  ( f i g u r e s 10,  arrowheads), as w e l l as i n f i x e d m a t e r i a l ( f i g u r e arrowhead).  Cytochemical  26,  t e s t s c a r r i e d out a t the  microscope l e v e l have shown t h a t the c e l l w a l l  11, light  ingrowths  s t a i n a g r e e n i s h c o l o r a f t e r t o l u i d i n e blue 0 and deep b l u e a f t e r the Schorml's r e a c t i o n .  Cell wall  ingrowths  formation appears t o . c o i n c i d e w i t h an i n c r e a s e i n a c t i v i t y of  the G o l g i apparatus,  as i n d i c a t e d by the  appearance of the G o l g i - d e r i v e d v e s i c l e s  loaded  (figure  141).  Discharge of m e t a b o l i t e s i n t o the e x t e r n a l medium a l s o seems to occur D)  THE OF  (figure  144).  SENESCENT CELLS—ULTRASTRUCTURAL FEATURES "TRANSITIONAL 2-3"  CELLS  The degree of d i f f e r e n c e between the " c e l l type and  " c e l l type #3"  type #1"  and  types "2" and  #2"  i s j u s t as d i s t i n c t as t h a t between  " c e l l type #2".  "cell  T r a n s i t i o n a l stages between  "3". c e l l s are, t h e r e f o r e , t o be expected.  These  t r a n s i t i o n a l stages, however, must be v e r y ephemeral, s i n c e they were d i f f i c u l t  t o f i n d and only on a few o c c a s i o n s were  they d e t e c t e d i n e l e c t r o n microscopic, o b s e r v a t i o n s .  For the  most p a r t , the t r a n s i t i o n between the type"#2" and type c e l l s was  abrupt  (figure  146).  The g e n e r a l morphology of one of these  transitional  "#3"  31 c e l l s i s shown i n f i g u r e 145. A t h i g h m a g n i f i c a t i o n , morphology does not seem t o be s t r i k i n g l y d i f f e r e n t  cell  from  t h a t i n " c e l l type #2", but a c l o s e r examination shows t h a t the  cytoplasm has .a h i g h e r degree of a u t o l y s i s  147,  164).  D i s r u p t i o n o f t h e t o n o p l a s t i s common  165, arrowheads). to  (figures;; (figure  The E.R. system i s more and more  difficult  d e t e c t , although s m a l l p r o f i l e s are s t i l l r e c o g n i z a b l e  ( f i g u r e 177) . Nucleus.  The nucleus i s r a r e l y observed i n c e l l s a t t h i s  stage of senescence.  When p r e s e n t , i t shows unmistakable  signs of d i s o r g a n i z a t i o n of the  the n u c l e a r envelope  ( f i g u r e 170).  Disorganization  (arrows) seems t o occur through  v e s i c u l a t i o n of i t s membranes.  T h i s process does not  proceed u n i f o r m l y throughout the n u c l e a r boundary  since,  as can be observed i n f i g u r e 170, the n u c l e a r envelope i s no longer d e t e c t a b l e i n c e r t a i n p l a c e s .  I t should be noted  t h a t the p o r t i o n o f the n u c l e a r envelope s t i l l t h a t f a c i n g a dictyosome.  intact i s  I n t e r n a l l y t o the remains o f the  n u c l e a r envelope, a d i s o r g a n i z e d  granular-fibrillar  m a t e r i a l c o n s t i t u t e s the remains o f the nucleoplasm.  No  chromatin or n u c l e o l u s can be r e c o g n i z e d . G o l g i complex.  S i m i l a r l y t o the n u c l e u s , dictyosomes are  rarely detected. atypical.  When p r e s e n t ( f i g u r e 170, D), they are  No steps i n the d i s o r g a n i z a t i o n of these o r g a n e l l e s  c o u l d be f o l l o w e d .  Nuclear-envelope-dictyosome  associations  cease t o e x i s t a t t h i s stage o f c e l l u l a r senescence; no t r a n s f e r o f m a t e r i a l i s d e t e c t a b l e between the two membrane systems. Mitochondria.  These o r g a n e l l e s seem to.be more numerous a t  t h i s stage of c e l l u l a r senescence than i n " c e l l type #2". They a r e u s u a l l y round i n p r o f i l e and s m a l l i n dimensions ( f i g u r e s 147, 164).  C r i s t a e are.conspicuous; some o f them  d i s p l a y a c o n c e n t r i c arrangement  ( f i g u r e s 174, 175). The  matrix shows a r a t h e r homogeneous and dense  appearance.  32 Chloroplasts.  These o r g a n e l l e s . p o s s e s s a morphology  r e m i n i s c e n t of t h a t found i n " c e l l type #2".. of  Extensive stacks  t h y l a k o i d s u s u a l l y occupy most of the c h l o r o p l a s t body  ( f i g u r e s 167,  169,  and 172)..  O c c a s i o n a l l y , . patches of  i n t r a p l a s t i d m e t a b o l i t e s are seen In a s s o c i a t i o n w i t h t h y l a k o i d s t a c k s ( f i g u r e 177, white  arrow).  An i n c r e a s e i n the number and s i z e of p l a s t o g l o b u l i seems t o take p l a c e ( f i g u r e s 166,  167,  169).  The most conspicuous changes o c c u r r i n g i n the c h l o r o p l a s t s are the budding o f f of p o r t i o n s of the p l a s t i d s . D i f f e r e n t steps i n the f o r m a t i o n and r e l e a s e of these p l a s t i d - d e r i v e d bodies are shown i n f i g u r e s 169, 179,  180,  182,  185, and 186.  172,  178,  I t i s apparent from most of  these p i c t u r e s t h a t t h y l a k o i d s p a r t i c i p a t e i n the formation of such b o d i e s .  Release i n t o the cytoplasm of p l a s t i d - d e r i v e d  bodies i s supported by s t u d y i n g f i g u r e s 167,  169,  176,  181  (arrowheads), where formations s i m i l a r to those r e p o r t e d above are found e i t h e r near p l a s t i d s or elsewhere i n the cytoplasm. Release of these formations seems t o i n v o l v e a process of budding 180  ( f i g u r e s 169,  172,  178,  185).  Figures  and 182 suggest t h a t f u s i o n of the outer membrane  d e l i m i t i n g these bodies w i t h the c h l o r o p l a s t membrane may  envelope  a l s o be important i n a c h i e v i n g t h e i r  release.  The f a t e of these bodies i n the cytoplasm c o u l d not be f o l l o w e d , but the sequence of events and the m o r p h o l o g i c a l as w e l l as enzymatic  c h a r a c t e r i s t i c s of " c e l l type  suggest t h a t they become degenerated cytoplasmic i n c l u s i o n s .  #3"  along w i t h a l l the other  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 r e d u c t i o n i n p l a s t l d . volume.  The  chloroplast  boundary becomes e x t r e m e l y . v a r i a b l e . While some.micrographs still  suggest the presence of both the envelope membranes  and the c h l o r o p l a s t E.R.  ( f i g u r e 177), o t h e r s show no  i n d i c a t i o n . o f the c h l o r o p l a s t E.R.,  o u t s i d e the usual, double  33 membrane envelope  ( f i g u r e s 185,  186).  At other p l a c e s ,  o n l y one membrane o f the c h l o r o p l a s t envelope ( f i g u r e 182).  remains  There i s a l a r g e number of v e s i c l e s  a s s o c i a t e d w i t h such a membrane, suggesting, the v e s i c u l a t i o n of  the outer membrane of the c h l o r o p l a s t  Pyrenoid.  envelope.  Pyrenoids are observed a t t h i s stage of  ( f i g u r e s 166,  167,  " c e l l type #3"  169).  senescence  Although they were not found i n  (see next s e c t i o n ) , the steps i n v o l v e d i n  p y r e n o i d d i s o r g a n i z a t i o n c o u l d not be f u l l y  elucidated at  p r e s e n t , due t o the s c a r c i t y w i t h which t r a n s i t i o n a l were found.  C e r t a i n micrographs  ( f i g u r e 168,  stages  arrowheads),  however, suggested the p o s s i b i l i t y of pyrenoids becoming is.olated from c h l o r o p l a s t s . 183)  Other micrographs  (i.e.,  showed p y r e n o i d a l s t r u c t u r e s i n s i d e v a c u o l e s .  theless, i t i s d i f f i c u l t  figure  Never-  t o a s c e r t a i n i f such images r e p r e s e n t  t r u e stages i n p y r e n o i d d e s t r u c t i o n or are simply the r e s u l t of  p e c u l i a r planes of s e c t i o n i n g .  Cell wall.  The c e l l wall, shows most of the f e a t u r e s a l r e a d y  mentioned i n " c e l l type E)  THE  #2".  SENESCENT CELLS—ULTRASTRUCTURAL FEATURES  OF THE This c e l l  "CELL TYPE  #3"  type r e p r e s e n t s the l a s t step i n  and leads t o t o t a l degeneration and c e l l N u c l e i and dictyosomes reduced t o remnants.  senescence  death.  are never d e t e c t e d .  The E.R.  C h l o r o p l a s t and m i t o c h o n d r i a are  r e c o g n i z a b l e , but show unmistakable  is still  s i g n s of d i s o r g a n i z a t i o n .  The background cytoplasm i s a l s o h i g h l y d i s o r g a n i z e d ( f i g u r e s 187, of  ribosomes  188,  189,  196,  201, and 202).  The  presence  cannot be determined with.accuracy.  At very  advanced stages the plasmalemma-seems t o have vanished ( f i g u r e 206), and the disappearance of the c y t o p l a s m i c matrix i s almost complete of  a c i d phosphatase  ( f i g u r e s 191,  206).  a c t i v i t y show a uniform  Studies  distribution  34 of r e a c t i o n products throughout the c e l l c a v i t y 191,  206). R e a c t i o n products are absent from the c o n t r o l  sections  ( f i g u r e 203).  Mitochondria. 196, in  (figures  M i t o c h o n d r i a l remnants are observed  (figures  197, 201, 202). These are round i n ' p r o f i l e . a n d  s i z e , w i t h none or very l i t t l e m a t r i x .  envelope i s d i s r u p t e d  i n places  Vesicular  many s t i l l  structures,  small  The m i t o c h o n d r i a l  ( f i g u r e s 201, 202). attached t o the i n n e r  membrane, r e p r e s e n t the remains o f the c r i s t a e  ( f i g u r e s 19 6,  197, 202). Chloroplasts.  The morphology o f the p l a s t i d s  a c c o r d i n g t o the plane o f s e c t i o n i n g 189),  ( f i g u r e s 187, 188,  and/or the degree o f degeneration  198).  varies  ( f i g u r e s 191,  As senescence proceeds, these o r g a n e l l e s  show a  tendency t o round up ( f i g u r e 191). The stroma has almost completely disappeared  ( f i g u r e s 190, 191), except i n  c e r t a i n areas between t h y l a k o i d s remains o f the c h l o r o p l a s t membrane as d e s c r i b e d  ( f i g u r e s 193, 194). The  envelope c o n s i s t o f a s i n g l e  i n the l a s t s e c t i o n  ( f i g u r e s 190, 198).  In more advanced stages o f degeneration t h i s envelope membrane breaks down i n p l a c e s ,  leaving  thylakoids  c o n t a c t w i t h the remains o f the cytoplasm The but  3-thylakoid  band p a t t e r n  is still  Subsequently, t h y l a k o i d  recognizable,  ( f i g u r e s 192, 200).  breakdown begins  198). Conspicuous d e p o s i t i o n  observed  ( f i g u r e s 193, 194,  of p l a s t o g l o b u l i i s a l s o  ( f i g u r e s 188, 189, 195, 199).  v a r i a t i o n i n the e l e c t r o n o p a c i t y seen  ( f i g u r e 191).  there i s a tendency f o r the bands t o clump and form  stacks o f up t o 40 or more t h y l a k o i d s 195,  In a d d i t i o n , a  o f p l a s t o g l o b u l i can be  ( f i g u r e s 195, 199). Genophore-like r e g i o n s 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 but p y r e n o i d s are t o t a l l y Cell wall. but  i n direct  ( f i g u r e 200, g ) ,  absent.  Plasmodesmata possess a c i d phosphatase a c t i v i t y ,  enzyme a c t i v i t y i s absent from c o n t r o l s e c t i o n s (see  35 f i g u r e s 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 e x h i b i t s features, s i m i l a r t o those r e p o r t e d  i n " c e l l type #2"  of c e l l w a l l degeneration.  ( f i g u r e 188) , there are s i g n s  In some c e l l s , a l o o s e n i n g o f  the packing o f the w a l l f i b r i l l a r m a t e r i a l leads t o the appearance o f . l a c u n a - l i k e spaces  ( f i g u r e 184).  the d i s o r g a n i z a t i o n of the outer  layer of t h e . c e l l wall i s  apparent  ( f i g u r e 207).  Figure  208 i l l u s t r a t e s  In other  cells  another  step i n c e l l w a l l degeneration, where only the i n n e r l a y e r of the w a l l seems t o remain. F)  ENZYME LOCALIZATION  In order  t o gain a b e t t e r understanding o f the  processes o f 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 s e v e r a l enzyme systems was c a r r i e d out' a t the u l t r a s t r u c t u r a l l e v e l i n the v a r i o u s c e l l types d e s c r i b e d .  The enzyme  systems s t u d i e d i n c l u d e d a c i d phosphatase, a l r e a d y elsewhere, c a t a l a s e , p e r o x i d a s e , and adenosine The  reported  triphosphatase.  f o l l o w i n g are the r e s u l t s o f the study.  Catalase.  I t appears t h a t i n young c e l l s  ( c e l l type #1),  c a t a l a s e r e a c t i o n s were p r i m a r i l y l o c a l i z e d i n microbodyl i k e structures  ( f i g u r e s 209, 210), although m i t o c h o n d r i a  a l s o showed some d e p o s i t i o n of a m i n o t r i a z o l e - t r e a t e d  ( f i g u r e 212).  Control  sections  m a t e r i a l showed no d e p o s i t i o n of  r e a c t i o n products i n m i c r o b o d i e s , but m i t o c h o n d r i a had some ( f i g u r e 211). Microbody-like  organelles  are s i n g l e membrane-  bounded and possess a g r a n u l a r ..matrix . ( f i g u r e s 211, 215). In some cases,, a n o n - c r y s t a l l i n e . core..is a l s o apparent ( f i g u r e 215, arrow). ... When, .the ..cor.e...is„,present,. r e a c t i o n products seem.to be p r e f e r e n t i a l l y a s s o c i a t e d w i t h i t as w e l l as w i t h the d e l i m i t i n g membrane.(figure 249).  Itis  i n t e r e s t i n g t o note the mlcrobody-mitochondrion a s s o c i a t i o n ( f i g u r e s 209, 211, 213).  Microbody-like  structures  also  36  seem t o b e p r e s e n t  i n "cell  type  #2". ( f i g u r e  head) , b u t w e r e never, d e t e c t e d i n " c e l l  213,  type  #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 deposition results  of reduced i ti s likely  enzyme a c t i v i t y Peroxidase. primarily  In "cell  type  detected  i n "cell  type  The p r e s e n c e  wall  and  (figures  OsO^.  214, 2 1 6 ) .  conclusions.  inhibition 1-2"  of  activity i s Figure  on t h e m a t e r i a l .  Deposition of reaction  i n the "transitional  result  b e t w e e n DAB  s u b s t a n t i a t e such  i s found.  similar  (figure 211),  #1" p e r o x i d a s e  o f KCN  shows  209), but  i s not.the  associated with the c e l l  experiments  reaction  that this  b u t due t o a r e a c t i o n  218 shows t h e e f f e c t found  (figure  are obtained i n control material  therefore  Control  products  arrow-  cells.  products  No  No  are also  depositioni s  #3."  of peroxidase  i n "cell  type  #2"  cannot  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 large cell and in the  amounts o f o s m i o p h i l i c wall  fibrillar  222 show p e r o x i d a s e "cell  type  Adenosine  respectively,  F i g u r e s 221,  and c o n t r o l  thus  further  experiments  demonstrating  i n determination of the existence of Material + -++  c o n t a i n i n g K - N a -Mg of reaction  products  (cell  -ATP  type  shows  (figures  experiment..  #1)  on t h e plasma  223, 225).  incubated  conspicuous  223, 2 2 5 ) , on m i t o c h o n d r i a l membranes  i n thylakoids  control  144).  localization  triphosphatase. +  a medium  (figures  (figure  activity.  depositions and  #2",  difficulties  peroxidase  in  material  m e t a b o l i t e s i n between t h e  membrane (figure 224),  F i g u r e 228 shows t h e  The. a b s e n c e o f r e a c t i o n  products i s  apparent. Deposition of reaction "transitional activity certainty  1.-2" c e l l s  i n "cell  type  products, i s observed i n  (figure  #2"  cannot  2 2 9 ) , b u t t h e enzyme be determined: w i t h  due t o t h e c h a r a c t e r i s t i c s  230 s h o w s a c o n t r o l  for "transitional  of these 1-2"  cells.  cells;  the  Figure  37 a b s e n c e o f r e a c t i o n , products., i s . e v i d e n t . . A T P a s e . a c t i v i t y was  absent The  from  Mg  "cell  type  #3".  -ATP i n c u b a t e d m a t e r i a l shows ..in  r e a c t i o n p r o d u c t s .within the m i t o c h o n d r i a  " c e l l , type  (figure  plasmalemma and. i n t h y l a k o i d s ( f i g u r e . 227.) .  #1"  226), on t h e  However, t h e  amount o f d e p o s i t i o n d o e s n o t seem to. be s o i n t e n s e as i n t h e case of the K -Na -Mg -ATP-dependent +  +  of  reaction products  in  a medium w i t h o u t ATP  are apparent but  #2".  (figure  G)  232)..  i n the " t r a n s i t i o n a l  of " c e l l  Reaction  1-2"  cells  products  (figure  to confirm t h e i r presence  Reaction products  organelles  Deposition  s e a s e s when, t h e m a t e r i a l i s i n c u b a t e d  again i t i s d i f f i c u l t  type  system.  + +  type  219), i n "cell  are missing i n the c e l l  #1".  ULTRASTRUCTURAL IDENTIFICATION OF AGING PIGMENT  Many i n c l u s i o n s  found  present i n Ectocarpus material  i n lysosome-like  resemble  (aging pigment),  the s o - c a l l e d  1961;  i n both protozoan  Strehler, In  and a n i m a l  cells  adapted  o f the pigment  (Hendy, 1971)  to  A t the. e l e c t r o n microscope  #2"  aged  231).  Reactions  (figure  l e v e l the  modified,, p r o c e d u r e .showed, t h e l a b e l l i n g  take p l a c e p r e f e r e n t i a l l y  type  as t h e c e l l  i n c r e a s e i n lipofuscin-reacting... substances  of Fontana's  (figure  were u s e d i n  study.  o c c u r r e d , ( f i g u r e . . 26) .. use  Rudzinska,  for ultrastructural  L i g h t m i c r o s c o p e . s t u d i e s showed t h a t a striking  (e.g.,  f o r such m a t e r i a l i n a g i n g c e l l s o f  E c t o c a r p u s , two t e c h n i q u e s the p r e s e n t  features of the aging  1962).  order, t o t e s t  identification  lipofuscin  w h i c h i s c o n s i d e r e d t o be one o f  t h e most i m p o r t a n t m o r p h o l o g i c a l process  structures  233).  i n .vacuolar, i n c l u s i o n s  increase significantly Furthermore,  labelling  in "cell i s restricted  38 to v a c u o l a r i n c l u s i o n s and does not.occur i n the. p l a s t i d drived metabolites  ( f i g u r e 233, arrows).  Schorml's m o d i f i e d  prodedure increases, the e l e c t r o n o p a c i t y of. some, v a c u o l a r inclusions  ( f i g u r e 235).  a f t e r formaldehyde  F i g u r e 234 shows v a c u o l a r i n c l u s i o n s  fixation.  39 DISCUSSION PART I — T H E YOUNG CELLS (CELL TYPE  #1)  C e l l s of Ectocarpus w i t h i n a f i l a m e n t e x h i b i t p r o g r e s s i v e i n c r e a s e s i n the tendency to b i n d osmium from the apex downward, f o l l o w e d by sudden l o s s e s i n the for  affinity  osmium towards the b a s a l p a r t of the f i l a m e n t s .  Light  and e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s , supplemented by c y t o c h e m i c a l and enzymatic  s t u d i e s , have r e v e a l e d major and  p r o g r e s s i v e d i f f e r e n c e s i n the morphology and c o n d i t i o n s of c e l l s i n each f i l a m e n t . are r e c o g n i z e d .  While " c e l l type #1"  metabolic  Three main c e l l  displays ultrastructural  f e a t u r e s c h a r a c t e r i s t i c of non-senescent c e l l s 1965;  Bourne and C o l e , 1968;  1970;  B i s a l p u t r a e t a l . , 1971), " c e l l type #2"  type #3" The  show unmistakable  and  (Bouck, 1965;  1968;  L i d d l e and Neushul, 1969;  "cell  Ectocarpus  Bourne and C o l e , 196 8; Cole and L i n , C o l e , 1970;  B i s a l p u t r a et a l . ,  In the r e s t i n g stage, the nucleus i s t y p i f i e d by  inconspicuous  appearance of chromatin.  more apparent  as c e l l s approach n u c l e a r d i v i s i o n  Lin,  1969,  i s s i m i l a r to t h a t d e s c r i b e d i n other brown  algae 1971).  Cole,  senescence.  o v e r a l l morphology of the nucleus of  " c e l l type #1"  ( c f . Bouck,  Cole and L i n , 1968;  s i g n s of  types  1968;  C o l e , 1969).  Chromatin becomes  L i d d l e and Neushul  t h a t u l t r a c e n t r i f u g a t i o n . s t u d i e s of Zonaria oogonium i n d i c a t e d t h a t two  the  (Cole and  (1969) r e p o r t e d farlowii  g r a n u l a r phases, d i f f e r i n g i n  s i z e and d e n s i t y , c o u l d be r e c o g n i z e d i n the n u c l e o l u s . McCully  (1968) r e p o r t e d only one g r a n u l a r component i n Fucus  n u c l e o l i . . Bourne and Cole  (19 68) d e s c r i b e d  Phaeostrophion  i r r e g u l a r e n u c l e o l i as g r a n u l a r . s t r u c t u r e s , w h i l e the n u c l e o l i of Eudesme were d e s c r i b e d as i r r e g u l a r dense s t r u c t u r e s (Cole, 1969). .In Ectocarpus  sp., n u c l e o l i have two  distinct  components, which i n morphology and dimensions c l o s e l y  40 resemble  those d e s c r i b e d f o r n u c l e o l i of h i g h e r p l a n t s .  (Lafontaine and Chaourdinard,  1963).  A similar, organization  has been r e p o r t e d i n n u c l e o l i of. L a m i n a r i a . m e r i s t o d e r m a t i c c e l l s "(Davies e t a l . , 1973). The a s s o c i a t i o n between chromatin and the i n n e r membrane of the n u c l e a r envelope i s observed a t many s i t e s around  the n u c l e u s .  T h i s a s s o c i a t i o n has been found i n  other m a t e r i a l t o occur w i t h the a n n u l i of the n u c l e a r pores complex  (Comings and Okada, 1970a; DuPraw, 1970).  Neverthe-  l e s s , evidence a l s o e x i s t s t h a t such r e l a t i o n s h i p s c o u l d take p l a c e a t s i t e s other than the a n n u l i (Comings and Okada, 1970b). nucleoplasm of  The f i b r i l l a r m a t e r i a l d e t e c t e d i n the  i n c l o s e r e l a t i o n s h i p w i t h the i n n e r membrane  the n u c l e a r envelope  c o u l d , a c c o r d i n g t o i t s dimensions,  be homologous t o chromatin f i b e r s of i n t e r p h a s e n u c l e i ( Z i r k i n and Kim, The  1972).  f u n c t i o n a l s i g n i f i c a n c e of chromatin-nuclear  envelope a s s o c i a t i o n s has long been debatable. authors 1970)  Several  (e.g., Comings and.Okada, 1970a, 1970b; DuPraw,  have p o s t u l a t e d t h a t such an a s s o c i a t i o n c o u l d be  important i n determining arrangements of the  chromatin  f i b e r s p r i o r t o t h e i r condensation i n t o chromosomes; thereby i n f l u e n c i n g n u c l e a r d i v i s i o n .  Other  authors  suggest t h a t t h i s r e l a t i o n s h i p c o u l d have an e f f e c t o r g a n i z a t i o n and d i s t r i b u t i o n of n u c l e a r pores 1971; The  T h a i r and Wardrop, 1971;  on  (Maul e t a l . ,  T e i g l e r and Baerwald,  1972).  l a t t e r h y p o t h e s i s c o u l d be important i n e x p l a i n i n g  n u c l e a r pore arrangements i n Ectocarpus.. In E c t o c a r p u s , as w e l l as i n D i c t y o t a (Neushul and Dahl, 1972a), f r e e z e etched p r e p a r a t i o n s .of the n u c l e a r envelope show.zones of u n i f o r m l y arranged pores- a l t e r n a t i n g w i t h p o r e - f r e e . r e g i o n s . Recent  s t u d i e s on brown a l g a l , n u c l e i  (Neushul and  Dahl,  1972a). have shown, t h a t the s t a i n i n g p r o p e r t i e s of the p e r i p h e r a l nucleoplasm vary w i t h changes i n n u c l e a r  41 metabolism.  I t i s p o s s i b l e , therefore,, t h a t the  internal  a r c h i t e c t u r e of the nucleus i n f l u e n c e s t h e . p a t t e r n n u c l e a r pores d i s t r i b u t i o n .  The  of  absence of n u c l e a r  pores  from the r e g i o n of the n u c l e a r envelope a s s o c i a t e d w i t h n u c l e o l u s i s . a l s o known i n other m a t e r i a l  (LaCour  Wells,  ubiquitous  1972).  Although n u c l e a r pores are  s t r u c t u r e s , t h e i r f i n e o r g a n i z a t i o n has completely  e l u c i d a t e d , and  been c u r r e n t l y proposed Wunderlich and  the  and  not y e t been  s e v e r a l s t r u c t u r a l models have  (e.g., LaCour and W e l l s ,  Speth, 1972).  1972;  Furthermore, Franke  (1970) has  suggested t h a t s t u d i e s on n u c l e a r pore s t r u c t u r e should extended to the 2 nm drawn.  l e v e l before  conslusions  can  be  D i s c u s s i o n of n u c l e a r pore o r g a n i z a t i o n i n Ectocarpus  i s t h e r e f o r e unwarranted at As  any  be  present.  i n the case of D i c t y o t a  (Neushul and Dahl, 1972a) ,  f r e e z e - e t c h s t u d i e s of the n u c l e a r envelope r e v e a l i n Ectocarpus two outer and  d i s t i n c t f r a c t u r e f a c e s , one  f r e e z e - e t c h images d i f f i c u l t . complicated  The  (DaSilva and Branton, 1970;  f a v o r s the l a t t e r h y p o t h e s i s . f r a c t u r e planes  i n t e r f a c e or passes  of the membranes.  T e i g l e r and Baerwald, 1972;  T i l l a c k and  Monneron e t a l . ,  are a t the cytoplasm-membrane i n t e r f a c e ,  Robinson and Preston,  Lewis  (1971—see a l s o  1972).  of membranous m a t e r i a l by the  envelope seems t o be  fairly  (e.g., Cole and L i n , 1968; Walker, 1971;  1972)  However, the p o s s i b i l i t y  r e v i v e d by T h a i r and Wardrop  Production  Recent Marchesi,  already been defended by Northcote and  (1968), was  the  on whether the f r a c t u r e  along the o r g a n e l l e - c y t o p l a s m  through the hydrophobic regions  which has  i n t e r p r e t a t i o n of  The  problem i s f u r t h e r  by c o n f l i c t i n g o p i n i o n s  plane occurs  1970;  the  the other the i n n e r membrane of the envelope.  s c a r c i t y of data, however, makes any  evidence  representing  Neushul and  nuclear  common i n brown a l g a l McCully, 1968;  Dahl, 1972a).  Bouck, In the  cells 1969;  present  that  42  s t u d y e i t h e r v e s i c u l a r o r more complex membranous...structures are  observed, but t h e i r  functions  arid f i n a l  fates, are  uncertain. Continuity  between t h e o u t e r  e n v e l o p e and t h e p e r i n u c l e a r Perinuclear Massalski has the of  and L e e d a l e , that  intense  since  portions  1972).  (e.g.,  Palandri  (1972)  T h i s m i g h t a l s o be  sections with  crystalline  such  deposits  arrangements inside  o f t h e E.R. b e t w e e n t h e E.R. and t h e o u t e r  the mitochondrial  as w e l l  algae  E.R. a r r a n g e m e n t m i g h t  activities.  t h e E.R. u s u a l l y c o n t a i n Continuity  of  the p e r i n u c l e a r  case f o r Ectocarpus,  dilated  i n other  1969; P a l a n d r i ,  metabolic  of the nuclear  E.R. i s f r e q u e n t l y , o b s e r v e d .  E.R. h a s . b e e n d e t e c t e d  suggested  indicate  membrane  membrane  envelope has been o b s e r v e d i n E c t o c a r p u s  as i n h i g h e r  plant  and a n i m a l c e l l s  (Bracker  and  G r o v e , 1971; M o r r e e t a l . , 1971; F r a n k e and K a r t e n b e c k , Since  details  chondrial Grove that  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-  a s s o c i a t i o n s were f u l l y  discussed  (1971) and F r a n k e and K a r t e n b e c k  considerations  will  be n e c e s s a r y h e r e .  all  the c i s t e r n a l  the  cisternal  cytomembranes s h o u l d  by B r a c k e r and  (1971) , no d e t a i l e d They p o i n t e d o u t  the concept of a continuous channeling  system i n v o l v i n g  be e x t e n d e d t o i n c l u d e  compartment b o r d e r e d by t h e o u t e r  inner mitochondrial valid,  membranes.  and t h e  I f s u c h an a s s u m p t i o n i s  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  to include chloroplast  envelope,  The.structures  since  i t s c o n t i n u i t y with the s p a c e i s o b v i o u s in.. E c t o c a r p u s .  designated  as o s m i o p h i l i c  bodies are abundant.in c e r t a i n c e l l s . possible  concept  t h e s p a c e b e t w e e n t h e two membranes o f t h e  c h l o r o p l a s t E.R. c i s t e r n a l  associated  1971).  Their  structured origin.is  closely  w i t h t h e c h l o r o p l a s t . E.R. .... I n some. s e c t i o n s  to follow  However, t h e i r  their  function  release  i n t o the paramural  i s unknown.  space.  These s t r u c t u r e s  correspond t o the s o - c a l l e d physodes o f other  i t is  authors.  could  43 (FeId-man and G u g l i e l m i ,  19 72).  Pore- and b r i d g e - l i k e formations s i m i l a r , t o those r e p o r t e d i n other p l a n t m a t e r i a l . (Franke.and  Scheer, 1972;  Franke e t a l . , 1972). a r e found i n Ectocarpus  dictyosomes.  In a l l other aspects the Ectocarpus dictyosomes are m o r p h o g i c a l l y s i m i l a r t o those f©und i n other brown algae (Bouck, 1965; Bourne and C o l e , 1968; Cole and L i n , 1968; McCully, 1968; C o l e , 1969, 1970; B i s a l p u t r a e t a l . , 1971). A d i s t i n c t a s s o c i a t i o n between dictyosomes i s very much i n evidence. perinuclear  and n u c l e i  D i r e c t c o n t i n u i t y between the  space and dictyosomes  can be seen, but i n g e n e r a l ,  the r e l a t i o n s h i p between these two membrane systems i s i n d i c a t e d by the t r a n s f e r o f s m a l l v e s i c l e s from the outer membrane o f the n u c l e a r envelope dictyosomes  t o the formative face o f  (Bouck, 1965; Bourne and C o l e , 1968; Cole and  L i n , 1968; C o l e , 1969, 1970; B i s a l p u t r a e t a l . , 1971). a d d i t i o n dynamic i n t e r a c t i o n s take p l a c e between and pyrenoids and vacuoles  ( f i g u r e s 60, 63), m i t o c h o n d r i a ( f i g u r e 112). Of p o s s i b l e  the dictyosome-pyrenoid  In  dictyosomes  ( f i g u r e 61)  s i g n i f i c a n c e are  i n t e r a c t i o n s , which c o u l d 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 t r a n s f e r o f photosynthates t o dictyosomes.  I t i s possible  t o assume t h a t such a d i r e c t  t r a n s f e r route c o u l d a l s o a f f e c t m e t a b o l i t e composition i n r e l a t i o n t o the photosynthates  t r a n s f e r r e d v i a the  c h l o r o p l a s t E . R . - p e r i n u c l e a r space-dictyosome (Bouck, 1965) . perinuclear authors  pathway  Dictyosomes i n Ectocarpus are e x c l u s i v e l y  i n p o s i t i o n , a f e a t u r e t h a t a c c o r d i n g t o some  (Bouck, 1965; .Cole and L i n , 1968; C o l e , 19 69) may be  of p h y l o g e n e t i c s i g n i f i c a n c e . The f i n e s t r u c t u r e o f m i t o c h o n d r i a in. a c t i v e  cells  of Ectocarpus. i s s i m i l a r , t o that, r e p o r t e d in. o t h e r brown algae  (Bouck, 1965; Bourne and Cole, 1968,; Cole and L i n , 1968;  Cole, 1969; L i d d l e and Neushul, Ectocarpus c h l o r o p l a s t  1969).  fine structure i s also  44 s i m i l a r to that reported Bourne and Cole,  f o r other brown algae  (Bouck, 1965;  1968; Cole and L i n , 1968; Evans, 1968;  B i s a l p u t r a and B i s a l p u t r a , 1969; C o l e ,  1969, 1970; L i d d l e and  Neushul, 1969; B i s a l p u t r a e t a l . , 1971), except f o r the presence o f f i l a m e n t s  and pores i n the t h y l a k o i d s .  Filament-  l i k e elements are seen j o i n i n g t h y l a k o i d s o f the same o r neighboring position.  t h y l a k o i d bands as w e l l as i n an i n t r a l o c u l a r So f a r as I am aware, t h i s i s the f i r s t r e p o r t o f  these s t r u c t u r e s i n brown a l g a l c h l o r o p l a s t s . Staehelin  (1966), a p p l y i n g  freeze-etching  Nevertheless,  techniques,  able t o r e p o r t i n C h l o r e l l a , the presence of f i b r i l s  was 4 nm i n  diameter running from the plasmalemma through the ground cytoplasm i n t o the c h l o r o p l a s t .  In a l a t e r work, the same  author was able t o show t h a t i n Cyanidium caldarium fibrils  linked photosynthetic  filaments  also traversed  ( S t a e h e l i n , 1967).  lamellae  t o one another.  study, although  elements were found t o be v a r i a b l e ,  the mean o f the averages c l o s e l y approach those by S t a e h e l i n  (1966, 1967).  Cole,  reported  In Ectocarpus as w e l l as i n  other brown a l g a l c h l o r o p l a s t s and  The  the l o c u l i o f the t h y l a k o i d s  In the p r e s e n t  dimensions o f f i b r i l l a r  similar  (e.g., Bouck, 1965; Bourne  1968; Cole and L i n , 1968; Cole,  1969) the space  between t h y l a k o i d s i n each band and between t h y l a k o i d bands as w e l l as the r e l a t i o n s h i p of the p e r i p h e r a l band t o the c h l o r o p l a s t envelope show s t r i k i n g u n i f o r m i t y .  Iti s ,  t h e r e f o r e , p o s s i b l e t h a t the f i b r i l l a r  described  elements  above c o u l d p l a y an important r o l e i n m a i n t a i n i n g r e g u l a r i t i e s w i t h i n the c h l o r o p l a s t s . expressed a s i m i l a r p o i n t o f view.  Staehelin  space (1967) has  T h i s p o i n t o f view  would f i t i n t o a broader concept, where establishment  of  s t r u c t u r a l u n i f o r m i t y by membrane t o membrane l i n k a g e s may be p h y s i o l o g i c a l l y advantageous  (e.g., Franke e t a l . , 1971a,  1971b, 1972). P o r e - l i k e i n t e r r u p t i o n s are observed In the  45  t h y l a k o i d membranes.  These i n t e r r u p t i o n s are p r o b a b l y  not  r e s t r i c t e d . t o Ectocarpus c h l o r o p l a s t s , but t h e i r presence has  not y e t been r e p o r t e d .  I t i s not known i f . t h e y are  permanent f e a t u r e s .of the t h y l a k o i d membranes. Freeze-etch  s t u d i e s 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 t h y l a k o i d membranes were r e c e n t l y c a r r i e d out  (Neushul, 1971) .  Based on these s t u d i e s , two  f r a c t u r e faces are r e c o g n i z e d Freeze-etch  types of  i n the brown a l g a D i c t y o t a .  images of Ectocarpus t h y l a k o i d s seem to  s i m i l a r to those i n D i c t y o t a . e a r l i e r studies  The  be  exposed f a c e s , as shown i n  (Meyer and Winkelmann, 1969;  Neushul, 1970),  seem t o r e s u l t from s p l i t t i n g of the t h y l a k o i d membranes. Further  comments on the s i g n i f i c a n c e of these f i n d i n g s  seem unwarranted here, s i n c e the s u b j e c t was considered  by Neushul  Concentric  thoroughly  (1971).  l a m e l l a r bodies of probable p l a s t i d  were found i n the cytoplasm of the red algae and Weier, 1970) . (1969) r e p o r t e d  Dawes and  Rhamstine  origin  (e.g. Brown  (1967) and  Sabnis  the presence of s p h e r i c a l l a m e l l a r bodies  i n the cytoplasm of the green a l g a Caulerpa p r o l i f e r a , whose c h a r a c t e r i s t i c s a l s o p o i n t towards a p l a s t i d S t r i k i n g thylakoid patterns Leathesia al.,  have been r e p o r t e d  difformis sporelings  1968), but t h i s was  process of p l a s t i d In the present  origin. i n p l a s t i d s of  (Cole and L i n , 1968;  found to be r e l a t e d to a s p e c i a l  division. study, c o n c e n t r i c l a m e l l a r bodies were  shown to have a p l a s t i d o r i g i n . c o n c e n t r i c bodies i n t o . v a c u o l e s  Incorporation  of  massive t r a n s p o r t of p l a s t i d m a t e r i a l to the where, a f t e r d i g e s t i o n i n l y s o s o m a l - l i k e products can be recovered  and  the  w i t h a s s o c i a t e d a c i d phos-  phatase a c t i v i t y suggests t h a t t h i s p r o c e s s c o u l d fashion,  r e u t i l i z e d by the  (3 thylakoids/band) were recorded  facilitate  cytoplasm,  in  the  cells.  A l t e r a t i o n s of the b a s i c p a t t e r n of t h y l a k o i d arrangement  Cole e t  t  46 Chorda f H u m  ( Bouck,1965 ), Fucus serratus.and  canaliculata  ( Evans, 1968: ) , L e a t h e s i a J d l f formls.. ( dole  Lin,1968 ).. In E c t o c a r p u s , d e v i a t i o n s pattern  Pelvetia  from  a l s o occur. These seem to be due  and  the.basic  e i t h e r to  the  exchange of t h y l a k o i d s between adjacent bands or to  the  b i f u r c a t i o n and merging of n e i g h b o r i n g bands. The  c l o s e r e l a t i o n s h i p between t h y l a k o i d membranes  and p l a s t o g l o b u l i i n d i c a t e s the p o s s i b i l i t y of involvement i n p l a s t o g l o b u l a r p r o d u c t i o n , known i n chromoplasts 1970  ) and  a phenomenon w e l l  ( e. g. Harris,1970;  p l a s t i d senescence  thylakoid  Lichtenthaler,  ( e. g. L i c h t e n t h a l e r , 1 9 6 8 ).  In both cases, however, p l a s t o g l o b u l i formation seems to the r e s u l t of t h y l a k o i d breakdown. In Ectocarpus " c e l l #1"  no  signs of t h y l a k o i d breakdown are e v i d e n t ,  type  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 excess of l i p i d p r o d u c t i o n assembly. The by  over the r a t e of  be  an  thylakoid  l i p i d nature of p l a s t o g l o b u l i i s  corroborated  t h e i r d e s t r u c t i o n a f t e r permanganate f i x a t i o n . However,  Magne (1971) has  r e c e n t l y shown t h a t i n a wide range of  p l a n t s , i n c l u d i n g Ectocarpus c r o u a n i i , i n s o l u b l e saccharides Therefore,  are p r e s e n t at the p l a s t o g l o b u l a r  poly-  sites.  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 r e l a t e d to t h y l a k o i d assembly t h e i r r o l e i n c h l o r o p l a s t metabolism more e l a b o r a t e d previously  and  than  supposed.  Nowhere e l s e has  such a wide range of v a r i t i e s  of  c h l o r o p l a s t d i v i s i o n been observed i n the same p l a n t as i n Ectocarpus  . T h i s could be the r e s u l t of an e x t e n s i v e  of a l l the c e l l s i n sequence along the f i l a m e n t s e r e c t and  the p r o t r a t e . s y s t e m s o f . E c t o c a r p u s sp.,  of both  i s . t h a t images.suggesting  and  plastid  d i v i s i o n . , i n most.cases r e f l e c t p e c u l i a r planes of s e c t i o n i n g through m u l t i l o b e d  chloroplasts  the  rather  than l i m i t i n g the s t u d i e s to. c e r t a i n , c e l l s . The. other more l i k e l y e x p l a n a t i o n  study  ( figure.4  ),  47 where s e v e r a l i n t e r p r e t a t i o n s are possible.. f i g u r e s 102  and  103  are without q u e s t i o n  However,  v i s u a l i z a t i o n s of  t r u e stages of chloroplas.t d i v i s i o n . . ..This c o n c l u s i o n based not only on the  is  study, o f . E c t o c a r p u s •material i n  s e r i a l s e c t i o n s , but a c t u a l l y these.images match c h l o r o p l a s t d i v i s i o n images.in the brown a l g a . S p h a c e l a r i a . and B i s a l p u t r a , 1970) .  (Bisalputra  I t seems apparent from the  present  s t u d i e s t h a t an i n t e r p r e t a t i o n of images suggesting c h l o r o p l a s t 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 a c c u r a t e evidence without i n t e n s i v e The  general  that described for  a  morphology of the pyrenoid  i n other brown algae  studies.  agrees w i t h  (see G r i f f i t h s ,  1970,  review). The  pyrenoid  body contains  i s highly reminiscent pyrenoids  f i b r i l l a r m a t e r i a l which  of t h a t of P y l a i e i l a  (Gibbs, 1962a).  In the p r e s e n t m a t e r i a l ,  have a c l e a r l y p o s i t i v e p r o t e i n r e a c t i o n 19 70).  No  DNA  or RNA  littoralis pyrenoids  (see a l s o Rosowski,  r e a c t i o n s were detected  i n the  pyrenoids  of E c t o c a r p u s , although the presence of both substances have been r e p o r t e d Arnott,  1970)  and  i n Tetracystis excentrica i n other a l g a l pyrenoids  (Brown  (Esser,  and 1967;  Simon, 19 54) . Pyrenoids appear i n Ectocarpus as p r o j e c t i n g b o d i e s , which u s u a l l y a r i s e from the s i d e of the f a c i n g the nucleus. have p h y l o g e n e t i c Bourne and  Cole,  This type of pyrenoid  implications 1968;  .In.Ectocarpus sp.,  stalked chloroplast  i s suggested to  (e.g., Evans, 1966,  1968;  H o r i , 1971). u s u a l l y only one  per c h l o r o p l a s t . .Cases, of two  pyrenoid  i s found  or.more pyrenoids o c c u r r i n g  i n the same c h l o r o p l a s t appear., to. be. .related, to. .the process of pyrenoid  division.  I t i s apparent from. this.study, t h a t  sometimes a very c l o s e r e l a t i o n s h i p e x i s t s between f i b r i l l a r , elements of the pyrenoid membranes.  The  matrix and  the  the thylakoid  meaning of t h i s r e l a t i o n s h i p r e q u i r e s  48 e l u c i d a t i o n ; the p o s s i b i l i t y e x i s t s t h a t a p h y s i o l o g i c a l involvement of t h y l a k o i d s i n p y r e n o i d development might occur. T h i s r e l a 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 t r e a t e d m a t e r i a l , a treatment which seems t o s t i m u l a t e pyrenoid d i v i s i o n  ( Manton,1966a).  B a i l e y and B i s a l p u t r a  (19 69) have d e s c r i b e d the  u l t r a s t r u c t u r e o f the c e l l w a l l i n E c t o c a r p u s acutus. The p r e s e n t f i n d i n g s support t h e i r  description.  Lomasomes and plasmalemmasome's 1968  ( Marchant and  Robards,  ) were found i n Ectocarpus sp. d u r i n g s p o r e l i n g develop-  ment ( u n i l l u s t r a t e d data ) as w e l l as i n sporophytes. Although these s t r u c t u r e s seem t o be w e l l r e p r e s e n t e d i n algae  ( see Cole and Lin,1970,  f o r a review ), t h e i r  presence i n the brown algae has only r e c e n t l y been r e p o r t e d i n s p o r e l i n g s of P e t a l o n i a d e b i l i s Bracker  ( Cole and Lin,19 70 ).  (1967) suggested 9 d i f f e r e n t f u n c t i o n s f o r the  lomasomes. As i n d i c a t e d by Cole and L i n (1970), evidence concerning the r o l e of these s t r u c t u r e s i s mostly circumst a n t i a l , and t h e r e f o r e , d i f f i c u l t  t o s u s t a i n without  f u r t h e r i n v e s t i g a t i o n . Recent a u t o r a d i o g r a p h i c s t u d i e s , however, i m p l i c a t e these s t r u c t u r e s i n the d e p o s i t i o n of m a t r i x m a t e r i a l s i n the c e l l w a l l Plasmddesmatal i n the brown algae was  ( Cox and Juniper,1973  ).  c o n t i n u i t y between n e i g h b o r i n g c e l l s shown by B i s a l p u t r a  (1966). S i n c e the  p u b l i c a t i o n of t h i s work f u r t h e r evidence showing plasmodesmatal c o n t i n u i t y i n these algae has r a p i d l y  accumulated.  The o r g a n i z a t i o n of these s t r u c t u r e s i n the c r o s s w a l l s a d j o i n i n g n e i g h b o r i n g c e l l s has been suggested as phylogenetically significant  ( e. g. Cole and Lin,1968; Hori,1971  Plasmodesmata are a l s o observed i n Ectocarpus sp. where the l a c k of o r g a n i z a t i o n . i n t o p i t . a r e a s i s apparent. In  c o n c l u s i o n , the u l t r 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 t h a t r e p o r t e d i n other brown a l g a e . The young c e l l s are t y p i c a l l y m e r i s t e m a t i c  ).  49 or the immediate d e r i v a t i v e of €hese. These c e l l s are c o n f i n e d t o the f i r s t  6-8  c e l l s - i n each f i l a m e n t . They  are c h a r a c t e r i z e d by a dense cytoplasm, w i t h a w e l l organized nucleus, mitochondria, c h l o r o p l a s t s ,  stalked  p y r e n o i d s , e x c l u s i v e p e r i n u c l e a r l o c a t i o n of the G o l g i apparatus, and t h e . n o n - o r g a n i z a t i o n of plasmodesmata i n t o pit  fields. S e v e r a l of these f e a t u r e s ( s t a l k e d p y r e n o i d morpho-  l o g y , p e r i n u c l e a r l o c a t i o n of non o r g a n i z e d i n p i t f i e l d s e a r l i e r authors  dictyosomes,  plasmodesmata  ) support the h y p o t h e s i s of  ( e.g. F r i t s c h , 1 9 4 5 ; Smith,1955 ) t h a t  Ectocarpus i s p r i m i t i v e among the brown a l g a e . N e v e r t h l e s s , other authors  ( e. g. B i s a l p u t r a e t al.,1971;  Chi,1971;  Neushul and Dahl,1972a ) have warned t h a t c a r e f u l use of the above mentioned c h a r a c t e r i s t i c s as p h y l o g e n e t i c markers i s necessary u n t i l more i n f o r m a t i o n r e g a r d i n g t h e i r behaviour and occurrence i s o b t a i n e d a t a l l developmental stages. PART I I - TRANSITIONAL "1-2"  CELLS  V a c u o l a t i o n . 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"  i n v o l v e s the process of v a c u o l a t i o n . Vacuoles  were r e p o r t e d t o a r i s e i n a v a r i e t y of ways, i n c l u d i n g d i l a t i o n s of the E.R.,  local  s p e c i a l a c t i v i t y of the G o l g i  apparatus or o t h e r s t r u c t u r e s such as m i t o c h o n d r i a , or combinations  of these  ( e. g. Marinos,1963; Barton,1965;  Ueda,1966; Mesquita,1969; M a t i l e and Moor,1968; B e l i t s e r , 1972;  Berjack,1972  ). The evidence from Ectocarpus  t h a t vacuoles are the r e s u l t of E.R. dictyosomes  activity,  indicates  although  are a l s o i n v o l v e d .  In a d d i t i o n t o t h e . w e l l known r o l e of vacuoles as r e s e r v o i r s of waste products i n p l a n t c e l l s  ( e. g. Robards,  19 70 ), the d i s c o v e r y of a wide range of a s s o c i a t e d lysosomal  50 enzymes ( e.g. led  Gahan,1969; M a t i l e , 1968,  to the concept t h a t vacuoles may  as a lysosome e q u i v a l e n t  1969a, 1969b )  function i n plant  ( e. g. Matile,1969a; H a l l  Davie,1971 ). In Ectocarpus remnants of cytoplasmic  has cells  and structu-  res i n s i d e vacuoles as w e l l as a s s o c i a t e d a c i d phosphatase a c t i v i t y are c l e a r i n d i c a t o r s of the r o l e of vacuoles lysosome  as  equivalents.  Autophagy. S e v e r a l mechanisms have been d e s c r i b e d  in  the l i t e r a t u r e to e x p l a i n the o r i g i n of v a c u o l a r i n c l u s i o n s ; t h a t i s , to e x p l a i n how i n s i d e vacuoles  m a t e r i a l becomes trapped  ( see review by Fineran,1971 ). In Ectocarpus  sp. more than one cytoplasmic  cytoplasmic  mechanism i s i n v o l v e d i n t r a p p i n g  regions  i n s i d e vacuoles.  One  of the mechanisms  i n v o l v e s the i s o l a t i o n of pockets of cytoplasm by the E. The  same process of c e l l u l a r autophagy was  other p l a n t s 1972;  reported  ( e. g. Buvat,1968; Mesquita,1972;  Coulomb,1973; Marty,1973 ). The  in  Villiers,  other mechanism of  autophagy found i n Ectocarpus i n v o l v e s i n v a g i n a t i o n o f tonoplast with  subsequent i n c o r p o r a t i o n of  m a t e r i a l i n t o the vacuoles.  cytoplasmic  ( Coulomb and  M a t i l e and Moor,1968; Wardrop,1968; M a t i l e and Belitser,1972;  the  T h i s process i s found to occur  f r e q u e n t l y i n other p l a n t c e l l s 1971;  R.  H a l l and  Buvat,1968; Winkenbach,  Davie,1971; Coulomb,1973 ).  There i s a l s o evidence suggesting  t h a t the cytoplasm might  take an a c t i v e p a r t 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 1972  described  i n Fraxinus  excelsior ( V i l l i e r s ,  ). I t i s q u i t e p o s s i b l e t h a t i n any mechanism of  i n c o r p o r a t i o n of cytoplasmic the t o n o p l a s t the process  and  m a t e r i a l i n t o vacuoles both  the cytoplasm may  take an a c t i v e p a r t i n  ( Fineran,1971 ).  Subsequent to accumulation of m a t e r i a l s  inside  vacuoles there i s . a marked r e a c t i o n of a c i d phosphatase a c t i v i t y . Progressive  a l t e r a t i o n s of the v a c u o l a r  are observed; l e a d i n g to the appearance of two  inclusions  types of  51 remnant materials..  The  geneous and comprised  f i r s t of these m a t e r i a l s i s h e t e r o -  of... a. g r a n u l a r component i n t e r m i n g l e d  w i t h a m y e l i n - l i k e component.  The second  i s simply g r a n u l a r .  M o r p h o l o g i c a l l y , t h e . " o s m i o p h i l l c s t r u c t u r e d b o d i e s " and mixed vacuole remnants are much a l i k e , although o r i g i n s are q u i t e d i s t i n c t In both time  the  their  (the "OSB"  appears  i n almost every young c e l l b e f o r e autophagy becomes d e t e c t a b l e ) and space c h l o r o p l a s t E.R.  (the "OSB"  o r i g i n a t e s from  w h i l e the mixed remnants are the r e s u l t of  autophagy and lysosomal a c t i v i t y ) . of  the  N e v e r t h e l e s s , the m a j o r i t y  e l e c t r o n - d e n s e i n c l u s i o n s accumulated  i n these  transi-  t i o n a l c e l l s can be b r o a d l y c l a s s i f i e d as " r e s i d u a l b o d i e s " , which c l o g the v a c u o l e s .  The reason f o r the c l o g g i n g of the  vacuoles i s not understood. possible explanations.  Recent work has suggested  Tappel  some  (1968) f i n d s t h a t p r o t e i n s  and membranes are l e s s 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 ,  p r o p e r t y c o n t r i b u t e s t o lysosomal engorgement. of  this  Another  evidence shows t h a t the h y d r o l a s e content of a c e l l  v a r i e s due  to the h e t e r o g e n e i t y of the lysosomal enzyme  population  (e.g., Hayashi,  Jacques,  1971;  understandable of  line  1967;  M a t i l e , 1968;  Hanker e t a l . , 1972).  S c h u l t z and  I t i s , therefore, quite  t h a t lysosomal s t r u c t u r e s may  be i n c a p a b l e  d i g e s t i n g the i n c l u s i o n m a t e r i a l , e s p e c i a l l y when t h e i r  enzyme complement i s not a p p r o p r i a t e .  This s i t u a t i o n  can  l e a d t o a permanent or temporary engorgement of v a c u o l e s . Comolli. e t a l . (1972) have a l s o suggested  t h a t lysosome  engorgement c o u l d r e s u l t from changes i n the enzymes brought  lysosomal  about.by m o d i f i c a t i o n s i n t h e i r r a t e s of  s y n t h e s i s and/or d e g r a d a t i o n . A c i d phosphatase a c t i v i t y was. found ...in.. Ectocarpus to be a s s o c i a t e d w i t h b o t h the E.R. E . R . - a s s o c i a t e d a c i d phosphatase was of  p l a n t s (e.g., Camefort, 1966;  and G o l g i apparatus. demonstrated i n a number  Catesson and  Czaninsky,  52 1968;  Figier.,1968;  Roland, 1969;' Robards and  Zee',1.969; Poux,1970; Coulomb, and G e z e l i u s , 19.72 phosphatase 1969;  ) , as was  Kidway,1969;  Coulomb., 1971.; F i g i e r , 1 9 7 2 ;  the. dlctyosome-assoclated  acid  ( e. g. Poux,1962a, 1962b; F i g i e r , 1 9 6 8 ;  Coulomb,  Halperin,1969; Roland,1969; Poux,1970; Coulomb  Coulon,1971.; F i g i e r , 1 9 7 2 ;  M i c a l e f , 1972;  Rougier,1972 ) . I t .  i s not known, however, i f the E.R.-associated tase and  and  a c i d phospha-  t h a t a s s o c i a t e d w i t h the dictyosomes possess  d i f f e r e n t h y d r o l y t i c p o t e n t i a l i t i e s ; a n d t h e r e f o r e , i f these f i n d i n g s bear any vacuolar  r e l a t i o n s h i p t o the c l o g g i n g of  apparatus.  C h l o r o p l a s t . A l s o a s s o c i a t e d with of the the  the  " c e l l type #1"  " c e l l type #2"  the  differentiation  morphology i n t o t h a t c h a r a c t e r i s t i c  of  i s a tendency f o r c h l o r o p l a s t t h y l a k o i d s  to form l a r g e s t a c k s . An i n c r e a s e i n t h y l a k o i d number w i t h aging was  a l s o r e p o r t e d i n other p l a n t s  ( e. g. McLean,1968,  Dodge,1970; Messer and Ben-Shaul,1972 ). C e l l W a l l . The  e x i s t e n c e of o s m i o p h i l i c  among the c e l l w a l l m i c r o f i b r i l s and periphery  seems to support  Armstrong and Boalch metabolites  metabolites  at the c e l l  Fogg and Boalch  wall  (1958) and  (1960) r e s u l t s f o r the r e l e a s e of  i n t o the c u l t u r e medium.  During these t r a n s i t i o n a l stages, s o - c a l l e d c e l l w a l l ingrowths Simultaneous with  (cwi)  formation  seems to reach  ingrowths formation,  of  the  a peak.  both e l e c t r o n  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 i n s o l u b l e carbohydrates by the P.A.S. r e a c t i o n ) show i n t e n s e d i c t y o somal a c t i v i t y , f u r t h e r s u p p o r t i n g  other authors r e s u l t s  t h a t dictyosomes are i n v o l v e d i n c e l l w a l l development i n the brown algae general  ( Cole,19 69)  as w e l l as i n p l a n t m a t e r i a l i n  ( e. g. Pickett-Heaps,1967a, 1967b; Wooding,1968;  Barton,1968 ).  53 PART I I I — T H E  "CELL TYPE # 2"  S e v e r a l authors c o n s i d e r i n t r a c e l l u l a r of  accumulation  l i p i d m a t e r i a l t o be symptomatic of senescence  animal 1972)  (e.g., Takah.ashi.et.al., 1970; and p l a n t c e l l s  Howse and  (e.g., McLean, 1968;  19 68; P a l i s a n o and Walne, 1972).  As one  i n both,  Welford,  Schuster e t a l . ,  s t u d i e s the Ectocarpus  c e l l s f u r t h e r away from the a p i c a l t i p , i t can be  observed  t h a t s i m u l t a n e o u s l y w i t h an i n c r e a s i n g ' t e n d e n c y to b i n d osmium there i s an i n c r e a s i n g a f f i n i t y f o r Sudan b l a c k B. microscope  s t u d i e s r e v e a l a v a r i e t y of i n c l u s i o n s  the cytoplasm.  Electron crowding  A f t e r permanganate f i x a t i o n some of these  i n c l u s i o n s are not p r e s e r v e d , w h i l e others are o n l y p a r t i a l l y preserved; f u r t h e r s u g g e s t i n g t h a t l i p i d m a t e r i a l i s p r e s e n t i n many of these i n c l u s i o n s . these c e l l s as senescent  The d e s i g n a t i o n of  seems, t h e r e f o r e , j u s t i d i e d .  i s f u r t h e r c o r r o b o r a t e d by the morphology of t h e i r  This  cell  organelles. The n u c l e a r boundary i s u s u a l l y i r r e g u l a r , a common f e a t u r e of s e n e s c i n g c e l l s i n both p l a n t s (Shaw and Manocha, 1965;  B u t l e r , 1967;  Berjack and V i l l i e r s ,  B r i a r t y e t a l . , 1970; 1972)  and animals  1972a, 1972b;  F a b b r i and P a l a n d r i , 1970;  Villiers,  (e.g., Sohal and A l l i s o n , 1971;  and C r i s t o f a l o , 1972). detect.  1970,  Lipetz  Nuclear pores are very d i f f i c u l t  to  I t i s not known from the p r e s e n t data i f t h i s i s  due t o an i n c r e a s i n g d i f f i c u l t y i n d i s t i n g u i s h i n g the pores or  i f t h i s i s the r e s u l t of a r e d u c t i o n i n the number of  pores due  to the low m e t a b o l i c s t a t e of these  cells.  V a r i a t i o n s i n the number of n u c l e a r pores due  to  the m e t a b o l i c s t a t e of the c e l l s were r e p o r t e d i n other b i o l o g i c a l m a t e r i a l (e.g... A f z e l i u s , 1955; 1959; et  Grasso.et a l . , 1962;  a l . , 1965;. Franke,  1967;  Barnes and  Davies,  Moor.and M u h l e t h a l e r , 1963; Franke and Scheer,  1970;  Wiener Wunderlich  54 Speth, 1972) . stood.  The reason for. t h e v a r i a t i o n s is. not  under-  However, as suggested by some authors, (e.g. T h a i r  and Wardrop, 1971;  La Fountaine and La Fountaine,  1973),  these v a r i a t i o n s m a y . r e f l e c t the s y n t h e t i c a c t i v i t y of the nucleus and s i m u l t a n e o u s l y i n d i c a t e the degree of exchange t h a t takes p l a c e between the nucleus and the cytoplasm.  The  evidence seems a l s o to I n d i c a t e t h a t c e l l s having a low m e t a b o l i c a c t i v i t y have a lower number of n u c l e a r pores (e.g. Barnes  and Davies, 1959;  M u h l e t h a l e r , 19 63).  Grasso e t a l . ,  1962;  Moor and  P a r t i c u l a r l y p e r t i n e n t are the r e s u l t s  obtained by Moor and Muhlethaler,(1963), who  have shown  decreases i n n u c l e a r pore number to be a s s o c i a t e d w i t h the aging process i n y e a s t c e l l s .  The n u c l e a r envelope  to produce v e s i c l e s or o t h e r membranous s t r u c t u r e s .  ceases Nucleoli,  when observed, are s i m i l a r i n o r g a n i z a t i o n t o those found in  " c e l l type #1".  by l i g h t microscope w i t h rearrangement  The presence of n u c l e o l i i s c o r r o b o r a t e d cytochemistry.  Increased n u c l e o l a r  of the n u c l e o l a r components has been  observed i n s e n e s c i n g n u c l e i i n other p l a n t s  (Fowke and  S e t t e r f i e l d , 1968;  Jordan  Chapman, 1971;  size  Chapman and Jordan, 1971;  Jordan, 1972).  and  No s i m i l a r c o r r e l a t i o n c o u l d  be done i n E c t o c a r p u s . Some e l e c t r o n micrographs chromatin.  suggest the presence of  T h i s i s confirmed by l i g h t microscope  T h e r e f o r e , even when o t h e r c e l l o r g a n e l l e s (e.g.  data. E.R.,  c h l o r o p l a s t s ) a l r e a d y d i s p l a y a g r e a t l y a l t e r e d morphology, nuclei s t i l l  g i v e a conspicuous DNA  reaction.  N u c l e i have  been c o n s i d e r e d very r e s i s t a n t t o d i s r u p t i o n d u r i n g senescence. ( B u t l e r , 1,9 67; Roux and.McHale, 1968; M i t t e l h e u s e r 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  l e a v e s have l o s t most of t h e i r RNA  and,some p r o t e i n ,  l e v e l s remained  high.  A decrease i n l e v e l s of both RNA  and p r o t e i n i s  DNA  55 observed i n these c e l l s cytochemical data).  (see summary o f l i g h t  microscope  The p r e s e n t data, however, suggest  t h a t such decreases a f f e c t p r i m a r i l y the p a t t e r n o f d i s t r i b u t i o n o f "these substances. of  A r e d u c t i o n .in the s i z e  RNA and p r o t e i n s t a i n i n g - a r e a s i s observed,  apparent e f f e c t on s t a i n i n g i n t e n s i t y . ( c f .  without"any  f i g u r e 15 w i t h  f i g u r e 16, and f i g u r e 18 w i t h f i g u r e 19). Reductions i n s t a i n e d areas are e a s i l y u n d e r s t o o d . a f t e r e l e c t r o n  microscope  s t u d i e s , which show the r e d u c t i o n t o be mainly due t o i n c r e a s i n g v a c u o l a t i o n and autophagy.  The s t a b i l i t y i n  s t a i n i n g i n t e n s i t y i s i n t e r e s t i n g and i n d i c a t e s t h a t i n the remaining cytoplasm RNA and p r o t e i n contents are comparat i v e l y high.  C e r t a i n c h a r a c t e r i s t i c s o f the n u c l e u s , i . e . ,  n u c l e o l a r morphology, DNA content, together w i t h h i g h RNA and p r o t e i n content i n d i c a t e the c a p a c i t y o f these  cells  for  that  active protein synthesis.  T h i s does not imply  there i s no a l t e r a t i o n i n the p r o t e i n s b e i n g s y n t h e s i z e d . Protein synthesis i s a nucleic  acid-directed  phenomenon, although much l e s s i s known about the mechanisms involved i n regulating t h e i r synthesis. to  There i s evidence  suggest t h a t a v a r i a t i o n i n gene e x p r e s s i o n occurs w i t h  d i f f e r e n t m e t a b o l i c c o n d i t i o n s , t h a t i s , some genes are expressed a t one stage o f the c e l l others  (e.g. C a r r and Pate, 1967).  type o f p r o t e i n s b e i n g produced  life  span but not a t  T h i s would a f f e c t the  and subsequently  development, d i f f e r e n t i a t i o n and senescence  cell  (e.g. Heslop-  H a r r i s o n , 1967; Shannon, 1968; S c a n d a l i o s , 1969, Spencer and T i t u s , 1972). .Such a p o s s i b i l i t y has. l e d Woolhouse (1967) to  t h i n k o f d i f f e r e n t i a t i o n and - senescence  as two stages o f  the same p r o c e s s , which i s the r e s u l t of. a c o n t r o l l e d gene expression  (see a l s o C a r r and.Pate, 1967).  Alterations  i n the amount and. type of p r o t e i n s b e i n g s y n t h e s i z e d and d i s r u p t i o n o f normal c e l l  f u n c t i o n s can..be the r e s u l t o f  causes other than those mentioned above. "Pelc (1970) t h i n k s of  aging as the r e s u l t of an accumulation o f damaged DNA  56 copies  ( u n t i l no c o r r e c t copies are l e f t ) , r a t h e r than the  r e s u l t of a c o n t r o l l e d e x p r e s s i o n . o f a l s o remains  (Johnson and  genes.. The  S t r e h l e r , 1972)  possibility  t h a t aging  may  r e s u l t from l o s s of genes coding  f o r ribosomal  RNA.  Aging-  dependent p r o t e i n v a r i a t i o n s may  a l s o be the r e s u l t of  mechanisms not d i r e c t l y r e l a t e d to p r o t e i n s y n t h e s i s Simon, 19 67;  Whatever the e x p l a n a t i o n ,  the  f a c t remains t h a t aging i s dependent upon the presence  and  production  Cherry, 1967) .  (e.g.  of c e r t a i n s p e c i f i c p r o t e i n s  Winkenbach, 19 71).  An  i n c r e a s e i n the amounts of  enzymes d u r i n g aging i n both p l a n t and r e p o r t e d by d i f f e r e n t authors Elliott Schuster  and Bak,  1964;  e t a l . , 1968;  Wattiaux, 1969; sp., as the c e l l can be detected  Blum, 1965;  and  lytic  animal c e l l s  (Klamer and F e n n e l l ,  was 1963;  Sommer and Blum,  Grusky and Aaranson, 1969;  C r i s t o f a l o , 1970;  Winkenbach, 1971;  (e.g. M a t i l e  C o m o l l i , 1971;  P a l i s a n o and Walne, 1972).  1965;  Elens  and  Matile  and  In Ectocarpus  ages, an i n c r e a s e i n a c i d phosphatase by cytochemical  means.  activity  However, i n c r e a s e s  i n enzyme a c t i v i t y are not n e c e s s a r i l y r e l a t e d to de novo s y n t h e s i s of enzymes (Klamer and F e n n e l l , 1963). i n c r e a s e i n enzyme a c t i v i t y can be due l a t e n t enzymes  (e.g. Simon, 1967).  The  to a c t i v a t i o n of  More r e c e n t  evidence  seems to suggest t h a t the i n c r e a s e c o u l d , at l e a s t i n p a r t , be the r e s u l t of an aging-dependent r e d u c t i o n i n the of enzyme degradation S r i v a s t a v a , 1969;  (e.g. Mainwaring, 1968,  rate  1969;  C o m o l l i e t a l . , 1972).  Walne et a l . , (1970) and P a l i s a n o and Walne  (19 72)  have suggested t h a t i n p l a n t .as w e l l as i n a n i m a l . c e l l s , l i p o f u s c i n - l i k e pigments accumulate as the r e s u l t , of In Ectocarpus sp. " c e l l type #2"  some of the  aging.  cytoplasmic  i n c l u s i o n s are morphologically, reminiscent, of the s o - c a l l e d l i p o f u s c i n i n c l u s i o n s of aging animal t i s s u e s (e.g. Takahashi et  a l , 1970;  Sohal and  Sharma, 1972).  (1965) proposed t h a t such lysosomal  Samorajski e t a l .  i n c l u s i o n s should  be  57 c o n s i d e r e d as l i p o f u s c i n i n c l u s i o n s when myelin or laminated f i g u r e s c o u l d be v i s u a l i z e d . However, as r e c e n t l y by Hasan and Glees  reviewed  (1972), m o r p h o l o g i c a l 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 i n c l u s i o n s can be q u i t e v a r i a b l e . T h e r e f o r e , 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 t o be a v a l i d one  ( see a l s o Zeman,1971 ). Of the d i f f e r e n t h y p o t h e s i s  advanced t o e x p l a i n the o r i g i n of l i p o f u s c i n  ( see Toth,1968,  and Zeman,1971, f o r a r e v i e w ) , the one r e l a t i n g them t o lysosomes  i s the most w i d e l y accepted  Novikoff,1960;  ( e. g. Essner  and  S t r e h l e r and Mildvan,1962; Koenig,1963;  Samorajski e t a l . ,  1964,  1965;  Frank and Christensen,1968;  Goldfisher et  Hirsch,1970;  1972a ). Since most of " c e l l type #2"  Brunk and  Ericsson,  cytoplasmic i n c l u -  s i o n s seem t o be of lysosomal o r i g i n , two adapted by Hendy  al.,1966;  techniques  (1971) f o r the u l t r a s t r u c t u r a l  identifica-  t i o n of l i p o f u s c i n were a p p l i e d t o the p r e s e n t m a t e r i a l . T o l u i d i n e blue s t a i n i n g show an i n c r e a s e i n the amount of g r e e n i s h - t u r q u o i s e - s t a i n e d i n c l u s i o n s w i t h aging. These i n c l u s i o n s are, t h e r e f o r e , probably p o l y p h e n o l i c i n nature  ( e. g. McCully,1966; Fontana's  Evans and Holligan,1972  silver 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 t o i d e n t i f y l i p o f u s c i n m a t e r i a l . S i l v e r n i t r a t e , however, i s a l s o known t o p h e n o l i c compounds solution  ( e. g.  impregnate  Chadefaud,1936 ).  ( Hendy,1971 ) uses a mixture of i r o n c h l o r i d e  potassium f e r r i c y a n i d e t o s p e c i f i c a l l y l a b e l i n c l u s i o n s . P h e n o l i c vacuoles p o s i t i v e l y to i r o n s a l t s Fritsch  Schorml's and  lipofuscin  ( e. g. physodes ) respond  ( e. g. Chadefaud,1936 ). However,  (1945) reported..that .no. response  could..be r e c o r d e d  w i t h i r o n c h l o r i d e . C u l l i n g . (1963) considered. Schorml s 1  method t o b e . q u i t e s p e c i f i c f o r the i d e n t i f i c a t i o n o f l i p o f u s c i n . In a d d i t i o n , an i n t e n s e Sudan b l a c k B c h a r a c t e r i z e s " c e l l type #2". Sudan b l a c k B  response  Physodes do not r e a c t t o  ( e. g. Chadefaud,1936; Evans and  Holligan,  58 1972) , b u t l i p o f u s c i n - l i k e . m a t e r i a l .gives, ...at.,least d u r i n g c e r t a i n phases stain  of i t s formation,,a s t r o n g response t o the  (e.g. C u l l i n g , 1963;  Pear.se, 1972) . ' I t seems, t h e r e -  fore,: t h a t the evidence p o i n t s t o the ' p o s s i b i l i t y -of some of the i n c l u s i o n s . found i n aging, c e l l s of Ectocarpus t o be. lipofuscin  related.  Gifford  (1968) suggested t h a t p l a s t i d - d e r i v e d  inclusions  b e l i e v e d to c o n t a i n both l i p i d s and p h e n o l i c compounds accumulated  inside.vacuoles.  Pearse  (1972) a l s o  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  m a t e r i a l s r e s u l t i n g from t r a n s f o r m a t i o n of p h e n o l i c compounds inside vacuoles.  From the p r e s e n t s t u d i e s , i t i s i m p o s s i b l e  to determine whether i n Ectocarpus p h e n o l i c compounds e x i s t independently or are mixed w i t h l i p o f u s c i n m e t a b o l i t e s . The i m p l i c a t i o n s 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 t o c e l l u l a r metabolism obscure.  have remained  largely  The i n c l u s i o n s have been suggested t o r e p r e s e n t  i n e r t waste products  (e.g. B j o r k e r u d , 19 64), but most  c o n s i d e r them t o i n t e r f e r e w i t h the p h y s i o l o g y of the l e a d i n g e v e n t u a l l y t o c e l l death 1964, 1972).  1968;  Raychaudhuri  (e.g. Samorajski e t a l . ,  and D e s a i , 1971;  Hers and van Hoof  cells,  Sohal and Sharma,  ( c i t e d i n Zeman, 1971) p o s t u l a t e d  t h a t c e l l u l a r d y s f u n c t i o n was  the r e s u l t of mechanical  d i s t u r b a n c e s c r e a t e d by i n c r e a s i n g accumulations of r e s i d u a l m a t e r i a l i n the cytoplasm of the c e l l s .  Accumulation of  l i p o f u s c i n i n c l u s i o n s , known t o c o n t a i n r e a c t i o n products of l i p i d p e r o x i d a t i o n and other f r e e r a d i c a l (e.g. Tappel, 1965.; B j o r k e r u d , 1964), was  reactions  shown t o produce  r a p i d . d e t e r i o r a t i o n of the membrane systems of s e v e r a l o r g a n e l l e s , i n c l u d i n g lysosomes. (Packer, e t al..,  1967).  Membrane d e t e r i o r a t i o n , e s p e c i a l l y lysosomal, membranes, was  suggested b y . H o c h s c h i l d (1971) t o . l e a d t o the leakage  of h y d r o l y t i c enzymes i n t o the cytoplasm; hence p r o f o u n d l y a f f e c t i n g c e l l metabolism.  Recent evidence  (Sullivan  and  59 Debusk, 1973)  seems t o support t h i s hypothesis, and  t h a t c e l l u l a r aging, may consequent  r e s u l t from membrane-damage and  s y n t h e s i s of a l t e r e d p r o t e i n s .  (1972) have proposed  suggests the  Sohal and Sharma  t h a t the e f f e c t s of l i p o f u s c i n accumula-  t i o n on the p h y s i o l o g y of the c e l l could.be the r e s u l t of 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  two  the  amount of n a t i v e cytoplasm i s reduced, d i s r u p t i n g normal c e l l u l a r f u n c t i o n s , and 2) the accumulation of c r e a t e s a new. environment  around  influence genetic a c t i v i t y .  lipofuscin  the nucleus t h a t might  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 E c t o c a r p u s . ' In E c t o c a r p u s , most of  the accumulation of i n c l u s i o n s c o i n c i d e s w i t h v e s i c u l a t i o n  of  the E.R.  Since t h e r e i s evidence t o i n d i c a t e t h a t the  nucleus might be Important the E.R.  i n m a i n t a i n i n g the i n t e g r i t y of  (e.g. F l i c k i n g e r , 1968), a p o s s i b l e  relationship  might e x i s t i n Ectocarpus between i n c l u s i o n accumulation 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  o f t e n been noted i n o t h e r s e n e s c i n g p l a n t m a t e r i a l and been i n t e r p r e t e d as a s i g n of E.R. degenerency Manocha, 1965; et  a l . , 1967;  Bain and Mercer, Villiers,  1972;  1966;  has  (Shaw arid  B u t l e r , 1967;  Treffry  Potapov and Krishnamurthy,  The d i s o r g a n i z a t i o n of the E.R.  1972).  i s a l s o expected t o  a f f e c t the ribosomal p o p u l a t i o n of these c e l l s .  Shaw and  Manocha (1965) r e p o r t e d t h a t disappearence of the E.R. simultaneous w i t h t h a t of ribosomes.  and  was  Fukazawa and H i g u c h i  (19 66) p r o v i d e d evidence f o r the e x i s t e n c e of a c o r r e l a t i o n between a r e d u c t i o n i n the ribosomal. p o p u l a t i o n , and a r e d u c t i o n i n the E.R.  However, M i t t e l h e u s e r and  Steveninck . (1971) have shown t h a t ribosomes  van  possess a g r e a t e r  s t a b i l i t y than other c e l l .organelles.. Berjack.and  Villiers  (1972b) r e p o r t e d t h a t even.in s t a g e s . o f e x t r e m e : d i s o r g a n i z a t i o n monosomes could.be found randomly s c a t t e r e d throughout cytoplasm  (see a l s o Gpik,. 1966;  Mia, 1972)..  B e r j a c k and V i l l i e r s ,  the 1970;  On. the c o n t r a r y , P a l a n d r i (1972) showed' t h a t  60 although the E.R.  i s s t i l l w e l l r e p r e s e n t e d , most o f the  ribosomal p o p u l a t i o n has disappeared from seneseing of Halimeda - tuna.  cells  In Ectocarpus,. s m a l l . c l u s t e r s o f p a r t i c l e s  are s t i l l  found here and there d u r i n g e a r l y stages of  type #3".  However, t h e i r ribosomal nature c o u l d not  determined  and so the p a t t e r n of ribosomal behaviour i s  difficult  be  to e s t a b l i s h .  The nucleus-dictyosome apparent  "cell  association i s s t i l l  a t t h i s stage of senescence  dictyosome-derived dictyosomes  and p r o d u c t i o n of  v e s i c l e s from the maturing  is. also detectable.  very  face of  These v e s i c l e s seem t o be  loaded w i t h m e t a b o l i t e s , but t h e i r f a t e i s d i f f i c u l t ascertain.  to  T h i s i s not o n l y due t o the c h a r a c t e r i s t i c s of  the background cytoplasm, but a l s o t o the v e s i c u l a t i o n of the E.R. of  t h a t makes i t d i f f i c u l t  to d i s t i n g u i s h e i t h e r  type  vesicle. C e l l w a l l t h i c k e n i n g i s a s s o c i a t e d w i t h aging i n  d i f f e r e n t p l a n t m a t e r i a l s (e.g., James, 1966; P a l a n d r i , 1968, Ectocarpus  1969;  senescence  McLean, 1968;  F a b b r i and  P a l a n d r i , 1972).  i s a l s o accompanied by w a l l t h i c k e n i n g  with l o c a l i z e d formation of c e l l w a l l ingrowths. w a l l ingrowths  In  These c e l l  are made of two d i f f e r e n t components:  1)  m e t a b o l i t e s , probably of v a c u o l a r o r i g i n , and 2) c e l l w a l l m a t e r i a l s which i s o l a t e the m e t a b o l i t e s from the plasma membrane.  The m e t a b o l i t e s c l o s e l y resemble some of the  m a t e r i a l accumulated  i n the paramural  r o o t c e l l s of Glechoma hederacea  space of seneseing  (Bowes, 1972).  This  author has a l s o shown, the m e t a b o l i t e s t o be v a c u o l a r i n o r i g i n and possible.lomasomal  i n nature.  In Ectocarpus  the  v a c u o l a r o r i g i n of the c e l l w a l l ingrowth m e t a b o l i t e s i s at  l e a s t p a r t i a l l y apparent, b u t - t h e i r m o r p h o l o g i c a l  c h a r a c t e r i s t i c s h a r d l y f i t i n t o a lomasdme One  concept.  can say t h a t t h i s p r o c e s s r e p r e s e n t s i n E c t o c a r p u s  a mechanism f o r the c e l l t o get r i d o f excess waste metabolites.  However, t h i s h y p o t h e s i s i s c i r c u m s t a n t i a l  61 and  f a i l s to a c c o u n t . f o r  does not occur u n i f o r m l y  the reason(s)  why  the phenomenon  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 e v i d e n t at p l a c e s . C e l l w a l l ingrowths a l l o w f o r . t h e easy r e c o g n i t i o n of t h i s c e l l type under the l i g h t microscope.  Similar  s t r u c t u r e s seem to e x i s t In other brown algae  (Chadefaud,  19 36), but have never been d e s c r i b e d at the e l e c t r o n microscope l e v e l .  Cytochemical  t e s t s c a r r i e d out at the  l i g h t microscope l e v e l showed these  s t r u c t u r e s to g i v e  p o s i t i v e r e a c t i o n s f o r both p h e n o l i c compounds with t o l u i d i n e blue 0--McCully, 1966) blue with Schorml's—Hendy, 1971). was  a l s o recorded  by Chadefaud  and  lipofuscin  Their phenolic  (1936).  response could be r a t h e r s i g n i f i c a n t . be i n t e r p r e t e d as evidence  (deep  nature  T h i s double s t a i n i n g Indeed, t h i s  f o r the occurrence  i n c l u s i o n of both p h e n o l i c and  (greenish  could  i n the same  lipofuscin-like  substances.  I t i s i n t e r e s t i n g t h a t as l o c a l i z e d ingrowths enlarge  at  the c r o s s w a l l l e v e l , symplast c o n t i n u i t y between c e l l s does not seem t o be d i s r u p t e d .  This observation could  be  p a r t i c u l a r l y p e r t i n e n t f o r the advance of senescence, s i n c e as proposed by Simon (1967), the r a t e of senescence c o u l d dependent upon the e x i s t e n c e of a s i n k " c e l l type  #1"  be  ( i n Ectocarpus  c o u l d a c t as a sink) f o r m a t e r i a l s  exported  from aging p a r t s of the p l a n t s . An a n a l y s i s of the m i t o c h o n d r i a l p o p u l a t i o n as w e l l as m i t o c h o n d r i a l here,  f e a t u r e s i n " c e l l type  #2"  seems unwarranted  s i n c e these o r g a n e l l e s are very d i f f i c u l t  At f i r s t one  gains the impression  that.the  mitochondrial  p o p u l a t i o n might have d r a s t i c a l l y decreased senescence.  Two  f a c t s argue against.such.an  these c e l l s possess.a  very crowded cytoplasm  of i n c l u s i o n s which make the mitochondria distinguish  (see a l s o . S c h u s t e r  to detect.  at t h i s stage  of  hypothesis:  1)  with a l l sorts  very d i f f i c u l t  e t a l . , 1968), and  2) more  advanced stages of senescence have l a r g e m i t o c h o n d r i a l  to  62 populations,  which, .become, v i s i b l e when. the.. number of  i n c l u s i o n s - i n . . t h e ground cytoplasm have decreased, r e v e a l i n g more e a s i l y -the remaining., s t r u c t u r e s . Conspicuous stacks type #2"  chloroplasts..  of t h y l a k o i d s . a r e  A s i m i l a r phenomenon, was  other aging p l a n t m a t e r i a l  (e.g. McLean, 1968;  Messer and Ben-Shaul, 1972), and s i g n of r e d u c t i o n  visible in  has  "cell  noticed  Dodge,  been considered  of p l a s t i d a c t i v i t y  in  1970; as a  (McLean, 1968).  In  Ectocarpus a very c l o s e r e l a t i o n s h i p e x i s t s between t h i s type of t h y l a k o i d arrangement and  the formation of m e t a b o l i t e s  of high e l e c t r o n d e n s i t y , whose response to the osmium  and  permanganate f i x a t i o n s as w e l l as to the Sudan B l a c k B indicates a possible l i p i d amounts of l i p i d m a t e r i a l Harnischfeger  content.  The  appearance of  i n p l a s t i d s was  hypothesized  (1972) as a s i g n of m e t a b o l i c c o n d i t i o n s  r e s u l t i n damage of the e l e c t r o n t r a n s p o r t therefore  i n reduced p l a s t i d  Phenolic  compounds  (or p h e n o l i c  H o l l i g a n , 1972;  by which  and  activity.  were suggested to be p l a s t i d d e r i v e d Evans and  chain  large  compound  precursors)  (e.g. G i f f o r d ,  Davies et a l . , 1973).  1968;  The  p o s s i b i l i t y t h a t the Ectocarpus p l a s t i d m e t a b o l i t e i s phenolic  i n nature i s not supported by  reaction  (Chadefaud, 1936) , s i n c e i t f a i l s  i n t r a p l a s t i d metabolite  ( f i g u r e 233,  the s i l v e r n i t r a t e  arrows).  i n t e r e s t i n g to note t h a t n o n - l a b e l l i n g of m e t a b o l i t e s or s i m i l a r m a t e r i a l  to l a b e l the It i s also  intraplastid  found i n the cytoplasm support  the concept t h a t 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  i n c l u s i o n s of lysosomal, origin... The. p o s s i b i l i t y , a l s o e x i s t s t h a t i n t r a p l a s t i d . m e t a b o l i t e s are the r e s u l t . o f f i x a t i o n artifacts.  Other.evidence seems to i n d i c a t e , however, t h a t  i n t r a p l a s t i d i n c l u s i o n s are. s i g n i f i c a n t i n ..senescence. ly,  First-  these i n c l u s i o n s . a r e a s s o c i a t e d w i t h a s p e c i a l arrange-  ment of the t h y l a k o i d membranes.  Secondly, i n permanganate  f i x e d m a t e r i a l as w e l l as f r e e z e - e t c h i n g  similar  preparations  63 b e a r i n g the same r e l a t i o n s h i p t o t h y l a k o i d s a r e a l s o d e t e c t a ble. Discharge of p l a s t i d m e t a b o l i t e s i n t o the cytoplasm i s apparent and t h e r e f o r e c o n t r i b u t e s t o the e s t a b l i s h m e n t of  the ground cytoplasm c h a r a c t e r i s t i c s o f " c e l l type PART IV - "TRANSITIONAL As senescence  #2".  2-3" CELLS  continues the cytoplasm becomes i n c r e a -  s i n g l y a u t o l y t i c t o the p o i n t where very few o r almost no i n c l u s i o n s are p r e s e n t . A t the l i g h t microscope transparency o f the c e l l s becomes The  l e v e l the  apparent.  s t r i k i n g d i f f e r e n c e s 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 t o assume the e x i s t e n c e of i n t e r m e d i a t e stages. These t r a n s i t i o n a l stages were seldom d e t e c t e d and i n most cases the t r a n s i t i o n was abrupt. Loss o f v a c u o l a r compartmentation plant c e l l 1967;  senescence  i s conspicous i n  ( e. g. Shaw and Manocha,1965; B u t l e r ,  Roux and McHale,196 8; Halperin,1969;  De  Vecchi,1971;  Habeshaw and Heyes,1971; M a t i l e and Winkenbach,1971; Stearns and Wagenaar,1971; B e r j a c k and V i l l i e r s , 1 9 7 2 a , 1972b ) . Gahan(1965) and Barton that  (1966), among o t h e r s , have suggested  the rupture o f the t o n o p l a s t marked the b e g i n n i n g of  the sequence o f events l e a d i n g t o the massive condition  autolytic  o f the c e l l s . The a u t o l y t i c c o n d i t i o n i s most  l i k e l y due t o the massive  r e l e a s e of t o x i c  substances  ( e. g. Shaw and Manocha,1965; Robards,1970 ) and l y t i c enzymes  ( e. g. Matile,1969a  ), which are known t o be  p r e s e n t i n the c e l l sap. Other authors  ( e. g. Shaw and Manocha,1965; B u t l e r ,  1967 ) maintained t h a t the vacuole content  might d i f f u s e  through the t o n o p l a s t . The l a t t e r p o s s i b i l i t y seems.to be --. supported by r e c e n t c y t o c h e m i c a l evidence  ( Brunk and  64 E r i c s s o n , 1972b).  D i f f u s i o n through the t o n o p l a s t of  cell  sap substances, may  thus, t r i g g e r an ..irreversible., c h a i n of  events u l t i m a t e l y l e a d i n g t o c e l l death; and the r u p t u r e of the t o n o p l a s t w i l l be a consequence r a t h e r than the  cause.  The causes of m o d i f i c a t i o n i n t o n o p l a s t p e r m e a b i l i t y 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 a n a l y s i s of them i n r e l a t i o n  to Ectocarpus r e s u l t s w i l l be presented. Accumulation  of l i p o f u s c i n i n c l u s i o n s such as those  i n Ectocarpus has been suggested  to cause damage t o lysosome  membranes, a f f e c t i n g p e r m e a b i l i t y and r e s u l t i n g i n leakage of  h y d r o l y t i c contents i n t o the cytoplasm ( G a b r i e l e s c u ,  1970;  H o c h s c h i l d , 1971;  Villiers  Brunk and Brun,  1972).  B e r j a c k and  (1970) thoroughly d i s c u s s e d the p o s s i b i l i t y  i n c o r p o r a t i o n i n t o vacuoles of dictyosome-derived c a r r y i n g carbohydrate m e t a b o l i t e s u l t i m a t e l y b u r s t i n g of lysosomal membranes. that l y t i c in  formation  vesicles  causes  The p o s s i b i l i t y a l s o e x i s t s  enzymes of e x t r a l y s o s o m a l o r i g i n may  " c e l l type #3"  play a role  ( L i n and Fishman, 1972).  Whatever the t r u e e x p l a n a t i o n , the evidence suggests once a massive  that  that  a u t o l y s i s i s i n i t i a t e d , t r a n s f o r m a t i o n of the  c e l l content must proceed w i t h extreme speed s i n c e i n most of  the cases s t u d i e d the t r a n s i t i o n between the " c e l l  type  #2" morphology and t h a t 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 a l s o B e r j a c k and V i l l i e r s ,  1970,  1972b; Woolhouse,  1967). A l l steps i n v o l v e d i n n u c l e a r d i s o r g a n i z a t i o n c o u l d not be f o l l o w e d , but a v a i l a b l e , information, shows many . s i m i l a r i t i e s w i t h the.same s i t u a t i o n i n other s e n e s c i n g plants  (e.g. Shaw and Manocha, 1965;  Barton, 1966;  Wooding,  1966) . The number of mitochondria.seemed  to.have i n c r e a s e d .  One must, however, be c a u t i o u s i n i n t 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 s t u d y i n g  " c e l l type #2" m i t o c h o n d r i a , of mitochondria  and secondly,  multiplication  a t t h i s stage o f senescence would be  d i f f i c u l t to explain.  D e s p i t e t h i s , an i n c r e a s e i n the  m i t o c h o n d r i a l p o p u l a t i o n was. r e p o r t e d i n . a g i n g c e l l s of Halimeda tuna tubers  (Lee a'hd Chasson, 1966) .  exceptions cases  ( P a l a n d r i , 1972) and aging s l i c e s o f p o t a t o These o b s e r v a t i o n s are  r a t h e r than the r u l e .  Indeed, i n most o f the  s t u d i e d , aging i n p l a n t c e l l s has not been c o r r e l a t e d  with i n c r e a s e s i n the m i t o c h o n d r i a l p o p u l a t i o n and  (see B u t l e r  Simon, 1971). At t h i s stage o f senescence, the m i t o c h o n d r i a l  envelope remains i n t a c t and the matrix  i s darker.  c r i s t a e a r e l e s s numerous than i n " c e l l type  The  "1" and o c c a s i o n -  a l l y assume c o n c e n t r i c arrangements, a c o n f i g u r a t i o n a l s o d e t e c t e d i n aging wheat leaves  (Shaw and Manocha, 1965).  S i m i l a r f e a t u r e s have been i n t e r p r e t e d as an i n d i c a t i o n o f m i t o c h o n d r i a l senescence i n other p l a n t m a t e r i a l  (see B u t l e r  and Simon, 1971, f o r a r e v i e w ) . I t i s known t h a t a d e c l i n e i n . r e s p i r a t o r y e f f i c i e n c y u s u a l l y accompanies senescence  (e.g. Drapper and Simon, 1971).  Lund e t al.,(1958) have r e p o r t e d t h a t a l t e r a t i o n of the 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 r e f l e c t s decreases i n r e s p i r a t o r y efficiency.  Data f o r Ectocarpus  and not backed up by experimental  are p u r e l y  morphological  evidence.  Moreover, a  drop i n r e s p i r a t o r y e f f i c i e n c y c o u l d 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).. i n t e r e s t i n g to consider.  The problem, i s , Varner  nevertheless,  (1961) has suggested t h a t  changes i n r e s p i r a t o r y metabolism,, p a r t i c u l a r l y i n o x i d a t i v e phosphorylation, due  could be a major cause o f senescence,  t o the many c e l l u l a r a c t i v i t i e s , d i r e c t l y , or i n d i r e c t l y  dependent upon i t . Of a l l the c e l l o r g a n e l l e s , c h l o r o p l a s t s undergo the  66 most s t r i k i n g changes. s t i l l resembles  In.some cases p l a s t i d . morphology  t h a t c h a r a c t e r i s t i c o f . " c e l l type #2".  As  p r o d u c t i o n o f i n t r a p l a s t i d m e t a b o l i t e s cease,.changes i n t h y l a k o i d organization.become.more apparent. an apparent  There i s a l s o  i n c r e a s e i n the number of p l a s t o g l o b u l i .  i n t e r e s t i s the budding the p l a s t i d body.  Of  o f stroma containing, s t r u c t u r e s from  M o r p h o l o g i c a l l y s i m i l a r phenomena were  r e p o r t e d i n other p l a n t m a t e r i a l , but not i n connection w i t h senescence Although  ( G u l l v a g , 1968; Schotz e t a l . , 1971).  the s i g n i f i c a n c e o f . t h e p l a s t i d budding  might not be completely understood,  phenomenon  i t seems reasonable t o  assume t h a t i t p l a y s a major r o l e i n the r e d u c t i o n of p l a s t i d volume (see a l s o Dodge, 1970; F a b b r i and P a l a n d r i , 1970).  Indeed, a decrease  i n p l a s t i d s i z e seems t o occur  i n Ectocarpus, p a r t i c u l a r l y i n " c e l l ..type #3" F r i t s c h , 1945).  A decrease  (see a l s o  i n p l a s t i d size i s rather  common i n seneseing p l a n t m a t e r i a l (Ikeda and Ueda, 1964; Barton, 1966; L j u b e s i c , 1968; Dodge, 1970, De V e c c h i , 1971; Stearhs and Wagenaar, 1971). Since pyrenoids were never found i n " c e l l type it  #3",  seems s a f e t o assume t h a t they might have disappeared  d u r i n g these t r a n s i t i o n a l stages.  However, as a l r e a d y p o i n t e d  out, images suggesting p y r e n o i d breakdown were very difficult  t o i n t e r p r e t and the e l u c i d a t i o n of the mechanism  of p y r e n o i d d i s o r g a n i z a t i o n must await f u r t h e r r e s e a r c h . PART V — T H E  "CELL TYPE #3"  It i s clear that t r a n s i t i o n a l very a l t e r e d c e l l u l a r a r c h i t e c t u r e .  "2-3" c e l l s possess a ..The ground. cytoplasm  shows extreme a u t o l y s i s ; . the. m i t o c h o n d r i a and. p l a s t i d s are senescent;  the t o n o p l a s t has. broken, .down i n p l a c e s ; the  nucleus i s d i f f i c u l t  t o d e t e c t .and when p r e s e n t h i g h l y  d i s o r g a n i z e d ; the dictyosomes  are r a r e l y d e t e c t e d and  67  abnormal i n apperance. A l l t h e s e f e a t u r e s a r e unmistakable s i g n s o f c e l l n e c r o s i s . T h e r e f o r e , " c e l l type #3" should be c o n s i d e r e d as a phase i n the f i n a l and not separated from the The  process o f c e l l  autolysis  " t r a n s i t i o n a l 2-3" c e l l s .  a u t o l y t i c appearance o f the background  cytoplasm  in  " c e l l type #3" c o r r e l a t e s d i r e c t l y w i t h the d i s t r i b u t i o n  of  a c i d phosphatase a c t i v i t y . R e a c t i o n product i s seen a l l  over the cytoplasm,  a s i g n t h a t the compartmentation o f  lysosomes no longer e x i s t s and the c e l l c a v i t y r e p r e s e n t s a s i n g l e l a r g e lysosomal u n i t . A l l the other enzymes t e s t e d seem to have ceased t o f u n c t i o n and the l i g h t data g i v e f u r t h e r p r o o f o f c e l l N u c l e i and dictyosomes  microscope  degeneration.  were never observed  i n "cell  type #3". T h i s i s not s u r p r i s i n g s i n c e the n u c l e i , as p r e v i o u s l y shown, begin t o d i s o r g a n i z e d u r i n g  the " t r a n s i -  t i o n a l 2-3" s t a g e s . The E.R., i f p r e s e n t , i s reduced  t o very  a l t e r e d and almost u n i d e n t i f i a b l e membrane p r o f i l e s . T h e plasmalemma i s d i s c o n t i n u o u s , the t o n o p l a s t no longer r e c o g n i z a b l e ; a l l i n c l u s i o n s , w i t h the e x c e p t i o n o f m i t o c h o n d r i a and p l a s t i d s remains have disappeared. M i t o c h o n d r i a l p r o f i l e s are s t i l l m i t o c h o n d r i a l envelope  numerous, but the  i s d i s r u p t e d i n s e v e r a l p l a c e s . The  c r i s t a e and the m a t r i x o f the m i t o c h o n d r i a are no l o n g e r d e t e c t a b l e . These f e a t u r e s a r e s i m i l a r t o those found i n degenerating m i t o c h o n d r i a De Vecchi,1971;  i n other senescent p l a n t s ( e. g.  B u t l e r and Simon,1971 ) .  C h l o r o p l a s t s are most o f t e n d e l i m i t e d by a s i n g l e membrane, a remnant o f the c h l o r o p l a s t envelope. They are almost devoid o f stroma,  and f o r the most p a r t o n l y l a r g e  stacks o f lamellae, remain. As d i s o r g a n i z a t i o n . p r o c e e d s the l a m e l l a r system.becomes greatly..reduced and t h e envelope membrane i s a l s o absent. The f i n a l of  steps i n d i s o r g a n i z a t i o n  Ectocarpus p l a s t i d s are, t h e r e f o r e , s i m i l a r t o those  r e p o r t e d f o r other p l a n t s ( e. g. Shaw and Manocha,1965;  68 De V e c c h i ,  1971;  Messer and Ben-Shaul, 1972).  In a s s o c i a t i o n  with, thylakoid.breakdown conspicuous depositions, of p l a s t o p g l b u l i are d e t e c t a b l e  (e.g. Ikeda and Ueda, 1964;  Barton,  1966;  F a b b r i and  1971;  Stearns and. Wagenaar ,..1971) . . In some cases,  the impression  P a l a n d r i , 1970;. Dodge,\197.0: B u t l e r and  t h a t the p l a s t o g l o b u l a r p o p u l a t i o n  one  Simon, gains  of degenera-  t i n g c h l o r o p l a s t s i s heterogeneous i n t h e i r e l e c t r o n opacity. Palandri  A s i m i l a r s i t u a t i o n was  reported  by F a b b r i  (1970) i n R i c i n u s communis senescing  Stearns and Wagenaar (1971) a l s o r e p o r t e d i n senescing  and  cotyledons.  s i m i l a r phenomena  c h l o r o p l a s t s of autumn leaves  and  suggested  t h a t v a r i a t i o n s i n e l e c t r o n o p a c i t y of p l a s t o g l o b u l i were due  to t h e i r r o l e as waste baskets f o r breakdown  Butler  products.  (1967) concluded t h a t although p l a s t o g l o b u l i i n  senescing  c h l o r o p l a s t s were the r e s u l t of an  of membrane breakdown products, as storage  accumulation  they probably a l s o  bodies f o r i n s o l u b l e l i p i d m a t e r i a l s  acted  not  n e c e s s a r i l y a s s o c i a t e d w i t h membrane breakdown. P l a s t i d s seem to be senescing  the most r e s i s t a n t o r g a n e l l e s  Ectocarpus c e l l s .  the same s i t u a t i o n was  in  I t i s i n t e r e s t i n g to note t h a t  found i n n e c r o t i c c e l l s of Ascophyllum  nodosum (Rawlence, 1972). D i s s o l u t i o n of the c e l l w a l l r e p r e s e n t s the l a s t stage i n c e l l u l a r d e s t r u c t i o n . occurs i n other p l a n t s H a l p e r i n , 1969; B a l and  (e.g. Clowes and  B a l and  C e l l w a l l breakdown Juniper,  1968;  Payne, 1972).  Payne (1972) found a c o r r e l a t i o n between  w a l l breakdown and E.R. Juniper,  i n Ectocarpus  1968).  development  (see a l s o Clowes  cell and  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 c a v i t y .  A c i d phosphatase„is_..pr.esen.t...inside the  but d e p o s i t i o n of r e a c t i o n . p r o d u c t s the c e l l w a l l .  De Jong  cells  i s .not conspicuous i n  (1966) r e p o r t e d  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 a c i d phosphatase a c t i v i t y , d i d b l o c k  the c e l l w a l l r e a c t i o n s t h a t were  69 otherwise e v i d e n t  i n ...unfixed c e l l s .  Other r e p o r t s  (e.g.  H a l p e r i n , 1969), show t h a t 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 w a l l s , but even i n - these, cases.no f u n c t i o n a l r e l a t i o n s h i p between-its. presence and could be demonstrated.  c e l l w a l l breakdown  Horton.and Osborne (1967)  have shown t h a t as senescence proceeds an i n c r e a s e i n c e l l u l a s e a c t i v i t y i s a l s o observed. was  C e l l w a l l breakdown  r e l a t e d to h i g h 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 c u l t u r e d  carrot c e l l s study other  ( H a l p e r i n , 1969).  No  attempts were made to  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 w a l l d i s o r g a n i z a t i o n 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: a f i b r i l l a r component, and  2) a cement-like  1)  (matrix)  m a t e r i a l which seems to be the f i r s t to d i s o r g a n i z e . et  a l . (1958), and Myers and  Preston  Cronshaw  (1959a, 1959b) r e p o r t e d  t h a t c e l l w a l l s of marine algae were apparently  composed of  a network of f i b r i l s embedded i n an amorphous m a t r i x .  Recent  evidence supports the occurrence of a s i m i l a r c o n s t r u c t i o n p a t t e r n f o r the c e l l w a l l of Fucus 1971).  The  cellulosic carboxylated  (Fulcher and  McCully,  f i b r i l l a r component of the w a l l i s probably ( P e r c i v a l , 1968), but i t might a l s o be polysaccharide,  a  probably a l g i n i c a c i d , which  i s known to assume a m i c r o f i b r i l l a r c o n f i g u r a t i o n 196 8) .  The  (McCully,  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)  shown i t to be p o s s i b l y a s u l f a t e d p o l y s a c c h a r i d e . (1969), Lamport  (1965), and.Thompson and.. Preston  has  Preston (1969)  suggested t h a t the o r i e n t a t i o n of t h e . . m i c r o f i b r i l s and s t a b i l i z a t i o n . o f the c e l l w a l l s t r u c t u r e c o u l d be on r e s p o n s i b i l i t y of p r o t e i n s .  the  the  70 PART V I — E N Z Y M E LOCALIZATION DURING DIFFERENTIATION AND The for  SENESCENCE  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  an a n a l y s i s o f e n z y m a t i c d a t a .  tions regarding  Since  reserved  detailed  a c i d p h o s p h a t a s e a c t i v i t y and  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 , c o n s i d e r a t i o n s w i l l be  considera-  lipofuscin  the  present  concerned w i t h the a n a l y s i s  adenosine triphosphatase, Adenosine triphosphatase  c a t a l a s e , and (ATPase).  of  peroxidase  Recently  results.  a number o f  papers have appeared which d e a l w i t h p l a n t ATPases Coulomb and  C o u l o m b , 1972;  e t a l . , 1972;  H a l l and  1972b; M a i e r and and  M a i e r and  1972;  (1972),  Thompson, 1 9 7 2 a ,  Coulomb' and  Coulomb  for instance, reported  d e p e n d e n t A T P a s e a t t h e p l a s m a membrane l e v e l . reported  t h a t ATPase a c t i v i t y  i o n s , but ions  inhibited  ( A t k i n s o n and  P o l y a , 1967).  green alga Mougeotia e v i d e n c e was  i s s t i m u l a t e d by  detected  the  s y s t e m was  see  l e v e l of the  s a i d t o be  +  a l s o R a t n e r and  provided  authors  divalent K+  the  Further  f o r the occurrence of monovalent  K -Na -ATPase a c t i v a t e d systems +  Mg  i n t h e p l a s m a membrane o f  i o n s s t i m u l a t e d ATPases a t the although  (1972)  monovalent  A pH-dependent  (Sundberg e t a l . , 1973).  presented  The  Other  ( u n d e r c e r t a i n c o n d i t i o n s ) by  s t i m u l a t e d A T P a s e was  Hodges  Sundberg e t a l . , 1973).  conflicting.  Maier  G i l d e r , 1972;  D a v i e , 1971'; L a i and  Maier,  r e p o r t s seem t o be  C r o n s h a w and  (e.g.  plasmalemma,  d i f f e r e n t from  the  ( e . g . Hodges e t a l . , 1972;  J a c o b y , 1973) .  L a i and  Thompson  evidence f o r the e x i s t e n c e of a K -Na +  (1972a)  -stimulated  A T P a s e a s s o c i a t e d w i t h t h e p l a s m a l e m m a , 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 , h o w e v e r , t h a t  p l a n t A T P a s e t h e y s t u d i e d had  different properties  the K -Na -ATPase system of animal +  +  ATPases are a s s o c i a t e d w i t h functions of the c e l l  the  from  cells.  some o f t h e m o s t  (e.g. Heyden, 1969).  A  important  study  71 of ATPase l o c a l i z a t i o n i n - Ectocarpus,,, t h e r e f o r e , 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 :  p h y s i o l o g i c a l status..  The results.. ;for:. Ectocarpus have shown  t h a t the p a t t e r n of r e a c t i o n product, deposition..was the same f o r the M g - and K -Na -Mg -ATPase systems. ++  monovalent ions  +  (Na , +  +  However,  ++  K*) seem t o have enhanced the 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 t both, the plasmalemma and the thylakoid levels.  Involvement o f monovalent ions dependent  ATPases i n s t i m u l a t i o n o f i o n .transport has been s t r e s s e d in plant c e l l s  (e.g. H a l l and Davie., 19 71;  Coulomb, 1972; Maier and Maier, 1972).  Coulomb and  Ratner and Jacoby  (1973) found t h a t i n t h e i r m a t e r i a l monovalent s a l t e f f e c t s on ATPase were not c a t i o n s p e c i f i c , and t h e r e f o r e not r e l a t e d t o the c e l l p o t e n t i a l f o r c a t i o n  absorption.  Whether or n o t the enhancement o f r e a c t i o n product  deposition  i n Ectocarpus r e f l e c t s c a t i o n s p e c i f i c i t y remains t o be determined.  However, other  r e q u i r e d i n order monovalent ions 1970;  authors have shown t h a t M g  f o r p l a n t ATPases t o be s t i m u l a t e d by  ( F i s h e r and Hodges, 1969; F i s h e r e t a l . ,  r e a c t i o n of the m i t o c h o n d r i a i s probably due t o  the M g - a c t i v a t e d system only, without being + +  monovalent i o n s . also reported  that M g  + +  above c o n s i d e r a t i o n s  and " c e l l type #2".  Novikoff.et.al.(1958)  ions p r e f e r e n t i a l l y s t i m u l a t e d the  ATPase system o f m i t o c h o n d r i a l The  a f f e c t e d by  Indeed, d e p o s i t i o n o f r e a c t i o n products i s  conspicuous i n both systems assayed.  fractions. h o l d f o r b o t h . " c e l l type  In " c e l l , type #3", however, none  of the systems assayed l e d to. deposition...of....reaction The  is  Leonard and Hanson, 1972). The  #1"  + +  products.  apparent lack, of.ATPases .in t h e s e . c e l l s - l e a d s one t o  conclude t h a t they are no longer  functional... The absence o f  ATPase a t t h e plasma ..membrane., implies,, .deeply, a l t e r e d p h y s i o l o g i c a l p r o p e r t i e s i n . 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  ( f i g u r e 191).  T h i s i s i n agreement  w i t h r e p o r t s showing t h a t as senescence proceeds changes i n permeability, are observed i n cells,, (e.g. E i l a m , Sacher, 1967; Catalase.  Fergusson and  Biochemical,  1965;  Simon, 1973).  cytochemical,  and  ultrastructural  s t u d i e s have,during the .past few y e a r s , p r o v i d e d  enough  evidence f o r the i d e n t i f i c a t i o n In p l a n t s of a c l a s s of organelles usually described et a l . , 1966;  as."microbodies"  F r e d e r i c k e t a l . , 1968;  (Molhenhauer  F r e d e r i c k and Newcomb,  1969a) or under more s p e c i f i c c o n d i t i o n s as peroxysomes ( F r e d e r i c k and Newcomb, 1969b; Marty, 1969,  1970;  et  Beevers,  a l . , 1971)  and glyoxysomes  Gerhardt and Beevers, 1970; al.,  ( V i g i l , 1970;  Gruber e t a l . , 1970;  Trelease 1970;  Trelease  et  1971). Cytoplasmic o r g a n e l l e s whose u l t r a s t r u c t u r a l  ( s i n g l e membrane boundary, g r a n u l a r matrix,  features  occasional  c o r e - l i k e s t r u c t u r e s ) resemble those, of the s o - c a l l e d microbodies are found i n a l l Ectocarpus s p o r o p h y t i c with the e x c e p t i o n like  of " c e l l type #3".  These microbody-  s t r u c t u r e s are i d e n t i f i e d c y t o c h e m i c a l l y  response to diaminobenzidine  (DAB)  cells,  by  incubation.  their Under these  c o n d i t i o n s heavy d e p o s i t i o n of r e a c t i o n products i s a s s o c i a t e d with  the m i c r o b o d i e s .  those incubated  i n a m i n o t r i a z o l e medium, show the r e a c t i o n  to be due  to the presence of c a t a l a s e  Novogrodsky, 1958;  Margoliash  enzyme marker f o r microbodies corroborated optimum pH  C o n t r o l s e c t i o n s , mainly (Margoliash  e t a l . , 1960)  and  which i s the  ( T o l b e r t , 1971)..  This i s  by the parameters of i n c u b a t i o n , i . e . ,  i n the a l k a l i n e , range.,, incubation, temperature  (37°C) (e.g. C z a n i n s k i . and Catesson, 1970,  1971;  Poux,  1972a, 1972b, 1972c). A c o n s i s t e n t and  c l o s e a s s o c i a t i o n was  found i n .  Ectocarpus between microbodies and m i t o c h o n d r i a . w i t h c h l o r o p l a s t s , although d e t e c t a b l e , was  not so  Association evident.  73 The mitochondrion-microbody  a s s o c i a t i o n i s not r e s t r i c t e d t o  the E c t o c a r p u s m a t e r i a l (e.g., H i l l i a r d e t a l . , 1971,  1972b; Tourte, 1972).  1971;  Poux,  T h i s a s s o c i a t i o n a l s o occurs i n  y e a s t and Tetrahymena c e l l s , where i t s occurrence i s r e l a t e d to the d i s t r i b u t i o n of g l y o x y l a t e c y c l e enzymes 1971).  I t i s i m p o s s i b l e , however, without  (Tolbert,  biochemical  evidence,to make any c o n c l u s i o n about the s i g n i f i c a n c e of such an a s s o c i a t i o n i n Ectocarpus  sp.  Reaction products were a l s o found i n m i t o c h o n d r i a . The e x p l a n a t i o n f o r m i t o c h o n d r i a l d e p o s i t i o n of r e a c t i o n products i s s t i l l debatable.  Since r e a c t i o n products  are  p r e s e n t even a f t e r i n c u b a t i o n i n a m i n o t r i a z o l e , one may out the p o s s i b i l i t y of c a t a l a s e involvement,  rule  although  c a t a l a s e has been s a i d t o be p r e s e n t i n . m i t o c h o n d r i a (Herzog and Fahimi, 197.2) .  Diaminobenzidine  i s known t o be  o x i d i z e d by hemoproteins other than c a t a l a s e , f o r i n s t a n c e , peroxidase and cytochrome oxidase Todd and V i g i l ,  1972).  (Seligman e t a l . , 1968;  Peroxidase i s i n d i c a t e d as a p o s s i b l e  cause of m i t o c h o n d r i a l r e a c t i o n s (Rothman, 196 8; C h i l d s and M i l l e r , 1971;  Gerhardt and Berger, 1971).  In E c t o c a r p u s ,  however, a f t e r i n c u b a t i o n under c o n d i t i o n s f a v o u r i n g optimum l o c a l i z a t i o n of p e r o x i d a s e , no obvious m i t o c h o n d r i a l r e a c t i o n s were found.  The data of P l e s n i c a r e t a l . (1967)  showed the absence of any k i n d of p e r o x i d a t i c a c t i v i t y i n p l a n t mitochondria.  The p o s s i b i l i t y then remains t h a t  m i t o c h o n d r i a l d e p o s i t i o n of r e a c t i o n products i s very l i k e l y due e i t h e r to cytochrome oxidase a c t i v i t y and Karnovsky, 1968;  Gerhardt and Berger, 1971;  (Seligman Kataoka,  1971)  or, as i n d i c a t e d by..Beard, and N o v i k o f f .(1969) ,-Novikoff and Goldfisher  (1969)., N o v i k o f f  and Poux (1972a), Peroxidase.  (1970), N o v i k o f f e t a l . (1971),  t o cytochrome c a c t i v i t y .  An i n c r e a s e i n peroxidase a c t i v i t y was  to be c h a r a c t e r i s t i c of the ageing process and Davies, 1969).  In Ectocarpus  reported  (e.g., G a l s t o n  " 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 p o s s i b l e t h a t i n " c e l l type #2". t h e r e i s p e r o x i d a s e . 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 v e r y d i f f i c u l t e v a l u a t e the p a t t e r n and i n t e n s i t y of the r e a c t i o n . any comparison w i t h data a v a i l a b l e . f o r the  to Therefore,  " c e l l type #1" or  t r a n s i t i o n a l stages ..immediately f o l l o w i n g . i s . not p o s s i b l e . Peroxidase a c t i v i t i e s In p l a n t s have been l o c a l i z e d ,  u s i n g the DAB/I^O,, procedure, i n many p a r t s o f the c e l l wall  (e.g. Ridge and Osborne,  1970; H a l l and Sexton,  1972;  Poux, 1972a, 1972b, 1972c); dictyosomes  1969,  1972a; H a l l and Sexton, 1972); vacuoles (e.g.  Coulomb, 1971;  cells:  H a l l and Sexton, 1972;  Poux, 1972a, 1972b, 1972c); ribosomes  (e.g. Poux  Hanzely and V i g i l ,  1972;  (e.g. Poux, 1972a,  1972b, 1972c); E.R.,  p e r i n u c l e a r spaces and plasma membranes  (e.g.  In E c t o c a r p u s , d e p o s i t i o n o f r e a c t i o n  Poux, 1972a).  products i s found i n the paramural space and i n the wall.  cell  That d e p o s i t i o n of r e a c t i o n p r o d u c t s . i s due t o p e r o x i -  dase a c t i v i t y can be i n f e r r e d from the experimental c o n d i t i o n s (e.g.  Poux, 1972a, 1972b, 1972c) as w e l l as from the  f o l l o w i n g c o n t r o l experiments:  1) a m i n o t r i a z o l e i n c u b a t i o n ,  which does not i n h i b i t d e p o s i t i o n of r e a c t i o n p r o d u c t s , and 2) potassium cyanide i n c u b a t i o n which i n h i b i t s of  deposition  r e a c t i o n products (Strum and Karnovsky, 1970;  Poux, 1972a).  The absence of r e a c t i o n products i n other i n t r a c e l l u l a r l o c a t i o n s does not n e c e s s a r i l y prove i t s absence.  Existing  evidence shows t h a t p e r o x i d a s e s , although u b i q u i t o u s i n d i s t r i b u t i o n , possess an unprecedented ..degree of heterogeneity  (e.g. Shannon, 1968;  and.Drawert,  1970)..  D e l i n c e e and Radola., 1970;  Rucker and Radola  Radola  (1971) have shown t h a t ,  i n tobacco t i s s u e c u l t u r e s , a number ..of. p e r o x i d a s e isoenzymes occur which c o u l d be separated ..into...3 groups on the b a s i s of  t h e i r i s o e l e c t r i c p o i n t s . . H a l l and .Sexton  (.1972) have  s t u d i e d the e f f e c t of pH on p e r o x i d a s e . a c t i v i t y and.found wide v a r i a t i o n i n pH-dependent responses.  The evidence  a  75 shows, t h e r e f o r e , t h a t d i f f e r e n t i s o p e r o x i d a s e s  might  r e q u i r e i n d i v i d u a l i n c u b a t i o n parameters to be. v i s u a l i z e d , as a r e c e n t The  study seems to i n d i c a t e (Poux, 19 72a).  present  data suggest: t h a t p e r o x i d a s e . a c t i v i t y i n  Ectocarpus i s mainly a c e l l w a l l a s s o c i a t e d phenomenon. Although r e c e n t . y e a r s biochemical little Osborne  have seen I n c r e a s i n g  and p h y s i o l o g i c a l aspects of  peroxidases,  i s known about t h e i r r e a l f u n c t i o n s .  Ridge  and  (1970) have suggested t h a t w a l l peroxidases might  f a c i l i t a t e hydroxylation  of p r o l i n e i n the w a l l p r o t e i n s ,  hence a f f e c t i n g c e l l w a l l e x t e n s i b i l i t y 1971).  i n t e r e s t i n the  Any  (Ridge and  r o l e of peroxidases i n c e l l w a l l  Osborne,  formation  i n Ectocarpus must, however, wait f o r f u r t h e r knowledge i n w a l l composition i n t h i s  alga.  76 CONCLUSION The p r e s e n t study has two o b j e c t i v e s . The  first  intends t o p r o v i d e a more d e t a i l e d knowledge of the morphology of non-senescing  cellular  v e g e t a t i v e c e l l s of the sporo-  p h y t i c g e n e r a t i o n of E c t o c a r p u s . H o p e f u l l y t h i s w i l l to  a b e t t e r understanding of the s i m i l a r i t i e s and  lead  the  d i f f e r e n c e s e x i s t e n t among the brown algae and between these and other organisms of  i n the p l a n t kingdom. The second  objective  the p r e s e n t work i s concerned with the study of the  senescing process as i t occurs i n Ectocarpus under c u l t u r e c o n d i t i o n s . C u l t u r e c o n d i t i o n s are known t o a f f e c t the metabolism  of the organisms  ( e. g. C a r o l e t al.,1972  ) and  so the i n t e r p r e t a t i o n of the r e s u l t s cannot be taken as d u p l i c a t i o n of f i e l d events. N e v e r t h l e s s , i t p r o v i d e a u s e f u l and a p p r o p r i a t e mean of understanding changes i n development and senescence way  important  i n a reproducible  . In  s t u d y i n g the aging process of any organism  the  i d e n t i f i c a t i o n of a s i n g l e i n i t i a l change c o u l d p r o v i d e a l e a d f o r a b e t t e r understanding of the events of N e v e r t h l e s s , as p o i n t e d out by B u t l e r  senescence.  (1967), i n i t i a l  damage t o the c e l l c o u l d be d i r e c t r e s u l t of a number of f a c t o r s a c t i n g alone and i n c o n c e r t , and t h e r e f o r e even a s m a l l change would have an accumulative e f f e c t .  Identifi-  c a t i o n of a s i n g l e i n i t i a l a l t e r a t i o n a t 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  t a s k . T h e r e f o r e , any attempt  l o c a l i z e c h r o n o l o g i c a l l y and s p a t i a l l y ted  to  senescence-associa-  phenomena must be regarded as an approximation t o the  r e a l sequence. B e a r i n g t h i s i n mind, a summary of o r g a n e l l e c h a r a c t e r i s t i c s and m o d i f i c a t i o n s o c c u r r i n g in. Ectocarpus from " c e l l type #1" f o l l o w i n g page.  t o c e l l death i s presented i n the  I  77 From the.data i t can be s a i d t h a t i n c r e a s e s i n v a c u o l a t i o n and autophagy as w e l l as c h l o r o p l a s t m o d i f i c a t i o n s seem t o be the f i r s t  d e t e c t a b l e s i g n s o f aging i n  E c t o c a r p u s . A f u l l cdmparision  o f t h e order of events i n  r e l a t i o n t o the data p r o v i d e d by senescence s t u d i e s i n other p l a n t s i s unwarranted here; not only because o f d i f f i c u l t i e s i n e s t a b l i s h i n g w i t h p r e c i s i o n the a c t u a l order o f events, b u t a l s o because, as d i s c u s s e d by Roux and McHale  (1968), v a r i a t i o n s i n the order o f appearance o f  a l t e r a t i o n s might be s p e c i e s dependent. It  i s apparent,  however, from t h i s work t h a t the  p a t t e r n o f c e l l u l a r d i s o r g a n i z a t i o n i n Ectocarpus  has many  p o i n t s i n common w i t h the events r e p o r t e d d u r i n g senescence in  other p l a n t s . The p o s s i b l e presence  of l i p o f u s c i n metabolites  d u r i n g the aging o f Ectocarpus  a l s o shows t h a t a common  denominator o f aging might e x i s t from algae t o man as suggested  by P a l i s a n o and Walne (1972) d u r i n g t h e i r study o f  senescence i n Euglena  g r a n u l a t a . Other changes a s s o c i a t e d  with aging i n other p l a n t and animal c e l l s suggestion  : i . e. l o b i n g of the nucleus  C r i s t o f a l o , 1 9 7 2 ; Munnell  ( L i p e t z and  and Getty,1968 ) , mitochondria  denser matrix and fewer c r i s t a e 1972;  support such a with  ( L i p e t z and C r i s t o f a l o ,  Weinbach e t al.,1967 ) , i n c r e a s e s i n c e l l u l a r autophagy  ( Sohal and A l l i s o n , 1 9 7 1 ; L i p e t z and C r i s t o f a l o , 1 9 7 2 ) . In c o n c l u s i o n , i t can be s a i d t h a t no s i n g l e t e c h n i c a l approach w i l l e l u c i d a t e such a complex phenomenon as i s the case o f senescence. to  Each t e c h n i c a l approach i s able  p r o v i d e p a r t i a l i n f o r m a t i o n . T h i s information, must then  be compared and c o r r e l a t e d w i t h other data, obtained by d i f f e r e n t means, and from a l a r g e number o f t e s t organisms. 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OF SYMBOLS ac  apical  cell  ce  c h l o r o p l a s t envelope  cer  c h l o r o p l a s t endoplasmic  Ch  chloroplast  cw  cell  cwi  c e l l w a l l ingrowth  D  dictyosome  Dv  dictyosome v e s i c l e  ER  endoplasmic  reticulum  er  endoplasmic  reticulum  g il  genophore i n n e r l a y e r of the c e l l  L  lomasome  M  mitochondrion  m  microbody  N  nucleus  nl  nucleolus  ol  outer l a y e r o f the c e l l  OSB  o s m i o p h i l i c s t r u c t u r e d body  Pg  plastoglobuli  pm  paramural space  pns  perinuclear  Py  pyrenoid  Pys  p y r e n o i d sac  r  ribosomes  V  vacuole  reticulum  wall  space  wall  wall  10 9 Figure  1  Phase  contrast observation  i n s i t u embedded f i l a m e n t  2  ( prostrate  system ). A p r o g r e s s i v e  i n c r e a s e i n the  of a f f i n i t y f o r osmium  i s observed from  the a p i c a l c e l l Figure  of an  (a.e.) downwards. X 750  Phase c o n t r a s t o b s e r v a t i o n . o f embedded f i l a m e n t  an i n s i t u  ( e r e c t system ). A  sudden l o s s i n a f f i n i f y f o r osmium occurs i n c e l l s of the o l d e r p o r t i o n o f f i l a m e n t s , X 1,000 Figure  3  In s i t u embedded f i l a m e n t  ( prostrate  system ).The l o s s of a f f i n i t y f o r osmium i s sudden  ( c e l l s l o c a t e d a t the o l d e r end  of the f i l a m e n t  ). N o t i c e  t h a t the c e l l  between the dark and the l i g h t  wall  cell is  convex. X 1,000 Figure  4  Multilobed  c h l o r o p l a s t . The p y r e n o i d i s  i d e n t i f i e d by an arrowhead. Phase c o n t r a s t observation Figure  5  Bright f i e l d  o f l i v i n g m a t e r i a l . X 1,500 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 types  ( older p o r t i o n of a filament  i d e n t i f i e d by the c e l l w a l l  cell )is  convexity.  X 1,000 Figure 6  Phase c o n t r a s t o b s e r v a t i o n  of l i v i n g ...  m a t e r i a l . P o s s i b l e phases o f c h l o r o p l a s t division  ( arrowheads ). X 1,500  110 Figure 7  Phase c o n t r a s t o b s e r v a t i o n  of l i v i n g  m a t e r i a l . C h l o r o p l a s t morphology.. X 800 Figure 8  Phase c o n t r a s t o b s e r v a t i o n  of l i v i n g  m a t e r i a l . Two d i f f e r e n t stages d i v i s i o n are observed Figure 9  ( arrowheads ). X 900  Phase c o n t r a s t o b s e r v a t i o n  of l i v i n g  m a t e r i a l . A c h l o r o p l a s t with i s depicted F i g u r e 10  of p y r e n o i d  3  ( arrowheads ) . X  Phase c o n t r a s t o b s e r v a t i o n  pyrenoids 1,500  of l i v i n g  m a t e r i a l . A t arrowheads l o c a l i z e d of the c e l l w a l l are shown. X F i g u r e 11  Phase c o n t r a s t o b s e r v a t i o n  ingrowths  1,000  of l i v i n g  m a t e r i a l . A c e l l w a l l ingrowth i s seen a t 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 s t r u c t u r e s , a f t e r  RNA  extraction. F i g u r e 12  " C e l l type #1".  Intense s t a i n i n g  of the nucleus,  especially  apparent a t arrowheads. X F i g u r e 13  " C e l l type #2". Nucleus p e r f e c t l y s t a i n e d . Nucleolus stained  F i g u r e 14  1,200  ( arrowhead ). X  un1,200  " C e l l type #3". Absence of DNA staining characterizes this type. X  1,200  cell  Ill Figures  15, 16, & 17  Cytochemical i d e n t i f i c a t i o n of  RNA-containing  structures, after  DNA  extraction. Figure  15  " C e l l type #1". stained  Nucleus  (N). Intense s t a i n i n g  i s found a l l over the plasm. X Figure  16  cyto-  1,200  " C e l l type #2". stained  Nucleus  (N). S t a i n i n g  Figure  17  un-  restri-  c t e d t o the p e r i n u c l e a r X  un-  region.  1,200  " C e l l type #3".  The  almost  absence of s t a i n i n g c h a r a c t e r i z e s t h i s c e l l type. X Figure  18  1,000  Cytochemical i d e n t i f i c a t i o n of p r o t e i n staining regions.  " C e l l type #1".  Intense  s t a i n i n g i s seen almost a l l over the cytoplasm, p a r t i c u l a r l y a t the (Ch) and pyrenoids  chloroplasts  (Py) l e v e l s . X  800  112 F i g u r e s 19 .& 20  Cytochemical i d e n t i f i c a t i o n of protein s t a i n i n g regions.  F i g u r e 19  " C e l l type #2".  Chloroplasts  (Ch) and pyrenoids  (Py) show  intense s t a i n i n g .  Cytoplasmic  s t a i n i n g i s r e s t r i c t e d to c e r t a i n areas. F i g u r e 20  " C e l l type #3".  X  800 The  almost  absence of s t a i n i n g from the cytoplasm of these c e l l s i s characteristic. F i g u r e s 21 & 22  X  1,200  Cytochemical i d e n t i f i c a t i o n of insoluble  F i g u r e 21  carbohydrates.  C e l l b e l o n g i n g t o the " t r a n s i t i o n a l 1-2"  stages.  S t a i n i n g i s observed i n the perinuclear region a r e a ) , paramural (arrowhead), (cw). F i g u r e 22  X  (Golgi  space  and c e l l  wall  1,200  " C e l l type #1".  Section  through the c e l l w a l l showing intense s t a i n i n g properties. X, 1,000 F i g u r e s . 23, 24 & 25  Cytochemical study of distribution  Figure  23  lipid  (Sudan B l a c k B ) .  " C e l l type #1".  Very  few  113 s i t e s w i t h a f f i n i t y f o r Sudan. X 1,000 F i g u r e 24  " C e l l type #2.Conspicuous staining  a l l over the c y t o -  plasm. X 1,000 F i g u r e 25  " C e l l type #3". An almost absence of s t a i n i n g  characte-  r i z e s t h i s c e l l type. X 1,000 F i g u r e 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 w i t h the Schorml's  reagent i s shown i n t h i s  picture.  X 750 F i g u r e 27  " C e l l type #2". T o l u i d i n e b l u e 0. Some o f the c y t o p l a s m i c i n c l u s i o n s  show a green  to t u r q u o i s e c o l o u r i n d i c a t i v e o f p o l y p h e n o l i c content. X 750  114 F i g u r e 28a  General u l t r a s t r u c t u r e o f a young  cell  ( c e l l type #1) b e l o n g i n g t o the p r o s t r a t e system o f E c t o c a r p u s sp. X 18,000 F i g u r e 28b  Plasmalemmasomes. The r e l a t i o n s h i p o f one of  these s t r u c t u r e s t o the plasmalemma  i s shown a t arrowhead. X 22,000 F i g u r e 29  S e c t i o n t a n g e n t i a l t o the n u c l e a r envelope showing n u c l e a r pores. Observe t h a t a hollow c e n t r a l core r a d i a t e toward  (arrowheads)  from spokes  the o u t e r r i m .  X 100,000 F i g u r e 30  Fibrillar  aspect o f chromatin. X 90,000  Note: To b e s t d e t e c t the i n t r a n u c l e a r f i b r i l l a r system t h i s p i c t u r e must be observed i n the d i r e c t i o n i n d i c a t e d by the arrowhead.  1  115 F i g u r e s 31 & 32  General u l t r a s t r u c t u r a l morphology of e r e c t system c e l l s . #1".  X 18,000  "Cell  type  116 F i g u r e 33  General u l t r a s t r u c t u r a l morphology  of a  young p r o s t r a t e system c e l l . X 18,000 F i g u r e 34  D i r e c t c o n t i n u i t y between the n u c l e a r envelope and the dictyosome i s d e p i c t e d at arrowhead. X 16,000  F i g u r e 35  A s s o c i a t i o n between the endoplasmic reticulum  ( E . R . ) and the mitochondrion  (M) i s shown a t arrowhead. X 30,000  117 F i g u r e 36  Freeze-etch. r e p l i c a o f the n u c l e a r A many-particulate particulate  envelope  (A) and a s p a r s e l y  (B) s u r f a c e s are r e c o g n i z e d .  X 60,000 F i g u r e 37  Freeze-etch  r e p l i c a o f the n u c l e a r envelope  Pore c o n t a i n i n g areas are permeated by pore f r e e r e g i o n s . X 30,000 F i g u r e 37a  Freeze-etch  r e p l i c a of the p e r i p h e r y o f a  young c e l l . Plasmalemmasomes are observed i n the paramural space  (pm). X 26,000  118 Figure  38  P o r t i o n of. the nucleus and p e r i n u c l e a r r e g i o n o f a young c e l l . The c l o s e t i o n between the' n u c l e o l u s  associa-  (nl) and the  i n n e r membrane of the n u c l e a r  envelope i s  apparent. D i r e c t c o n t i n u i t y o f the p e r i nuclear  space w i t h the E.R. i s observed.  At other p l a c e  ( empty arrowhead ) a  m u l t i v e s i c u l a r s t r u c t u r e occupies a p o s i t i o n shared by both the E.R. and the p e r i n u c l e a r space. Other f e a t u r e s are E.R. fenestrae  ( arrowheads ) and m i t o c h o n d r i a  (M). The s m a l l arrowhead p o i n t s t o a mitochondrion DNA-containing area. X 55,000 Figures  39 & 40  V e s i c u l a r - l i k e i n c l u s i o n s are seen  i n s i d e the p e r i n u c l e a r space  (arrowheads ).  X 18,000 Figure  41  The r e l a t i o n s h i p o f the p e r i n u c l e a r i n c l u s i o n s t o the n u c l e a r  envelope mem-  branes i s shown a t arrowheads. X 40,000 Figure  42  A vesicular structure  ( arrowhead ) i s  observed i n a s s o c i a t i o n w i t h the outer membrane o f the n u c l e a r dictyosome  envelope. A  (D) i s a l s o d e p i c t e d .  X 25,000  119 Figure  43a  Freeze-etch. p r e p a r a t i o n  of a c h l o r o p l a s t ,  "g" i n d i c a t e s the p o s i t i o n o f the genophore.-Thylakoid a s s o c i a t e d are d e p i c t e d  a t small  fibrils  arrowheads and a t  nearby p o s i t i o n s . A f e n e s t r a - l i k e i s shown i n the c h l o r o p l a s t E.R.  structure ( big  arrowhead ). X 40,000 Figure  43b  D e t a i l o f the area l a b e l l e d "A" i n f i g u r e 43a. and  Both the c h l o r o p l a s t envelope (ce) ( probably ) the c h l o r o p l a s t  (cer) are shown. A p o r e - l i k e  E.R.  structure i s  observed a t arrowhead. X 30,000 Figure  44  Endoplasmic r e t i c u l u m  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 i n s i d e . the E.R.  ( i ) . The arrowhead p o i n t s  f e n e s t r a - l i k e s t r u c t u r e . X 60,000  to a  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 p e r i n u c l e a r  space  endoplasmic r e t i c u l u m  ( pns ) and  ( er ) inclusions  i s shown i n t h i s p i c t u r e . A " Structured  Osmiophilic  Body " (OSB) i s seen i n  a s s o c i a t i o n w i t h the c h l o r o p l a s t (Ch). X 90,000 Figure  46  Freeze-etch preparation The  pattern  of a chloroplast.  o f t h y l a k o i d arrangement and  the p o s i t i o n o f the c h l o r o p l a s t  envelope  (ce) i n r e l a t i o n t o the c h l o r o p l a s t (cer) are shown. Other f e a t u r e s  E.R.  include  the presence o f a t h y l a k o i d  f r e e area ( F ) ,  and  i n the  of v e s i c u l a r structures  narrow c y t o p l a s m i c r e g i o n  found i n  between the "ce" and the "cer" arrowhead ). X 36,000  ( big  121 Figure  47a  A p o r t i o n of a c h l o r o p l a s t i s depicted; "g" i n d i c a t e s the p o s i t i o n o f the genophore. The e x i s t e n c e  of d i r e c t  between the c h l o r o p l a s t E.R.  continuity  (cer) c i s t e r -  n a l space and the c h l o r o p l a s t envelope (ce) i s shown a t arrowhead. A l s o apparent i s an initial  step i n the formation o f a " Os-  m i o p h i l i c Structured  Body " (OSB). Meta-  b o l i t e s o f high e l e c t r o n o p a c i t y are seen to be discharged (pm). Figure  47b  i n the paramural space  X 42,000  A step i n the process o f formation o f a " OSB " i s d e p i c t e d . the  The r e l a t i o n s h i p o f  " OSB " t o the c h l o r o p l a s t E.R. (cer)  i s apparent. X 40,000 Figure  48  P o r t i o n o f a c h l o r o p l a s t showing a genophore-like  region  (g). D i r e c t  luminal  c o n t i n u i t y between the c h l o r o p l a s t E.R. (cer) c i s t e r n a l space and the c h l o r o p l a s t envelope The  (ce) i s apparent  ( arrowhead ) .  c l o s e a s s o c i a t i o n between the c h l o -  r o p l a s t and the mitochondrion  (M) i s a l s o  shown. X 42,000 Figure  49  An almost t a n g e n t i a l s e c t i o n through the r e g i o n o f the c h l o r o p l a s t E.R.  (cer) shows  the i n t r i c a t e p a t t e r n o f arrangement o f the  "cer" d e r i v e d  tubular  Lomasome-like s t r u c t u r e s  structures. (L) are p r e s e n t  i n the paramural space. Membrane are seen i n s i d e the vacuoles  residues  (V). X 24,000  Figure  50  V e s i c u l a r - l i k e i n c l u s i o n s are present i n s i d e the c h l o r o p l a s t E.R; space  Figure  51  cisternal  ( c e r ) . X 36,000  Osmiophilic Notice  s t r u c t u r e d body morphology.  t h a t a non s t r u c t u r e d component i s  i n t e r m i n g l e d w i t h a m y e l i n - l i k e one. X 60,000 Figure  52  An o s m i o p h i l i c s t r u c t u r e d body (osb)  is  observed i n the space shared by the c h l o r o p l a s t E.R. space Figure  53  (cer) and the p e r i n u c l e a r  (pns). X 30,000  The a s s o c i a t i o n between a o s m i o p h i l i c s t r u c t u r e d body (osb) and the p y r e n o i d (Py) sac  Figure  54  i s shown.  A p o r t i o n o f the p y r e n o i d  (Pys) i s a l s o d e p i c t e d . X 36,000  The d i s c h a r g e o f o s m i o p h i l i c bodies  structured  (osb) i n t o the paramural space  (pm) i s shown. X 36,000 Figure  55  The n u c l e a r  envelope-dictyosome r e l a t i o n -  s h i p i s shown. A l s o d e p i c t e d  i s the  a s s o c i a t i o n o f the dictyosome  (D) w i t h  a o s m i o p h i l i c s t r u c t u r e d body  (osb).  X 50,000  123 F i g u r e 56  P o r t i o n of a young c e l l  ( cell  showing p e r i p h e r a l l o c a l i z a t i o n mitochondria. Osmiophilic bodies  ( osb)  type #1 ) of  structured  are found e i t h e r i n a s s o c i a -  t i o n w i t h the c h l o r o p l a s t  (Ch) or they l i e  i n the cytoplasm without any  apparent  r e l a t i o n s h i p t o these o r g a n e l l e s .  An  e a r l y stage i n the process of formation of a c e l l w a l l ingrowth d e p i c t e d . X 30,000  (cwi) i s a l s o  124 F i g u r e 57  Dictyosome.morphology. I n t e r r u p t i o n s be r e c o g n i z e d i n the c i s t e r n a e  can  o f the  dictyosome. These seem t o be o f two t y p e s : 1) a narrow t u 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. I n t e r r u t p i o n s r e c o g n i z a b l e i n the c i s t e r n a e dictyosome ptions  of the  ( arrowheads ). These i n t e r r u -  seem t o be more;abundant i n the  innermost c i s t e r n a e . terna  are  displays  (D). X 70,000  The outermost c i s -  a h y p e r t r o p h i e d appearance  125 Figure  59  Dictyosome morphology. B r i d g e - l i k e elements are observed i n between the c i s t e r n a e o f the dictyosome as w e l l 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 e x i s t e n c e  o f a very c l o s e  between the dictyosome (Py) Figure  61  association  (D) and p y r e n o i d  i s observed. X 30,000  Dictyosome  (D)-mitochondria  t i o n i s depicted.  X 30,000  (M) a s s o c i a -  126 F i g u r e 62  I n c o r p o r a t i o n o f dictyosbme-derived cles  vesi-  ( b i g arrowheads ) i n t o vacuoles (V).  Three p o s s i b l e  cases o f m i t o c h o n d r i a l  d i v i s i o n are shown a t s m a l l arrowheads. X 36,000 F i g u r e 63  Dictyosome  (D)-pyrenoid  (Py) a s s o c i a t i o n .  X 20,000 F i g u r e 64  A stage i n the formation o f a lomasomel i k e structure hypertrophied  (L) i s seen i n s i d e an dictyosome c i s t e r n a  ( c. f .  with f i g u r e 49, showing a p o t e n t i a l lomasome-like s t r u c t u r e  'L' i n s i d e the  paramural space ). X 24,000 F i g u r e 65  Multimembranous i n c l u s i o n occupies the outermost c i s t e r n a o f a dictyosome. The mode o f formation  of t h i s  through the i n v a g i n a t i o n peripheral  structure o f the c i s t e r n a  membrane i s d e p i c t e d a t the  arrowhead. X 80,000 F i g u r e 66  Tubular-like  i n c l u s i o n s are seen i n s i d e  a dictyosome-derived  vesicle  (Dv). X 80,000  127 F i g u r e 67  Mitochondrion.morphology.  X 20,000  F i g u r e 68  D e t a i l o f the mitochondrion  cristae  I n c l u s i o n s are shwon a t arrowhead and i n the i n s e t . X 50,000 I n s e t X 120,000 F i g u r e 69  Mitochondrion  (M)-nucleus  (N) a s s o c i a t i o n ,  X 24,000 F i g u r e 70  F r e e z e - e t c h p r e p a r a t i o n showing the assoc i a t i o n o f a mitochondrion chloroplast  F i g u r e 71  (M) w i t h a  (Ch). X 22,000  Mitochondrion  (M)-pyrenoid  (Py) a s s o c i a -  t i o n . X 24,000 F i g u r e 72  Mitochondrion-mitochondrion a s s o c i a t i o n ( arrowheads ). A t arrow the mitochond r i o n - E.R. a s s o c i a t i o n i s d e p i c t e d . X 30,000  F i g u r e 73  High m a g n i f i c a t i o n o b s e r v a t i o n o f t h y l a k o i d s . B i g arrowhead p o i n t s t o an i n t e r r u p t i o n . Small arrowheads i n d i c a t e the presence o f b r i d g e - l i k e elements. X 120,000  F i g u r e 74  Freeze-etch preparation of a p o r t i o n of the t h y l a k o i d system o f a c h l o r o p l a s t . Arrowheads i n d i c a t e the presence o f b r i d g e - l i k e elements. X 120,000  128 F i g u r e 75  D e v i a t i o n s from the normal p a t t e r n o f t h y l a k o i d arrangement  F i g u r e 76  are shown. X 60,000  T h y l a k o i d i n t e r r u p t i o n s a r e seen a t arrowheads  "a". Arrowheads "b" p o i n t t o  b r i d g e elements. Arrowheads . "c" i n d i c a t e p o r e - l i k e i n t e r r u p t i o n s i n the c h l o r o p l a s t envelope. X 80,000 F i g u r e 77  High m a g n i f i c a t i o n o b s e r v a t i o n o f the arrowhead  labelled  "a" i n f i g u r e 78.  X 100,000 F i g u r e 78  S e c t i o n t a n g e n t i a l t o the t h y l a k o i d membranes  showing the presence o f pore-  l i k e i n t e r r u p t i o n s ( arrowheads F i g u r e 79  ). X 50,000  High m a g n i f i c a t i o n o b s e r v a t i o n o f the thylakoid architecture.  Arrowheads p o i n t  to b r i d g e - l i k e elements. X 90,000 F i g u r e 80  Freeze-etch  preparation of thylakoids  showing the p a r t i c u l a t e nature o f the exposed s u r f a c e s . A many p a r t i c u l a t e (B) and a s p a r s e l y p a r t i c u l a t e (A) s u r f a c e s are r e c o g n i z e d . X 60,000  129 F i g u r e 81  Freeze-etch  preparation of a chloroplast.  The p a r t i c u l a t e nature o f exposed s u r f a c e s i s d e p i c t e d . A many p a r t i c u l a t e (B) and a s p a r s e l y p a r t i c u l a t e (A) s u r f a c e s are observed. X 60,000 F i g u r e 82  C h l o r o p l a s t a r c h i t e c t u r e . The p a t t e r n o f b i f u r c a t i o n and merging o f n e i g h b o r i n g t h y l a k o i d bands i s d e p i c t e d . N o t i c e  that  the p e r i p h e r a l band of t h y l a k o i d s i s not continuous, i n s t e a d i s made up o f p o r t i o n s of the c e n t r a l l y p l a c e d bands heads ). X 40,000  ( arrow-  130 Figure  83  Tangential  s e c t i o n through a dictyosome.  Interruptions 2) n a r r o w  : 1) l a r g e  ( arrowheads "a" ) ,  ( a r r o w h e a d s "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 Figure  84  Mitochondrial  80,000  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 the c h l o r o p l a s t (Ch), pore-like  interruptions are detected i n  the  thylakoids  the  chloroplast  X  75,000  ( a r r o w h e a d s " a " ) and i n e n v e l o p e ( a r r o w h e a d "b" ),.  131 F i g u r e s 85, 86, 87, and 88 formation  In the  and-detachment from, c h l o r o p l a s t s  of c o n c e n t r i c F i g u r e 85  D i f f e r e n t stages bodies.  the arrowhead p o i n t s t o the p l a c e where b i f u r c a t i o n o f one t h y l a k o i d has o c c u r r e d . X 45,000  Figure 8 6  C o n c e n t r i c arrangement o f t h y l a k o i d s i s observed i n s i d e the c h l o r o p l a s t . X 40,000  F i g u r e 87  C o n c e n t r i c body becomes d e t a ched from the p l a s t i d  (arrow-  head) . X 36,000 F i g u r e 88  C o n c e n t r i c body l i e s f r e e i n the cytoplasm. X 26,000  F i g u r e 89  A c i d phosphatase a c t i v i t y i s found a s s o c i a t e d with c o n c e n t r i c bodies they are p r e s e n t  once  i n s i d e vacuoles.  X 30,000 F i g u r e 90  A c i d phosphatase a c t i v i t y  (control  p r e p a r a t i o n ) . N o t i c e the absence o f . r e a c t i o n product. F i g u r e 91  X. 30,000  C o n c e n t r i c body I n s i d e X 30,000  vacuole.  P o r t i o n o f a c h l o r o p l a s t showing a t h y l a koid free region  (F). B r i d g e - T i k e  elements  are found between the outermost t h y l a k o i d of the p e r i p h e r a l band and the c h l o r o p l a s t envelope. X 80,000 The  c l o s e r e l a t i o n s h i p between the  pyrenoid  matrix f i b r i l s and the surroun-  ding t h y l a k o i d membranes i s observed ( m a t e r i a l h e l d f o r 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 c l o s e a s s o c i a t i o n w i t h t h y l a k o i d s . X 50,000 Figure  95  Freeze-etch preparation  of a c h l o r o p l a s t .  F i b r i l l a r elements are seen i n a s s o c i a t i o n w i t h the t h y l a k o i d s A plastoglobulus  ( arrowheads ) .  (Pg) i s a l s o  depicted.  X 36,000 Figure  96  S e c t i o n p a r a l l e l t o the t h y l a k o i d membranes.  F i b r i l l a r elements are seen t o run  i n between t h y l a k o i d s X 90,000  ( white arrows ).  134 Figures.97,  98, and 99  Plastoglobuli architecture.  N o t i c e the f i b r i l l a r o r g a n i z a t i o n o f these  s t r u c t u r e s . A t p l a c e s the f i b r i l l a r  elements t h a t composed to  these bodies  seem  be l i n k e d t o the t h y l a k o i d membranes  ( arrowheads ). One o f t h e micrographs ( f i g u r e 99 ) shows the f i b r i l l a r o f one p l a s t o g l o b u l u s t o be  phase  continuous  with the f i b r i l l a r phase o f another.  F i g u r e 100  F i g u r e s 97, and 98  X 100,000  Figure  X  99  E.R.- vacuole  80,000  continuity i s depicted at  arrowheads. X 25,000 F i g u r e l o i Laminaria  type o f c h l o r o p l a s t d i v i s i o n .  The c o n s t r i c t i n g r e g i o n o f the c h l o r o p l a s t i s i n d i c a t e d by an arrowhead. X 20,000 F i g u r e s 102, and 103  S p h a c e l a r i a type o f c h l o r o p l a s t  division. F i g u r e 102  Plasmalemmasome-like  (pm)  s t r u c t u r e s are a l s o d e p i c t e d . X 12,000 F i g u r e 103  Arrowhead p o i n t s t o the p l a c e o f the p e r i p h e r a l lamellar bridge X 18,000  formation.  135 F i g u r e 10 4  B l e b - l i k e s t r u c t u r e produced by a c h l o r o p l a s t o f dark t r e a t e d  material  ( 5 days i n t o t a l darkness ). X 60,000  136 Figures  105, and 106.  Non c o n s e c u t i v e  serial  sections  showing a c o n s t r i c t i n g c h l o r o p l a s t .  Figure  107  Figure  105  X 16,000  Figure  106  X 26,000  C h l o r o p l a s t w i t h two d i s t i n c t c o n s t r i ction sites  ( b i g arrowheads ) . Geno-  phore i s i n d i c a t e d by small  arrowheads.  X 24,000 Figure  108  C h l o r o p l a s t w i t h two p y r e n o i d s . pyrenoid  The  on the r i g h t s i d e o f the  micrograph shows a c l o s e r e l a t i o n s h i p t o t h y l a k o i d s . X 24,000  F i g u r e 109  T h i s f i g u r e d e p i c t s a stage i n the process o f p y r e n o i d d i v i s i o n ; n o t i c e both the c h l o r o p l a s t envelope  (ce)  and  the c h l o r o p l a s t E.R. ( c e r ) . X 100,000 F i g u r e 110  Cross s e c t i o n o f a p y r e n o i d d e p i c t i n g the r e l a t i o n s h i p e x i s t e n t among the d i f f e r e n t membrane systems which surround  F i g u r e 111  the p y r e n o i d . X 40,000  Cross w a l l a d j o i n i n g two c o n s e c u t i v e c e l l s . N o t i c e t h a t the plasmadesmata are not organized i n t o a p i t - l i k e  area.  X 30,000 F i g u r e 112  D i r e c t c o n t i n u i t y between the p y r e n o i d sac space X 22,000  (Pys) and the vacuole ( v ) .  Figure  113  C e l l w a l l o r g a n i z a t i o n . Two r e g i o n s are recognized . i n t h e c e l l , w a l l : 1) an Inner l a y e r  (II) showing a p a r a l l e l  arrangement o f i t s f i b r i l l a r 2) an outer  layer  elements,  (ol) r e t i c u l a t e i n  appearance. X 60,000 Figure  114  Longitudinal section passing  through  the r e g i o n o f d i v i s i o n o f a  pyrenoid.  X 50,000 Figure  115  A t arrowheads, one process o f autophag o c y t o s i s , i n v o l v i n g the i n v a g i n a t i o n o f the t o n o p l a s t ,  i s depicted.  Provacuole-  l i k e s t r u c t u r e s are i n d i c a t e d by arrows. T h e i r o r i g i n from the E.R. i s apparent. X 36,000 Figure  116  D e t a i l o f a p o r t i o n o f the cytoplasm showing p r o v a c u o l a r tendency t o with vacuoles  s t r u c t u r e s and t h e i r  merge w i t h themselves and (arrowheads). X 40,000  F i g u r e 117  F r e e z e - e t c h p r e p a r a t i o n o f the c e l l w a l l , showing the b i l a y e r e d o r g a n i z a t i o n . X 30,000  F i g u r e 118  Freeze-etch  preparation of a region of  the cytoplasm  showing  provacuolar  s t r u c t u r e s , and t h e i r tendency t o fuse with each other F i g u r e 119  ( arrows ). X 40,000  The involvement o f E.R. elements i n the i s o l a t i o n of cytoplasmic  territories i s  shown. N o t i c e a t arrow the b i f u r c a t i o n of the E.R. X 40,000 F i g u r e s 120, and 121  Aspects o f the i s o l a t i o n o f  cytoplasmic  t e r r i t o r i e s i n v o l v i n g the  p a r t i c i p a t i o n o f the E.R. X 60,000 F i g u r e s 122, and 123 process  Two d i f f e r e n t stages i n the o f t r a n s f o r m a t i o n o f E.R.  membranes and i s o l a t e d m a t e r i a l s t y p i c a l vacuoles F i g u r e 124  The  into  are shown. X 60,000  t r a p i n g o f c y t o p l a s m i c m a t e r i a l by  t o n o p l a s t i n v a g i n a t i o n i s shown. X 45,000  140 F i g u r e s 125, and 126  A c i d phosphatase a c t i v i t y i s  a s s o c i a t e d w i t h v a c u o l e s . X 30,0.00 F i g u r e 127  Vacuole showing remnants of d i s o r g a n i zed membrane i n c l u s i o n s . X 30,000  F i g u r e 128  I n c l u s i o n s crowding the cytoplasm transitional  F i g u r e s 129, and 130  " 1-2 " c e l l .  of a  X 6,000  A c i d phosphatase a c t i v i t y i n  dictyosomes  ( f i g u r e 129 ) and E.R.  ( f i g u r e 130 ) .  F i g u r e 131  F i g u r e 129  X 10,000  F i g u r e 130  X 20,000  A c i d phosphatase a c t i v i t y  ( control  p r e p a r a t i o n ). N o t i c e the absence o f r e a c t i o n product. F i g u r e 132  X 12,000  T r a n s i t i o n a l " 1-2 " c e l l . N o t i c e the p a t t e r n o f t h y l a k o i d arrangement. X 25,000  F i g u r e 133  N o t i c e t h a t n e i t h e r the p l a s t o g l o b u l i (Pg) nor the r e g i o n o f i n t r a p l a s t i d metabolite  ( b i g arrowhead ) are p r e s e r v e d by  permanganate f i x a t i o n . X 20,000 F i g u r e 134  Permanganate e f f e c t s on the p r e s e r v a t i o n of c y t o p l a s m i c  i n c l u s i o n s . Notice  that  w h i l e some i n c l u s i o n s are p r e s e r v e d "1"; others are only p a r t i a l l y "2", and s t i l l others are not preserved X 17,500  a t a l l "3".  F i g u r e s 135, and 136  T r a n s i t i o n a l " 1-2 " c e l l s .  Two types o f vacuolar. I n c l u s i o n s seem to be p r e s e n t i n these c e l l s . One type (a) i s g r a n u l a r i n appearance, the other i s m y e l i n - l i k e (b). X 36,000  142 F i g u r e 137  High: m a g n i f i c a t i o n o b s e r v a t i o n o f . v a c u o l a r i n c l u s i o n s found i n t r a n s i t i o n a l " 1-2 " c e l l s . A mixed, m y e l i n - l i k e , inclusion  (B) and g r a n u l a r I n c l u s i o n s (A)  are d e p i c t e d . X 80,000 F i g u r e s 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 o f a c i d phosphatase a c t i v i t y X 12,000 F i g u r e 140  T r a n s i t i o n a l "1-2 " c e l l . A c i d phosphatase a c t i v i t y  ( c o n t r o l p r e p a r a t i o n ).  N o t i c e the absence o f r e a c t i o n product. X 10,000 F i g u r e 141  T r a n s i t i o n a l " 1-2 " c e l l .  Aspect o f the  G o l g i r e g i o n . Dictyosome-derived v e s i c l e s (Dv)  are f i l l e d with  X 20,000  metabolites.  Figure  142  T r a n s i t i o n a l " 1.-2 " c e l l . Notice  Cell wall.  the presence o f e l e c t r o n dense  metabolites fibrils  among the c e l l w a l l micro-  and a t the w a l l  periphery.  X 36,000 Figure  143  " C e l l type #2". General u l t r a s t r u c t u r e of a p r o s t r a t e system c e l l . A t b l a c k arrows s e v e r a l c h l o r o p l a s t involved i n metabolite  regions  p r o d u c t i o n are  i n d i c a t e d . White arrows p o i n t t o a h i g h l y v e s i c u l a t e d E.R. r e g i o n . X 18,000 Figure  144  " C e l l type #2". C e l l w a l l . N o t i c e the presence o f conspicuous patches o f metabolites fibrils  among the c e l l w a l l micro-  and a t the w a l l  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 o f an e r e c t system  cell.  X 8,000 Figure  146  T h i s p i c t u r e d i s p l a y s the abrupt t i o n between " c e l l  transi-  type #2" and " c e l l  type #3" morphologies. X 8,000 F i g u r e 147  Transition aspect  " 2-3 " c e l l s .  General  o f the background cytoplasm.  X 26,000  144 F i g u r e 148  " C e l l type  #2". General u l t r a s t r u c t u r e  of an e r e c t system c e l l . A t arrows several c h l o r o p l a s t regions involved i n metabolite production  are I n d i c a t e d .  X 14,000 F i g u r e 149  High m a g n i f i c a t i o n o b s e r v a t i o n o f a c h l o r o p l a s t region involved i n metabolite p r o d u c t i o n . X 60,000  F i g u r e 150  High m a g n i f i c a t i o n o b s e r v a t i o n o f the background cytoplasm #2". X 60,000  of a " c e l l  type  145 F i g u r e 151  " C e l l type #2". General u l t r a s t r u c t u r e of a p r o s t r a t e system c e l l . c h l o r o p l a s t regions production  Several  engaged i n m e t a b o l i t e  are i n d i c a t e d  ( arrows ) . A  r e g i o n o f E.R. d i s o r g a n i z a t i o n i s a l s o depicted. Figure  152  X 20,000  " C e l l type #2". C e l l wall-ingrowths are observed. The a l t e r n a t e d i s p o s i t i o n , o f ~ metabolite  ( !, 3, 5 ) and w a l l  material  ( 2, 4 ) are i n d i c a t e d . X 40,000  146 F i g u r e 153  " C e l l type #2". The nucleus nucleolus  (nl) are d e p i c t e d .  (N) and the Mitochondria  (M) are a l s o " observed. A. c e l l , w a l l ingrowth i s seen and i t s double c o n s t i t u t i o n i n d i c a t e d ( l a b e l s 1, and 2 ). Black arrows i n d i c a t e the s i m i l a r i t y e x i s t e n t between the c e l l w a l l i n c l u s i o n m a t e r i a l and the vacuole  inclusion  m a t e r i a l . X 50,000 F i g u r e s 154, and 156  High m a g n i f i c a t i o n  observa-  t i o n o f c h l o r o p l a s t zones o f m e t a b o l i t e production.  F i g u r e 155  F i g u r e 154  X 70,000  F i g u r e 156  X 36,000  Release o f i n t r a p l a s t i d  metabolite  i n t o the cytoplasm. X 18,000  )  F i g u r e 157  Release o f i n t r a p l a s t i d m e t a b o l i t e s  into  the cytoplasm. X 32,000 F i g u r e 158  P o r t i o n o f a c r o s s w a l l s e p a r a t i n g two " c e l l type  #2" n e i g h b o r i n g  c e l l s . The  l a y e r i n g c o n s t i t u t i o n o f one o f the c e l l w a l l ingrowths i s i n d i c a t e d . At arrowheads plasmodesmata are i n d i c a t e d . X 40,000 F i g u r e 159  Freeze-etch  preparation of p o r t i o n of a  c h l o r o p l a s t d e p i c t i n g a region of metabolite production  ( arrowhead ).  X 34,000 F i g u r e 160  " C e l l type  #2". General u l t r a s t r u c t u r e  of an e r e c t system c e l l . The morphology of the c e l l w a l l i s a l s o d e p i c t e d . X 10,000 F i g u r e s 161, and 162  F i g u r e 163  C e l l w a l l ingrowths morphology.  F i g u r e 161  X 12,000  F i g u r e 162  X 20,000  T r a n s i t i o n a l " 1-2 " c e l l . An e a r l y stage i n the formation  of a c e l l w a l l  ingrowth i s shown. X 30,000  Figure  164  T r a n s i t i o n a l " 2-3 " c e l l .  General  aspect, o f the background cytoplasm. X 12,000 Figure  165  T r a n s i t i o n a l " 2-3 " c e l l . T o n o p l a s t i s absent a t c e r t a i n p l a c e s  ( arrowheads ) .  X 50,000 Figure  166  T r a n s i t i o n a l " 2-3 " c e l l . morphology. N o t i c e  Chloroplast  a t arrowhead the  p r o t r u s i o n from the c h l o r o p l a s t body of a stroma c o n t a i n i n g  s t r u c t u r e . .Notice  also that t h i s structure i s d i s t i n c t from the p y r e n o i d  (Py), but s i m i l a r t o  other  s t r u c t u r e s seen i n the cytoplasm o f  other  cells  ( c. f ; f i g u r e s 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 .  C h l o r o p l a s t and pyrenoid Notice  morphology.  the l a m e l l a r nature o f the r e g i o n  l a b e l l e d "A". P l a s t i d - d e r i v e d 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 . p l a s t envelope  The c h l o r o -  (ce) i s c l e a r l y  around the p y r e n o i d  body  defined  (Py). At the  arrowhead the a b s c i s s i o n o f the p y r e n o i d from the c h l o r o p l a s t seems t o be occurring. X 36,000  149 F i g u r e 170  T r a n s i t i o n a l " 2-3 " c e l l . N o t i c e the n u c l e u s  that  i s i n the process o f  d i s o r g a n i z a t i o n . The n u c l e a r envelope i s no longer r e c o g n i z a b l e a t p l a c e s , w h i l e i n others seems t o be.degenerating ( arrows ). The p o r t i o n o f the n u c l e a r envelope longer remaining  i n t a c t seems  to be the one f a c i n g the dictyosome (D). X 50,000 F i g u r e 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  o f a c e l l w a l l ingrowth  ( cwi ) i s d e p i c t e d . D e p o s i t i o n o f new w a l l m a t e r i a l i s seen a t arrowheads. X 38,000 F i g u r e 172  T r a n s i t i o n a l " 2-3 " c e l l .  Chloroplast  morphology. N o t i c e the l a m e l l a r nature of the area l a b e l l e d  "A" and the pheno-  menon o f a b s c i s s i o n o f p l a s t i d s t r u c t u r e s "B". X 50,000  derived  F i g u r e s 173, 174, and 175  T r a n s i t i o n a l " 2-3 " c e l l s .  M i t o c h o n d r i a l morphology. N o t i c e the c i r c u l a r arrangement o f the m i t o c h o n d r i a l cristae  ( f i g u r e s 174, and 175 ).  X 30,000 F i g u r e 176  P l a s t i d - d e r i v e d body. X 36,000  F i g u r e 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 -  s h i p o f m e t a b o l i t e t o the c h l o r o p l a s t lamellae i s s t i l l picture F i g u r e 178  apparent i n t h i s  ( white arrowhead ) . X 36,000  T r a n s i t i o n a l " 2-3 " c e l l .  Abscission  of c h l o r o p l a s t - d e r i v e d s t r u c t u r e s from the p l a s t i d body. X 42,000 F i g u r e 179  T r a n s i t i o n a l " 2-3 " c e l l .  The i n v o l v e -  ment o f t h y l a k o i d s i n the i s o l a t i o n o f stroma r e g i o n s i s d e p i c t e d X 50,000  ( arrowheads )  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 o f stroma, regions  from the main p l a s t i d body i s  shown. X 20,000 Figure  181  T r a n s i t i o n a l " 2-3 " c e l l . d e r i v e d bodies  Figure  182  Chloroplast  ( arrowheads ) are seen  in  the cytoplasm. M i t o c h o n d r i a  at  "A". X 16,000  a r e seen  T r a n s i t i o n a l " 2-3 " c e l l . A p o r t i o n o f a p l a s t i d showing an area of i s o l a t i o n o f stroma m a t e r i a l i s d e p i c t e d . A t arrowhead the membrane o f one o f the i s o l a t e d areas has  fused w i t h the c h l o r o p l a s t envelope  membrane, which i s s i n g l e membrane i n :  c o n s 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 . structures vacuoles.  F i g u r e 184  Pyrenoid-like  (Py) are observed i n s i d e X 24,000  " C e l l type #3". Aspect of the c e l l w a l l . X 60,000  152 F i g u r e s 185, and 186  T r a n s i t i o n a l " 2-3  " cells.  S e r i a l s e c t i o n s showing the r e l e a s e o f p l a s t i d d e r i v e d bodies i n t o the cytoplasm. X 36,000  F i g u r e s 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. F i g u r e s 187, and 188  P r o s t r a t e system c e l l s . X 6,000  Figure F i g u r e 190  189  " C e l l type  E r e c t system c e l l . X 6,000 #3". C h l o r o p l a s t morphology.  X 30,000 F i g u r e 191  " C e l l type  #3". A c i d phosphatase  ( e r e c t system c e l l t i o n product  activity  ). Notice that reac-  i s distributed  the c e l l c a v i t y . X 20,000  throughout  F i g u r e 192  " C e l l type. #3". D e t a i l of a t h y l a k o i d s t a c k . X 70,000  F i g u r e s 193, and 194  " C e l l type #3". P o r t i o n s o f  c h l o r o p l a s t s showing s i g n s o f t h y l a k o i d d i s o r g a n i z a t i o n . X 30,000 F i g u r e 195  " C e l l type #3". P o r t i o n o f a . c h l o r o p l a s t showing e x t e n s i v e d i s o r g a n i z a t i o n o f the t h y l a k o i d system. X 24,000  F i g u r e 196  " C e l l type #3". Aspect o f the d i s o r g a n i z t i o n o f the cytoplasm and m i t o c h o n d r i a (M). X 36,000  F i g u r e 197  " C e l l type #3". M i t o c h o n d r i a l  (M)  remnants showing d i f f e r e n t stages o f d i s o r g a n i z a t i o n . X 36,000  F i g u r e 198  " C e l l type;'#3". C h l o r o p l a s t showing widespread s i g n s of t h y l a k o i d d i s o r g a n i t i o n . X 36,000  F i g u r e 199  " C e l l type #3". Accumulation  of plastoglo-  b u l i i n s i d e the c h l o r o p l a s t . X 26,000 F i g u r e 200  " C e l l type #3". D e t a i l o f t h y l a k o i d arrangement. A genophore-like r e g i o n (g) i s apparent. X 30,000  F i g u r e 201  " C e l l type #3". Aspect o f d i s o r g a n i z a t i o n of cytoplasm and m i t o c h o n d r i a (M). X 28,000  F i g u r e 202  " C e l l type #3". M i t o c h o n d r i a l X 42,000  remnant.  F i g u r e 203  " C e l l type #3". A c i d phosphatase  activity  ( c o n t r o l p r e p a r a t i o n ) . N o t i c e the. absence o f r e a c t i o n product. F i g u r e 204  " C e l l type  X 12,000  #3". A c i d phosphatase  Reaction product  activity.  i s apparent i n plasmo-  desmata and cytoplasm. X 36,000 F i g u r e 205  " C e l l type  #3". Acid-phosphatase  (control preparation r e a c t i o n product  activity  ). N o t i c e t h a t no  i s found a s s o c i a t e d  with plasmodesmata. X 30,000 F i g u r e 206  " C e l l type  #3". A c i d phosphatase  Reaction product  i s distributed  activity. throughout  the c e l l c a v i t y . X 12,000 F i g u r e s 207, and 208  " C e l l type  #3". Aspects o f  d i s o r g a n i z a t i o n o f the c e l l w a l l . F i g u r e 207  X 30,000  F i g u r e 208  X 45,000  F i g u r e s 209, and 210 ted  " C e l l type  #1".  M a t e r i a l incub  f o r determination., of - c a t a l a s e a c t i v i t y  Microbody-like  organelles  (m) show d e p o s i -  t i o n o f r e a c t i o n product. F i g u r e 209  X 30,000  F i g u r e 210  Arrow p o i n t s t o c o r e - l i k e structure.  F i g u r e 211  " C e l l type  #1".  X 36,000  Aminotriazole  (catalase c o n t r o l ) .  incubation  No r e a c t i o n product  i s observed i n the microbodies (m). X 22,000 F i g u r e 212  " C e l l type  #1".  determination t i o n product (M). F i g u r e 213  M a t e r i a l incubated f o r  of catalase a c t i v i t y .  Reac-  i s observed i n mitochondria  X 30,000  " C e l l type  #2".  determination  M a t e r i a l incubated f o r  of catalase a c t i v i t y .  Arrow  head p o i n t s t o zone o f d e p o s i t i o n o f r e a c t i o n products. F i g u r e 214  " C e l l type  #1".  X 30,000  Peroxidase  observed i n the c e l l w a l l . F i g u r e 215  " C e l l type  #1".  activity i s X 16,000  A microbody showing a  c o r e - l i k e s t r u c t u r e (arrow) i s d e p i c t e d . X 36,000 F i g u r e 216  "Cell type#l".  Peroxidase  apparent i n the c e l l w a l l mural space.  X 13,000  activity i s (cw) and p a r a -  F i g u r e s 217, and 220 . " C e l l type  #3". A c i d phospha-  tase 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 a v i t y . X 26,000 F i g u r e 218  " C e l l type  #1". Peroxidase  t r o l preparation  activity;  ( KCN ) . Notice, the  absence o f r e a c t i o n product. F i g u r e 219  X 12,000  T r a n s i t i o n a l " 1-2 " c e l l . ATPase activity  ( Mg -dependent system ). N o t i c e ++  d e p o s i t i o n o f r e a c t i o n product mitochondrion F i g u r e 221  " C e l l type  i n the  ( arrow ). X 18,000  #2". M a t e r i a l incubated f o r  l o c a l i z a t i o n o f peroxidase  activity.  X 30,000 F i g u r e 222  con-  " C e l l type  #2". Peroxidase  activity  ( c o n t r o l p r e p a r a t i o n ). X 30,000  159 F i g u r e s 223, and 225 " C e l l type #1". ATPase a c t i v i t y + + ++ ( Na -K -Mg. - dependent system ) . D e p o s i t i o n o f r e a c t i o n product i s conspicuous a t the plasmalemma and i n a s s o c i a t i o n with thylakoids.  F i g u r e 224  F i g u r e 223  X 16,000  F i g u r e 225  X 36,000  " C e l l type #1". ATPase a c t i v i t y ( Na -K -Mg  - dependent system ) .  D e p o s i t i o n o f r e a c t i o n product i s observed i n the mitochondrion (M). X 32,000 F i g u r e 226  " C e l l type #1". ATPase a c t i v i t y dependent system  ( Mg ++  ). D e p o s i t i o n o f r e a c t i o n  product i s apparent i n the mitochondrion. X 40,000 F i g u r e 227  " C e l l type #1". ATPase a c t i v i t y dependent system  ( Mg ++  ) . R e a c t i o n product i s  observed i n a s s o c i a t i o n w i t h the plasmalemma and t h y l a k o i d s . The r e a c t i o n , however, does n o t seem so i n t e n s e as i n the case o f the NIa - K - M g - dependent +  +  ++  system. X 12,000 F i g u r e 228  ATPase a c t i v i t y system  ( Na -K -Mg - activated +  +  control preparation  ++  ) . Notice  the absence o f r e a c t i o n product. X 14,000  160 F i g u r e s 229, and 230  ATPase a c t i v i t y  ( Na -K -Mg +  +  ++  a c t i v a t e d system ) . F i g u r e 229  Deposition of reaction product  i s observed i n asso-  c i a t i o n w i t h the plasmalemma and F i g u r e 230  t h y l a k o i d s . X 14,000  Control preparation ( i n c u b a t i o n medium minus ATP ) . N o t i c e the absence of r e a c t i o n X 12,000  product.  161 F i g u r e s 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 -  like material  ( Hendy,1971, m o d i f i c a t i o n  of the Fontana' s technique ')'••* N o t i c e  that  l a b e l l i n g i s p r i m a r i l y found i n v a c u o l a r inclusions. F i g u r e 231  X 20,000  F i g u r e 233  N o t i c e t h a t zones o f i n t r a p l a s t i d metabolite  formation  are f r e e o f l a b e l l i n g ( arrows ), and so are s i m i l a r i n c l u s i o n s present i n the cytoplasm. X 20,000 F i g u r e 232  ATPase a c t i v i t y  ( Mg ++ - a c t i v a t e d system;  control preparation of r e a c t i o n product. F i g u r e 234  Lipofuscin  ). N o t i c e the absence X 18,000  ( c o n t r o l p r e p a r a t i o n ).  X 18,000 F i g u r e 235  Schorml's m o d i f i c a t i o n f o r the u l t r a s t r u c t u r a l of l i p o f u s c i n . X 18,000  ( Hendy,1971 ) identification  

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