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A morphological and cytochemical analysis of bud initiation and development in the filamentous brown… Burns, Alan Robert 1981

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A MORPHOLOGICAL AMD CYTOCHEMICAL ANALYSIS  OF BUD  INITIATION AND DEVELOPMENT IN THE FILAMENTOUS BROWN ALGA SPHACELARIA FURCIGERA  by ALAN ROBERT BURNS B.Sc,  The U n i v e r s i t y  A THESIS  SUBMITTED  of  British  Columbia,  1978  IN PARTIAL FULFILMENT OF  THE REQUIREMENTS  FOR THE DEGREE OF  MASTER OF SCIENCE  in  the  Depa rtrnent of  Botany  We a c c e p t  this  thesis  r equ i r e d  THE UNIVERSITY  as  conforming  standard  OF BRITISH  October  (c)Alan Burns  to  1981  COLUMBIA  the  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia,  I agree that  the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this  thesis  for scholarly purposes may be granted by the Head of my Department or by his representatives.  It is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Depa rtment The University of B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  6  Ab s t r a c t A  study  on  development furcigera and  in  localized c e l l  the  (Kutz)  electron  was  the  forms  synthesized  presented peroxidase,  of of  only  development c e l l  its  structural  wall  the  alginate  contrast  axial an  layer,  the  layer  into  is  found  in  i n i t i a l ' s a  thick  in  This fucan  is  ii  a  by  early  two  in  the  The  c e l l  c e l l  c e l l  wall  only  layer.  c e l l  wall  overlying fucan  alginate a  of  maintains  change  another  has  alginate  the  pre-existing  layer,  inner-most  inner  wall  original  an  wall  the  m i c r o f i b r i l s .  overlies  c e l l  to  incorporation  c e l l  turn  is  phosphatase  during  the  newly  localized,  related  there  This  Evidence  acid  is  outer  wall  c e l l .  in  and  the  p a r t i a l  a c t i v i t i e s  characterized  four  terminates  and  c e l l  mother  wall.  A  outer  of  the  Continued  the  outer-most  bud  with  wall  However,  the  the  which  deposition  activity  i n i t i a l .  i n i t i a l of  of  f i n a l l y  fucan  bud  of  initiation.  c e l l  c e l l  integrity.  instead the  bud  material  appearance  composed  and  the  bud  enzyme  light  the  characterizes  triphosphatase  the  of  c e l l  together  three  of  cytochemistry.  combined  existing  the  of  of  new  of  of  peroxidase  lysis / loosening  wal 1  the  techniques  as  and  Sphacelaria  protrusion  mother  the  i n i t i a t i o n alga  using  well  stage  adenosine  a c t i v i t y ,  layers  axial  bud  brown  subsequent  m i c r o f i b r i l s  that  of  out as  through  lysis/loosening  of  and  detectable  protrusion  wall  carried  microscopy,  of  earliest  process  filamentous  thickening  wall  layered  the  an layer  layer. thin  is  In  outer  Concommitant  with  the formation  loss  of  mass  of  cytoplasmic  and  density  the c e l l vacuoles,  and  an  The  endomembrane  dictyosomes  and  the d e r i v a t i v e  bud  expansion.  behind after the of  migration  and a  to  the ax.ial  karyokinesis , daughter and  a  nuclei  sister  vacuolated quiescent vacuolated,  cross  axial a  of  becomes  They  the  proliferates.  The  bud  however,  lags  protrusion  only  between  mother  axial  the  As  base  is  characteristic  of  however,  cell.  between of  a  After the  bud  two cell  cell  is highly  the  adjacent  resembles cell  iii  with  established.  formation  sister  cell.  pace  t h e bud p r o t r u s i o n ,  deposited  the  of  reticulum,  are confined,  axial  is  in  The  bud  position  structurally  feature  the  the t i p of  wall  cell.  cells.  number  (endoplasmic also  is a  cytoplasmic  the  the n u c l e u s ,  medial and  resulting  axial and  a  re-appear.  region a  of  cytoplasm  cell  vacuoles  in  in  vesicles)  towards  free"  mother  basal  system  movement  approaches  increase  there  t h e bud p r o t r u s i o n k e e p s  i t migrates  nucleus  the  The  "vacuole  cytoplasmic  into  an  protrusion,  increase  organelles.  Organelle  wall  dense a  and  non-  mefistematic  TABLE  OF  CONTENTS  Abstract TABLE  i i  OF CONTENTS  iv  LIST  OF F I G U R E S  v i  LIST  OF T A B L E S  x i  ACKNOWLEDGEMENTS  x i i  Introduction  1  Materials  4  Table  and Methods  1  9  Results Part Light  10 1  Morphology  .  10  microscopy  Electron  10  microscopy  Quiescent  axial  11  cell  11  Stage  I  12  Stage  II  13  Stage  III  14  Stage  IV  15  Stage  V  17  Stage  VI  18  Stage  VII  19  Part Light  2  Cytochemistry  microscope  Cytochemistry  Polarized  light  Cell  extractions  wall  Ultrastructural  21 21  microscopy  23  & Calcofluor  localization  Peroxidase  fluorescence  o f enzyme  activity  ...  23  ...  24 24  iv  Adenosine Acid  tri-phosphatase  phosphatase  (ATPase)  25  (AcPase)  25  Discussion Part  1  27 Morphology  27  Nucleus  27  Chloroplasts  and M i t o c h o n d r i a  30  Endomembrane  system  31  Vacuoles  32  Cell  Wall  33  Part  2  36  Light  Cytochemistry microscope  Cytochemistry  Polarized  light  Cell  extractions & Calcofluor  wall  Ultrastructural  36  microscopy  41  localization  fluorescence  o f enzyme  activity  ...  42  ...  44  Peroxidase  44  ATPase  45  AcPase  47  Summary  49  Phase  I  49  Phase  II  51  Phase  III  52  Table KEY  2  FOR  ..  54  FIGURES  55  FIGURES Literature  56 Cited  69  v  LIST  Figure  1  Light of  Figure  2  Figure  3  Figure  4  Figure  5  Figure  6  Light of  Figure  7  Light of  Figure  8  of Figure  9  10  56 (Nomarski) 56 (Nomarski) 56 (Nomarski) 56 (Nomarski)  bud  VI  VII  56 (Nomarski)  bud  56 (Nomarski)  bud  56  E l e c t r o n micrograph axial  Figure  V  micrograph  stage  (Nomarski)  bud  micrograph  stage  Light  IV  micrograph  stage  56  I I I bud  micrograph  stage  cell  I I bud  micrograph  stage  Light of  (Nomarski)  I bud  micrograph  stage  Light of  micrograph  stage  Light of  micrograph  FIGURES  quiescent axial  Light of  OF  of  quiescent  cell  57  E l e c t r o n micrograph  of  stage  I  bud Figure  11  E l e c t r o n micrograph of  Figure  12  57  stage  of  cell  wall  1  57  E l e c t r o n micrograph  of  stage  II  bud Figure  13  58  E l e c t r o n micrograph bud  of  stage  III 58  vi  Figure  Figure  14  15  Electron  micrograph  of  I I I bud  stage  Electron  of  cell  wall 58  micrograph  of  stage  IV  bud Figure  Figure  16  17  Electron  micrograph  of  IV  18  Figure  19  20  IV  cell  wall  bud  59  micrograph  stage  of  (bright  field)  bud  59  Light  micrograph  White  f l u o r e s c e n c e ) of  IV Figure  stage  Light of  Figure  59  (Calcofluor stage  bud  59  Electron  micrograph  of  IV  stage  Electron  of  cell  wall  bud  59  micrograph  of  stage  V  bud Figure  21  60  Electron serial and  Figure  22  centriole  serial  Figure  Figure  23  24  25  of  stage  micrograph  section  centriole  of  stage  of  VI  Electron  of  first nucleus VI  bud  60  second nucleus VI  bud  61  centriole  bud  61  micrograph  microtubules  Electron  of  through  micrograph  stage  of  through  Electron  and Figure  section  Electron  and  micrograph  of  of  micrograph  centriole  stage of  bud  VI  stage  bud  61  VI 61  vii  Figure  26  E l e c t r o n micrograph  of  stage  VII  62  bud Figure  27  E l e c t r o n micrograph of  stage  VII  of  crosswall 62  bud  Figure  28  Schematic  drawing  of  stage  I bud  63  Figure  29  Schematic  drawing  of  stage  I I I bud  63  Figure  30  Schematic  drawing  of  stage  VI  63  Figure  31  Schematic  drawing  of  stage  VII  Figure  32  Schematic  drawing  of  chemical  composition cell Figure  33  of  Schematic  initial 34  Light TBO  Figure  35  drawing  Figure  36  0.5  of  well  initial  chemical advanced  of  bud  after 65  staining of  bud  after 65  staining  micrograph  methylation  and  of  bud  Safranin  after 0 65  staining Figure  37  Light PAS  Figure  38  Fast Figure  39  micrograph  of  bud  after 65  staining  Light  Light  micrograph Green  FCF  Blue  of  bud  after 65  staining  micrograph  Aniline  bud 64  wall  micrograph  pH  Light  of  cell  6.8  pH  63  64  micrograph  Light TBO  bud  bud  wall  composition  Figure  early  bud  of  Black  bud  after  staining.  vill  65  Figure  40  Light  micrograph  Ruthenium Figure  41  Light 2  Figure  Figure  42  43  44  after  staining  micrograph  IKI/H S04 Figure  Red  o f bud  65  of bud  after  staining.  6  Electron  micrograph  activity  of e a r l y  Electron  micrograph  activity  of advanced  Electron  micrograph  of p e r o x i d a s e  bud i n i t i a l  45  bud i n i t i a l  46  Electron  Electron  micrograph of stage micrograph  intracellular  of p e r o x i d a s e  sites  of p e r o x i d a s e V I I bud of  Figure  Figure  Figure  47  48  49  50  of ATPase  Figure  51  micrograph  activity  within  Electron  micrograph  activity  of the plasma  Electron  micrograph  activity  within  Electron  micrograph  Electron control  Figure  52  67  Electron  control  66  general  activity Figure  66  66  acitivity Figure  66  of p e r o x i d a s e  control Figure  5  of ATPase  a dictyosome of ATPase membrane  micrograph  micrograph  activity  plasma  67  of ATPase omission)  67  of ATPase  (boiling)  Electron  67  of ATPase  a mitochondrion  (substrate  67  67 of AcPase  membrane  ix  68  Figure  53  E l e c t r o n micrograph activity  Figure  54  55  of  AcPase  a mitochondrion  E l e c t r o n micrograph activity  Figure  of  of  bud  of  AcPase  cell  E l e c t r o n micrograph  68 of  AcPase  control Figure  56  Figure  57  68  E l e c t r o n micrograph activity  68  of  AcPase  in cytoplasmic vesicles  E l e c t r o n micrograph  of  AcPase  activity  in additional cytoplasmic  vesicles  to  those  of  Fig.56  x  68  68  LIST Table  1  Summary  of l i g h t  OF  TABLES  microscope  cytochemistry Table  2  Summary  of a l l major  occurring and  9  during  bud  events initiation 54  development  xi  ACKNOWLEDGEMENTS  I  first  people.  My  chance  to  providing for  learn me  never  extended  t o D r . L.  for  h i s endless  all  f o r h i s moral  criticism Cole,  that  me  in  Oliveira  readings  of  i t was  I  needed  my  work  me t h e for  i t and  thesis.  were  always  laboratory.  special  microscopy,  when  o f my  two  for giving  completing  and  his  to  electron  support  into  thesis  working  gratitude  of  interpretations  this  Finally,  art  financial  pressuring and  express  D r . T. B i s a l p u t r a ,  the  with  preparing  experience  to  supervisor,  suggestions in  wish  His  invaluable  an  Thanks  enjoyable are  also  for his infinite  suggestions,  my  and most  thesis  drafts  of  support. I  am  sincerely  I received  Dr. P.J. Harrison  from  grateful  my  a n d D r . R.  f -xii  f o r the  committee DeWreede.  members,  helpful D r . K.  Introduction  Sphacelaria apical  growth  1959).  Each  upon  a  and  possesses  division  cells,  'superior'  not  expand  furc igera  (Kutz),  secondary  The  inferior  with  which  The  review). these  other  underlying  In  in this  fungal  studies.  branch  formation  1979b), alga  to the  the  may  of  (see  judging Studies  on  et  in  non-fungal  moss  Funar ia  bladderwort U t r i c u l a r i a  Batrachospermum  ( A g h a j a n i a n and  1  report, to  the  divide (Fritsch  and  The  rarely  regularity has  been  1980).  Trinci  is  1979  is biased  absence  organisms  S.  in Sphacelar i a  ultrastructural  (Scmiedel  In  formation  and  these  1978).  process  branch  virtual  the  formed  1959)  al.  a  segment  filament  literature  the  undergo  proceed  1977).  that  proximally.  this  this  Burnett  the  by  in  (Fritsch  (Doworetsky  respect,  Once  (laterals)  undergoes  cell  then  Wynne  (Ducreux  process  (Fritsch  secondary  multiseriate  cells  fungi  two  then  superior  authors  in  used  branches  furc igera  organisms  confined  from  segment  S.  characterized  of  branched  and  and  species  a  displays  cells  'inferior'.  (Bold  cells  that  apical  segment  elongate  one  producing  arises  from  by  the  production  invariably  noted  and  alga  prominent  results  size  segment  longitudinally 1959).  which  in  a  cells  brown  copiously  primary  segment  transverse  do  usually  produces  primary  one  filamentous  is  axis  division  These  is  have  and  (Fineran  of  well for  a  towards any  non-  process  of  been  mostly  Schnepf  1 979a,  1980),  Hommersand  the  red  1980),  and  the  green  algae  Bulbochaete  O e d o c l a d ium  (Pickett-Heaps  Even  though  the  that  the  number  process  different  of  organisms  understanding The  of  In in  the  with  so,  bud  microscopy and  are  numerous  with  the  algal  cell  wall  cell bud  and  wall  the  same  been  (Bartinicki-Garcia 1973, 1977,  1974,  Werz  Gooday  Novotny  1979,  important  alginates  during  enzymes,  The  and  Gooday to the  peroxidase,  of  to  its  course  of also  2  associated during  be  and a  the  observed.  using  light Novotny  studies major  have brown  "alginates". two  fold  turgor  structural  role force  integrity  Bartinicki-Garcia 1975,  Trinci  the  two  driving  1972,  monitor  adenosine  the  combine  1970,  these  the  and  can  play  Forman  literature  better  variations  place  1968,  of  to  Lippman  of  techniques.  wall  "fucans"  maintain  and  therefore,  development.  Most  yield  a  to  algae  1966,  postulated  time  cell  brown  detection  i t must  range  for  is  taking  polysaccharides,  has  need  development  1 976).  Toth  apparent  wide  concerning  the  of  a  cytochemical  (McCully  chemical  expansion;  at  of  alterations  studies  a  i t is  process.  information  and  to  study  with  composition  1 975 ,  Forman  this  initiation  dealt  in  of  morphological of  is  1979).  (Ducreux  small,  common  morphogenetic  significant  Cytochemical  The  there  hence  Chara  is  and  observations  chemical  process  studies is  purpose  doing  and  branching  this  ul t r a s t r u c t u r a l  1 97 7 )  of  1974),  (Pickett-Heaps  Pickett-Heaps 1980).  behavior bud  of  It  fucans  initiation  suggests  triphosphatase  is,  that and  and and  three acid  phosphatase synthesis 1980,  the  activities role,  also  involved  (Halperin  Giordani  permit  the  are  in  1969,  1980).  Henry  if  the  any,  pertinent  of to  wall  Thus,  enzymes  in  Coupe  techniques  detection  cell.  degradation  1979,  Cytochemical  ultrastructural within  cell  of  and  these  wall  understanding  D'Auzac  exist  which  enzymatic  information  cell  and/or  concerning  restructuring  bud  initiation  is and  development. Sphacelar ia studying a  this  regular  has  not  furcigera  developmental  branching  been  pattern  extensively  is  an  process and  b)  excellent because i t is  investigated  cytochemically.  3  an  a)  system it  for  possesses  organism  which  u1trastructural1y  or  Materials  and  Methods  Sphacelaria obtained  from  University Austin)  the  (now  and  for  and  in  12hr/day  qualities  at  an  of  natural  Light  microscopy  fluorescence  mercury  beam  filter. of  the  II  and  with  ST*  this  study  development  of  has  The  lights  material  permitted  cultures  (Chihara  were  1968)  fluorescent  mimic  The  at  tubes  spectral  very  closely  staining live  were  using  BP  field a  an  method  exciting  and  brightener  an  4  LP  employed and  (Waaland  propagules  50W  with  at  vital and a  a  Dialux super  EB  used  an  RKP  suppression  modified  similar  20  was  filter,  Waaland  light  pressure  and  dye  bud  Zeiss  reflected  43 0 a  of  Nomarski  using  illumination  340-380  study  or  Leitz  HBO  the  mirror,  made  optics  bright  provided a  Observations  contrast  latter  fluorescence  fluorescent  White  at  watts/m^.  material:  with  the  splitting  The  live  live  which  conjunction  400  of  For  lamp  9.1  Indiana Texas  sporangia  Lite'(TM)  of  of  was  sunlight.  microscopy  microscope.  The  medium  fluorescent  interference  Photomicroscope  800).  'Vita  Algae,  University  marine  intensity  using  differential  of  furc igera.  by  these  those  LB  as  Chihara's  of  initiation  #  the  unilocular  illuminated  Sphacelariaceae)  Collection  at  culture  identification  20°C  in  Culture  hairs  maintained  (Phaeophyta,  located  (stock  propagules, its  sp.  version  Calcofluor 1975).  In  stage  of  development solution in  in  fresh  bud  culture  were  for a  terval  placed  culture  propagules grow  were  medium,  medium,  period  of  washed  to  up  were  minutes  the  to  2  f o r 10  and  and  v/v  dye  minutes  each  photographed.  Some  incubator  weeks.  removed  i n a 0.005%  3x  observed  returned  propagules  and  During  allowed  this  examined  to  time i n -  for  signs  of  initiation.  Electron  microscopy:  for  transmission  hrs  at  0.05M w/v  room  fixation  was  electron  cacodylate  caffeine by  a wash carried  cacodylate  (Sigma  R-2751  and  and  in  Reichert  0mU3  (2x0.5mm)  copper  Observations Zeiss  Light  EM  10  the  Epon  same  1%  grade)  1961) .  acetate were  and  carried  electron  microscopy  coated  plus  0.1%  lead out  Formvar citrate  with  j_ caffeine  free  ruthenium red i n darkness in  buffer  the m a t e r i a l were  cut  was on  a  on  single  and  post-stained  (Reynolds  either  was Post-  washes  collected  1%  This  a t 4°C  Sections  with  contained  a  caffeine.  w/v  series,  1/2 in  n  several  a methanol  used  for 2  1978).  v / v OsO^  After  fixed  which  f o r 16hrs  u1tramicrotome, grids  7.0  buffer  plants  g 1 u t a r a l de h y d e  Greenwood  containing  (Luft  adult  were  v/v  a t pH  and  1977).  through  embedded  uranyl  buffer  buffer  Rascio  1.6%  out u s i n g  practical  dehydration  with  in  branched  microscopy in  (Mueller  sodium  (Colombo  Copiously  temperature  sodium  followed  a  f o r 30  a Zeiss  slot  1963). EM  9S o r  microscope.  of sectioned  material:  5  Copiously  branched  adult  plants  processed  were  for  glycol  a  were  in  cut  knives.  n  or  on  a  Du  microscope  Pont  were  slides. Staining  Observations  d  was  One  Sorval  The  the  red. by  media  microtome  with  distilled  using  air  Leitz  in  sections  water  summarized  a  a  (Po1yscience, thick  then  of  embedment  J.B.4  are  those  Dehydration  micron  were  to  omission  t o two  onto  procedures  manner  followed  slides  made  with  embedding  floated  were  identical  ruthenium  J.B.4  Pennsylvania).  Sections  stained.  a  series  methacrylate  an  microscopy  OsO^  methanol  Warrington,  in  electron  post-fixation through  fixed  glass  on  clean  dried  in  and  Table  1.  20  EB  Dialux  microscope.  Enzyme  localization  Copiously  branched  temperature cacodylate (Mueller in 4°C  and  Peroxidase :  at  buffer  of  M  the  plants  v/v pH  Greenwood  0.05  composition  adult  1.6%  buffer  t h e same in  in  at  1%  7.0  which The  briefly  the p r e - i n c u b a t i o n  and  0.05  medium  pH  2 mM  aminotriazole  1 mM  diaminobenzidine  stored  was:  7.0  1%  was  at  level:  fixed  contained  buffer  tris-maleate,  6  in  material  caffeine  tris-maleate  50 mM  were  glutaraldehyde  1978).  plus  u1trastruetura1  at M  room  sodium  caffeine  then  washed  overnight pH  7.0.  at The  Adenos ine  50  mM  triphosphatase:  1 mM 0.3  phosphatase:  3 mM  pr e - i n c u b a t i o n s at  incubation  room  medium  Peroxidase:  7.0  t r i s - m a l e a t e , pH  5.0  mM  50 mM  minutes  pH  MgSO^  Acid  All  tris-ma1eate,  Pb(N0 ) 3  PbCNO^j were  carried  temperature.  out The  in  the  composition  Pre-incubation  medium  plus  of H a l l  and  Pre-incubation  triphosphatase:  ( m o d i f i c a t i o n of G i l d e r  and  Ac i d  Pre-incubation  plus  phosphatase:  Beta-g1ycerophosphate . Barka  at  room  room  at pH  were  medium  carried  plus  medium  and A n d e r s o n  incubation,  cacodylate temperature  sodium  30 the  out  3 mM  l^c^.  1972)  ATP.  Cronshaw  8  mM  1973)  sodium  (modification  of  1962)  i n the dark  f o r 60  minutes  temperature.  After sodium  of  0.3%  Sexton  Adenos i n e  incubations  for  was:  (modification  All  dark  cacodylate  4°C 7.0.  followed The  a l l material  buffer in  at  1.6%  buffer i n 1%  material  pH  v/v  was  i  7.0,  7.0. n  re-fixed  i n 0.05  for 2 in  a  M  hrs at 0.05  M  P o s t - f i x a t i o n overnight  0.05  then  7  washed  glutaraldehyde  a t pH  OsO^  was  M  sodium  routinely  cacodylate processed  at for  electron  microscopy.  Contol  preparations  Peroxidase:  were  Material H  20  2  as  was  incubated  Material  triphosphatase:  f o r 5 minutes at  pH  was  in  Ac i d  Material  was  phosphatase:  of  Cynamid  10  mM  boiled i n 50  7.0.  incubated  by  in  the  absence  of  •  Adenosine  1 Donated  follows:  mM  Some the  Inc.,  8  to  incubation  tris-maleate material  absence  incubated  sodium  Canada  prior  of  was  also  ATP.  in  the  fluoride.  Baie  buffer  d'Urfe,  P.Q.  presence  Table  C y t o c h c n i c e t Techniques  I n d i c a t i c r.  CorboxyI  Four  6  SUI(3 ZC  1  Ccl I  Wat I  Two  C e l l  Wall  Layered  Pattern  Layered  P a t t e r n  of  . C e l Is  of  I n i t i a l  Axial  £ u d  cwi cwa cwa cv/4  CHO  Pu +  P  .  Pi +  Pu +  Pi +  CVI  L  Pi +  groups T80  pi!  Q.5  S o f r o n i n pK  0  D.5  Evens  &  Ho L I i  gon  1972,1376  s u l f a t e groups  Pu +  Pi +  Pu +  —  s u l f a t e  ++  •  ++  +  ++  +  ++  -  -  Pc +  Pi +  McCul 1S70  +  S p i c e r  groups  ly  1960  A l c i a n  Blue  c a r t o x y t  CCS  2 . 4  s u l f a t e  pli  Source  G  Spi  cer  1960  Croups ALcian GGS  Glue  pK  C . 5  S a f r a n i n  0  a f t e r  c o r -  boxy I  n e t h -  y lati  s u l f a  pH  a f t e r  —  +  . +  Steednan  -  1950  s u l f a  to  +  +  +  Cook  groups  1S77  BIua  su t fa t e  2 . 4  groups  y la tl  no  +  +  ' +  Cook 1377  t h -  on ++  A c r i di no  a d d  0 range  nuc i n s  +  ++  +  Hicks  &  Hatthaei  7.4  1950  Acr i di ne 0 range pH  *  COP-  boxy L  pli  +  ta  groups  on  A l c i a n 8CS  -.  4-  su L f a t e groups  +  1  ++  -  .+  Cook 1977  0 . 5  Periodi  c  Acid  S c h i f f s  Reset  ion  , 2 - g l y c o l  +  ++  +  ++  Foder  groups  L  O ' G r i e n 1EEG  eel lu  P c l a r i z ad  lose  ce LI u l o s e a l g i n a t e s  Light Calcc f luor tthite ET  +  -  £  +  -  -  +  Jensen 1 96 2  —  +  -  +  Andc  rsen  1056  b c t o - 1 , 3 £*, 1,4 Linked p o l y s a c c h a r i d e s  +  a l g i n a t e s  +•  HeslepHa r r i s o n  £.  He&lopHarri  son  1961 ' F u c a n*  -  -  +  X  V/hytc  oxt ract i on fcI  toned  al  ot  1281  by  CcIco fluo r .White  ST  •Alcjlncte' e x t r a c t i o n f c I lowed by CcIco fIuor White CT  fucans  ro-  noi n  crc  S,  cniun  a c i d i c  Reo  .022  p o l y -  7S  C luo  1K  y  X  Movotny  £  Forr.on 1970  ++  +  ++  +  ++  Cur r 1EG5  sacchor1 A n i l i n e  +  u n s to i n ed  Ruth  Black  -  +  dec  p r o t e i n  +  +  •  -  Fl  in  shcr  1SCC  a c e t a t e  Fast FCF  Green pH  p r o t e i n  •  •  +•  Fcdcr  2 . 0  &  0 ' U r i on 1S6C  + X  = =  p o s i t i v e , not t e s t e d .  -  =  negative  ,  Pu  =  purple  ,  Pi  =  pink  ,  *  »  a o d i f i c d  ,  /  Results :  Light  Part  mother  axial  cell  first  sign  stage  I,  wall  in  is  time  while  position  between  development time  stage  bud  and  V  basal  a  VII,  is  cell  and  the  4  of  Fig.  whereby 20%  of  cell  while  to  expand  as  II  wall  in  remains  Stage  stage  Initiation  II,  of  the  of  the  III  the  length  in  and  nucleus  height  the  as  mother  1).  the  text  cell  the  cell  The  the  defined of  with  is  nucleus  portion bud  maximum  of  in  by  thought  the  the  stage to  of  assumed axial  nuclear  VI  the  since  formation  of  sister  medial cell  During  the  completion The  IV  protrusion  (Fig.7).  occur,  by  a  mother  Bud  (Fig.8).  subtending  10  has  (Fig.6).  characterized  a  quiescent  the  axial  the  height.  cytokinesis  resulted  the  an  p r o t r u s i o n c h a r a c t e r i z e s stage  developed  reaches  in  protrusion  of  stage  its  stage  the  i.e.  has a  in  A  from  comparison.  to  position  wall,  bud  karyokinesis  crosswall,  cells,  the  for  of  area  a  cell  doubled  (Fig.5),  well  as  2-8.  stages  approximately  towards  the  the  Figs.3  equals  has  small  initial  protrusion continues  protrusion  is recognized  migration  process  bud  stages  (Compare  protrusion  a  developmental  these the  Figs.  referred  quadrant  the  a bud  included  as  right  longitudinal  stage,  also  of  in  initiation,  upper  to  protrusion  this  depicted  is  through  unchanged  outer  is  bud  With  relation  development  recognized  During  however,  cell  of  passes  III.  The  (Fig.l)  the  (Fig.2).  and  Morphology  microscopy:  axial  and  1  next of  a  cytokinetic two  axial  daughter cell.  At  this  time,  it  is  evident  denser  cytoplasmic  apical  meristematic  cell  has  while  re-established  its  organization  vegetative  axial  bud  cells.  cell  the  bud  organization cell,  cytoplasmic  the  that  has  resembling  the  subtending  that  is and  taken that  vacuolated,  Growth  would  cell  of  sister less  the  a an  axial  compact  characteristic  subsequent  constitute  on  of  division  latter  phase  of of  branching.  Electron  microscopy  Quiescent  axial  ultrastructure exhibits  an  position and  evident.  the  arrangement.  dispersed in  number  the  cytoplasm.  inclusions, (Matile  inclusions  the  Within  abound  in  cytoplasm  the of  (ER)  central  preferential  In  paramural  nucleolus are  these  cells,  which  causes  a  peripheral  elements  are  location of  within  activity,  electron  with  are  membranous  autophagic  addition,  and  Dictyosomes  remnants  space  also  chloroplasts  (Fig.9).  extensive  11  assume  the  vacuoles  observed. the  to  ensheathing  no  of  vacuolation  reticulum  display  are  of  mitoc.hondr i a  partially  and  a  material  degree  and  nucleus  occupies  feature  general  The  heterochromatic  indicative  1974),  A  conspicuous  throughout  few  and  cell.  developed  Endoplasmic  both  axial  the  well  high  chloroplasts  shows  profile  cytoplasm.  most the  Fig.9  quiescent  dispersed  is  observed  a  irregular  The  however, the  of  within  evenly  cell:  the  dense  majority  of  them  close  lying  adjacent  examination  layered  pattern.  CW1  CW4)  to  cuticle  by  material  i n which  seems  be  to  electron  wall  dense  microfibrils.  Wall  transverse axial  restricted  to  plasmodesmata confined  Stage  position  nucleolus  In  vesicles  be  and In  the  walls  containing occupy  active  nucleus  axial  and  are  Numerous  are e x c l u s i v e l y  is  occupies  Its  a  profile  cells.  A  sectioning  of c e n t r i o l e s  medial is  less  conspicuous  reveals  a pair  12  with  of the CW2  wall.  these  less  oriented  are continuous  axis)  face  a  fibrillar  heterochromatin  a distinctive  to the f o r m a t i v e  dense  component  show  branch  transfer  char-  electron  CWl  thin  (Fig.9).  dispersed  to the f u t u r e  a  are  distinctive  layers  (Fig.10).  serial  CW3  CW4  longitudinal  the  by  longitudinal wall  wall  quiescent  addition,  and  CW4  observed;  cell  randomly  envelope  the  stage  in  Dictyosomes favoring  also  this  than  respect  nuclear  can  and  A  (Fig.9,  longitudinally  the inner  outer  covered  CWl  more  and  while  the  within  irregular  (with  cell,  CW3  recognized  is  CW2  wall.  distinctive  microfibrillar  layers a  a  amorphous  of  to the t r a n s v e r s e  I:  evident.  and  c a n be  an  and  layers  walls  reveals  defined  background  longitudinal  layers  of  Wall  consisting  quiescent  wall  a poorly  composition  the  wall  outermost  presence  embedded.  outer  layers  the  C u ) . The  the  the  this  which  (Fig.9,  acterized  of  Four  of  to  are a  inpocketing  perinuclear  of  nuclear  of  the dictyosomes  also  lateral of the  (Fig.10). position,  envelope  derived (Fig.10).  Mitochondrial noticeable  profiles  decrease  concurrent  These  to  The  four  in  wall  the  distinct CWl  and  Stage is  In  the  The  position  same in  as  in  the  size  addition,  a  inability  is by  and  a  density  CW4).  also  organelles axial  the  are  cell.  in  the  outer  i s , however,  innermost  Within  noted  de-  distribution  There  of  takes  of  the  distinct  protrusion.  to  number  and  protrusion.  In  I.  this  between  and  organelles  the  case  stage  there  nucleolar  layer, region,  wall  layers  at  this  seems  the to  13  definite  is  frequently  the  larger  Equally  a  nucleolus  striking  material. lateral  A  is pair  inpocketing  stage.  occupy of  a  is  body  heterochromatic with  (Fig.12)  nucleolus.  sectioning.  and  II  the  from  associated  to  in  nucleus  mitochondrial  seem the  nucleus However,  both  detect  envelope  Chloroplast  the  serial  the  these  to  independent  of  nuclear  of  second  This  demonstrated  of  c e n t r i o l e s remains  of  vacuoles and  quiescent  thickening  stage  of  in  CWl  the  a  (Fig.11).  encountered. as  the  d i s c o n t i n u i t i e s are  II:  increase  of  while  cellular  remain  (Fig.11,  localized  mass  Otherwise,  remaining in  of  number,  characteristic  1960).  layers  region  CW2  the  are  observed  in  number  cytoplasmic  the  wall  conspicuous  CW4,  of  those  longitudinal a  (Esau  morphology  similar  in  increased  the  events  differentiation and  in  increase  place.  have  profiles  the  have  cytoplasm  chloroplasts, be  oriented  increased  of the  the long  parallel  to  bud axis the  portion  of  the p r o t r u d i n g  extensive  reorganization  dictyosomes observed,  and  and  protrusion.  A  further in  decrease  cytoplasmic  The  basal  four  Closer  to  layers  can  and  CW3  CWO,  in  S tage of, have inner  wall  nuclear become  of  and  i s the  this  stage  cisternae  The  within  these  the  events,  and  an  a  increase  previously  now  only  acquired  (Fig.12,  CWl,  on  occurs  migration. displaced  In  wall.  prior  Fig.13  from  the  i t s original  Otherwise  14  two  cell  the  number  no  CW4). wall of  layer,  (Fig.12).  has  with  the  CWl,CW2  wall  increased  layer  longitudinal cell  just  in to  outer  hand,  outer  CWl  structures  continuous  the outer  the  of l a y e r s  dense  the other  remaining  stage  in  the convergence  numerous h o l e - l i k e  of  only  reduction  electron  of  recognizable  protrusion  This  single  layer, while  characteristic  the  from  a new,  longitudinal  ER  of  vacuoles  protrusion  observed.  portion  of  also  of the  with  of  is  i n the r e g i o n  t h e ER  detected.  are  the  apex  III : This  of  layers  displays  thickness basal  is  results  innermost  the  some  feature  (ER,  is maintained.  of  be  into  which  The  cell  vesicles)  concentration  An  system  proliferation  i n t h e number  wall  the  layers  the  fashion.  endoraembrane  Concurrent  mass  portion  wall  of  density  longitudinal  the  characteristic  (Fig.12).  cytoplasmic  in a consistant  associated  of  their  appearance  protrusion  of  their  consisting  dictyosomes  stacked  wall  CW4  of  wall.  t o , or a t the onset nucleus  appears  position visible  near  changes  to the in  nuclear  morphology  encountered negate the  in  that  Supportive were  number  this in  The  of  electron  dense  previous  Stage  few,  stage  (Fig.15).  The  noted  of  as  in a  envelope.  and  the  the  bud  reappearance  nuclear  stage  of  bud  appears  to  centrioles  development.  be  The  organelles  i t i s apparent within  t h e bud  the  attached  thinner  the  tip still  However,  loosely  by  analysed.  the  remaining of  not  expained  were  now  layers.  seems  be  not  does  cellular  by  vacuoles  wall  this  that  a l lother  migration  of  the  to  may  sections  is characterized  nucleus  portion  confined  of  i n the r e g i o n  which  nuclear  revealed,  absence  although  CWO ,  but  were  outer  to  than  the  i n the  (Fig.13).  This  of  studied  fact  of  cell  layer,  stage  portion  wall  two  distinct  basal  next  Centrioles  the  i f any,  cell  layer  IV:  the  is  unaltered,  only  wall  idea  morphology  are  consists  inner  smaller  virtually  cytoplasm.  I  a  and  there  sections Their  observed  remains  detected.  presence.  of  arrangement  that  the  therir  fact  were  towards  extends axial  by  beyond  mother  protrusion.  stages,  laterally No  absence  of  changes  feature  of  this  The  pattern  of  the w a l l  pair  the  of  the  enlarged by  the  sectioning of  centrioles  inpocketing  i n other  cytoplasmic  dominant  into  of  initial  confines  Serial a  positioned  major  bud  i s accompanied  heterochromatin. previous  the the  cell  This  the occurrence  of  the  organelles  are  vacuolation  remains  a  stage. layering  15  is similar  to t h a t  of  stage  III,  number outer  but  of cell  the  the  wall  outer  outer  CWl,  is  now  CW3  ,  the  layer  in  as and  outer  change  layer,  CW4  the  at  developmental same  bud  (Fig.18).  The  represents  and as  the  obtained  the  (Waaland  and  i t approaches suggest  protrusion,  material.  The  the  of  base  through  of  the  this that  the  consist  sequence the  bud  protrusion  axis  longitudinal  d i r e c t i o n . The  exhibits  random  orientation  (Fig.  the  or 16).  bud,  the  of  somewhat  the  innermost protrusion, of  layers  portion  is  of  the  obtained a  bud of  microscopy  cell  bud wall  convergence fluorescence  These  observations  CWO  and  CWl  of  synthesized  wall  orientations,  from  in a  relation  to  transverse  protrusion,  transverse pattern  of  wall  newly  from  of  micrograph  the  layers  upwards  t i p of  16  the  protruding  Note  wall  portion  the  the  (old)  shifts  This  thin  representing  microfibrillar  longitudinal  a  of  of  as  layering  material.  mainly  become  non-labelled  1975).  cell  bud  understanding  of  new,  wall  the  fluorescence  original  new  of  thickness  with  tip  Waaland  the  now  micrograph  of  to  of  wall  (Fig.17)  addition  confined  Meanwhile,  better  non-f1uorescing  disappearance  strongly  IV  the  non-protruding  cell  brightfield stage  has  region  basal  the  in  area  combined  A  increase  non-protruding  wall.  wall. of  the  layer  the  the  pattern  comparing  material  of  in  apical  as  an  remain  the  cell  longitudinal the  of  the  be  which  wall  CWl,  thick  by  the  of  to  CWO,  This  longitudinal  wall  ,  layer,  (Fig.16).  cell  CW2  appears  discontinuities  protrusion as  there  of  the to  a  however,  microfibril microfibril  orientation following  Stage  Here  cell  general  consistently  stages  V:  mother  is  o f bud  the  and  appearance  earlier  stages,  within  a  remains  in  fairly  profiles.  extensive  the  associated  with  peripherally  initial  remains  centrioles  throughout  cytoplasm  and  conspicuous  feature  of  this  cytoplasmic  vacuolation.  appear  t o be  smooth  ER  unchanged obvious  the  from  granular of  with  of  (Fig.19)  from  resulting and  be  ER  closely  Chloroplasts  remain  longitudinal  axis  protrusion. is  the  The m o s t  resumption  tend  mother  ER  followed  membranes. and  vacuoles  one a n o t h e r  pattern  rupture  is  matrix  autophagic  located  distributed  to  vacuoles axial  As i n  to  of  occupy  cell.  via 1 1 that  Many  wall  dilation vacuoles  membranous  creating  of stage  17  a  (Matile  layering  i n the outer  by  in  stage  CWO  and often  They  also  vacuoles. V  IV, a l t h o u g h layer  by  vesicles  1974). larger  a  Vacuoles  the s e q u e s t r a t i o n of cytoplasm  This  the smooth  to fuse  The  an  of a  indicative appear  derived  elements.  separation contain  within  stage  The  endomembrane  also  their  the expanding  The  The  appear  with  still  dictyosome  and d i c t y o s o m e s .  axial  unchanged.  are  of  position  the  (Fig.20).  are  of  the w a l l  basal  of  the  the cytoplasm  profiles  positioned  to  bud  distribution  t h e ER  a l l  between  inpocketing.  Mitochondrial  throughout  parallel  of  nuclear  even  mid-way  the nucleus  pair  lateral  system a  a  lies  protruding  of  in  development.  nucleus  the  observed  of  remains there i s t h e bud  protrusion uous  (Fig.20).  decrease  which  the  eventually  underlying that  in  This  i s not  gives  a  be  thickness  cytoplasm.  this  could  way  the of  this  under  Hox^ever,  consistent  result  of  contin-  pressure  should  feature  a  outermost  the  it  of  be  this  layer of  the  pointed  out  or  subsequent  s tages.  Stage  VI :  probably was  not  at  This  is  the  occurs.  The  complete  resolved.  opposite  that  the  These  the  An  are  electron  to  tubules  and  This  the  migration  lies  the  be  occurred.  with  of  nuclear the  the  to  anchor  and  observed  base  nuclear  Extranuclear  centrioles can  suggested  between  appears  envelope.  centrioles  had  associated  material  envelope  the  division  21-24)  membrane  near  nuclear  of  outer  nuclear  division  nuclear  (Figs.  material  the  nuclear  of  presence  nucleus  dense  and  positioned the  the  distinctly  (Fig.24).  centriole  the  wherein process  replication  centrioles  envelope  towards  of  centriolar  division. of  However,  poles  events  stage  the  micro-  radiating  at  this  time  (Fig.24). It  is  budding  cell  compared  to  (Fig.25). in  also  obvious  the  possess those  to  basal  change  can  the no  stage  that a  of  Otherwise,  relation  within  evident  V  be  chloroplasts  reduced  number  adjacent  quiescent  other except  portion  the  of  detected  18  differences that  there  the  axial  in  the  within  of  are  thylakoids axial  can  the  be  detected  more  mother  cells  vacuoles cell.  organization  of  No the  cell  wal1.  Stage  VII:  This  formation. the  nucleolus  each an  in  closely  in  crosswall  between  organelles  large  of  structural  the  meristematic cell  CWI, (CW4) and  of of CW3  the  vacuoles  the of  condition  cell  the  containing  a  the  one  is  also  reveals  that  two  located  within  (Fig.26). nuclei  The  indicates  two  daughter  axial  cell  in  the  consequently position  and  seems  of  cell  B  is  characteristic  Cell  A  fact  that  of - i t s A  possess  (Fig.26,  this  is  most  (Fig.26,  to  cells  ).  a  Cell  larger  ).  Overall  reminiscent  of  the  of  dominant  filament.  reveals  sister  the  organelles  examination  bud  in  organization.  and  peripheral  each  cells  envelope  the  quiescent  organization  of  close  daughter  between  cytoplasmic  the  A  a  centrioles  cytoplasmic  a  resulting  than  sectioning  bud  occurred.  vacuolated  occupy  lacks  amount  also  their  highly  apical  has  resembles  is  of  nuclear  dissimilarity  reflected  'B'  a  pair  each  in  Heterochromatin  serial  the  cytokinesis The  it  of  a  phase  occurred  smaller  stage.  and  final  nuclei,  considerably  nuclei  inpocketing  the  already  daughter  possesses  of  has  previous  both  nucleus  two is  the  in  presence that  of  that  observed  represents  Karyokinesis  formation  visible  stage  of  that is  only  cell the  continuous  axial  sister  the  cell  axial  19  walls  innermost with  cell  (Fig.26). cell  of  extend  the  wall wall  Layers only  two  layer, layer  CWl  ,  4 CW2  slightly  beyond  the  boundary newly  level  between  formed  plasmodesmata transverse  of the  the two  crosswall and  wall  of  that  crosswall cells.  is  that  It  is  already  this  20  branch  apparent  traversed  crosswall  the f u t u r e  designates  by  represents  axis  that  the the  numerous the  (Fig.27).  first  Results :  Light of  a  Part  2  Cytochemistry  microscope  Cytochemistry:  bud  initial  from  an  axial  microscope  i t is  possible  within  outer  cell  wall  observed  with  the  to  the  that  Electron pattern this  microscopic may  is  using  reflect  the  outer CW2,  most CW3,  cross  and  walls  case  the  are  referred cell of  the  only to  the  two as  x<rall  a  cell  In  wall  the  cells  CWl.  with  cell  The  layers  wall  be  of  CW3  the  the CWl,  assuming  in  the  wall  evident  layer,  position  From  into  are  wall  axial  extend  In  and are  If  tested  designated  CW4  mentioned  layering  composition.  are  layers  the  present.  and  pattern  microscope.  could  cell  lamella'.  wall  outer  that  light  similarity  electron  they  CW3  the  layering  striking  are  layer  development  with  chemical  layers  'middle  the  a  hypothesis  layers  cell  layer,  in  this  axial  of  a  suggest  most  The  position  initial,  inner  inner  CW4. of  bud  all  then  distinct  the  bears  transmission  studies.  four to  that  the  cell  distinguish  images  case,  cell  to  studying  mother  differences  cytochemical  mother  In  this  of  and  CWO,  the  the they  and  the  orientation  of  illustrated  in  Figs.28-31 . Based Table are  1,  it  a  ,  CW4  with and  series  appears  composed  protein, CW2  on  of  that  mainly perhaps CWl  polysaccharides,  cell  of a  appear with  cytochemical wall  layers  sulfated  trace to  some  of  protein  summarized  CWl,  CW3  and  polysaccharides  cellulose  consist  21  tests  of in  in  beta CWl.  CW3.  CWO and  Whereas  linked In  in  brown  acidic algae  sulfated  polysaccharides  and  acidic  The  d i s t r i b u t i o n of  the  polysaccharides  cell  wall  schematically The (two  of  results  of  the at  a  the  a  other aspects detail  of in  From cell the  initial. CWO cell  is  layer  that wall  in  have  been  procedures  brown  by  algal  has  been  of  (McCully reveals  made TBO,  will  is  said  interpreted  acid  be  TBO  this  polysaccharides as  1970). that  with PAS  Figs.34-37.  staining  within  contradict  produced  must  in  microscopic  CWl,  CW2,  the  extreme and  some  Interpretative  be  from  it  assumes after  of  the  and  to  CW3 wall  the  the  seems a  observations  outer  continuation  layers  Instead  staining  literature  stains  1979).  dealt  with  in  discussion.  a  cell.  PAS  micrographs  synthesized  not  33.  sulfated  PAS  the  initial  and  alginic  presented  Evidence  wall  of  histological  layers  newly  study  light  bud  to  of  'fucans'  (Percival  stains  metachromasia presence  termed  polysaccharides  appear  these  electron  wall  used  of  are  the  and  presence  Sample  stains  TBO  with  of  acid'  major  Figs.32  reaction  careful  interpretation caution.  in  the  the  commonly  developing  widely  Red  positive  However,  two  first  characterize  indicating  cell  of  most  interpretation.  and  the  been  'alginic  these  presented  histology)  to  have  light of  appear  to  layer  CWO  the  outer  portion  be  synthesized position  convergence  22  outer  three  converge  upon  microscope  basal  peripheral  the  of  the  of  reveals  three of  the as  in  the  outer  the  a  bud that  converging bud  mother  subtending bud  initial  three  cell  wall  layers  inner  most  the  of cell  other  wall the  in  that of  presence both  protrusion  CWI  Cell to  seems  linked of  the  appears  fucans  periods  significant  changes  that  under  initial  of  the  equivalent  within  to  the  cell  wall  is  such  an  to  cell  that  of  cell.  revealed  the  cell  bud  CWI  layered  light  and  to  two  Polarized  Calcofluor CaC^  the  wall  initial.  layers  of This  CW2 ,  results.  of in  time the  these  fluorescence:  and Only  at  alginates  CW4,  conditions,  dye),  i.e.  to  affect  on  1) .  23  to  of  CaC^ the  these  the  out  produced  cell  wall.  removes  beta-  staining  'alginates',  Na2CC>3  carried  temperatures  stainability  (according  Attempts  with  extractions  high  Calcofluor no  bud  The  i s , on  However,  mother  layers  with  polysaccharides  have  CW4 .  initial  the a x i a l  cell  &  interesting  long  of  completed.  1) .  extractions  extract  is  t h e bud  remains of  confined  (see Table  produced  It  wall  mother  was  of  the  microscopy:  axial  wall  over  cell  cell  thickness  birefringent  birefringence and  of  bud  of  the  region  overall  light  CWI,  extension  the  layered  Polarized  mother  layer,  an  the  the  four  axial  wall  hand,  developed extent  the  reaction  while  'alginates'  (see  Na^CO^ Table  Ultrastruetura1  localization  Peroxidase:  results  My  restricted of  to  electron  (Figs.  42  the  and  oxidase  activity  Mueller  1981).  consistent cell cross  walls  activity the  bud  the  the  walls  is  a l l  the  into  the  cell.  peroxidase  outermost  partially  the  cell  bud  layer,  layer,  CWl  (Fig.43).  generally  lacks  peroxidase  sub-layers  of  activity the axial the  cross cell cross  can  reaction be  wall  walls  of  The  the  2 4  in  of  in  axial  the  layer  be  but  wall  of  the  of  layer entire  the  i t  the  extends  subtending of  occassionaly  The  middle  cell  from  activity  cells  bud  within  portion  of  of  located  detected  of  of  region  also  and  that  peroxidase  cell  indicative  bud  of  onset  the  is  remaining  peroxidase  other  localized  the  in  CWO,  (Fig.43).  similar the  still  activity  separating  a  expansion  portion  product  detected  displays  activity  wall  outermost  wall  The  can  the  cell  layers  activity  and  is  region  However,  wall  Olah  plasmodesmata  wall  further  into  is  appearance  most  cytochrome  activity  protrusion  inner  mitochondria  1981,  and  is  appearance  of  lamella  numerous  cell  Upon  the  result  cells  activity  The  Fahimi  middle  by  the  42-45).  peroxidase  (Fig.42). of  and  (Fig.42).  marked  of  region  mother  initial, the  inside  cross  extending  axial  the  peroxidase  the  a l l axial in  protrusion  beyond CW4  in  of  activity  within  likely  wall  CWl,  initiation  (Figs.  product  most  Cell  and  the  that  (Angermuller  layer  traverse bud  is  feature  wall  wall  reaction  45)  enzyme  suggest  cell  dense  of  CWl thin  peroxidase lamella the to  (Fig.45).  of  sister that  of  Figure  44  is  of  peroxidase  the  presented  absence  Adenosine appears  to  cristae membrane  consistently  the  process  intracellular The  product  that and  at  the  stages this  time  endoplasmic  same of  nuclear  intense  ATPase  Control substrate was  Acid  and  were  preparations was  destroyed  reaction  (Fig.46)  are  by  it  heat,  to  and which  showed  of  the  a  plasma  (Fig.48).  activity have  more  interesting  the  47). cell  than to  No  in  note  the  signs  wall. which  enzyme  definite  reaction  exhibit  51),in the  to  dictyosomes  also and  some  seems  initiation  which  50 in  mitochondrial  perinuclear  within  reticulum,  development  (Figs.46  observed  or  and  is  profiles  omitted  envelope,  activity  bud  many  (Figs.  nuclear  ATPase  of  deposits  frequently  and  ATPase  appears  stages  is  surface  of  early  there  in  activity  endoplasmic  initiation  reticulum  activity  ATP  exhibits  envelope  reaction  It  the  inner  bud  incubated  ATPase  dictyosomes,  re-distribution  during  later  of  with  The  absence  E^C^.  chloroplast  reticulum,  the  was  intracellular.  and  (Figs.46-49) .  material  (ATPase):  association  envelope  demonstrates  the  i.e.  strictly  in  and  when  substrate,  be  endoplasmic  the  control  activity  of  chloroplast  occur.  a  tri-phosphatase  observed  During  as  the  activity  decrease  in  product.  phosphatase  intracellular  (AcPase):  localization.  AcPase  also  Deposition  25  seems of  to  exhibit  reaction  an  product  is  confined  57),  inner  This  course  surface  control  of  activity  inclusions (Fig.53)  membrane  pattern  not appear  bud  does  initiation  were  activity,  and  detected (Fig.55) was  sodium  used  detected.  26  and  (Figs.  the c e l l fluoride,  no  AcPase  and to  52 a n d 5 4 ) .  to change  development.  within  (Figs.56  and p r i m a r i l y  of the plasma  preparation  AcPase  cytoplasmic  of m i t o c h o n d r i a  distribution  AcPase  of  some  the " c r i s t a e  the  the  to  No  signs  wall. an  during of  In the  inhibitor  activity  was  Discuss ion :  The  general  similar 1977)  Part  to  (see  restrict that  of  other  Russell my  bear  Morphology  u11rastructure  that  and  1  Sphacelaria  brown  1973  algae  for  discussion  a more  of Sphacelar i a  described  a  review).  to  direct  tribuloides  those  furcigera  (Galatis  in  the  significance  of  a l .  literature  Therefore,  features  et  is  I  S.  shall  furc igera  to the p r o c e s s  o f bud  initiation.  Nuc l e u s :  It  Funar i a ,  side  influenced axial  and  position  behavior periaxial  the  accepted  spindle  of  is  the  in  that  the  red  27  are  1979a,  of  the  nucleus  nucleus  This  remains a midway  type of  of of the  Batrachospermum  i n the gland  (Fineran  after  i n the  portion  formation  alga  and  direct  i t occupies  the  My  a  protrusion  protruding  i n the  1979b).  suggest  orientation  determined  orientation  initial.  during  Utricularia  moss  the  time the  1980)  the  of  bud  the  the  position of  and  observed  Hommersand  bladderwort  generally and/or  and  the  in  the nucleus  not  location  basal  cell  been  do  IV a t w h i c h  the  initial  (Aghajanian  The  by  Schnepf  initiation  stage  mother has  between  the  between  axial  and  that  and  occupied  observations,  cell.  until  formation  (Schmiedel  of  mother  unaltered  of  cell  site  demonstrated  position  relationship  the  axial  the  the  microscope  spatial  been  branch  by  mother  light  has  1980). of  the  the  initials It  is  nucleus  nucleus  has  completed  its  1980).  study  My  orientation of  the  by  rather  than  subtending event The not  took  with  filaments  tubules  not  the upon and  reported  a  'A')  cell  the  case,  in  plate then  stage  VII  must  have  The  basal,  centrioles suggests  1980).  other  migration although  streaming  It  is  in  the  that  in  the  such  In the  in  suggests  brown  S^.  note  Trinci  and et  is  have  1967,  run  micro-  al .  that  1981) micro-  between  the  f urc i g e r a . Whether  migration that  envelope and  the  is  association  is also  the  1973)  of  dependent  association resembles and  or  unknown.  microfilaments  envelope  (Russell  28  to  to  furc igera .  nuclear  authors  1 980 , B a s s e l  microtubules S.  nucleus  microtubules  nuclear  nuclear  the  other  interesting  envelope  report  of  of  of  (Bracker  observed  role  with  with for  is  nuclear  the  karyokinesis.  Heinzmann  nuclear  integrity  centrioles  of  orientation  (Cell  study,  and  recent  Bornens  nuclear  final  observed  the  the  commonly  play a  original  The  of  involvement  factors.  centrioles the  cell  our  the  and  they  However,  in  were  centrioles  plate  to  Furuya  pre-mitotic migration  this  nuclear  behind  (Schnepf  probable  i f  position  protoplasmic  and/or  the  the  and  place.  mechanism  suggested  cell  axial  as  orientation  prior  lateral,  (Mineyuki  initial.  and  the  just  sister  dealt  1979)  that  90°  bud  the  the  view  during  1980)  of  indicate  shifted  this  the  Furuya  orientation  would  as  into  influences  and  migration  maintained  nucleus  (Mineyuki  an  supports  was  orientation  the  pre-mitotic  green  (Maro of  the that  algae  (Atkinson  et  Robinson proper  1971,  1980).  axis  of  during  bridging  the  to  similar  finding the  hyphae  procedure  the  the  centrioles  with  respect  to  the  which  enhances  electron  dense  bridging  on  the  nuclear specified  was  however,  (Maro  Borens  division  Consistent  duplication  of  migration  to  karyokinesis initial  while  migration  of  hyphae  (Scmiedel  with  and  lack  the  place  the  (Trinci Schnepf  the  A  a  bridging  regarding that  this  i t  recent  is  a  report  that  part  attachment  most  the  of was  probably  and of  nucleus  nucleus  the  fixation,  suggested  interpretation  poles  other  of  the not  1980).  opposite  the  of  suggests  of  dense  envelope.  the  reports  type  centrioles  sister  base  non-1ipoprotein  the  more  Saprolegnia,  structure. A  the  one  the  during  of  prior  electron  fungus  association  takes this  an  nuclear  the  just  becomes  visibility  preserved  on  of  the  material  envelope,  Nuclear  fungal  The  anchored  and  of in  the  centriole-nucleus  centriole  VI.  poorly  nucleus  connect  glycerinated  1978).  a  to  reported  (Heath  and/or  the  observation  membrane  material  rarity  of  interpretation  appears  were  and  in  added  was  Caceres  assist  rotation  outer  1 973 , may  that  the  al .  association  This  material  centriole  after  with  et  an  the  karyokinesis.  attractive  Wilson  Such  positioning  nuclear to  al .  is  the  their the  remains  within  basipetally.  has  reported  1979)  and  1979a).  29  in  moss  Following  in bud  stage  observed subsequent  nucleus.  migrates been  in  After the  bud  Basipetal elongating initiation  cytokinesis  each  daughter  cell  containing although in  the  possesses  a  no  nucleolus.  significant  nuclear  Chloroplasts bud  and  chloroplasts  of  bud  and  filament  and  If  though  the  cytoplasmic  germinating  done  seldom  1980,  hard  the  Here  the  of  been  to  detected  along  more  the  chemical  30  this  is  Uwate  and  not  components  in  observed  in  1965),  the  other  the  recent  of  (Howard  method  does  orgenelles.  been  light  1980,  protoplasmic  other  has  substitution  routine  1980,  various  in  bud  streaming  nucleus  accuminatum  within  How  the  (Aufderheide  the  (Larson  cell  microtubule/micro-  the  process  F u s a r ium  into  through  with  stages  organelles.  migrate  Fuller  why  number  daughter  these  suggested  attractive  in  protoplasmic  and  during  early  migration.  t r a n s p o r t of  freeze  by  to  the  each  of  occurs  grains  mold  that  trend  increase  and/or  imagine  microtubules  preserved  1980)  streaming  seems  the  again  Taylor  differential  on  existence  have  pollen  alternative  each  small,  otherwise  during  seem  and Lin  migrate  Even  is  nuclear  migration  is  simultaneously  1979).  the  and  Satir  i t  ,  rather  general  complement  unclear  Uwate  1980).  streaming,  work  to  involvement  Bradley  The  ensuring  mitochondria  is  1965,  nucleus  are  are  mitochondria  thus  prior  accomplished  Lin  and  sufficient  protrusion  (Larson  differences  f ur c i g e r a ,  initiation  Chloroplasts  own  nucleoli  Mitochondria: in  of  a  The  its  morphology.  development  receives  therefore  and  revealed  hyphal  apex  fixation.  In  that  Aist the are  addition,  mitochondria associated  were  with  furc igera, only  it  is  microtubules  cytoplasmic  appear  nuclear  frequently  to  unwise  involvement  from  Based  to  on  rule  out  system:  in  of  such  a  envelope, membrane another Morre  membrane  way  that  ER,  Golgi  i n both al .  1 97 9 ) .  and As  development  of  onset  o f bud  initiation  of  such  ER  throughout it  that  known  engaged  Robertis  Dictyosomes and  they  exhibit  be  pinching  contributing  and  membrane  are  of  and  membrane  (Morre  combines  differentiation  various  (nuclear  transitional  there  system.  I I i t has a l r e a d y R o u g h ER is a  seems  with  1974,  after  the  proliferation  extended  itself  to predominate  characteristic of  one  i s increased  Soon  i s an e x t e n s i v e  protein  and  f e a t u r e of  f o r export  (De  1980). in  the  stage  outer  material  dictyosomes.  system  and M o l l e n h a u e r  scarce  from  microtubule  the system  I  a perinuclear position.  off  of  time  synthesis  Robertis  the  o f F_j_ a c c u m i n a turn  associated  this  the  towards  intimately  there  stage  encountered,  II  endomembrane  that  in  a n d De  associated  by  I n S.  it  t h e bud m a t u r e s  the c y t o p l a s m .  is well  cells  the  rarely  endomembrane  apparatus,  space  1979).  centrioles  components  are  closely  o f o r g a n e l l e s i n S_;_ f u r c i g e r a .  flow  the  be  Aist  possibility  II  elements)  et  the  The  II  the  to  and  the r e s u l t s  i n the m i g r a t i o n  Endomembrane  concepts  (Howard  microtubules,  radiate  envelope.  observed  to  Similar  31  o f bud  development  Vesicles  nuclear  appear  membrane  the forming observations  faces have  to and  of the been  reported  in  Lofthouse II,  other  and  there  profiles  of  ER  found  concept  Lin  that  ER  in  the  network.  to  dictyosomes  and  This  arise  i s not  et  of  al .  by  stage  parallels suprising  ER  the since  vesicles  in keeping the  1973,  dictyosome  transition  from  1974 , M o r r e  However,  this  dictyosomes  can  Russell  number  associated  the  Mollenhauer  1977).  cytoplasm  and  1969,  with  (Green  1979 ,  the 1969,  Uwate  and  morphologically  the  1 980) .  The  dynamic the  cells.  vacuoles  organelle  earliest  appears  highly With  vacuoles It  the  was  DNA  occurs  in  Interestingly in  serial  replication  cytoplasm  of  the  bud  yeast  cells,  shrink  and  leading  the III  failed  nucleus occurring  32  to in  net cell  (Wiemken  bud to  stage  et in  reveal  (supportive  of  thereby  water  loss  volume a l .  and  1970).  vacuolation  development  since  the  duplication  a  decrease of  by  fragment  reduction  density  and  the  axial  initial  eventually disappear  during  is  the  expansion and  the  initiation  initiation.  quiescent  sectioning  within  bud  the  pressure  II  during  in  cytoplasmic  stage  be  i t does  necessitates a  enough,  heterochromatin DNA  shrink  vacuoles  turn  furc igera,  increasing  osmotic  bud  as  when  which  encountered  of  that,in  the  increase  stage  to  to  encountered  noted  reducing in  proved  vacuolated  appear  III.  that  the  adjacent  and  most  an  the  (Cole  Galatis  increase  profiles  Vacuoles:  At  an  algae  1975,  throughout  numerous  Morre  Capon  is  development  were  brown  any of  i n j5.  signs the  of  idea  heterochromatin  decondenses very of  prior  dense.  the  Renewed  axial  continues  until  cytoplasm  appears  (Fineran  the  the  vacuole  of  the  dense  the  basal at  the  the  in  the  the  V  and  sub-tending  than  of  was  portion  stage  vacuoles  observed  changing  manipulation  being  water  (Wiebe  1978),  1975,  that  the  direction  the  anisotropic  vacuolar  of  the  and  bud  its  initial  bladderwort  gland  initial  the  bud  been  growth  is  the  taken  (unequal)  same by  yielding  driving  as  It  force  pressure,  absorbed  (Buller  1977).  probably  Turgor  implicated  elongation  pressure  of  mechanical initial.  Pickett-Heaps  turgor  pattern  osmotically  has  plant/fungal  out  the  in  cytoplasm  initial  formation  of  Forman  two  the  the  Therefore,  is  of  and  Cell  bud  electron  expansion  behind  Green  the  most  situation  during  result  force  less  constantly  represents  the  and  1980).  The  behind  of  receives  similar  Utricularia  cell  cytokinesis.  cell  A  replication)  v a c u o l a t i o n occurs  mother  daughter  cell.  to  into  the  the  driving  1958,  Novotny  must  be  pointed  in a l l d i r e c t i o n s  the of  cell the  must  wall  and  reside  (Green  in  1962,  1980)  Wall: fold;  The  role  i t must  same  time  (Bartinicki-Garcia 1973,  Werz  1977,  Gooday  1974, 1979,  of  yield  the to  cell the  maintain and Novotny Gooday  wall  driving its  Lippman and and  33  in  bud  turgor  expansion  is  force  at  structural 1 972 ,  Forman Trinci  and  integrity  Bar t i n i c k i - G a r c i a  1975,  1980).  Pickett-Heaps Such  conditions  of  elongation  and  it  appear  also  seems  earliest  sign  protrude  (yield)  new  material  wall  maintain expansion. wall  was  to  of  the  to  be  universal  operate  bud  there  adjacent  to  not  behind  investigated  at  existing  cell  authors  (Bar t i n i c k i - G a r c i a  1976,  1976,  Toth  in  initiation.might the  other  new  has  on  Fevre  1979).  material  Golgi  a  deposition  of  cell a  wall  1 974 ,  the  other et  al. of  throughout  further  (see  by  of  appearance  event.  have  cell  lysis  Bal  The  wall  awaits  algae  of  helps during  However,  lytic  apparatus  which  postulated  Werz  brown  the  been  of  to  yielding  1 973 ,  indicative  seen  be  the  time.  protruding  wall  studies of  the  be  cell  involvement  1977,  Fevre  discontinuities  of  components  fungi the  can  be  of  the  this  and At  plasmalemma  integrity  mechanism  wall  wall to  the  plants  furcigera.  the  appears  structural  The  within  initiation  and  among  The  bud  origin  study,  but  implicated Russell  the  1973  for  a  review). Morphologically, initial  is  of  initiation,  the  outer  it  converge  upon  and  thickness  of  that  of  becomes a  wall.  the  the  of In  the  wall  However, that  the  as  the  layer  accepts  newly  such  way  wall cell  of  the  wall  protrusion of  34  the  3  CWO  ,  protrusion  and  layers  that  synthesized the  remains  adjacent  of  within  wall  that  bud  stages  exist  bud  outer  layer,  a  early  the  wall  in  expanding  layers  outer  thickens the  defined  evident  new  wall  interest.  well  sub-tending  material  to  four  longitudinal  develops  cell  particular  bud  innermost  the  axial  the wall  overall  equivalent cells.  With  respect  microfibrillar CW3  during  cell  o r i e n t a t i o n of bud  transverse agreement  to with  wall  expansion  generally  that  microfibrils growth  by  slipping  Roelofsen  1954,  Green  expansion  continues,  consists only  of only  possible  hypothesis random  there  of is  a  this  passive axis  explanation study  cell  material  only. with  This the  which  concept view  filamentous  the  that  algae  wall  the and  t i p and Then,  as in  bud.  The  further  cell  at  wall  t i p growth  and f u n g i  One  hypothesis  1979).  of is  wall  and  As bud  layer,  CWl,  microfibrils. multi-net  The  growth  i s deposited the  t h e bud  initial  electron support  of the  from the  deposition the  expansion  grows  micrographs  comes  t i p of  in  tubular  the d i r e c t i o n  is in  1977).  o f new  protrusion  is characteristic  (Pickett-Heaps  3 5  concerning  not along  that  the  in  (Houwink  material  demonstrates  occurs of  the  is  i n the d i r e c t i o n  cell  using  re-alignment of  Calcofluor wall  at  cell  the p r o t r u s i o n .  longitudinal favour  new  this  oriented  a  1954).  and V i a n  inner  from  This  another  1 9 6 9 , Roland  explanation  i s that  of  aligned  one  t h e new  shift  Roelofsen  passively  longitudinally  orientation  portion  and  past  CWl,CW2, a n d  hypothesis"  assumptions,  become  layers  direction.  growth  (Houwink  accepted  of the bud, the  gradually  longitudinal  the. " m u l t i - n e t  axis  the w a l l  development  a  the  of  to the l o n g i t u d i n a l  keeping of  many  Discuss ion :  Light  Part  2  Cytochemistry  microscope  cytochemical behavior  Cytochemistry:  studies  during  bud  of  the  have  revealed  within  outer  cell  and  the  the  protruding  layered  pattern  Correct  employed results data  and  the  and  a  main  algal  fucoidan,  galactose,  mannose,  Fucans  can  1979).  Different (Haug affect the  of  et the  an  method  cytochemical  and from  algae  ratio  b)  contain  of  acid  36  the  of  composed acid  (fucoidin,  glucuronoxylofucan) heteromolecu1es proportions acid  uronic  of  (Percival  acid  (Percival  chemically  different  age  tissue  manuronic polymer  acid  fucans  varying  3-20%  walls  L-guluronic  glucuronic  and  cell  alginic  and  fucose,  a l . 1967)  alginic  on  morphological  the  polydisperse  to  contain  in  acid  sargassan,  xylose  brown  our  1,4-linked  D-manuronic  addition  1979).  within  occur  alpha  family  hinges  and  the  microscope.  cytochemical of  to  cell  walls.  of  ascophyllan,  pattern  comparable  biochemical  blocks  in  acid  correlation  light  mother  electron  each  polyuronide  containing  to  of  the  layered axial  and  chemical  with  results  a)  1,4-linked  its  out  is  the  the  polysaccharides  a  known  of  existing  comprising  alginates  that  with  cell  and  the  tissues;  contiguous beta  both  initial  careful  algal  carried  of  limitations  the  brown  Two  of  bud  wall  distinctive  wall  observed  of  with  on  brown  a  interpretation  awareness  cell  initiation  microscope  Morphological  of acid  the to  (Percival  is  guluronic 1979). A l l  brown  algae  contain  polysaccharides which the  an  alga  amount  since  i t  reduce  (Evans  Quillet walls  1974).  are  only  subjected  to  imprecise  nature  wonder any  a  set  of  one The  used the  CWO  consist  with  TBO  and  other of of  very  1968).  with  poorly, However, suggest  prompted  more  regarding  a  the  i f the  techniques  are  to  nature  of  are  to  the  it  be  is  no  applied  to  the  at  CWl,  CW3,  a  whereas  and  Toth  positive of  (see  PAS  into  and  positive CW2  ,  1976)  (McCully  other  the  metachromasia  and  1968,  If  interpret  polysaccharides  1965,  PAS,  question.  might  that  investigation  37  and  examined  1976)  opposite  TBO  TBO  in  one  1 968)  a l l , PAS  the  can  cell  also  this and  (purple  Toth  application  thorough  Add  are  stains  1 965 ,  (McCully  just  to and  algal  but  stains,  indicate  1968,  at  present, serves  brown  points  sulfated  TBO  on  (Lestang  staining  polysaccharides  of  air  that  stains  (McCully  consist  metachromasia are  PAS  bearing  nature  to  classical  two  1965,  to  results.  two  stains  degree  material  interpretations  histological  acidic  direct  alterations.  histology  the  with  CWl  the  these  (McCully  reaction  of  sulfated  the  diversified  histological  of  of  of  results  biochemically  algal  results  the  is  histological  brown  exclusion  apparent  variety  results  in  i t  environmental  that  a  i t s hygroscopic exposure  of  of  and  a i r has  during  Thus,  not  the  1972)  polysaccharide  that  desiccation  range  Callow  to  sulfated  thought  wide  and  i s exposed  of  is  a  CW4, (pink and 1965,  histological  Table the  staining  1).  This  literature reactions  and  their  significance.  TBO high  is  a  thiazin  molecular  sulfate  and  weight  exists  beta  groups  in  is  polymerization  and  charges  sulfated  polysaccharides)  charged and  polar  groups  polymerize  from  blue  and  1977).  methods  the  on  (Cook  upwards  inconsistent Cook  of  The  in  0.5  determined  red  Novotny  at  pH  red/pink (Lison  groups and  6.8.  It  is  (McCandless  1979, was  dye  the  the  1975)  often  (e.g.  of  to  and  but  are  Percival  demonstrated  absent 1979). in  38  colour  from Yet  sieve  shift  (Lison  is  1936,  suggest  that  and at  pink  ionized  of  point  pink  literature sulfated  to  that  groups  known  that  and  non-  terrestrial  tubes  pH  (McCully  groups  animals  red  at  which  i t is well in  form  1)  i n the  found  to  TBO  carboxyl  However,  positively-  various  pH  be  and  the  purple  is characteristic  are  acidic  Metachromasia  Table  reported  1965).  in a  factors  low  when  aggregates  with  (see  1977).  of  1977).  staining use  (Cook  the  many  supposed  polysaccharides plants  TBO  Forman  McCully  terrestrial  using  the  not  metachromasia  1936,  sulfated  are  paper  (orthochromatic  through  cause  (Cook  1977).  occur  results  by  carboxyl,  sufficient  obtained  are  by  to  This  red  this  polysaccharides  1970,  dye  results  sulfated  carboxyl  attract  is influenced  presented  (as  units  with  Cook  (red)  to  1977).  towards  1966,  chromotrope  the  free  alpha  gamma  monomeric  negative  having  forms,  thought  of  metachromasia  (McCully  three  (purple),  Metachromasia  exhibiting  compounds  phosphate  Metachromasia blue),  dye  plants  metachromasia and  companion  cells  of  higher  metachromasia  plants  in Pinguicula,  (Heslop-Harrison apparent, assuming TBO  (O'Brien  and  caution  of  sulfated  metachromasia.  colour  observed  appears  weight  and/or  negative  nothing  more.  metachromasia  Thus  the  which  suggest  other  that  primarily  CW4,  CWI  One question of  of  the  such  the  1:50,000 1936),  is  no  phycologists minute.  concentration  detail out  time  1:2,000  TBO  This  staining staining?  There  with  shown  in  CW3,  and TBO those  Table  and  CWO  1  are  while  staining and  CW2 ,  become  over-stained?  indicate f o r one  is recognized  recommend  How  by  this produces the  then that  f o r TBO  loss  should a  range  to  (Lison  Currently  dilution  that minute  1:10,000  given.  1:2,000  asked  non-specific,  recommended  a  the  duration  t o be  A  for staining  is  the  are questions  the s t a i n  39  molecular  with  agreement  the dye  specificity. VJe  the  results  TBO  of  using  on  polysaccharides.  originally  commonly  based  chromotrope  CWI,  concerning  studies of  my  when  metachromatic  polysaccharides  acidic  interval  becomes  exercised  of  the  layers  specimen  was  of  pink plants  It  the  techniques  wall  does  be  function  that  sulfated  point  specimen. and  of  the  are  Our  stained  cell  1981).  in perfect  procedure.  dilution  but  feel  was  carnivorous  fact,  a  concentration  staining  when  I  point  a s , a t what  i.e.  be  as  polysaccharides  charge  are p r i m a r i l y  final  of  should  In  histological  composed and  to  are, instead,  using  genus  that  presence  red/pink  a  a l . 1964),  Heslop-Harrison  therefore, the  et  f o r one elevated an of  one of  overfine carry dye  concentrations  and  durations  comparisons  be  variety  of  other  correct  concentration  be  best  and  a  made  other  of  In  time  dye  case  of  be  and  a  thirty  performed  obtained  techniques.  the  our  staining  results  histological  assessed. staining  with  of  the  In  with  concentration  seconds  a  wide  way  the  time  can  this  staining  seemed  and  of  1:10,000  to  give  the  bestresults. The acid  principle  will  carbon  bring  bond  derivatives with  about  in to  behind  1,2-glycols form  pararosani1ine It  positive which  to  has  state  been  specific  test  acid, a  a  group  complexes  galactose  as  sialic  acid,  often The  mannose,  al.  1957).  degree  of  carbohydrate-protein  monosaccharide groups  free  known  to  1979)  and  that  units to  thus  periodate  of  is  poorly  is  40  react  the  basic  rarely  referred  on  the  positive. the  (Leblond the  et  of  1,2-glycol fucans  are  (Percival  study  presence  as  amount  number  fucose One  to  and  probably  leave  enough, and  exist  contain  and  depends  to  PAS  sensitive  glucose  as  is  always  and  (Cook  reports  hexosamine  to  will  acid  a  which  in  alkylamino  alginic  galactose  reduced  or  compound  complex  PAS  carbon  coloured  Interestingly  be  the  with  compounds  so  periodic  combines  reactivity  protein,  should  and  of  additional  as  combined  react.  contain  well  that  aldehydes  technique of  is  amino  These  However,  PAS  fucose,  their  that  carbohydrate-protein and  cleavage  magenta  the  for  reaction  which  reported  1970).  that  or  dialdehydes.  form  (McCully  PAS  oxidative  fuchsin-sulfurous  1977 ) .  the  of  revealed alginic  acid  (Druramond  demonstrated and  fourth  that  be  (Conchie  with  presence  acidic  and  do  1,2and  meet  Leblond  et  as  1,2-glycol  unusual 1977 )  as  the  structural bud  role  in  the  cell  by  serve  but  and  and  can  is  the  practice, algae  Percival  1968).  reaction the  that  reaction  theory  O'Brien  than  of  can  1 950 ,  Therefore,  should  simple  as  Evans a  1977). in  their  not  be  demonstration  within  t h e bud  Callow  1972,  additional  inner  alginate  sub-layers and  initial  Medcalf  among  the  protein  wall may  layer play  a  to the  I t i s more  likely  enzymatic  intense  not  restriction  otherwise.  of  is  However,  are  "fucan"  staining,  linkage  abundance  suggests  the  structural  the  sub-layers  of  and  protein  demonstration wall  protein  histological  These  wall  protein  cytochemical  Fucus  assumption  PAS  In  positive  more  observed  cell  these  safe  (Conchie and  presence  initial.  initial  that  PAS  (Medcalf  were  bud  from  p o l y s a c c h a r i d e s of brown  e t a l . 1966,  i t may  sub-layers the  a  third  1950).  groups.  Feder  revealed  carbohydrates  of  of  every  extracted  positive  criterium  1 957 ,  with  study  groups.  (Larsen and  a  another  react  the o n l y  to  anything  Finally, layers,  that  this  while  could  Percival  glycol  significance  interpreted of  and  sulfated  a l.  196 2 ) ,  i n the f u c o i d i n  respect  of  both  the  unit  i s apparent  made  a l .  periodate  fucose  ves i c u l o s u s It  et  i n nature.  peroxidase  lends  support  The  activity to  this  hypothesis . Polarized  light  microscopy:  This  41  technique  fascilitates  the  localization  birefringent cellulose  are  CW4,  known  The  employed  layers  composed  not c e l l u l o s e .  confirms layers  the and  chemical alginic middle  Cell  CaC^  solution  polysaccharides conditions  of  with  beta  no  since  alginates  alpha-linked  was  and  beta  in  a 1%  2  This  CaCl and  s  and  that  i t was  our r e s u l t s .  The  that  under  to  a  t h e same to  the  inhibit  i s known  Calcofluor This  1%  beta-linked  the  to  does  be not  i s , however,  Calcofluor  has  been  demonstrated  to  react  polysaccharides  as o c c u r  42  acid  fucans.  1,3-linked  polysaccharides  supposed  of  that  'composed  contradictory  alginic  rather  with  extracts  is  i  2  major  reports the  solution  3  the  suggest  hours  Na C0  wall  fluorescence:  extractions  f o r 18  cell  is  (1956)  Calcofluor  wall  birefringence  three  acid'  i s i n agreement  cell  polysaccharides  that  these  found  specificity  investigated 1,4-  of  is possible  but  the  appear  layers  histological  three  acidic  and  1956).  wall  other  these  Andersen  wal 1  It  the  acid  (Andersen  to c e l l  'alginic  affect.  acid  since  extensively  i t would  &  60°C  soluble.  alginic  unlikely  this  whereas  results  extraction  stain  at  of  that  that  cell  has  expected  results  birefringence  the  confined  nature  extractions  of  was  possess  Alginic  birefringent  Indeed,  and  that  1970).  beta-linked  Thus  suggests  lamella'  alkaline  of  component.  wall  be  suggest  chemical  acid  results  to  CWI.  techniques  but  (McCully  i n our study  and  are  polysaccharides  properties  Birefringence CWI,  of  i n fucans  and n o t  (Haigler  et  al.  1980,  data is  Wood  are  in  not  conflict  really  extractions find  that  1970,  Novotny  fresh,  been  in  Thus  with  not of and  rest  that  extraction Forman  of i t  and  is  1975).  have  been  material  and  the  plastics  upon  the  thoroughly  seems  since  precise the  i t  the  suprising  techniques  live  embedment not  are 7-8%  extraction  1980).  the  extraction  the  r e s u l t s , but  is  known  i t i s not  that  In  these  affects extraction  of  those  to  (McCully  addition, from  this  uncommon  non-specific  adapted  investigated.  43  that  these used  on  fixation  and  techniques  have  U11rastruetura 1 Peroxidase : detected  localization  of  Intracellular  in  this  isozymes  study.  of  activity  may  from  peroxidase and  (Klisurska  and  Dencheva  techniques  may  not  result  have  different  and  and  sensitive  the  was  not  fact  that  different  pH  1980)  be  activity  peroxidase  This  specificities,  enzyme  substrate  temperature  therefore enough  to  optima  cytochemical detect  a l l  of  them. Peroxidase p l a s m o d e sma t a it  appeared  the  bud  activity  of  to  the  be  This  reports  on  wall  plants  from  1972,  Benayoun existing soften wall  al .  outer and  onset  The  of  protrusion  resulting  wall  of  the  initiation that  activity  (Henry occurs in  in  1979). as the  the  cell  wall  of  suprising  in  light  of  the  of axial  A  was  bud  may  protrusion  progressive  44  cell  the  bud  at  the  be  cell  cell  zone wall  continues  restriction  of  wall  involved  suggested  abscission in  new  of  the  the must  encountered of  and  cell of  expansion  cell  1979,  initiation,  mother  region  decrease  Henry  Deposition  peroxidase  the (Hall  1979,  bud  activity  as  within  groups  Fry  onset  the  the  and  the  continued  loosening/lysis  furc igera  in  the  in  and  intense  1975,  peroxidase  wall  S.  extensible.  for  indicates  peroxidase  activity  the  cell  of  taxonomic  At  more  intense  wall  tobacco  cell  the  localization  various  1981).  in  cell  not  Northcote  allows  bud  is  peroxidase  and  become  material  initial.  cell  Yung  et  mother  particularly  existing  Sexton  observed  axial  protrusion.  of  was  in  for  the  tissue  of  peroxidase to  expand  peroxidase  activity  to  initial  a  cell  suggestion in  cell  also  outermost  wall.  that  wall  This  loosening  microfibrils growth  wall  the  newly  The  fact  the  outermost  to  that  e.g.:  1980)  a  layer  activity  layers,  activity and  of  CWl  as  and  the  to  of  the  always  postulated  for  activity  is  the  active  cell i t  activity bud  the  resistant  is in  initial.  associated also  of  newly  Therefore,  may  blight  i t  direction  Conversely,  CWO,  the  and  deposited  in  CWl  involved  newly  peroxidase  is  previous  hypothesis.  subjected  lack  the  bud  growth  peroxidase.  wall  peroxidase  anti-microbial  not  the  expansion  the  1954).  of  primarily  multi-net  are  to  cell  Roelofson  cell  wall  to  that  observe  deposited  is  aligned  agents,  suprising  response the  CWO,  support  passively  microfibrils  loosening  (Menzel  and  layer,  activity  states  become  (Houwink  deposited  in  supports  hypothesis  wall  lends  peroxidase  indirectly  This  not  thin  with  reflect green  cotton  an  algae  (Venere  1980).  ATPase:  "ATPase  different  physiological  cells"  (Hall  Gilder  and  nucleic  1971).  acid  phosphorylation and  D'Auzac  the  plasma  lysosomes,  processes  These  Cronshaw  include  1973,  1980). membrane,  1971)  ATPase  ion  and  cell  activity  transport and  has  number and  1971, 1976),  oxidative  synthesis been  of  animal  (Hall  Cronshaw  1971), wall  a  plant  mitochondria,  endoplasmic  45  with  both  (Hall  nucleus,  dictyosomes,  in  Bentwood  metabolism (Hall  associated  (Coupe  localized  in  choloroplasts,  reticulum,  developing  vacuoles, Gilder  p l a smod e sma t a  and  D'Auzac  Cronshaw  changes These  1973,  1980).  development  The  in  of  and  _S.  the  Wheeler  fur c i g e r a  are  activity.  therefore  results  indicate  during  bud  initiation  ATPase  activity  is  found  plasma  membrane  bud  a  and  osmotic  number  of  absorption  of  site  This  for  1979,  1973 ,  may  be  and  D'Auzac  process  and  and and  cell  places  wall.  for  ATPase  van  1980)  and in  1980).  activity bud  der  and  cell  along  may the  and  and  46  which  the  1974 ,  of  and  activates  an  and the  assembly complexes and  that  synthesis the  an  maintain  Roland  membrane  development.  is  Furthermore, as  its  Cronshaw  exchange  suggests  explain plasma  of  to  multi-enzyme  growth  stage  and  serves  function  report  the  transport  to  al.  to  light  Gilder  1980).  recent  this  in  Ion  which  et  Thus  adjacent  developmental  using  wall  initiation  However,  (Ban  known  cell  found  development.  regulates  Woude a  appears  1976).  i n the  functions  pattern  1971,  phenomenon  also  activity  suprising  (Hall  substances  is  and  the  not  microfibrils  involved  of  of  pressure  some  wall  Young  ATPase  is  enzymes  membrane  (Roland  system  consistently  Cronshaw  biological  and  plasma  Coupe  initiation  distribution  in ion transport  Bentwood  important  its  regardless  initial.  role  pH  1971,  re-organizational  ATPase  unaltered  the  bud  likely  that  and  1973,  of  endomembrane  (Hall  a l . 1979,  involves  in trace 1 l u l a r l y  known  et  wall  '  Our  the  cell  process  nucleus,  structures  the  Vian ATPase (Coupe  rather during  high the  AcPase:  The  (Bentwood  and  hydrolyses  a  Suzuki  to  Blum  1 96 5 ,  cell  wall  that of  cell  wall  should  activity. activity 1967)  be  cell  recently  was  employed  be  interpreted  primarily reaction  S_.  this  with  vesicular  for  other  postulated  local  hydrolysis  in  to  cells  et  al. and  Only  was  not  cell  wall AcPase  block  AcPase  Jong  et a l .  different  1981).  cell  of  (De  different sub-cellular  in  However,  the  absence  that  was  substrates  locations  one  therefore  the  activity  appeared  of  substrate  results  must  caution.  associated  few  AcPase  assist  i s known  of  activity  furc igera.  the  tobacco  shown  study  furc igera,  deposits  of  i t was  (Oparka  in  imply  reports  activity  product  fixation  walls  between  activity  In  reaction  S_.  and  was  would  AcPase  of  Sommer  also  i t  and  phosphate  AcPase  i n the  which  a  to  are  and  role  1980).  Glutaraldehyde  distinguish  AcPase  a  walls  taken  1965,  Recently,  walls  cell  detect  i n the  and  1980).  play  (Crasnier  There  AcPase  (Akiyama  inorganic  (Blum  unclear  that  esters  associated  cell  might  the  not  and  polysaccharides  in to  1980).  remains known  in  activity  al.  sycamore  extension  failure  AcPase  Giordani  enzyme  is  deficiency  degradation 1969,  It  orthophosphate a  et  AcPases  1976).  of  that  Calleja  wall  detected  can  and  from  this  plant  Cronshaw  increased  (Halperin isolated  of  variety  1980)  leads  role  with  were  bodies.  organisms  AcPase the  detected Similar  (Oliveira  47  plasma in  the  findings 1975,  to  membrane.  Minor  mitochondria have  Bentwood  be  and  a  been  reported  and  Cronshaw  1 976 , this  Murray  1 980 ,  AcPase  activity  presented AcPase  here,  may  be  metabolites seems  assist  with  function, this  cells budding  i t does  i t  and  shows  plasma that  similar  non-budding  the  results  j>.  transport  of  membrane.  Therefore, of  i t  AcPase  furc igera  may  extracellular Whatever  activity  budding  that  extracellular  association  distribution  48  of  1980).  the AcPase  cells.  the  for  suggested  in  f o r both  function  on  have  membrane and  A  based  transport  that  plasma  is necessary  given  (Crasnier  suggest  seem  be  the  cell  the  a l. 1980,).  authors  in  hydrolysis  across  study as  to the  the  metabolites  in  the  et  not  other  involved  into  in  can  but  reasonable  acitivity  Tronchin  and  i t s  localized non-budding  patterns  in  both  Summary  Bud  initiation  furc igera  is  a  and  complex  differentiation  of  summarizes  the  major  during  process.  this  sub-divided  Phase of  I :  the  into  A  vicinity  that  are  revealed  in  involved  in  material  and  cell the  wall bud  new  a) b)  cell  wall  result 1980)  of is  expansion taken  by  for  being  probably of the  the cell,  the bud  simplification  and  subsequent  axial  mother  characterizes  it  is  protrusion  cell the  in  the  earliest  initiating  bud  development  are  However, and  they  deposition  expansion,  of  the is  the  (Henry  1979). into  initial initial. is  49  driving The  protrusion,  and  involvement  of  has  been  linked  pressure,  vacuole(s) force  on  of  deposited  direction  dependent  existing  that  Turgor the  the  wal 1  prove  being  activity  cell  study  possible  absorbed  however,  i.e.  not  probably  new of  Calcofluor indeed  are  of  1oosening/1ysis  Peroxidase  breakdown water  the  the  Results  activity.  encountered  factors  for  reveals  2  cytoplasmic  material  cytochemistry  Table  The  enzymatic  wall  de-  initiation.  synthesis  initial.  peroxidase  of  study.  allowing  cell  thickening  bud  this  cell.  alterations of  the  phases.  nucleus  responsible  Sphacelaria  involves  axial  purposes  wall  of  in  that  cellular For  the  stage  process quiescent  three  cell  of  detectable  a  localized  outer  development  the  the  (Wiebe  behind of  to  the  growth  anisotropic  (unequal) the  same  yielding in  of  the  a l l directions  After  the  initial  modifications  cytoplasmic  vacuoles  density.  There  chloroplasts  and  proliferation events axial bud  of  begins, basal  the  nucleus in  number and  the  of  the  the  of  bud  layered axial  of  force  noticeable  of  the  bud  in  protrusion  which  retains  cell.  reduction  volume  cytoplasmic  expansion  i t does  decrease  which  incorporation  the  cell  This  new  pattern pattern  mother  consists  a  cytoplasmic  longitudinal  protrusion. layered  cause  The  of  the  the  as  number  of  mother  inactive  mass  activity  nucleus  axial  of  However,  increase in  size  turgor  probably  perpetuates  and  pressure  takes  the  in  over  expansion  initial.  outer  integrity  in  the  loss  the  These  continued  of  a  a  synthetic  mitosis.  must  driving  Continued the  for  in  system.  into  established,  cytoplasmic  and  migration  the  in  increase  for  totally  vacuoles  main  allow  within  not  increase  increase  exception  position is  entail  overall  is  1980).  follow  marked  the  and  preparation  the  as  to  pressure  is  endomembrane  Organelle  with  its  size  the  cell  initial.  a  turgor  protrusion  mitochondria  contribute mother  an  as  1962,  that  and is  wall  (Green  wall  cytoplasmic  and  cell  of  cell wall  cell  which observed  cell.  The  four  cell  of  new  wall during  wall  cell  the  the  is  of  of  the  layers,  axial each  the  deposited  cell  into  structural  expansion  original  wall  50  maintains  material  significantly  the  wall  wall  material  differs in  cell  in  from wall  of  mother one  bud  made  a  the the cell up  predominatly  of  outer  layer,  fucan  another layer  fucan  "fucans"  layers inner  are  alginic  and  present,  the  a  layer  confined  and  fucoidan  to  the  and  Forman  this  amorphous highly  Phase  and  II;  the  Once  to  be  it  is  initial  been  the layers  then  alginate cell  and  wall  a  thick  reported  tha-t  areas  (McCully  electron  as  at  protein  cell  wall  continues. to  protrusion.  the  This  by  this  the  of  the  1966,  wall  Novotny  microscopic  study  fucan  layers  appear  fibrillar  point  i t was  for  be  accounted  the for  aligned  has  with  material  at  cell  in  the the  cell  very  appear  During the  and  of  wall  material  cell  wall  mother wall  direction  51  which  of  are  phase, the  the  as  deposition  bud  protrusion increasingly  wall  not  cell  the  likely  of  the  loosening/lysis is  a  commences.  appearance  becomes  cell  new  towards  plate  this  t i p of  position  axial  established  dense  ribosomes  that  existing i f  a  has  migration  activity  outermost implies  cell  future  synthesis.  deposited  as  mother nuclear  Peroxidase  restricted  passively  axial  invested  in  newly  the  cytoplasm,  richly  engaged  the  two  fibrillar  matrix  alginate  occupied  cytoplasm  new  The  only  an  organized.  The  of  amorphous  suggestion  "vacuole-free" place  the  inner  layer  has  with  layer,  an  fucan  It  begins  alginate with  outer  to  It  initial  (Fig.33).  is  confirms  an  bud  thin  acid  1975).  by  terminates  Within  alginate  "alginates".  followed  layer  (Fig.32).  or  as  wall.  of  important This  microfibrils growth  bud  may  become  without  the  aid and  of  cell  wall  Roelofsen  Phase  III:  future  the  After  nucleus  there  occur of  and  two  division.  The  organelles  hand, in  the  cell  and  sister  layer  of  likely  of  both  the to  function  longer  required study  understanding opened and the the  up  cell  bud  cell  1980,  for cell  wall  vacuoles,  studies  should  morphological  of  analysis  52  the  rich  other  and  has  performing 1980)  cell  poor  features  axial  as  ground  development.  future  a  On  a  divide,  become.  the outermost  the  the c e l l  provide  of  fucan  cell. an  It  anti-  i t may  no  loosening/lysis.  and  of  the  and  and  i t  sister by  of  with  vacuolated  Venere  provided  initiation  grow  to  the  cell.  poles  deposition  cell  cells  (Menzel  the p o s s i b i l i t y  morphometric  and  the  mother  cytological  confined  the  cytoplasmic  follows  branch.  are  of  i n an u n e q u a l  to  quiescent  i s now  of  i s non-vacuolated  is highly  these  has  bud  The  multicellular  protect  microbial  This  Houwink  site  opposite  soon  continue  the  activity  serves  be  a  and  characteristic  to  results  cytoplasm  axial  organelles  Peroxidase  by  the  the a x i a l  nuclei.  nuclei  will  forming  of  migration  daughter  bud  reached  karyokinesis  b e t w e e n t h e tx*o  ultimately  predicted  reappearance  portion  and  crosswall  in  as  has  is a  the b a s a l  duplication  nucleus  formation  the  plate  within  Centriole  agents  (1954).  cell  vacuoles  loosening  more of  studies.  work I t has  bud  also  Stereologica1  organelles, detailed  for  especially  approach  initiation  to and  development. if  any,  could  in be  The  determining studied  cytochalasin and if  by  various  isozymes  provide  these  a  enzymes  perhaps  degradation synthesis  more  and  acid  localized. variables  and  and  performed  to  environmental  roles, The  could  Rambourg  gain 'factors  a  light, better  affecting  development.  53  and of  if  of  in new  elucidated  and  Marinozzi techniques be  in  this  be  used  of  the This of and  cell  wall  cell  wall  using  the  1980)  and  whereby  ultrastructurally  manipulations and  role  behavior  be  can  the  development  any,  site  and  AcPase.  the  and  1974)  experimental  could  locations  picture  (Barsotti  temperature,  microfilaments  ATPase  initial  (Schnepf  elucidating  initiation  their  bud  colchicine  techniques  carbohydrates  Finally, as  and  complete bud  in  nucleus,  migration  sub-cellular  deposition.  (Thiery  glycoproteins  help  the  the  as  nuclear  peroxidase,  deposition  phosphotungstic Thiery  of  reveal  and  thus  other  during  better  block  of  of  such  cytochemical  the  position  development  microtubules  Additional  demonstrate  would  and  the  drugs  can  1980)  of  the  using  which  played  process. to  B  Heinzmann any  influence  nutrients  understanding branch  of could  such be  of  the  initiation  and  Table  Phase  I  2  Organelles  Cytoplasm  nuc1eus near bud wa 1 1 p r otru s ion  decrease in of vacuoles  increase in # of m i t o chondria and chloroplasts  increase in cytoplasmic m a s s and density  Cell  #  proliferation of ER  Wall  localized in region trusion. intense activity bud w a l l  thickening o f bud pro-  peroxidase throughout protrusion  r e d u c t i o n i n # of w a l l l a y e r s from 4 to 2 a t t i p o f bud protrusion  increase in nuclear size  Phase  Phase  II  III  nuclear migrat i o n towards future cell plate  "vacuole-free"  continued d e p o s i t i o n of new wall material i.e. f u c a n s and alginates  dense•cytoplasm  peroxidase activity f i n e d to o u t e r h a l f bud wal1  nucleu s at future cell plate  r eap pe a r a n c e o f vacuoles in in basal port i o n of axial mo t h e r cell  continued d e p o s i t i o n of o f new wall material and m a i n t a i n e n c e o f 2 l a y e r e d a p p e a r a n c e of bud wa 1 1  c e n t r i o l e duplication and migration to opposite poles of nucleus  dense c y t o p l a s m restricted to bud protrusion  pe r o x i d a s e a c t i v i t y o u t e r wal 1 l a y e r of protrusion only  karyok i n e s i s  "vacuole-free" and dense cytoplasm in bud cell  formation of resulting in and a s i s t e r  h i g h l y vacuolated sister axial cell  54  cross a bud axial  conof  in bud  wal1 cell cell  KEY  C,  chloroplast,  CVJ1 ,  cell  wall  layer  layer,  wall 3,  outer  vacuole,  P,  1,  cell  ML,  Ct,  CW2 ,  cell  wall  layer  cell  reticulum,  mitochondrion,  FIGURES  crosswall,  layer CW4,  CWO,  endoplasmic  CrW,  FOR  wall N,  middle  c e n t r i o l e , Cu, wall 4,  layer,  nucleus, lamella,  plasmodesmata .  55  layer CWl, D, Nu,  Mt,  2,  inner  cuticle, CW3 ,  cell  cell  wall  dictyosome,  ER,  nucleolus,  M,  microtubule,  V,  Figs.1  to  Nomarski  8  i n t e r f e r ence  differential  contrast  observations  of  initiation  bud  in  S. . f u r c i g e r a .  Fig.l  Quiescent a  axial  medial  cell.  position  The  nucleus  within  occupies  the  cytoplasm  (arrowhead) .  Figs.2  and  3  Early and  stages II).  cell I  Figs.4  to  7  in  Arrow  wall  initiation  denotes  protrusion  (stages  site  of  I  initial  characterizing  stage  (Fig.2).  Sequence  of  (stages  III  progressive of  to  results  the  the cell.  to  VI).  the  of  sister  in  in  in a  axial  position Arrows  transverse  56  position  of  the  of  'B'  'A'. the  of  (stage two  and  a  nucleus  between  the the  subtending indicate  within  position two  VII)  daughter  Arrowheads  indicate  in  bud.  division  formation  cell  wall  Observe  developing  cell  initiation  (arrowhead)  cell  bud  bud  the  nucleus  Completion  cells,  stages  change  the  relation  Fig.8  bud  of  cells.  each the  Quiescent  axial  normal  in  cytoplasm. can  a  Four  be  Axial  cell  mother  trusion occurs  of in  marks  the  inpocketing  vicinity  of  become  less  Note  the  four  wall  thickness  of  region  within  pro-  longitudinal  wall  bud  nucleus  and  initiation.  The  envelope a  pair  the c y t o p l a s m  magnification  cell  denote  slight  the  outer  (*) of  has  vacuolated.  Increased Fig.10.  CW4)  I). A  contain  that  to  the  nuclear  to  Note  (CWI  in  of  of  the  observed  centrioles.  the  outer  the beginning  the  vacuolated  (stage  the  a  highly  recognized wall.  displays  organelles  layers  wall  longitudinal  cell  of  complement  distributed  was  This  cell,  of  the  the  57  inset  of  increase  in  layer  protrusion.  wall  of  in  composition  the  wall  existence  cell  layer  and  cell  of  4  (CW4)  in  Arrowheads  discontinuities  layer  1  (CWl).  Axial  mother  layers  1,  observed  cell  2  in  the  longitudinal outer  cell  of  the  and  wall  wall  (CWO).  Axial  mother  portion  almost  Increased  the  (stage  the  absence  seems  to  exist  inner  (CWI)  loose between  cell  protrusion.  58  wall  CW3)  the  outer  of  in  a  the  region  wall  III).  The  most  this  stage  is  vacuoles.  inset  attachment the  denote cell  of  of  single  outer  of  magnification  depicting  and  Arrowheads  feature  total  wall  into  (CWO)  within  cell  Cell  CW2  converge  layer  characteristic  II).  (CWI,  protrusion.  layer  the  3  basal  discontinuities  the  (stage  outer  layers  at  in  Fig.13  (*)  that  (CWO) the  tip  and of  Fig.15  Axial  mother  cell  nucleus  occupies  a  cytoplasm  which  occurrence  of  bud  Fig.16  of  the  tip  15).  of  The  the  Bright  of  The  field  The  the  also  continued  of  CWl.  the  cell  in  (CWl)  at  Fig. wall  Note  the  microfibrillar  Arrowheads  point  to  CWO.  of  thinner a  wall  (inset inner  wall  covers  cytoplasm.  the  the  within  a  the  thickness.  cell  into  latter  into  micrograph  outer  (arrows)  migration  and  in  discontinuities  Fig.17  the  of  (CWO)  arrangement  material  of  protrusion  differ  random  indicative  from  detail  outer  layers  bud  Note  vacuoles  The  the  nuclear  Organizational  IV).  position within is  protrusion.  absence  (stage  a  bud  initial.  layers outer  thick  converge  wall  inner  layer.  wall  layer  ( a r r o w h ead s ) .  Fig.18  Calcofluor of  the  dotted the newly  old  White  same . c e l l line  fluorescence depicted  defines  fluorescing synthesized  wall.  59  the  in  microscopy Fig.17.  boundary  cell  wall  non-f1uorescing  The  between and  the cell  51  A  Fig.19  Magnified the  view  uninterrupted  wall  layer  deposited  Fig.20  CW4  cell  The  nucleus  the  basal  (stage  layer  inset  in  Fig.20.  Note  merging  of  the o l d  cell  with  wall  comes  portion  V)  rupture  the  of  and  layer  to of  the  of  the a x i a l  60  the  midway  the a x i a l  newly  of  between  mother  protrusion.  a t the t i p of  reappearance of  of  CWl.  rest  (arrowheads)  CWO  portion  that  the  cell  Note  the  cell  wall  the p r o t r u s i o n  vacuoles mother  i n the cell.  and  basal  Figs.21  and  22  Two  serial  centrioles  (arrowheads) appear  24  of  Fig.25.  the  in  with  and  of  observed  sides  Figs.23  sections  the n u c l e u s ,  Note  a  nucleus that  on  feature  the  opposite  associated  karyokinesis .  High  magnification  centrioles indicated  found in  occurrence  at  Figs.  and  microtubules  of  electron  (arrowheads)  21  the and  orientation  nuclear the  observation  and  dense  of  the  Note the  relation  cell  (stage  the  number  to  the  extra-  organization  bridging  in  the  positions  22. of  1  material  the  nuclear  V I ) . Note  the i n -  envelope.  Fig.25  Axial  mother  crease  in  vacuoles axial  within  mother  61  the  cell.  basal  of  cytoplasmic  portion  of  the  &l  A  Stage  VII  is  completion is  of  deposited  cell  subtending  the  position  of  by  vacuoles  abound  A  reveal  a  crosswall  cell  cell  centrioles  cell  'A',  denser,  'B'  and  'A'.  sectioning.  in  the  Note  (*)  as  Numerous  while  cell  virtually  non-  cytoplasm.  portion  Fig.26  the  by  A  bud  axial  serial  exhibits  separating  the  sister  revealed  vacuolated  division.  between  the  'B'  characterized  of cell  i s shown the  plasmodesmata  62  the 'A'  crosswall from  at higher presence (arrows).  cell  (CrW) 'B'  magnification of  in to  numerous  <oZ A  Figs.28  t o 31  A  schematic  in  diagram  appearance  morphological and  the  process The  newly details  wall)  bud  dotted  between  of  the  old  deposited see  observed  delineates cell  wall  cell  conclusion.  63  and  wall  change  prominent  (nucleus,  initiation  line  the  three  features  cell of  depicting  vacuoles  during  the  development. the  layers  boundary and  layers.  the For  Figs.32  & 33  Two  schematic  distribution  drawings  of  polysaccharides microscopy  the  depicting  two  major  identified  cytochemistry.  distribution  pattern  initial  (Fig.32)  advanced  bud  64  of  with  initial  cell by  that  (Fig.33).  early of  wall light  Compare an  the  a  the bud more  64- A  Fig.34  to  41  Light  Fig.34  TBO  Fig.35  TBO of  Fig.36  microscope  at  at  wall  cell  wa11  layers  CWl  me t h y l a t i o n. CW3  CWO  does n o t .  0.  Note  produced and  stain.  layer  wall  CWl  colour  green  CWO  the  within are  cell  idicative  protein.  Aniline  sub-- l a y e r s  wall  reacts  IKI/H so4. 2  present  Black/Safranin  Blue  Ruthenium CWO  and  absence  CWl.  and  FCF/Safranin  Green  dark  CWl  35.  the  layer  Cell  strongly  Fig.  group  layers  reaction,  cell  41  wall  Only  protein  Fig.  cell  0/carboxyl  of  Fig.40  in  with  Note  Safranin  Fast  Fig.39  0.5.  pH  staining  reacts  Fig.38  6.8. C o m p a r e  pH  PAS  Fig.37  cytochemistry.  the  of  Red.  (arrows)  bud  Note  strongly  Only  in c e l l  65  0.  wall  the  within  the  initial.  that  cell  whereas  a  Note  slight layers  CWl  wall  layer  does  not.  reaction CW3  and  CWl.  is  A  Figs.42  to  45  Demonstration  pero-iH-«;p  of  r  during  Fig.42  The  bud  cell  initiation  wall  peroxidase the  Fig.43  A  the  bud  more  cell  Fig.44  A  wall  absence  Fig.45  the  cell  of wall  Peroxidase  activity  lamella  the  cell  the  lack  of  the  inner  half  of  in  the  lack  of  incubated Note  within  crosswall from  cytokinesis.  66  that  Note  in  substrate.  of  than  CWI.  activity.  after  exhibit  extent  inital  preparation  of  bud  the  within  Fig.42.  peroxidase  the  Note  bud  activity layer  control  development.  protrusion.  in  peroxidase  J  p l a smod e s m a t a  product  advanced  presented  •, activity  - -U"iUose  and  activity.  reaction  of  and  , t  its  the  the  which  sister  middle seperates  axial  cell  (,U  Figs.46  Fig.46  to  51  Demonstration  ATPase  of ATPase  activity  nucleus,  is  activity.  associated  dictyosomes  and  ATPase  activity  within  Fig.48  ATPase  activity  associated  Fig.49  Fig.50  of  ATPase  activity  drial  cristae.  preparation  absence  of  Another enzymatic Note  the  control activity loss  67  with  the  by  the  inner  mitochon-  incubated  substrate.  i n ATPase  dictyosome.  membrane.  exhibited  Control  reduction  Fig.51  the plasma  the  chlorop lasts.  Fig.47  surface  a  with  Note  in a  the  general  activity.  preparation was  of ATPase  in  destroyed activity.  by  which heat.  A  Figs.52  t o 57  Fig.52  Demonstration  The  inner  exhibits  Fig.53  AcPase  Fig.54  surface AcPase  activity  cristae  of the  Intense  AcPase  the  entire  membrane  Fig.55  of AcPase  Fig.56  & 57  detected  activity  surface  in  carried  the  plasma  which out  in  sodium  (*).  by  the the  fluoride.  activity.  is exhibited  vesicles  68  of  along  cell.  of AcPase  activity  6  the  is displayed  of the i n h i b i t o r  cytoplasmic  within  mitochondria.  was  the l o s s  AcPase  was  preparation  presence  membrane  activity.  inner  incubatiion  Note  of the plasma  of a bud  Control  activity.  various  6fc A  Literature  Cited  A g h a j a n i a n , J . 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