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Ultrastructure studies in ustilago hordei (Pers.) Lagerh. Robb, Elizabeth Jane 1971

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ULTRASTRUCTURE  STUDIES  IN .USTILAGO HORDE I (PERS.) LAGERH.  BY  ELIZABETH JANE ROBB B.Sc.  Hons. B i o l , York U n i v e r s i t y , Toronto, 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Botany  We accept t h i s t h e s i s as conforming required  t o the  standard  THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1971.  N  In  presenting this  thesis  in p a r t i a l  fulfilment of  an advanced degree at the U n i v e r s i t y of B r i t i s h the L i b r a r y s h a l l I  f u r t h e r agree  make i t  freely available  that permission  for  the requirements f o r  Columbia,  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s of  this  written  representatives. thesis  It  for financial  is understood that copying o r p u b l i c a t i o n gain shall  permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  not be allowed without my  F o r out o f olde f e l d e s , as men seytb, Cometh a l t h i s newe corn from y e r t o y e r e , And out o f olde books, i n good f e y t b , Cometh a l t h i s newe s c i e n c e t h a t men l e r e . The Parlement o f F o u l e s G e o r r r e y Chaucer  i  ABSTRACT  A comparative  l i g h t and e l e c t r o n microscope  technique has  been used t o study the c y t o l o g i c a l changes accompanying spore  telio-  ( i . e . probasidium) g e r m i n a t i o n i n U s t i l a g o h o r d e i (Pers.)  Lagerh.  S p e c i a l emphasis has been p l a c e d on d e t e r m i n i n g the  u l t r a s t r u c t u r a l events i n v o l v e d i n k a r y o k i n e s l s , e s p e c i a l l y m e i o s i s , and c y t o k i n e s i s . The t h e s i s i s d i v i d e d i n t o f i v e p a r t s , o f which the f i r s t i s concerned w i t h pre-germinal d i f f e r e n t i a t i o n .  The g r e a t i n -  crease i n m i c r o a n a t o m i c a l complexity which occurs d u r i n g the pre-germinal stages i s due l a r g e l y to an i n c r e a s e i n the amount o f endoplasmic  r e t i c u l u m (ER) and t o the f o r m a t i o n o f "primary  hydration vacuoles."  E v i d e n t l y t h e n u c l e a r envelope g i v e s r i s e  to t h e new ER which i n t u r n d i l a t e s t o form the v a c u o l e s .  This  i s accompanied by an i n c r e a s e i n m i t o c h o n d r i a l s i z e and the development o f patches o f patches o f " f l o c c u l e n t  cytoplasm."  P a r t I I concerns t h e i n i t i a t i o n and subsequent o f t h e metabasidium ( i . e . promycelium)•  extension  I n i t i a t i o n involves  t h e l l o c a l i z e d d e g r a d a t i o n o f the i n n e r spore w a l l , and d e p o s i t i o n o f new w a l l m a t e r i a l .  The ER and spherosome-like  seem t o be a s s o c i a t e d w i t h these a c t i v i t i e s .  bodies  Once spore w a l l  r u p t u r e has o c c u r r e d the s t r u c t u r a l b a s i s o f p r o m y c e l i a l extens i o n i s unknown b u t changes i n the number, s i z e , and d i s t r i b u t i o n o f the spherosome-like  o r g a n e l l e s appear t o have profound  e f f e c t s on the d i f f e r e n t i a t i o n o f t h e organism.  ii  S e p t a t i o n , k n e e - j o i n t f o r m a t i o n , and budding are d i s c u s s e d i n part I I I .  E l a b o r a t e membrane complexes are a s s o c i a t e d w i t h  cross w a l l i n i t i a t i o n .  A membranous p l a t e i s completed across  the c e l l b e f o r e s e p t a l w a l l t h i c k e n i n g b e g i n s . of sporidia  The  initiation  ( i . e . basidiospores) involves a l o c a l i z e d  plasti-  c i z a t i o n o f the promyoelial w a l l f o l l o w e d by d e g r a d a t i o n o f the o l d w a l l and subsequent s y n t h e s i s o f new B r i d g e - f o r m a t i o n r e s u l t s when two  wall material.  adjacent c e l l s g i v e r i s e to  b u d - l i k e processes which grow t o g e t h e r and subsequently  fuse  to produce a p r o t o p l a s m i c b r i d g e . The  s t r u c t u r e and a c t i v i t i e s o f the m e t a b a s i d i a l n u c l e i  and t h e i r a s s o c i a t e d s t r u c t u r e s are d i s c u s s e d i n p a r t IV, Both m e i o s i s and m i t o s i s are unusual i n t h a t the two  chromatin  b o d i e s a p p a r e n t l y remain a t t a c h e d to the c e n t r i o l a r - k i n e t o c h o r e e q u i v a l e n t and a t l e a s t one o f the chromatin bodies i n a t t a c h e d to the n u c l e o l u s throughout  the d i v i s i o n c y c l e .  are compatible w i t h Brown and Stack's  The  results  (1971) model f o r somatic  n u c l e a r d i v i s i o n i n some f u n g i . Membrane complexes, resembling  those which I n i t i a t e  septa,  form i n a s s o c i a t i o n w i t h prophase n u c l e i and m a i n t a i n a s p e c i f i c r e l a t i o n w i t h the nucleus throughout  division.  In p a r t V the  s u g g e s t i o n i s made t h a t these complexes form p a r t o f a mechanism c o n t r o l l i n g the p o s i t i o n a l r e l a t i o n s h i p s o f n u c l e a r and d i v i s i o n s i n the promycelium.  cell  iii  TABLE OP CONTENTS Page ABSTRACT  i  TABLE OP CONTENTS  tiii  LIST OF TABLES  iv  LIST OF DIAGRAMS  iv"  LIST OP FIGURES LEGEND OF SYMBOLS ACKNOWLEDGEMENT GENERAL INTRODUCTION PART I :  v v i i viii 1  Pregermination Development i n H y d r a t i n g Teliospores  PART I I :  li+  I n i t i a t i o n o f the Promycelium and Promycelium E x t e n s i o n  I4.8  PART I I I : P r o m y o e l i a l S e p t a t i o n and S p o r i d i a l Formation PART IV:  Nuclear D i v i s i o n with S p e c i a l Emphasis on Meiosis  PART V:  91  117  The P o s i t i o n a l R e g u l a t i o n o f C e l l and Nuclear Division  157  GENERAL CONCLUSION  175  APPENDIX A  179  APPENDIX B  181  APPENDIX C  186  iv  LIST OF TABLES Page PART I :  TABLE I - Summary o f E l e c t r o n Techniques  PART I V :  PART V:  19  ••••  TABLE I - Summary o f L i g h t Techniques  Microscope  Microscope  ••••••••••••  TABLE I - Membrane Complexes and G o l g i 169  i n Fungi APPENDIX:  121  TABLE I - O s m o l a l i t y o f B r o t h , Broth p l u s M a t e r i a l , and 2% Glutaraldehyde  ,  185  LIST OF DIAGRAMS Page GENERAL INTRODUCTION:  DIAGRAM I - L i f e  Cycle o f  U s t i l a g o h o r d e l ..... PART I V :  3  DIAGRAM I (FIGURE 3k) Brown and Stack's Model f o r Somatic Nuclear D i v i s i o n i n Some Fungi .............  Hid  DIAGRAM I I (FIGURE 3 5 ) Model f o r M e i o s i s i n U s t i l a g o h o r d e l .......  11+9  V  LIST OP FIGURES INTRODUCTION: Figure  1.  A time l a p s e study o f t e l i o s p o r e germination.  Figures 2 - l i .  One-, two-, and t h r e e - c e l l e d ( l i g h t microscopy).  state  PART I : Figure  1.  Quiescent  teliospore.  Figures 2 -  Karyogamy ( l i g h t  Figures 5 - 6 .  Quiescent  Figures 7 - 8 .  Stage one t e l i o s p o r e .  Figures 9 - 1 2 .  Stage two t e l i o s p o r e .  Figure 1 3 •  Stage two t e l i o s p o r e .  ER-NE r e l a t i o n s h i p ,  F i g u r e 111.  Stage two t e l i o s p o r e . tionship.  ER-vacuolar r e l a -  Figures 15 - 1 7 .  Stage t h r e e t e l i o s p o r e .  microscopy).  teliospore.  Fully  activated.  PART I I : Figures 1  -  Promycelium i n i t i a t i o n .  Figure 5 .  Nuclear  Figure 6 .  Spore w a l l r u p t u r e and promycelium emergence .  FIGURE 7 .  Migration of organelles.  Figures 8 - 9 .  Spherosome-like  Figures 11 - ll|..  Spherosome and v a c u o l e s  Figure 1 5 .  ER  Figure 1 6 .  Pores i n the p r o m y o e l i a l  Figure  17.  and m i t o c h o n d r i a l  migration.  bodies. i n aging  tissue.  s t a c k s i n t h e promycelium.  Haploid  wall,  nucleus.  Figure 1 8 .  The  c e l l o f a f o u r - c e l l e d promycelium,  Figure 1 9 .  Spherosomal membrane  formation.  20.  Figure  The s i n g l e - c e l l e d  promycelium.  PART I I I : 1-6.  Figures  Figures 7 -  10.  Septation. Membrane complexes.  F i g u r e s 11 -  13.  Bridge  F i g u r e s Ui -  18.  Sporidium  formation. formation.  PART IV: Plate  1.  M e i o s i s i n TJ. h o r d e i  Plate  2.  M e i o s i s i n U. k o l l e r i ( l i g h t microscopy)  Plate  3.  M e i o s i s and m i t o s i s i n U. h o r d e ! ( l i g h t microscopy).  (light  F i g u r e $,  General  view o f a h a p l o i d  Figure  6.  Nuclear  pores.  Figure  7.  Migrating haploid 8-13.  Figures  microscopy).  nucleus.  nucleus.  The CKE.  F i g u r e s U4. -  16.  R e p l i c a t i o n o f t h e CKE.  F i g u r e s 17 -  18.  The n u c l e a r w e l l and the CKE.  F i g u r e s 19 -  21.  Microtubules.  F i g u r e s 22 -  26.  Meiosis I .  F i g u r e s 27 -  33.  M e i o s i s I I and p o s t - m e i o t i c m i t o s i s .  PART V: Figures  1-6.  Membrane complexes and t h e prophase I nucleus.  Figures  7-8.  Membrane complexes and the prophase I I nucleus.  Figure  9.  Membrane complex  Figure  10.  Membrane complex septum attachment.  formation.  vii  LEGEND OP SYMBOLS ch  —  chromatin  cl  - centriolar-kinetochore-equivalent - c e n t r a l l a m e l l a (septum)  ER  —  endoplasmic r e t i c u l u m  CKE  f  - flocculent  L  —  l i p i d body  M  —  mitochondrion  mc  —  membrane complex  cytoplasm  rat  - microtubule  mtoc  —  microtubule  N  —  nucleus  organizing  (dN - d i p l o i d n u c l e u s , hN - h a p l o i d n u c l e u s )  NP  - nuclear - nuclear  Nu  —  nucleolus  Pi  - perforations - p l a t e (septum)  PM  —  plasma membrane  NE  P  envelope pore  pW  - promyoelial  S  —  spherosome-like body  V  —  vacuole  ve W  —  centre  wall  vesicle spore w a l l  (Wl - outer w a l l l a y e r ) (W2 - middle w a l l l a y e r ) (W3 - inner w a l l l a y e r )  M a g n i f i c a t i o n s - The b l a c k  scale represents  0.5 u °n e l e c t r o n  micrographs and 1.0 u on l i g h t microscope p i c t u r e s unless  otherwise i n d i c a t e d .  viil  ACKNOWLEDGEMENT  I p a r t i c u l a r l y wish to thank my supervisor, Professor Clayton Person f o r h i s encouragement of independent research and f o r h i s guidance and valuable suggestions i n the preparation of t h i s t h e s i s .  Thanks are also due to Dr. T.  Bisalputra and other members of my thesis committee f o r i n valuable discussions concerning the preparation of the nuscript.  ma-  The author also wishes to acknowledge Dr. C.  Robinow (University of Western Ontario, Canada) and Dr. R. M. Brown (University of Texas at Austin, U.S.A.) f o r t h e i r h e l p f u l c r i t i c i s m and advice i n the preparation o f part IV. I would also l i k e to express my appreciation to Mr.  L.  I i . Veto f o r h i s expert teaching, t e c h n i c a l assistance and advice i n electron microsoopy and photography. To Margaret Shand and Carole Stanley, many thanks f o r providing assistance of many kinds and f o r your moral support i n times of " c r i s i s . "  The smooth running of the "smut" lab  i s l a r g e l y due to the patience, good humour and competence of these two people.  Thanks are also due to a l l the other mem-  bers of the "smut l a b " f o r t h e i r constant encouragement and h e l p f u l discussions. L a s t l y , and most importantly, I wish to thank my husband, Dave, whose encouragement, help, understanding,  and  i n f i n i t e patience enabled me to complete t h i s investigation and  manuscript.  1  GENERAL  INTRODUCTION  The h i s t o r i c a l development of s c i e n t i f i c i n t e r e s t i n the smut fungi centres on one o f Man's most pressing problems, - the World's food supply.  Many o f these t i n y plant pathogens  which constitute the order U s t i l a g i n a l e s (subclass Heterobasidiomycetidae) are p a r a s i t i c on cereal grains*  The crop  diseases which they cause greatly reduce not only crop y i e l d but also q u a l i t y ; as v i t a l agents of crop damage they are second only to rusts (Uredinales).  Since cereals constitute app-  roximately one-quarter o f the t o t a l food consumed by Man, the economic importance o f t h i s group of fungi i s clear (Fischer and Holton, 1958;  Christensen, 1963)»  n  o  t  sur-  p r i s i n g l y , much research has been devoted to i t s c o n t r o l . During the l a s t 20 to 30 years t h i s pursuit has l e d to the opening up o f new and complex realms o f study bent upon elucidating the physiological and genetical i n t e r r e l a t i o n s h i p s between host and parasite (Flor, 191+2 and Person,  1959).  1951+J  Halisky,  1965;  Such studies, while o f p o t e n t i a l economic value,  hold a t h e o r e t i c a l f a s c i n a t i o n o f t h e i r own.  In addition smut  fungi, p a r t i c u l a r l y members o f the genus Ustllago. have found favour as purely genetic tools being used i n studies of mutat i o n (Hood, Kozar,  1968), recombination  1969)*  (Holliday, 1961 and  h a p l o i d i z a t i o n (Day and Jones,  t i c complementation (Dinoor and Person,  1969)*  1969).  I96I4.;  and gene-  The species  most extensively used are Ustllago mavdis. Ustilago  violaceae.  2  and U s t l l a g o h o r d e l . C o n s i d e r i n g the importance o f the smuts both  economically  and as a r e s e a r c h t o o l , and c o n s i d e r i n g the energies which have been devoted to c e r t a i n aspects of smut p h y s i o l o g y  and  g e n e t i c s , amazingly l i t t l e o y t o l o g i o a l i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g any one  species.  F i s c h e r and H o l t o n have reviewed  the c y t o l o g y of the smuts up to 1957*  Almost a l l knowledge of  the s t r u c t u r e , and l i f e c y c l e of these organisms has been  ob-  t a i n e d by l i g h t miorosoopy; i n g e n e r a l v e r y l i t t l e i s known about the u l t r a s t r u c t u r e of the U s t i l a g i n a l e s .  Even more  amazing and annoying from the g e n e t i c i s t s ' p o i n t of view, i s the p a u c i t y of i n f o r m a t i o n concerning the n u c l e i of these f u n g i , e s p e c i a l l y at m e i o s i s . in  t h i s t h e s i s was  The major purpose of the work presented  to o b t a i n i n f o r m a t i o n on ohromosome number,  n u c l e a r s t r u c t u r e , and mechanism of d i v i s i o n d u r i n g the m e i o t i c and p o s t - m e i o t i c stages i n U s t i l a g o h o r d e i  (Fers.) Lagerh.  comparative l i g h t and e l e o t r o n microscope teohnique employed f o r t h i s purpose. smuts, the m e i o t i c process  been  In U s t l l a g o h o r d e l , as i n o t h e r i s i n e x t r i c a b l y associated with  o t h e r c e l l u l a r events p e r t a i n i n g t o the germination s e x u a l spore  has  A  - the t e l i o s p o r e .  The  of the  scope of the work  was,  t h e r e f o r e , broadened t o i n c l u d e a o y t o l o g i c a l a n a l y s i s of t e r l i o s p o r e a c t i v a t i o n and germination, and  of b a s i d i o s p o r e  of germ tube  extension,  formation.  A d e t a i l e d d e s c r i p t i o n of the v a r i a t i o n s i n the l i f e c y c l e s of  d i f f e r e n t s p e c i e s of the U s t i l a g i n a l e s oan be found  B i o l o g y and C o n t r o l of the Smut Fungi F o r convenience,  i n The  ( F i s c h e r and Holton, 1957 )»•  the l i f e c y c l e of U s t i l a g o h o r d e i i s p r e -  3  DIAGRAM I L i f e cyle of Ustilago hordel A schematic r e p r e s e n t a t i o n o f the l i f e c y c l e o f U s t i l a g o h o r d e i . H a p l o i d n u c l e i are denoted by n and d i p l o i d n u c l e i by 2 n . Erratum:  diakaryon  should read  dikaryon.  aented schematically i n Diagram I .  Ustilago hordei i s parasi-  t i c on barley during the major part o f i t s l i f e c y c l e .  The pa-  r a s i t i c stage i s i n i t i a t e d only a f t e r the fusion of two comp a t i b l e haploid c e l l s  (sporidia) o f d i f f e r e n t mating type (+  and -) to form a dikaryon.  The dikaryotic state i s maintained  u n t i l the p a r a s i t i c stage culminates with the fusion i n pairs, o f compatible haploid n u c l e i and subsequent ploid teliospores.  production o f d i -  On teliospore germination, the d i p l o i d f u -  sion nucleus undergoes meiosis within the promycelium, each teliospore giving r i s e to four haploid c e l l s .  Each c e l l then  usually begins t o bud i n y e a s t - l i k e fashion to form a clone o f haploid c e l l s (basidiospores or s p o r i d i a ) .  There are two clones  of each mating-type per promycelium* The term f t e l i o s p o r e s " i s commonly applied to the sexual spores o f rusts and smuts; however, notice should be taken that these structures are now more properly referred to as "probasidia".  Germinating teliospores give r i s e to specialized  tub-like c e l l s o f l i m i t e d growth which are commonly c a l l e d "promycella (Hawker, 1966),  In keeping with the new terminology  the term "promycelium" should be replaced by "metabasidium". Throughout t h i s thesis these terms w i l l be used interchangeably. The most complete study to date on teliospore germination and basidiospore production i n Uatilago hordei i s that o f D, T, Wang (193if).  In her studies Masig observed most o f the funda-  mental events which characterize t h i s stage.  The r e s t i n g spore  contains a single large nucleus which undergoes d i v i s i o n shortly a f t e r the metabasidium i s formed.  In the p a r t i c u l a r s t r a i n ob-  served by Wang t h i s f i r s t nuclear d i v i s i o n usually takes place  INTRODUCTION. F i g u r e s l a - 0.  PLATE 1  S e r i a l photographs of t e l i o s p o r e s of U s t i l a g o h o r d e i germinating i n a t h i n l a y e r of complete b r o t n between a g l a s s s l i d e and a cover s l i p . 1 a and 1 b r e p r e s e n t r e s t i n g spores and spores a f t e r one hour of h y d r a t i o n r e s p e c t i v e l y . Figures l c - 0 were taken a t h a l f - h o u r i n t e r v a l s ( T o t a l = 6^g n r . ) . The arrow i n l i i n d i c a t e s the f i r s t septumj the arrows i n l j i n d i c a t e the second and t h i r d s e p t a . In the promycelium on the l e f t the i n i t i a t i o n of the f i r s t two spord i a 6an be d e t e c t e d i n l j , the t h i r d s p o r i d i a i n 11 and the f o u r t h s p o r i d i a i n I n . The p r o mycelium on the r i g h t i s undergoing K n e e - j o i n t formation, ca, X 1,000. Note: each s c a l e d i v i s i o n r e p r e s e n t s one micron ( i . e . t o t a l s c a l e l e n g t h i s 10 m i c r o n s ) .  5 w i t h i n the spore, one o f the daughter n u c l e i subsequently  migrat-  ing  i n t o the promycelium, but she noted t h a t sometimes the s i n -  gle  nucleus w i l l f i r s t migrate  vide.  i n t o the germ tube and then d i -  The l a t t e r i s almost always the case i n the s t r a i n o f  Ustilago hordel studied i n this t h e s i s . subsequently dently.  The two daughter n u c l e i  d i v i d e a g a i n , e i t h e r s i m u l t a n e o u s l y o r indepen-  A c c o r d i n g t o Wang, t h r e e septa a r e l a i d down s e -  p a r a t i n g the f o u r n u c l e i .  In the s t o c k c u l t u r e used i n t h i s  t h e s i s the three septa a r e not l a i d down t o g e t h e r . septum i s l a i d down immediately  a f t e r the f i r s t n u c l e a r d i v i s i o n  d i v i d i n g the promycelium i n two. med  immediately  ing  The promycelium i t s e l f c o n t a i n s three  t h e chambers, the f o u r t h chamber l y i n g i n the spore. A l -  most immediately of  The o t h e r two s e p t a a r e f o r -  f o l l o w i n g the second n u c l e a r d i v i s i o n , one on  e i t h e r s i d e o f the f i r s t . of  each c e l l g i v e s r i s e to a sporidium, and each  t h e n u c l e i d i v i d e s a g a i n , one o f the daughter n u c l e i i n t o the bud, the o t h e r remaining  As  t h a t two  the chambers sometimes anastamose v i a a b r i d g e which by-  passes a septum. branch.  The b r i d g e then extends a p i c a l l y to form a  The two u n d i v i d e d n u c l e i o f the j o i n e d c e l l s pass i n t o  the branch t o i n i t i a t e a d i k a r y o n . v i o u s l y observed of  pass-  i n the parent c e l l .  an a l t e r n a t i v e to s p o r i d i a l p r o d u c t i o n Wang observed of  The f i r s t  These events which were p r e -  by Wang and which c o n s t i t u t e the major p o r t i o n  t h i s t h e s i s are summarized p h o t o g r a p h i c a l l y i n the i n t r o -  d u c t o r y F i g u r e s l a - o and 2 - l i .  I n a d d i t i o n to h e r o b s e r v a t i o n s  on the nucleus Wang a l s o s t u d i e d the "cytome" which i s now r e f e r r e d to as the m i t o c h o n d r i a , the "ergastome" c o n s i s t i n g o f  6 osmiophilic l i p i d bodies, and formation of vacuoles i n the germinating spore.  V a r i a t i o n s on the pattern of teliospore  germination and basidlospore production i n the smut fungi are described i n The Biology and Control of the Smut Fungi by Fischer and Holton  (1957).  At present very l i t t l e u l t r a s t r u c t u r a l information i s available pertaining to the heterobasidiomycetes i n general, and to the U s t i l a g i n a l e s i n p a r t i c u l a r . used scanning electron microscopy  Several workers have  ( H i l l e and Brandos,  or surface r e p l i c a techniques (Khanna et a l . , 1966) serve the surface features of smut t e l i o s p o r e s , Vaissalo  (I96I4.) made  1956)  to ob-  Kukkonen and  a very preliminary attempt to investigate  teliospore formation i n Anthracoldea aapersa,  Freeze-etching  has revealed some of the f i n e s t r u c t u r a l features of the r e s t i n g teliospores of T i l l e t i a contraversa (Hess and Weber,  1970)  and T i l l e t i a caries (Allen et a l . , 1971), and of the s p o r i d i a o  f  Patilago hordei (Hlgham, personal communication).  The cy-  tology of mycelial mutant of Ustilago hordei has been studied i n some d e t a i l by Stein (1970).  F u l l e r t o n (1970) has i n v e s t i g a -  ted several aspects of the i n t r a c e l l u l a r hyphae of eleven species of smut i n t h e i r corresponding hosts.  Among other hetero-  basidiomycetes studies include those on b a s l d i a l development i n the tremellaceous fungus E x i d i a nucleata (Wells, 196i|.a and 1964b), budding i n Tremella mesenterica (Bandoni and Bisalputra, 1971)* and those on uredospore germination (Ehrlich and E h r l i c h , 1969;  Manocha and Shaw, 1967;  Sussman et a l , , 1969* Williams  and Leglngham, 1962j.) and various aspects of the host-parasite  7 r e l a t i o n s h i p among rusts (Bracker, 1967; E h r l i c h and E h r l i c h ,  1963;  Van Dyke and Hooker,  1969)*  Some information i s also  available concerning the Rhodotorulas  (Marchant and Smith,  1967) which have now been r e c l a s s i f i e d as heterobasidiomycetes (Banno,  1967).  by Bracker  Fungal u l t r a s t r u c t u r e has recently been reviewed  (1967);  i n many respects the cytology o f the hetero-  basidiomycetes i s very s i m i l a r to that o f other fungi. The l i t e r a t u r e pertaining to the metabolism  (Allen, 1965)  and cytology (Bracker, 1967) o f fungal spores i s now quite v o l uminous.  Excellent reviews of many aspects o f quiescent and  germinating fungus spores are to be found i n Spores:  Their  Dormancy and Germination (Sua a man and Halvorson, 1966), The Fungi. An Advanced Treatise (ed. Ainsworth, G.C. and Sussman, A, S.,  1965)*  and The Fungus Spore (ed. Madelin, 1966).  However,  even though c y t o l o g i c a l investigations have been extensive, almost a l l studies have considered only quiesoent spores and spores i n whloh germination has occurred.  Throughout t h i s study  the term "germination" w i l l be applied to the formation o f the metabasidium.  L i t t l e information i s available concerning  the very important events which occur i n the a c t i v a t i n g pregerminal spore and which lead up to the process o f germination itself.  An attempt i s made i n t h i s study to determine the  sequential morphological changes which take place i n the t e l i o spore and metabasidium before and a f t e r germination.  Such  studies give r i s e to c e r t a i n i n t e r e s t i n g conclusions concerning the changing numbers and d i s t r i b u t i o n o f organelles.  Parti-  cular attention has been paid to the nature and o r i g i n s o f the  INTRODUCTION.  PLATE 2  Figure  2.  The o n e - c e l l e d s t a t e as seen with phase o p t i c s i n a l i v i n g promycelium growing i n a t h i n l a y e r of comp l e t e b r o t h between a g l a s s s l i d e and a cover s l i p . The l a r g e d i p l o i d nucleus (dN) and a "spherosomel i k e body" i s i n d i c a t e d , c a . X 4,600.  Figure  3.  The t w o - c e l l e d s t a t e as seen w i t h phase o p t i c s i n a promycelium f i x e d i n 2$ g l u t a r a l d e h y d e i n 0.01 M cac o d y l a t e g u f f e r . The f i r s t septum (arrow) and one of the h a p l o i d n u c l e i (hN) are i n d i c a t e d . Note the condensed s t a t e of the n u c l e a r chromatin, c a . X 4,600.  Figure  1).. The f o u r - c e l l e d s t a t e as seen w i t h phase o p t i c s i n a l i v i n g promycelium grown as i n f i g u r e 2. Two of the three septa are v i s i b l e (arrows). A r e f r a c t i l e membrane complex (mc) i s a s s o c i a t e d w i t h the f i r s t septum. Three of the four h a p l o i d n u c l e i are obvious, c a . X lj.,600. Note: each s c a l e d i v i s i o n r e p r e s e n t s one micron, ( i . e . t o t a l s c a l e l e n g t h i s 10 m i c r o n s ) .  1  • • • •  1  1  '  1  •  1  8  endoplasmic r e t i c u l u m and the vacuoles tem,  and t o the p o s s i b l e f u n c t i o n s of the  bodies"  (S) which are r e a d i l y observable  sys-  "spherosome-like i n the l i g h t miorosoope  2).  (Pig.  S e v e r a l e x c e l l e n t reviews are a v a i l a b l e (Burnett, 1968; and  i n the germinating  B a k e r s p i e g e l , 1965).  is difficult  of meiosis and m i t o s i s i n f u n g i O l i v e , 1953;  The  and with the e x c e p t i o n of y e a s t n u c l e i , those t o observe and  i n i t i a l problem i s the s m a l l s i z e of the  (1.5-2.5 u)  Robinow  At best the study of f u n g a l n u c l e i  the smuts are perhaps the most d i f f i c u l t pret.  O l i v e , 1965;  •  of  inter-  nuclei  T h i s i s compounded by the p r a o t i o a l l y i n d e s t r u c -  t i b l e f u n g a l w a l l , and the g e n e r a l unresponsiveness  of the  p r o t o p l a s t to the u s u a l methods of f i x i n g and s t a i n i n g . more, the e a r l y m e i o t i c stages occur w i t h i n the  Further*  forming  t h i c k - w a l l e d t e l i o s p o r e which i s u s u a l l y embedded i n dead host t i s s u e - m a t e r i a l whioh i s impossible to squash, and  difficult  to s e c t i o n . Chromosomes and s p i n d l e s were f i r s t d e s c r i b e d i n the of  a s p e c i e s of U s t l l a g o by Harper ;(1898).  subsequently  confirmed  Rawitscher  nuclei  (1922)  the presenoe of a s p i n d l e i n d i v i d i n g  n u o l e i and d e s c r i b e d i t s i n t r a n u l e a r n a t u r e .  The t e l i o s p o r e  i s the s e x u a l spore, and presumably r e p r e s e n t s the o n l y d i p l o i d c e l l i n the l i f e c y c l e . oocurs  I t i s commonly assumed t h a t m e i o s i s  d u r i n g the f i r s t two  the t e l i o s p o r e germinates,  (or t h r e e ) n u c l e a r d i v i s i o n s  after  but the d e t a i l s of these n u c l e a r d i -  v i s i o n s are so u n c l e a r t h a t there i s s t i l l some doubt as to which r e p r e s e n t s the r e d u c t i o n division,(Sampson,  1939;  Hirsohhorn,  9  1945).  Wang (1934) and Sampson (1939) suggest t h a t t h e f i r s t  division i s reductional. and  T h i s has been supported g e n e t i c a l l y  i s now commonly assumed t o be the case; but H i r s c h h o r n  ( 1 9 4 5 ) , Das ( 1 9 4 9 ) , and even F i s c h e r and H o l t o n ( 1 9 5 7 )  support  the i d e a t h a t the second d i v i s i o n i s the r e d u c t i o n a l one. Kharbush ( 1 9 2 7 ) , Wang (1934), and H i r s c h h o r n  (1945)  collectively  s t u d i e d f i v e d i f f e r e n t genera i n c l u d i n g e l e v e n s p e c i e s o f U s t i lago.  From t h e i r chromosome counts a t m e i o s i s and m i t o s i s  concluded t h a t n = 2 .  With the e x c e p t i o n  they  o f Harper ( 1 8 9 8 ) and  Dickson ( 1 9 3 1 ) t h i s count has been comfirmed by most workers (Rawitscher, 1 9 2 2 ; Wang, 1943J  D a  s , 1 9 4 9 ; Person and Wighton,  1964). To the best o f the author's knowledge t h i s i s t h e f i r s t d e t a i l e d u l t r a s t r u c t u r a l study o f n u c l e a r a c t i v i t y i n a smut fungus.  I n v e s t i g a t i o n o f the m e t a b a s i d i a l  cell  and n u c l e a r  d i v i s i o n s i n d i c a t e s that c e r t a i n i n t e r e s t i n g r e l a t i o n s h i p s e x i s t between t h e two.  S p e c i a l c o n s i d e r a t i o n has been g i v e n t o  the problems o f chromosome number and mechanism o f n u c l e a r d i v i s i o n i n U s t i l a g o h o r d e i , and t h e data have been  reanalysed  i n the l i g h t o f the e l e c t r o n microscope o b s e r v a t i o n s  and the  c u r r e n t trends  i n fungal  cytology.  BIBLIOGRAPHY Ainsworth, G.G. and Sussman, A,S, (ed.) 1 9 6 5 . Advanced T r e a t i s e . I I . Academic P r e s s ,  The F u n g i ; An N.Y.  A l l e n , J.V., Hess, W.M., and Weber, D.J, 1 9 7 1 . Ultrastruct u r a l i n v e s t i g a t i o n s o f dormant T i l l e t l a c a r i e s t e l i o s p o r e s , Mycologia 6 ^ : l i | i i - l 5 6 , A l l e n , P.J, fungi.  1965, M e t a b o l i c aspects o f spore g e r m i n a t i o n i n Ann, Rev. P h y t o p a t h o l . 2L 313-311-2 • :  Bandoni, R.J, and B i s a l p u t r a , A,A, 1 9 7 1 . Budding and f i n e s t r u c t u r e o f T r e m e l l a mesenterioa h a p l o n t s . Can. J . Bot, 27-30. Banno, I . 1 9 6 7 , S t u d i e s on the s e x u a l i t y o f Rhodotorula. Gen. A p p l . M i c r o b i o l , 1^: 1 6 7 - 1 9 6 , Bracker, C.E, 1 9 6 7 . topathol. £ :  Ultrastructure of fungi.  314.3-37^.  Ann,  Rev,  J. Phy-  B u r n e t t , J.H, 1 9 6 8 . Nuclear d i v i s i o n . Fundamentals o f Mycol o g y , Edward A r n o l d ( P u b l i s h e r s ) L t d , London, pp, 3 o 0 Chrlstensen, J , J , 1963, Monograph # 2 , Am,  Corn smut caused by U s t i l a g o maydis P a t h o l , Soc,  Das, MIC, 19U-9. Morphology and c y t o l o g y o f Entyloma m i c r o sporum (Unger) Schroet, and U r o o y s t i s anemones (Pers.) Wint, on Ranunculus repens L~. Ind, P h y t o p a t h o l , 2,: 1 0 8 127. Day^ sAijW. , and Jones, J.E. 1 9 6 9 . Sexual and parasexual anal y s i s o f U s t i l a g o v i o l a c e a e . Genet, Res, 1J±.: 1 9 5 - 2 2 1 . Dickson, S, o f the effect tion,  1931. Experiments on the p h y s i o l o g y and g e n e t i c s smut f u n g i . C u l t u r a l c h a r a c t e r s . P t . I I , The o f c e r t a i n e x t e r n a l c o n d i t i o n s on t h e i r segregaProc. Roy, Soc, B, 1 0 8 : 3 9 5 - 4 3 2 ,  Dinoor, A,, and Person, C, 1969. Genetic complementation l n U s t i l a g o h o r d e l . Can, J , Bot, bJL: 9-H*-, E h r l i c h , H.G. and E h r l i c h , M.A. 1 9 6 3 , E l e c t r o n microscopy o f the h o s t - p a r a s i t e r e l a t i o n s h i p s i n stem r u s t o f wheat. Am. J . Bot. £ 0 : 1 2 3 - 1 3 0 . E h r l i c h , M.A, and E h r l i c h , H.G, 1969. Uredospore development i n P u c c i n i a g r a m i n i s . Can, J , Bot, lj/7_: 2 0 6 l - 2 0 6 l j . . F i s c h e r , G.W,, and H o l t o n , C.S, 1 9 5 7 . B i o l o g y and C o n t r o l o f the Smut F u n g i . The Ronald Press Co., N.Y,  11  1942* Inheritance of pathogenicity of Melampsora Phytopathology 3 2 : 653 - 6 6 9 .  F l o r , H.H. lini.  F l o r , H.H. 1954* Host-parasite i n t e r a c t i o n i n f l a x rust - i t s enetics and other implications. Phytopathology h 5 : 680-  g8 5 .  '  '  '  .  F u l l e r t o n , R.A. 1 9 7 0 . An electron microscope study of the i n t r a c e l l u l a r hyphae of some smut fungi ( U s t i l a g i n a l e s ) . Aust. J . Bot. l B : 2 8 5 - 2 9 2 . Halisky, P.M. 1 9 6 5 * Physiologic s p e c i a l i z a t i o n and genetics of the smut f u n g i . I I I . Bot. Rev. 3 1 : 1 1 4 - 1 5 0 . Harper, R.A. 1 8 9 6 . Nuclear phenomena i n the smuts. Wise. Acad. S c i . Arts L e t t . 5 : 4 7 6 - 4 9 8 .  Trans.  Hawker, L.E. 1 9 6 6 . Germination: morphological and anatomical changes. The Fungus Spore, ed. Madelin, M.F. Butterwprtbs, London• pp. l 5 l - i 6 l • Hess, W»M., and Weber, D.J. 1 9 7 0 . Ultrastructure of T i l l e t i a contra v e r s a t e l i o s p o r e s as revelled by f reeze-etcning. Am. J . Bot. 5 7 : 745 (Abstr.). H i l l e , M. and Brandes J . 1 9 5 6 . Electronenmlkroskopische untersuchung der sporenoberflache einiger Us111ago-Arten. Phytopathol. Z. 2 9 : 104-109. 1945.  Hirschhorn, E. 235.  Holliday, R. Res.  2:  196l. 204^230.  Several smut fungi.  Mycologia 3 7 :  The genetics of Ustilago maydis.  217-  Genet.  Holliday, R. 1 9 6 4 . The induction of mitotic, recombination by mitomycin C i n Ustilago and Saccharomyces. Genetics, Princeton 5 0 : 3 ^ 3 - 3 3 5 . Hood, C H . 1 9 6 8 . U.V. induced l e t h a l i t y and mutation i n synchronized cultures of Ustilago hordei s p o r i d i a . Mut. Res. 6 : 391-400.  '  Khanna, A., Payak, M.M., and Mahta, S.C. 1 9 6 6 . Teliospore morphology of some smut f u n g i . I Electron microscopy. Mycologia 5 8 : 5 6 2 - ^ 6 9 . Kharbueb, S.S. 1927* Contribution a 1'etude des phenomenes sexuels chez l e s Ustilaginee's. Ann. S c i . Mat. Bot. 9 : 285-297.  ""  Kozar, F. 1969. M i t o t i c recombination i n biochemical mutants of Ustilago hordei. Can. J . Genet. C y t o l . 1 1 : 9 6 1 - 9 6 6 .  12  Kukkonen, I * and V a l s s a l o , T . 196IL. An e l e o t r o n mioroaoope study on spore f o r m a t i o n i n a smut. Ann, Bot* Penn. 1 ;  236-249.  "  Madelin, M.P, 1966, The Fungus Spore, C o l s t o n Papers No, B u t t e r w o r t h s , London.  18,  Manocha, M.S., and Shaw. 1967. E l e c t r o n m i c r o s c o p y o f uredospores o f Melampsora l i n i and o f r u s t - i n f e c t e d f l a x . Can.  J . B o t . 2j5: 1575-1582.  Merchant, R., and Smith, D . 6 . 1967* W a l l s t r u c t u r e and bud f o r m a t i o n i n Rbodotorula g l u t i n i s . A r c h . M i k r o b i o l . 58* 248-256. — — 3  O l i v e , L.S. lei.  1953*  Bot.  The s t r u c t u r e and behaviour o f fungus nuc^ 3 9 - 5 8 6 .  19:  Rev.  O l i v e , L.S. 1965. N u c l e a r behaviour d u r i n g m e i o s i s . The F u n g i I The Fungal C o l l . e d . Ainsworth, G.C. and~Suss-~ man, A . s . Academic P r e s s , N.Y. pp. 143-161. Person^ C O . 1959. s i t e systems.  Gene-for-gene r e l a t i o n s h i p s i n h o s t - p a r a Can. J . B o t . 373 &101-1130.  Person, C and Wighton, D. 1964. The chromosomes o f U s t i l a g o . Can. J . B o t . 6_: 2 4 2 ( A b s t r . ) . Rawitacner, F.  II.  1922.  B e i t r a g e z u r k e n n t n i s des U s t i l a g i n e e n .  Z e i t . f u r Bot.  273-296.  Robinow, C F . and B a k e r s p i g e l , A. 1965. Somatic n u c l e i and forms o f m i t o s i s i n f u n g i . The Fungi I The Fungal C e l l , ed. Ainsworth, G.C. and Sussman, A . S . Academic p r e s s ,  N.Y.  pp. 119-139.  Sampson, K. 1939. L i f e c y c l e s o f smut f u n g i . M y c o l . Soc. £2: 1 - 2 3 .  Trans. B r i t .  S t e i n , C,W. 197p. An e l e c t r o n microscope study o f a m y c e l i a l mutant o f U s t i l a g o h o r d e i ( P e r s i ) • M.Sc. The s i s , U n i v e r s i t y o f B r i t i s n cjoiumDiaT B r i t i s h Columbia, Canada. Sussman, A.S. and Halvorson, H.Oi 1966. Spores: T h e i r Dogga mancy and Germination. Harper and Row, P u b l i s h e r s , 1 1 Sussman^'A*S*, Lowry, R.J., Durkee, T.L. and Maheshware R. 19o9. U l t r a s t r u c t u r a l s t u d i e s o f cold-dormant and g e r m i n a t i n g uredospores o f P u e c i n i a graminls v a r . t r i t i c i .  Can. J . B o t . 1^7} 2073-2077^  Van Dyke, C G . and Hooker, A.L. 1969. U l t r a s t r u c t u r e of host and p a r a s i t e i n i n t e r a c t i o n s o f Zea maya w i t h P u e c i n i a so sogfeli. Phytopathology 59: 19itfZT%'GT~'  13  Wang, C.S, 194-3. S t u d i e s on t h e c y t o l o g y o f U s t i l a g o c r a m e r i . Phytopathology j£: 1 1 2 2 - 1 1 3 3 . Wang, D.T. 1934* C o n t r i b u t i o n a l»etude des U s t i l a g i n e e s ( C y t o l o g i c du p a r a s i t e e t p a t h o l o g i e de l a c e l l u l e h o t e ) . Le B o t a n i s t e 26: 539-670. W e l l s , K. 1964a. The b a s i d i a o f E x l d l a n u c l e a t a I . s t r u c t u r e , Mycologia £ 6 : 3 2 7 - 3 4 1 . W e l l s , K.  ment.  1964b.  Ultra-  The b a s i d i a o f E x i d i a n u c l e a t a I I . Develop-  Am. J . Bot. £ 1 : 360-370.  W i l l i a m s , P.G. and Ledingham, G.A, 1964* F i n e s t r u c t u r e o f wheat stem r u s t uredospores. Can. J . B o t . l±2: l 5 0 3 - l f > 0 8 .  PART I P r e g e r m i n a t i o n Development i n H y d r a t i n g T e l i o s p o r e s  TABLE OP CONTENTS Page ABSTRACT  15  .  15  INTRODUCTION MATERIALS AND METHODS  16  .  C u l t u r e s and C u l t u r i n g  16  Sample T a k i n g  ••  P r e p a r a t i o n f o r E l e c t r o n Microscopy  18  •  20  OBSERVATIONS P r e p a r a t o r y Methods R e s t i n g Spores •  20  • •  •  •••  Stage One  27  • F u l l y A c t i v a t e d Spore  28 30  DISCUSSION  30  The Spore W a l l The P r o t o p l a s t CONCLUSION BIBLIOGRAPHY  22 26  Stage Two Stage Threes  17  ••  •••  32 IPk-3  15  PART I Pregermination Development i n H y d r a t i n g T e l l o a p o r e s  ABSTRACT An attempt has been made t o determine  the sequence of c y t o -  l o g i c a l changes o c c u r r i n g d u r i n g p r e g e r m i n a t i o n i m b i b i t i o n of Ustilago hordel teliospores.  The f i r s t  an i n c r e a s e i n the amount of endoplasmic  d e t e c t a b l e changes are r e t i c u l u m , the d e v e l o p -  ment of patches o f " f l o c c u l e n t cytoplasm", and a p e c u l i a r amoeb o i d a c t i v i t y o f the n u c l e u s .  S h o r t l y a f t e r , vaouoles b e g i n t o  appear i n the c e n t r a l r e g i o n s of the p r o t o p l a s t . d r i a i n c r e a s e i n s i z e throughout  The  mitochon-  the p r e g e r m i n a t i o n p e r i o d but  show no evidence of d i v i s i o n u n t i l s h o r t l y b e f o r e g e r m i n a t i o n . L i p i d does not seem t o decrease s u b s t a n t i a l l y .  Hypotheses are  put forward t o account f o r the o r g i n of the endoplasmic  reti-  culum and of "primary v a c u o l e s " ,  INTRODUCTION In  a r e c e n t review of f u n g a l u l t r a s t r u c t u r e , Bracker (196?)  s t a t e s the need f o r f u r t h e r i n f o r m a t i o n c o n c e r n i n g the c y t o l o g y • »  of  spore g e r m i n a t i o n , and p a r t i c u l a r l y the need f o r s t u d i e s i n  m a t e r i a l which i s amenable t o s p e c i f i c s t r u c t u r e - f u n c t i o n analysis.  With t h i s i n mind an attempt has been made t o study, l n  some d e t a i l , the s e q u e n t i a l c y t o l o g i c a l changes o c c u r r i n g during  the p r e - g e r m i n a t i o n development of i m b i b i n g t e l i o s p o r e s of  the smut fungus, U s t l l a g o hordei»  16  Hopefully more information w i l l be forthcoming.  Most f i n e  struoture studies of the germination of fungus spores have i n cluded only observations of spores i n the dormant or quiescent state, and spores during and after germination.  Very  little  attention has been paid t o the important events which lead up to germination i t s e l f  (Corfman, 1966; Hyde and Walkinshaw,  1966).  The teliosporea of Ustilago hordei belong t o the category of r e s t i n g spores which A l l e n ( 1 9 6 5 ) r e f e r s to as "environmental", that i s , they w i l l resume development immediately when the environment  permits.  In t h i s case only the presence  of free water i s required f o r germinations to proceed. teliospore i s also the sexual spore.  The  I t contains the only  d i p l o i d nucleus i n the entire l i f e cycle (Intro. Diagram I ) . At present very l i t t l e i s known about the u l t r a s t r u c t u r e of spores among the U s t i l a g i n a l e s .  Kukkonen and Vaissalo  (1961+) have studied teliospore formation i n Anthracoldea asperaa, and several workers have investiggted the surface f e a tures of mature spores ( H i l l e and Brandes, 1956).  Recently,  freeze-etching has been used t o study the teliospores of T i l l e t i a contraversa (Hess and Weber, 1970) and T i l l e t i a caries (Allen et a l . , 1971).  MATERIALS AND METHODS CULTURES AND CULTURING Ustllago horde! (Pers.) Lagerh. - A l l teliospores used i n these studies were the product of crosses between the two  17  wild-type haploid mating strains I j J and E3, i s o l a t e d by Thomas A l l were produced i n the f i e l d .  (196U)*  None of the samples  had been stored longer than twelve months, C u l t u r i n g, - I t i s known that when spore samples are germinated on complete medium a greater amount of synchrony oan be obtained than on d i s t i l l e d water (Bech-Hansen, unpublished). Hence, a l l teliospore samples were hydrated i n a shake culture consisting of a modified oomplete Vogel's broth prepared according t o Hood (1966) (see also Appendix A ) ,  The temperature  was maintained at 22° G, SAMPLE TAKING These studies show that the greater the i n i t i a l spore concentration, the f a s t e r i s the germination r a t e .  The i n i t i a l  spore concentrations employed were not c o n t r o l l e d .  During the  e a r l y studies the f i r s t promycelia were detected at approximately f i v e hours a f t e r the i n i t i a t i o n of hydration. In subsequent experiments where greater i n i t i a l concentrations were used, the f i r s t signs of germination began after two and a h a l f hours.  However, the sequence of stages seen i n a l l cases ap-  peared t o be i n d e n t i c a l .  Only the period of time spent i n each  stage was longer i n the former case. Each of the events described i s , therefore, a composite of a number of observations, each observation being made l n the context of the Individual experiment• During the early studies pre-germination samples were taken at o, h, 2h» and £ hours; i n l a t e r studies at 0, h$ 1, and 2 hours.  Each sample consisted of 3 m i l l i t r e s of spore suspen-  18  sion, PREPARATION POR ELECTRON MICROSCOPY (also see Appendix B) During the f i r s t , steps i n the preparation procedure the spores were recovered by centrifugation•  The material was f i x -  ed f a r electron microscopy, at room temperature, according t o one of the following procedures: 1.  1.5/6 KMnOj^ (aqueous) f o r 20 minutes.  2.  2.0$ glutaraldehyde l n 0.01 M cacodylate buffer at pH 7.0 or 7-2 f o r 12 t o 16 hours (the longer time was required by the r e s t i n g spores), followed by washing i n buffer and p o s t - f i x a t i o n i n 1.0 or 2.0$ OsO^ l n the same buffer f o r 3-3% hours.  The material was then washed i n e i t h e r d i s t i l l e d water  (pro-  cedure 1) or buffer (procedure 2 ) . Glutataldehyde-osmium  fix-  ed spores were subsequently stained i n 0.5$ aqueous uranyl acetate f o r 2-4 hours.  A f t e r p e l l e t i n g i n 2% water-agar the  spares were dyhydrated through a standard ethanol s e r i e s .  The  material was then either passed through a standard propylene oxide series and embedded i n Epon 812, or was d i r e c t l y embedded i n Spurr's p l a s t i c (Spurr, 1969).  The three basic preparatory  methods are outlined i n Table I . Sections were cut our on a S o r v a i l Porter-Blum MT-2 u l t r a microtome using glass or diamond knives, and were post-stained i n a saturated solution of uranyl acetate i n 70$ ethanol f o l l o w ed by lead c i t r a t e (Reynolds, 1963).  A l l sections were viewed  with an H i t a c h i HS-75 microscope operating at 5 0 KV.  TABLE I Summary of E l e c t r o n Microscope Technique Method  Fixation  Post  Fixation  l . f $ KMnOlj. aqueous B  Dehydration  Embedding  Ethanol-propylene oxide  Epon  2.0$ b u f f e r e d g l u teraldehyde  1-2$ b u f f e r e d OsC^  Ethanol-propylene oxide  Epon  2.0%  1-2$ b u f f e r e d OsO^  Ethanol  Spurr's  buffered g l u teraldehyde  H  20  LIGHT MICROSCOPY (see a l s o Appendix Thick sections  (0*25  C)  u) were made of r e s t i n g spores and  spores a f t e r one hour of h y d r a t i o n , which had been prepared a c c o r d i n g t o e l e c t r o n microscope method A s t a i n e d w i t h 1.0$  T o l u i d i n e b l u e i n 1,0$  microscope, equipped w i t h 51+6 mn all  (KMnO^). eorax.  They were  A Z e i s s photo-  i n t e r f e r e n c e f i l t e r , was used i n  studies,  OBSERVATIONS PREPARATORY METHODS Methods A, B, and C each p r o v i d e a s l i g h t l y d i f f e r e n t image of the i n t e r n a l contents of ungerminated  spores.  No doubt, each  r e f l e c t s some of the p r o p e r t i e s of the l i v i n g organism.  This  i s a s t r o n g argument f o r the n e c e s s i t y of employing more than one technique b e f o r e attempting t o V i s u a l i z e what the l i v i n g s t a t e of the c e l l might have been. the  U n l e s s otherwise s t a t e d a l l  events here d e s c r i b e d have been seen w i t h a l l t h r e e p r o c e -  dures . Method A appears t o produce the h i g h e s t l e v e l of a r t e f a c t . As has o f t e n been n o t e d , permanganate causes b o t h n u c l e i c  acid  and l i p i d t o l e a c h out; the chromatin, n u c l e o l u s , r i b s o m e s , and l i p i d b o d i e s b e i n g c o n s p i c u o u s l y absent. c y t o l o g i c a l contents a l s o o c c u r s . of  D i s t o r t i o n of the  The p r o t o p l a s t and many  the o r g a n e l l e s appear t o s h r i n k i n t o t a l s i z e ; the  els ter nal  spaces of the ER  intre-  and n u c l e a r envelope, and the  inter-  membranous space of the m i t o c h o n d r i a are u s u a l l y expanded. doubt, the grotesque shapes of the m i t o c h o n d r i a seen i n the  No  21  dormant spores i n F i g u r e 1 are a r t e f a o t s caused by KMn%  fixa-  tion* Such d i s t o r t i o n s never occur i n glutaraldehyde-osmium f i x ed m a t e r i a l *  N e v e r t h e l e s s , permanganate f i x a t i o n  consistently  g i v e s good membrane c o n t r a s t and p r o v i d e s s u f f i c i e n t information*  morphological  I t a l s o a l l o w s r a p i d and even p e n e t r a t i o n o f the  embedding p l a s t i c *  As has been noted by a number o f  authors  (Sussman, e t a l * , 1969)* t h i s l a t t e r p r o p e r t y i s o f c o n s i d e r a b l e importance i n d e a l i n g w i t h t h i c k - w a l l e d spores*  Because o f these  a s p e c t s , much o f the i n f o r m a t i o n c o n t a i n e d i n t h i s r e p o r t has come from t i s s u e prepared a c c o r d i n g to method A* Glutaraldehyde-osmium f i x a t i o n tends t o p r e s e r v e the  cells  i n a more n a t u r a l s t a t e , b u t the a p p l i c a t i o n i s more p r o b l e m a t i c * Both pH and o s m o l a r i t y appear t o be important B, Table I ) * 312,  factors  (Appendix  Method B, i n which m a t e r i a l i s embedded i n Epon  appears t o r e s u l t i n the l e a c h i n g out o f some p r o t e i n -  aceous m a t e r i a l , c a u s i n g an i n c r e a s e i n membrane v i s i b i l i t y * The major d i f f i c u l t y w i t h t h i s method as suggested by Sussman e t a l * (1969), i s improper p e n e t r a t i o n o f the p l a s t i c 6 and 1 5 ) .  Low  v i s c o s i t y Spurr's embedding p l a s t i c r e t a i n s  g r e a t e r p r o p o r t i o n o f the ground substance t i s s u e evenly*  (Figs*  and i n f i l t r a t e s  a the  However, t h e r e i s a g e n e r a l tendency towards  n e g a t i v e s t a i n i n g o f the membranes*  T h i s problem i s unique t o  ungerminated spores and does not occur once a promycelium has been formed*  Whether t h i s e f f e c t i s caused by p h y s i o l o g i c a l i n -  a c t i v i t y o f the membranes o r by masking o f the membranes by dense cytoplasm known*  (Walkinshaw e t a l * , 1967;  Weiss, 1963)  i s un-  22  RESTING SPORES Spore W a l l (W).  - Considering  t h a t a r e s t i n g spore  has  a t o t a l diameter o f 8 - 1 0 u, the spore w a l l , which i s approxi m a t e l y 1 u t h i c k (Ranges 0 . 6 3  -  1 . 2 i j . u ) , i s i t s most p r o -  minent c o n s t i t u e n t .  I t i s composed o f t h r e e d i s t i n c t  ( P i g s . 1 to 8 ) .  outermost l a y e r (Wl), which i s the t h i n -  nest  The  layers  (Average w i d t h = 80 rau), i s o f medium e l e c t r o n d e n s i t y  and  o f constant  w i d t h around the e n t i r e s u r f a c e .  nal  f a c e i s s l i g h t l y i r r e g u l a r but  pattern of organization.  I t s exter-  there i s no c h a r a c t e r i s t i c  T h i s l a y e r does not  v i s i b l e a f t e r p r e p a r a t i o n w i t h method B.  seem to  be  In F i g u r e 8 ,  patches  o f amorphous e l e c t r o n dense m a t e r i a l can be seen a d h e r i n g to the o u t e r The  surface. middle l a y e r (W2)  e l e c t r o n dense and  o f the spore w a l l i s  s t r u c t u r a l l y amorphous.  l a y e r i s v e r y v a r i a b l e ; on one  The  exceedingly thickness of  s i d e o f the spore i t may  l i t t l e wider than the outermost l a y e r (Range: 0 . 1 2 w h i l e on the o p p o s i t e  s i d e i t may  -  U s u a l l y the boundary between the middle l a y e r and inner-most l a y e r (W3)  i s abrupt.  dense o f the t h r e e , and  be  0.16  be as t h i c k as 0 . 6 2  -  a l s o the most s t r u c t u r e d .  After  mium f i x a t i o n , a s t a i n i n g g r a d i e n t a p p a r e n t l y  the  I t consists  O.33  exists i n this  are most d i s t i n c t w i t h methods A and  to 0 . 5 3  u thick.  spore  gluteraldehyde-os-  l a y e r and the d e n s i t y o f f i b e r s decreases towards the  is  u.  T h i s t h i r d l a y e r i s the l e a s t  embedded i n a homogeneous m a t r i x .  These f i b r i l s  u),  0.70  o f f i b r i l s o r i e n t e d p a r a l l e l to the circumference o f the and  this  C.  protoplast. This layer  I t s i n t e r n a l s u r f a c e i s uneven  and r i d g e s p e r i o d i c a l l y p r o j e c t f o r d i s t a n c e s o f approximately  23  60 mu i n t o the p r o t o p l a s t  ( F i g s . $b and  17).  Plasma Membrane (PM).- When t e l i o s p o r e s are f i x e d i n KMnOj^ the p r o t o p l a s t p u l l s away from the spore w a l l and the plasma membrane i s c l e a r l y v i s i b l e  ( F i g . l a ) , but w i t h g l u t -  araldehyde and osmium, the plasmalemma i s r a r e l y seen a t t h i s stage.  The average u n i t membrane t h i c k n e s s i s 121+ A . 0  The  plasmalemma i s r e l a t i v e l y smooth and the r e s u l t s w i t h methods B and C i n d i c a t e t h a t i n the l i v i n g c e l l i t i s p r o b a b l y c l o s e l y a p p l i e d to the spore w a l l except where "paramural b o d i e s " are present. I n the r e s t i n g spore, s m a l l q u a n t i t i e s o f amorphous materi a l are sometimes s i t u a t e d between the plasma membrane and the spore w a l l  ( F i g s . 1 , 1 1 , and 1 7 ) .  These simple "paramural bo-  d i e s " do not appear t o be membrane bound and show none o f the complex t u b u l a r o r v e s c i c u l a r forms commonly a s s o c i a t e d w i t h such s t r u c t u r e s  (Marchant and Rabards, 1 9 6 8 ) .  In a d d i t i o n ,  where the plasma membrane has p u l l e d away from the w a l l , t i n c t f i b e r s w i t h an average diameter o f 22 A  0  dis-  are v i s i b l e  tween the p r o t o p l a s t and the spore w a l l ( F i g s , l a , l b , and Endoplasmic R e t i c u l u m (ER) and Ribosomes. spores, the ER i s s p a r s e .  be15).  — I n resting  Usually a s e r i e s of short  cisternal  elements l i e j u s t beneath, and p a r a l l e l w i t h , the plasma membrane, w h i l e i n the c e n t r a l r e g i o n s a few s h o r t fragments o f i n d e t e r m i n a t e morphology  are s c a t t e r e d a t random.  Although  the amount o f d a t a on glutaraldehyde-osmium f i x e d t i s s u e i s l i m i t e d , i n t h i s r e s p e c t , the ER membranes appear to be smooth.  2k  The ribosomes o f U. h o r d e i measure 100 t o 15>0 A meter. packed.  0  i n dia-  I n the r e s t i n g p r o t o p l a s t these a r e v e r y d e n s e l y Most, i f n o t a l l , o f them appear to be f r e e .  Nucleus ( N ) . - D i p l o i d r e s t i n g n u c l e i  (dN) a r e p o s i t i o n e d  c e n t r a l l y i n t h e spore and appear t o be s p h e r i c a l t o o v o i d i n shape.  O f t e n the n u c l e u s appears t o be beaked on one s i d e .  The diameter ranges from 2.0 t o 2.7 u ( F i g s . 1 and 5 ) . s i o n a l l y a r e s t i n g spore has two h a p o i d n u c l e i  Occa-  (hN), each  h a v i n g a diameter r a n g i n g from 1.5 t o 1.8 u ( F i g . 6 ) .  Light  microscope o b s e r v a t i o n s on r e s t i n g spores and spores a f t e r One hour o f h y d r a t i o n i n d i c a t e t h a t n u c l e a r f u s i o n can o c c u r d u r i n g h y d r a t i o n ( F i g s . 2, 3 and !{.), b u t no I n f o r m a t i o n i s a v a i l a b l e at the u l t r a s t r u c t u r e l e v e l . In t h e r e s t i n g spore the n u c l e a r envelope (NE) tends to be p o o r l y d e f i n e d .  T h i s l a c k o f d e f i n i t i o n may be due t o i n -  adequate f i x a t i o n , o r t o a r e a l d i f f e r e n c e i n the p h y s i o l o g i c a l s t a t e o f t h e membrane, o r t o b o t h .  Only a h i n t o f t h e  f a m i l i a r double membrane i s observed l n F i g u r e 5 and few nuclear  pores can be seen.  I n glutaraldehyde-osmium f i x e d  t i s s u e the n u c l e o l u s  (Nu), appears t o be u n i f o r m l y g r a n u l a r  and e l e c t r o n dense.  In agreement w i t h the r e s u l t s o b t a i n e d  from hyphal n u c l e i  ( S t e i n , 1970) t h e n u c l e o l a r diameter mea-  sures approximately 1 u (Range 0.93 - 1»19 u) I n b o t h hapl o i d and d i p l o i d n u c l e i . M i t o c h o n d r i a (M). - The m i t o c h o n d r i a g e n e r a l l y a r e l o c a t e d randomly i n t h e r e s t i n g t e l i o s p o r e a l t h o u g h t h e r e may be a tendency t o aggregate i n t h e more c e n t r a l r e g i o n s ( F i g .  5)»  25  At  t h i s stage t h e i r c r o s s - s e c t i o n a l shape i s ovoid t o round.  No evidence has been found f o r the presence of elongate m i t o chondria.  As was  p r e v i o u s l y mentioned, the d i s t o r t e d  shapes  observed a f t e r permanaganate f i x a t i o n are b e l i e v e d t o be a r t e factual.  The average maximum l e n g t h o f the r e s t i n g  d r i a as determined 0.38  mitochon-  after,glutaraldehyde-osmium f i x a t i o n , i s  u (Range : 0.2° - 0.60  fi).  The c r i s t a e , which are  fairly  w e l l - d e v e l o p e d , are p l a t e - l i k e , and tend t o be arranged i n p a r a l l e l a r r a y s which may  or may not l i e i n the l o n g a x i s of  the m i t o c h o n d r i o n . L i p i d Bodies  ( L ) . - The r e s t i n g t e l i o s p o r e c o n t a i n s a  l a r g e number of l i p i d protoplast.  bodies randomly s c a t t e r e d throughout  the  A f t e r permanganate f i x a t i o n they appear as con-  spicuous e l e c t r o n t r a n s p a r e n t bodies which sometimes c o n t a i n t r a c e s of s e m i - e l e c t r o n dense m a t e r i a l ( P i g . l ) .  They are  bounded by an e l e c t r o n - d e n s e l i n e about liO A° t h i c k , which cannot be shown t o have u n i t membrane s t r u c t u r e even a t h i g h magnifications.  No analgous membrane-like s t r u c t u r e i s p r e -  sent a f t e r p r e p a r a t o r y methods B and C. osmium f i x a t i o n the l i p i d dense and homogeneous. s i z e from O.J.  t o 0.5  Spherosome-like semi-dense,  After glutaraldehyde-  bodies are u s u a l l y s l i g h t l y  electron  In the spore these bodies range i n  p. Bodies  (S).  A f t e r permanganate f i x a t i o n  membrane-bound b o d i e s are o f t e n observed .which  range i n s i z e from 0.1  t o 0.6  a s s o c i a t e d w i t h fragments  u (diameter).  of endoplasmic  They are u s u a l l y  reticulum.  Evidence  i s presented i n p a r t I I t h a t these b o d i e s are i d e n t i c a l w i t h  26  those membrane-bound bodies which c o n t a i n very e l e c t r o n - d e n s e ( P i g s . 5 and  7)«  spores these l a t t e r bodies range i n s i z e from 0.2 t o 1.9  u.  m a t e r i a l a f t e r glutaraldehyde-osmium In  fixation  STAGE ONE Approximately one-half t o one hour a f t e r the b e g i n n i n g of h y d r a t i o n the n u c l e u s of the t e l i o s p o r e undergoes a r a d i c a l change i n shape.  I t becomes l o b e d .  t i o n v a r i e s from a m i l d d i s t o r t i o n  The extent o f t h i s c o n d i ( P i g . 8)  t o an extreme  s t a t e i n which the nucleus i s deeply indented and sends put l o n g attenuated arms that bulge i n t o n u c l e a r l o b e s ( P i g . 7 ) » T h i s extreme s t a t e has o n l y been seen a f t e r KMn0j_^ f i x a t i o n ; however, the amount of data f o r glutaraldehyde-osmium  fixed  material i s limited. When the nucleus i s i n the lobed c o n d i t i o n l i p i d  bodies  appear t o become c l o s e l y a s s o c i a t e d w i t h the n u c l e a r envelope. F i g u r e 7 shows a l i p i d  body, w i t h i n a p r o t o p l a s m i c arm, exten-  d i n g deep i n t o a n u c l e a r c l e f t . are  At t h i s time l i p i d  bodies  a l s o o c c a s i o n a l l y seen i n c l o s e a s s o c i a t i o n w i t h the mem-  bren^-bound bodies c o n t a i n i n g e l e c t r o n dense m a t e r i a l ( S ) . In F i g u r e 8 a l i p i d  body appears t o be fused w i t h two such  o r g a n e l l e s ( P i g . 8,  arrow).  Another e a r l y c y t o l o g i c a l change found t o occur i n the a c t i v a t i n g t e l i o s p o r e i s an i n c r e a s e i n the amount of smooth ER i n the i n n e r r e g i o n s of the p r o t o p l a s t , p a r t i c u l a r l y s u r rounding and p a r a l l e l w i t h the n u c l e a r envelope.  The ER  appears t o be connected w i t h the n u c l e a r envelope a t some points  ( P i g . 7» arrow).  27  Shortly a f t e r hydration begins, well-developed, t r a n s p a r e n t zones develop i n the cytoplasm  electron  ( P i g s . 7 and  These zones, r e f e r r e d t o as " f l o c c u l e n t cytoplasm" r e l a t i v e l y l a r g e and appear t o have s t r u c t u r e .  8).  ( f ) , are  Occasionally,  a f t e r permanganate f i x a t i o n , l a r g e s e m i e l e o t r o n dense g r a n u l e s (Average diameter = 0.l8 u) are l o c a t e d i n , or around o f , the f l o c c u l e n t areas  ( P i g . 16).  the edges  S i m i l a r r e g i o n s have a l s o  been r e p o r t e d i n a g i n g hyphae of U. h o r d e i , but o n l y a f t e r glutaraldehyde-osmium STAGE  fixation  ( S t e i n , 1970)*  TWO  At approximately h a l f way  through the p r e g e r m i n a t i o n i n -  t e r v a l , complex who E l s of ER develop i n the cytoplasm ({Pig. 9 ) . A l l the i n f o r m a t i o n p e r t a i n i n g t o these whorls has been d e r i v e d from permanganate-fixed  m a t e r i a l ; the d e n s i t y of the p r o t o p l a s t  a f t e r glutaraldehyde-osmium  f i x a t i o n renders any o b s e r v a t i o n  of these membranes d i f f i c u l t .  They are c e n t r a l l y  located.  Whether t h e i r p r o x i m i t y t o the n u c l e u s i s f o r t u i t o u s , or whether the p r o x i m i t y r e f l e c t s some r e a l r e l a t i o n s h i p between the  two  i s not yet c l e a r . About -the same time, vacuoles w i t h f l o c c u l e n t appear i n the cytoplasm ( P i g s . 10 and by a unit,imembrane, ness of 96 A  0  11).  contents  They are bounded  the t o n o p l a s t , which has an average  (Range: 80-120 A ) . 0  thick-  A f t e r permanganate f i x a t i o n  they are very i r r e g u l a r i n shape and p r o j e c t outwards s h a r p l y , e x p e c i a l l y a t p o i n t s of c o n t a c t w i t h what appears ( P i g . 11, arrow).  t o be !.ER  In F i g u r e 10 s e v e r a l vacuoles are a p p a r e n t l y  i n t e r c o n n e c t e d and are p a r t i a l l y surrounded  by a complex* system  28  of  ER,  One  end o f these e a r l y v a c u o l a r systems always l i e s i n  the v i c i n i t y o f the nucleus i n the observed m a t e r i a l ( P i g s , 1 0 and  11). Spherosome-like  of  b o d i e s are o f t e n present i n the v i c i n i t y  d e v e l o p i n g v a c u o l a r complexes.  At t h i s stage they do not  always appear t o be a s s o c i a t e d w i t h ER  ( P i g . 10<)',  Well-deve-  l o p e d spherosomes commonly o c c u r , as w e l l , a l i g n e d a l o n g port i o n s o f the ER which appear to be a s s o c i a t e d w i t h d e v e l o p i n g v a c u o l e s ( P i g , ll).). As the spores approach g e r m i n a t i o n a l a r g e p r o p o r t i o n o f the n u c l e i are a s s o c i a t e d w i t h l o n g ER c i s t e r n a e  (Pig. 1 7 ) .  Sometimes a nucleus i s almost completely surrounded by s e v e r al  l a y e r s o f c i s t e r n a e which l i e p a r a l l e l w i t h the n u c l e a r  envelope  ( F i g . 13)*  In F i g u r e s 1 2 and 13 two  c l e a r l y connect w i t h the n u c l e a r envelope  such  cisternae  (Arrows).  FULLY ACTIVATED SPORE (STAGE THREE) F i g u r e s 15* 1 6 ,  and 17 i l l u s t r a t e the maximum s t a t e o f  d i f f e r e n t i a t i o n t h a t the spore a t t a i n s b e f o r e the metabasidium begins t o form.  C e r t a i n o f i t s components remain  unchanged from the dormant s t a t e  essentially  ( F i g s . 1 , 5 and 6 ) .  Although  the spore i n c r e a s e s s l i g h t l y i n volume d u r i n g i m b i b i t i o n , the spore w a l l does not show any n o t i c e a b l e change i n t h i c k n e s s , nor i s t h e r e any evidence o f s t r e t c h i n g . bodies are s t i l l present ( F i g . 1 7 ) .  Simple  paramural  A p p a r e n t l y the number,  s i z e , and p o s i t i o n o f both the l i p i d b o d i e s , and the some-like o r g a n e l l e s remain c o n s t a n t . of  However, a  sphero-  comparison  F i g u r e 1 w i t h F i g u r e 17 (method A) o r o f F i g u r e s 5 and 6  29  w i t h F i g u r e 15 (method B) i n d i c a t e s a much g r e a t e r degree o f c y t d l o g i c a l complexity i n t h e f u l l y a c t i v a t e d spores•  This  i n c r e a s e i n complexity has been achieved v i a the stages a l ready d i s c u s s e d .  The sum t o t a l o f these changes, p l u s the  f i n a l steps i n the p r e p a r a t i o n ,for g e r m i n a t i o n , can be summarized  as f o l l o w s ;  Endoplasmic Reticulum. - In the f u l l y a c t i v a t e d  spore,  b o t h the t o t a l q u a n t i t y , and the l e n g t h of i n d i v i d u a l c i s t e r n a e t h a t can be t r a c e d i n any one s e c t i o n i n c r e a s e c o n s i d e r a b l y , p a r t i c u l a r l y i n the p e r i n u c l e a r r e g i o n ( F i g 17).  Rather  late  i n t h e p r e g e r m i n a t i o n p e r i o d a f u r t h e r change occurs i n the d i s t r i b u t i o n o f ER.  The c i s t e r n a e tend t o s t a c k ( F i g . 16,  arrows). V a c u o l e s . - Vacuoles a r e not present i n dormant spores. By the time jthe hydrated it  spore reaches the stage o f g e r m i n a t i o n  c o n t a i n s a t l e a s t one l a r g e vacuole p l u s a v a r i a b l e number  of s m a l l e r v a c u o l e s .  I n F i g u r e 17 t h e r e a r e a number o f p a i r s  of these s n a i l vacuoles j o i n e d t o g e t h e r by what appears t o be short ER segments. H u e l e i . - The nucleus which i s once again more r e g u l a r i n shape has become e c c e n t r i c a l l y s i t u a t e d i n the spore 15 and 17).  In F i g u r e 17 the double n a t u r e o f the n u c l e a r en-  velope i s c l e a r l y v i s i b l e . presence  (Figs.  S e v e r a l f i g u r e s a l s o i n d i c a t e the  o f many simple n u c l e a r pores  13, 15* and 17).  (UP) ( F i g s . 9, 10,  11,  F u r t h e r d e t a i l s c o n c e r n i n g the m e i o t i c  n u c l e u s w i l l b s a d i s c u s s e d i n p a r t IV. M i t o c h o n d r i a . - F i g u r e 6 and F i g u r e 15 i l l u s t r a t e m a t e r i a l  30 prepared by method B and are a t the same m a g n i f i c a t i o n , p a r i s o n o f these two  A com-  f i g u r e s c l e a r l y shows the dramatic i n c r e a s e  i n the s i z e o f the m i t o c h o n d r i a d u r i n g h y d r a t i o n .  The  average  maximum l e n g t h o f the m i t o c h o n d r i a i n f u l l y a c t i v a t e d s p o r e s , a f t e r glutaraldehyde-osmium 1.2  u (Range: 0.7  -  u ) , a f i g u r e which i s almost t w i c e t h a t o f the r e s t i n g  mitochondria 15  f i x a t i o n , i s 0.75  (0.3d  u).  A comparison o f F i g u r e 5 w i t h Figure  i n d i c a t e s t h a t t h e r e i s no s i g n i f i c a n t change i n the  o f mature m i t o c h o n d r i a , o r i n the number, l e n g t h , and  shape  arrange-  ment o f c r i s t a e . The number o f m i t o c h o n d r i a does not appear to i n c r e a s e s i g n i f i c a n t l y throughout  p r e g e r m i n a t i o n development.  Only i n  t h e v e r y l a t e s t stages d e p i c t e d i s t h e r e evidence t h a t the m i t o c h o n d r i a are b e g i n n i n g to d i v i d e .  I n F i g u r e 17  the arrows  i n d i c a t e a m i t o c h o n d r i o n a p p a r e n t l y i n the process o f cons t r i c t i o n , and a double membrane-bound o r g a n e l l e which i s i n t e r p r e t e d as a c r o s s - s e c t i o n through an immature  mitochondrion.  The most conspicuous f e a t u r e o f t h e s e d i v i d i n g and  immature  o r g a n e l l e s i s the poor development o f the c r i s t a e . F l o c c u l e n t Cytoplasm. - The patches o f " f l o c c u l e n t c y t o plasm  11  which develop v e r y e a r l y i n the h y d r a t i o n process a r e  s t i l l present  ( F i g . 16).  any p a r t i c u l a r a r e a o f the  They do not seem to be l o c a l i z e d i n spore.  DISCUSSION THE  SPORE WALL T e l i o s p o r e s have very complex w a l l s - a f a c t which i s p r o -  31  (Allen, 1965).  b a b l y important i n t h e i r a b i l i t y t o s u r v i v e  No  doubt t h i s same complexity, compounded by an a s t o n i s h i n g t h i c k n e s s , i s r e s p o n s i b l e f o r the l a c k o f c y t o l o g i o a l i n f o r m a t i o n concerning  the development and  In agreement w i t h p r e v i o u s Hess and Weber, 1 9 7 0 )  s t u d i e s on smut f u n g i  The  spores.  (Graham, I960;  the t e l i o s p o r e w a l l of U s t i l a g o  c o n s i s t s of t h r e e major l a y e r . an i n t e r m e d i a t e  g e r m i n a t i o n of these  Graham (I960) a l s o  hordei  reported  cementing l a y e r i n T i l l e t i a c o n t r a v e r s a  amorphous e l e c t r o n dense m a t e r i a l adhering to the  s u r f a c e may  Kuhn. outer  be the c o l l a p s e d mucus-like j e l l y which surrounds  the developing  spore i n i t i a l s  (Kukkonen and  V a i s s a l o , 1961+).  In agreement w i t h the s t u d i e s of H i l l e and B r a n d e s ( 1 9 5 6 ) t e l i o s p o r e of U s t l l a g o h o r d e i smuts i n p o s s e s s i n g  appears to be  a smooth outer  e x c e p t i o n a l among  surface.  A l l non-motile f u n g a l spores possess at l e a s t one t r o n dense l a y e r , and the cytoplasm.  elec-  t h i s l a y e r i s never the most p r o x i m a l t o  I t s dense n a t u r e i s due  melanin which i s p o s t u l a t e d damage and  the  t o enzyme l y s i s  t o the presence of  to give resistance to r a d i a t i o n ( B a r t n i c k i - G a r c i a , 1969).  The  innermost w a l l l a y e r of U s t i l a g o h o r d e i i s the o n l y f i b r i l l a r layer. Tilletia  T h i s i s i n agreement w i t h b i o c h e m i c a l contraversa  the i n n e r l a y e r s but The  d e n s i t y of the  of the p r o t o p l a s t .  studies i n  which show t h a t c h i t i n i s predominant i n i s l a c k i n g i n the /outer  (Graham, I960).  " c h i t i n " f i b r i l s decreases i n the  A s i m i l a r g r a d i e n t has been noted i n the  i n n e r w a l l l a y e r s of Neurospora ascospores (Lowry and 196I4.).  The  vicinity  Sussman,  r i d g e s w h i c h p r o j e c t i n t o the p r o t o p l a s t are s i m i l a r  32  t o those demonstrated cells  by f r e e z e - e t c h i n g i n v e g e t a t i v e f u n g a l  (Bauer, 1 9 7 0 ; Hess, 1968), and c o n i d i a  Sassen e t a l . ,  (Campbell,  1969b;  1967)*  THE PROTOPLAST The plasma membrane o f U s t i l a g o h o r d e i appears t o be wider  (approximately 1 2 0 A ) than t h a t u s u a l l y r e p o r t e d f o r  fungi  ( 6 0 - 8 0 Ao),  0  Corfman ( 1 9 6 6 ) , however, g i v e s s i m i l a r  dimensions f o r the spores o f a myxomycete. membrane o f the hyphae o f U s t i l a g o  U n l i k e the plasma  ( S t e i n , 1 9 7 0 ) t h a t o f un-  germinated spores i s r e l a t i v e l y smooth,  A c r e n u l a t e plasma  membrane i s o f t e n a s s o c i a t e d w i t h a c t i v e growth (Marchant e t a l , , 1 9 6 7 ) o r w i t h a g i n g (Hawker, 1 9 6 5 ) i n f u n g i .  During the  development o f c o n i d i a i n A l t e r n a r i a b r a s s i c o l a the plasma membrane, which i s c o n v o l u t e d d u r i n g the p e r i o d o f w a l l f o r mation, becomes smooth i n t h e mature spore  (Campbell,  1969a).  The tendency f o r the plasma membrane t o p u l l away from t h e spore w a l l a f t e r permangante f i x a t i o n i s p r o b a b l y a r t e f a c t u a l . U s u a l l y paramural bodies a r e n o t found i n mature spores (Marchant and Robards,  1968),  The amorphous s t r u c t u r e s t h a t a r e  sometimes seen l y i n g between the plasma membrane and spore wall  ( F i g s . 1 and 1 7 ) do n o t appear t o be e i t h e r t u b u l a r o r  v e s i c u l a r and i t i s q u e s t i o n a b l e whether they a c t u a l l y s t i t u t e paramural b o d i e s .  However, the p o s s i b i l i t y  con-  exists  t h a t t h e s e simple i n c l u s i o n s may be r e s i d u a l breakdown p r o ducts o f paramural b o d i e s which were a c t i v e d u r i n g spore w a l l d e p o s i t i o n (Campbell, 1 9 6 9 a ; C a r r o l l , 1 9 6 9 ; Wilsenach and Kessel, 1965)*  The n a t u r e o f t h e f i b e r s which connect the  33  p r o t o p l a s t w i t h the spore w a l l i s unknown, but f u n c t i o n i s to m a i n t a i n  presumably t h e i r  c l o s e adherence o f these two  parts  o f the spore d u r i n g prolonged storage and d e s s i c a t i o n , Griffiths  ( 1 9 7 1 ) has d e s c r i b e d s i m i l a r f i b e r s i n the b a s i -  diospores  o f Panaeolus compaaaulatua (L,) F r ,  The  p a u c i t y o f endoplasmic r e t i c u l u m and  p o s i t i o n o f the ER  The  parietal  are f e a t u r e s which are common t o a number et a l , , 1 9 6 6 ;  o f r e s t i n g spores (Buckley 1969),  the  E h r l i c h and  Ehrlich,  short p a r i e t a l fragments are p o s t u l a t e d to be  remanent o f an ER-net which was  a c t i v e i n the t h i c k e n i n g o f  immature spore w a l l (Reeves, 1 9 6 7 ) , (1961+) c l a i m t h a t ER  i s pressed  Kukkonen and  the  Vaissalo  a g a i n s t the mucous w a l l s o f the  sporogenous hyphae s h o r t l y a f t e r spore w a l l f o r m a t i o n i n a smut fungus, A n t h r a c o i d e a  the  begins  aspersa.  An i n c r e a s e i n the amount o f endoplasmic r e t i c u l u m d u r i n g germination  appears to be almost u b i q u i t o u s  (Bracker. 1 9 6 7 ;  f o r exception  among f u n g i  see Sussman et a l . , 1 9 6 9 )  i s a l s o c h a r a c t e r i s t i c o f the dormant seed o f h i g h e r  and  plants.  T h i s i n c r e a s e r e f l e c t s a g e n e r a l i z e d a c c e l e r a t i o n o f the t a b o l i c r a t e which accompanies a c t i v a t i o n and (Allen, 1 9 6 5 ) .  With U s t i l a g o h o r d e l one  c a t i o n s o f i n c r e a s e d metabolic appearance o f s h o r t ER  germination  o f the f i r s t  i n the v i c i n i t y o f , and p a r a l l e l w i t h the n u c l e a r  continues  indi-  a c t i v i t y i n the spore i s the  elements i n the i n t e r i o r ,  During the p r e g e r m i n a t i o n  particularly envelope.  period t h i s nuclear-associated  to develop, and may  c i s t e r n a e almost completely  me-  ER  form s e v e r a l p a r a l l e l l a y e r s o f  surrounding  the nucleus ( F i g . 1 3 ) ,  S e v e r a l o t h e r r e p o r t s o f s i m i l a r nuclear-ER a s s o c i a t i o n s  e x i s t i n the l i t e r a t u r e of f u n g i (Buckley et a l . , and Greenwood, 1966; Wells, 1965)  Peat and Banbury, 1967;  1966;  Gay  Wells, 1964a;  and h i g h e r p l a n t s (Esau and G i l l , 1971).  and  Figures  12 and 13 i n d i c a t e t h a t d u r i n g the p e r i o d of maximal development of t h i s a s s o c i a t i o n , c l e a r connections e x i s t between the ER and the n u c l e a r envelope.  Such connections are w e l l -  documented i n f u n g a l hyphae a l t h o u g h r a r e l y i n spores (Namboodiri, The ing  1966).  o r i g i n of the abundant ER which forms i n spores  the f i r s t  dur-  few hours of h y d r a t i o n appears t o have p r e -  v i o u s l y been a mystery]  Linnane et a l , (1962) suggested  that  i n yeast the r e t i c u l a r system sometimes seems t o o r i g i n a t e the n u c l e a r envelope.  from  I n view of the f a c t t h a t i n U s t i l a g o  h o r d e i the f i r s t ER t o be formed l i e s i n the v i c i n i t y of the n u c l e a r envelope, and that n u c l e a r envelope-ER connections be demonstrated,  t h i s would appear t o be p r o b a b l e .  can  Abundant  evidence i n d i c a t e s t h a t i n animal c e l l s and more p a r t i c u l a r l y i n h i g h l y a c t i v e c e l l s such as eggs, the n u c l e a r envelope does serve as a source of r a p i d l y p r o l i f e r a t i n g ER membranes ( W i s c h n i t z e r , 1970). As g e r m i n a t i o n becomes imminent, smooth entoplasmic  reticu-  lum tends t o form s t a c k s i n the cytoplasm - p a r t i c u l a r l y i n a parietal position  ( F i g . 16),  Some authors have  suggested  t h a t these s t a c k s of membranes, f r e q u e n t l y seen i n f u n g i , r e semble simple G o l g i (Campbell, 1969b). of  However, the r e s u l t s  t h i s work tend t o support the view of W e l l s  (1964a  and  1 9 6 4 b ) t h a t s i n c e these s t a c k s are not always a s s o c i a t e d w i t h the nucleus or w i t h v e s i c l e p r o d u c t i o n , they cannot  justifi-  35  a b l y be r e f e r r e d t o as G o l g i .  Such aggregations o f ER a r e not  r e s t r i c t e d to f u n g i , and t h e i r p o s s i b l e s i g n i f i c a n c e i s s t i l l debatable.  Esau and G i l l  ( 1 9 7 1 ) review the l i t e r a t u r e on  this  s t a c k i n g phenomenon i n p l a n t and animal c e l l s , w i t h p a r t i c u l a r emphasis on the evidence l i n k i n g i t w i t h e i t h e r an a c t i v e o r a passive state.  I n most f u n g i the development o f s t a c k s o f  smooth membranes appears to be a s s o c i a t e d w i t h h i g h l y a c t i v e s t a t e s such as ascosporeawall f o r m a t i o n ( C a r r o l l , 1 9 6 9 ) ,  and  n u c l e a r d i v i s i o n i n mature b a s l d i a (Manocha, 1 9 6 5 ; W e l l s ,  1961ib).  I n U s t i l a g o h o r d e i they f o r m j u s t p r i o r to the  first  u l t r a s t r u c t u r a l s i g n s t h a t g e r m i n a t i o n has begun. Vacuoles are not present i n most r e s t i n g o r dormant spores (with the e x c e p t i o n o f the powdery mildews) but are formed d u r i n g the processes o f a c t i v a t i o n and g e r m i n a t i o n (Hyde and Walkinshaw, 1 9 6 6 ; Hawker e t a l . , 1 9 7 0 ; Hawker and Abbott, 1 9 6 3 ; Niederpruem Niederpruep, 1961+).  and Wessels, 1 9 6 9 ; Voelz and  Such v a c u o l a t i o n appears to be a n e c e s s a r y  adjunct o f h y d r a t i o n and g e r m i n a t i o n - so much so t h a t i f the v a c u o l e s are damaged, o r prevented from expansion, the spore ceases to germinate  ( M i t c h e l l and McKeen, 1 9 7 0 ) .  In s p i t e o f  the obvious importance o f the development o f these organe l l e s i n the spore no p r e v i o u s attempt has been made to  determine  their originJ S e v e r a l t h e o r i e s e x i s t to account f o r the o r i g i n s o f p l a n t vacuoles. branous  Vacuoles may  a r i s e v i a d i l a t i o n o f t h e intramem-  c i s t e r n a e of e i t h e r the endoplasmic  1 9 6 1 ) o r the G o l g i apparatus  r e t i c u l u m (Buvat,  (Marinos, 1 9 6 3 ) .  Prom h e r s t u d i e s  36  on the merlstems o f the h i g h e r p l a n t Anthoceros. proposed  Manton ( 1 9 6 2 )  t h a t vacuoles always a r i s e f r o m p r e - e x i s t i n g v a c u o l e s .  B e l l and Muhlethaler  (I96I4-) concluded t h a t they c o u l d a r i s e  the d e g e n e r a t i o n o f m i t o c h o n d r i a and p l a s t i d s . S p i c h i g e r ( 1 9 6 8 ) have more r e c e n t l y proposed  from  M a t i l e and  t h a t vacuoles a r e  formed by the development and coalescence o f l y a o s o m e - l l k e bodies.  The l a s t f o u r suggestions can be e l i m i n a t e d i n the con-  s i d e r a t i o n o f vaouole development i n the ungerminated spore o f U s t i l a g o h o r d e i because o f the f o l l o w i n g f a c t s : 1.  No o r g a n e l l e s which can be i d e n t i f i e d as G o l g i a r e  present i n U. h o r d e i . 2.  R e s t i n g spores o f t h i s fungus possess no v a c u o l e s .  3.  Evidence f o r m i t o c h o n d r i a l d e g e n e r a t i o n has been ob-  s e r v e d i n agirghyphae  but never i n h y d r a t i n g s p o r e s .  To the c o n t r a r y , t h e m i t o c h o n d r i a d u r i n g  pregerminatlon  development a r e seen to i n c r e a s e i n s i z e and number ( i . e. o n l y i n the f i n a l p r e g e r m i n a t l o n s t a g e s ) , ij..  Those membrane-bound dense b o d i e s which, during and  a f t e r g e r m i n a t i o n , appear to a c t as l y s o s o m a l e q u i v a l e n t s ( F t . I I ) , do n o t c o a l e s c e o r change i n number o r s i z e throughout  pregerminal  development.  At about t h e time when v a c u o l e s b e g i n t o appear i n some spores, "Whorls" o f endoplasmic  r e t i c u l u m occur i n o t h e r  spores.  Such whorls have not been seen i n spores which c o u l d be shown to possess a v a c u o l e . cisternae  O f t e n , a whorl has one o r more i n f l a t e d  ( F i g . 9 , arrow), the contents o f which a r e f l o c c u -  l e n t a f t e r permanganate f i x a t i o n , and resemble t h e f l o c c u l e n t  37  contents d e t e c t a b l e I n d e v e l o p i n g v a c u o l e s .  The e v i d e n c e ,  t h e r e f o r e , seems t o be most c o n s i s t e n t w i t h t h e h y p o t h e s i s o f Buvat.  I t i s suggested t h a t i n U s t i l a g o h o r d e i t h e "primary  vacuoles. the  11  which are r e s p o n s i b l e f o r t h e i n i t i a l s w e l l i n g o f  t e l i o s p o r e . a r i s e from d i l a t i o n o f t h e c i s t e r n a e i n  whorls o f ER.  F i g u r e s 9 , 1 0 , and 1 1 a r e r e p r e s e n t a t i v e o f the  p o s t u l a t e d developmental sequence.  As g e r m i n a t i o n approaches,  t h i s process seems t o become more g e n e r a l i z e d , so t h a t , as seen i n F i g u r e 1 7 , s m a l l fragments o f ER throughout the spore appear t o g i v e r i s e to s i m i l a r d i l a t i o n s .  The h y p o t h e s i s i s  f u r t h e r supported by t h e f a c t t h a t the t o n o p l a s t membrane (Average w i d t h = 9 6 A°) i s the same w i d t h as the u n i t membranes o f the ER (Average width = 9ij. A ° ) . McClure  Robinson, Park and  ( 1 9 6 9 ) have presented evidence t h a t v a c u o l e s , Induced  i n t h e hyphae o f A s p e r g i l l u s n i g e r by t h e v a c u o l a t i o n f a c t o r , a l s o form by expansion o f the ER lumen.  What i s now r e q u i r e d  i s t h e a p p l i c a t i o n o f the a p p r o p r i a t e h i s t o c h e m i c a l t e s t s 1.  e. f o r a c i d phosphatase) t o determine f i r s t whether v a c u o l e s  show l y s o s o m a l a c t i v i t y and second whether they a r e i n f a c t der i v e d from the ER. S i m i l a r hypotheses have r e c e n t l y been suggested f o r t h e o r i g i n and development of  o f vacuoles i n the meristematic regions  t h e shoot and a d v e n t i t i o u s r o o t s o f Glechoma hedeaoa L .  (Bowes, 1 9 6 5 ) .  and i n the r o o t meristems  (Mesquita, 1 9 6 9 ) .  o f Lupinus albua  Mesquita was a b l e to show t h a t the rough  ER, which g i v e s r i s e t o the v a c u o l e s , i s continuous with the n u c l e a r envelope, and t h a t t h e r e i s d i r e c t communication  be-  tween the i n t r a v a c u o l a r space and  the p e r i n u c l e a r c i s t e r n a .  Such a d i r e c t c o n n e c t i o n has not been observed  i n Ustilago  h o r d e i but i t has been mentioned t h a t the whorls  of ER are i  u s u a l l y i n c l o s e p r o x i m i t y w i t h the n u c l e a r envelope, t h a t one end  and  of the f i r s t primary vacuoles i s u s u a l l y a l s o  very near the n u c l e u s . A d e t a i l e d d i s c u s s i o n of the m e i o t i c n u c l e u s of U s t i l a g o h o r d e i i s presented i n p a r t IV, but a few p o i n t s , which perhaps have s p e c i a l r e l e v a n c e t o the germination deserve mention h e r e .  Normally,  process,  the t e l i o s p o r e s of smut  f u n g i are c o n s i d e r e d t o c o n t a i n a s i n g l e d i p l o i d  nucleus.  To the best of the author's knowledge t h i s i s the study t o suggest  t h a t t h i s i s not always the case.  t h a t the mature spore may ( P i g s . |2, 3, h*  first  and  The  fact  sometimes have two h a p l o i d n u c l e i  6) suggests t h a t the s y n c h r o n i z a t i o n of  karyogamy w i t h spore f o r m a t i o n i s l o o s e .  I t i s unkown  whether such spores, i n which karyogamy has e v i d e n t l y been d e l a y e d , are capable of undergoing A second  germination and m e i o s i s .  unusual f e a t u r e i s the apparent  p l a s t i c i t y o f nu-  c l e a r shape which develops very e a r l y i n h y d r a t i o n .  Corfman  (1966) r e p o r t e d a s i m i l a r occurrence s h o r t l y a f t e r the  be-  g i n n i n g o f h y d r a t i o n i n the spore of a myxomycete, F u l i g o septica  (L.)  Weber, and  suggested  t h a t i t s f u n c t i o n was  maximize the p o t e n t i a l f o r n u c l e o c y t o p l a s m i c In  to  interaction.  U s t i l a g o h o r d e i the lobed s t a t e seems t o be a s s o c i a t e d  w i t h the movement of the nucleus from a c e n t r a l t o a p e r i p h e r a l p o s i t i o n i n the s p r e .  T h i s suggests t h a t i t may  be  39  a m a n i f e s t a t i o n o f t r u e n u c l e a r amoeboid motion. apparent  Whether the  a s s o c i a t i o n o f l i p i d bodies w i t h the l o b a t e nucleus  i s f o r t u i t o u s , or f u n c t i o n a l i s unknown.  L i p i d bodies are a l s o  a s s o c i a t e d w i t h the n u c l e i o f some phycomycetes d u r i n g gamete f o r m a t i o n ( B l o n d e l and T u r i a n , I960), The o b s e r v a t i o n s r e p o r t e d here on the development o f the m i t o c h o n d r i a l p o p u l a t i o n d u r i n g g e r m i n a t i o n seem to be a t v a r i a n c e w i t h the commonly accepted views.  I n a l l cases so f a r  r e p o r t e d the quiescent m i t o c h o n d r i a are much l a r g e r than seen a f t e r g e r m i n a t i o n  those  (Hawker and Abbott, 1 9 6 3 ; Hawker, 1 9 6 6 ;  Lowry and Sussman, 1 9 6 8 ; Marchant, 1 9 6 6 ) .  Bracker  (1967)  no-  t e d t h a t the decrease i n m i t o c h o n d r i a l s i z e i s o f t e n accompanied by an i n c r e a s e l n the t o t a l number, suggesting t h a t t h e l a r g e m i t o c h o n d r i a d i v i d e t o form s m a l l e r ones.  During  t e l i o s p o r e g e r m i n a t i o n i n U s t i l a g o h o r d e i the exact o p p o s i t e seems to o c c u r .  The m i t o c h o n d r i a , which are o r i g i n a l l y q u i t e  s m a l l , double t h e i r average maximum l e n g t h d u r i n g h y d r a t i o n . The p o p u l a t i o n does not i n c r e a s e n o t i c e a b l y i n number and  fi-  gures which have been i n t e r p r e t e d as d i v i s i o n f i g u r e s ( F i g , 1 7 , arrows) o c c u r o n l y p r i o r to g e r m i n a t i o n .  During the  last  stage d i s c u s s e d h e r e , and i n subsequent s t a g e s , v e r y few  such  f i g u r e s o c c u r a t any one time, and the average  s i z e o f the  m i t o c h o n d r i a l n the p o p u l a t i o n , i n g e n e r a l , c o n t i n u e s t o i n crease u n t i l w e l l i n t o m e t a b a s i d i a e x t e n s i o n .  I t would be i n -  t e r e s t i n g t o know whether t h i s unusual behaviour  reflects  some d i f f e r e n c e i n the d e v e l o p i n g r e s p i r a t o r y p a t t e r n o f UstJilago h o r d e i t e l i o s p o r e s .  On the o t h e r hand i t may  simpler?  iiO  r e s u l t from the f a c t t h a t the p r o t o p l a s t of the t e l i o s p o r e does not a c t u a l l y i n c r e a s e g r e a t l y i n mass d u r i n g the f o r mation of the promycelium.  The r e s t i n g m i t o c h o n d r i a of t h i s  fungus a l s o seem t o be unique are well-developed  i n the f a c t t h a t t h e i r  cristae  and r e t a i n the p a r a l l e l grouping p r e v i o u s l y  r e p o r t e d f o r the hyphae ( S t e i n , 1-970). L i p i d i s p r o b a b l y the most common storage m a t e r i a l i n fungus spores, p a r t i c u l a r l y i n the spores of r u s t s and which germinate 1965).  at the expense of t h e i r pwn  A decrease i n the number of l i p i d  reserves  smuts  (Allen,  or o i l bodies d u r i n g  germination i s almost u b i q u i t o u s among f u n g i (Hawker et a l . , 1970 Manocha and Shaw, 1967; and  Walkinshaw et a l . ,  Ledingham, I96I1; McKeen, 1970).  h o r d e i a l s o appears  t o be unique,  1967;  Williams  In t h i s r e s p e c t U s t i l a g o  s i n c e no decrease i n the  number or s i z e of the l i p i d bodies i s d e t e c t a b l e .  However,  the o c c a s i o n a l a s s o c i a t i o n of l i p i d bodies w i t h the membrane- bound, l y s o s o m e - l i k e o r g a n e l l e s ( P i g . 8)  may  be  indi-  c a t i v e t h a t some u t i l i z a t i o n does occur.  I t seems l i k e l y  t h a t , because the spores were; germinated  i n a comparatively  r i c h medium, they may  be l e s s dependent on endogenous r e s e r v e s .  F l o c c u l e n t , or foamy, cytoplasm occurs i n the spores of s e v e r a l f u n g i , and v a r i o u s f u n t i o n s have been a t t r i b u t e d t o it.  Campbell (1969a) suggests t h a t i t r e p r e s e n t s dextran  accumulations;  Hyde and Walkinshaw (1966) and V o e l z  and  Niederpruem (I961i) suggest t h a t they are r e g i o n s i n which glycogen, or some other storage compound, has been leached out.  Sussman et a l . (1969) p o s t u l a t e t h a t such r e g i o n s serve  kl the f u n c t i o n of v a c u o l e s .  The  observation that i n U s t i l a g o  h o r d e i such r e g i o n s are o f t e n a s s o c i a t e d w i t h c l u s t e r s of l a r g e granules  ( P i g . 16)  supports the i d e a t h a t  "floccu-  l e n t " cytoplasm r e s u l t s from the l e a c h i n g out of some component.  No h i s t o c h e m i c a l i d e n t i f i c a t i o n was  attempted;  s i z e and appearance of the granules are compatible  the  with  suggestions t h a t they c o u l d be glycogen, d e x t r a n , or perhaps glucan.  Wynn and Gajdusek  germination of uredospores  (1968)  have shown t h a t d u r i n g the  of the bean r u s t , Uromyces p h a s e o l j ,  a p o r t i o n of the endogenous r e s e r v e s are t r a n s f e r r e d t o a temporary r e s e r v o i r of p o l y s a c c h a r i d e (probably g l u c a n ) , which is utilized  i n the r a p i d  e l e c t r o n microscope,  e l o n g a t i o n of germ tubes.  W i t h the  t h i s storage form i s s a i d t o be r e p r e -  sented by p a r t i c l e s which are s i m i l a r i n apperance t o glycogen.  CONCLUSION D u r i n g the p e r i o d of pregerm|nation  h y d r a t i o n , the  telio-  spore of U s t i l a g o h o r d e i undergoes a number of u l t r a s t r u c t u r a l changes commonly a s s o c i a t e d w i t h germination; the amount of endoplasmic  r e t i c u l u m i n c r e a s e s , temporary storage m a t e r i a l  accumulates,  and  vacuoles form.  However, the fungus  appears  to be unusual i n the f a c t t h a t the m i t o c h o n d r i a do not in  s i z e or i n c r e a s e n o t a b l y i n number, and  number and  the f a c t t h a t t h e  s i z e of l i p i d bodies does not decrease.  The  b i l i t y of s e q u e n t i a l p r e g e r m i n a t i o n s t u d i e s i s c l e a r . importance  decrease  feasiThe  o f s i m i l a r s t u d i e s i n other types of fungus spores  i s made m a n i f e s t by the f a c t t h a t many, i f not most, of the  42  m e t a b o l i c changes which r e s u l t i n g e r m i n a t i o n o c c u r w i t h i n the spore l o n g b e f o r e the event i t s e l f .  Any attempt  to c o r r e -  l a t e such m e t a b o l i c changes w i t h p o s t - g e r m i n a l c y t o l o g i c a l s e r v a t i o n s i s o f l i m i t e d v a l u e , and may  be m i s l e a d i n g .  ob-  I n the  author's o p i n i o n the p r e v i o u s "mystery" concerning t h e o r i g i n of  the endoplasmic  r e t i c u l u m and the "primary h y d r a t i o n v a -  cuoles" i s indefensible.  I t has, t e n t a t i v e l y , been  suggested  t h a t t h e ER i s d e r i v e d i n some manner from the n u c l e a r env e l o p e , and t h a t the primary vacuoles form by d i l a t i o n o f ER cisternae.  These s u g g e s t i o n s are c e r t a i n l y s t i m u l a t i n g , as  w e l l as b e i n g o f c r i t i c a l importance  t o any understanding  f u n g a l spore g e r m i n a t i o n , and are worthy o f f u r t h e r s t u d y .  of  I.  PLATE 1  Figure l a .  General view o f a q u i e s c e n t t e l i o s p o r e ( i . e . 0 h r . of g e r m i n a t i o n ) , showing the t h r e e w a l l l a y e r s (Wl, W2, and W3), the plasma membrane (PM), and the s t r u c t u r e d d i s t r i b u t i o n of l i p i d bodies ( L ) , spherosieme-like bodies ( S ) , m i t o c h o n d r i a (M), endoplasmic r e t i c u l u m (ER) and the d i p l o i d f u s i o n n u c l e u s (dN). Method A. c a . X 24,400.  Figure l b .  Part of f i g u r e 1 a showing the r e l a t i o n s h i p be- . twesn the ER and a spherosome-lke body. The p l a sma membrane (PM) i s shown c l e a r l y . Note t h e f i n e f i l a m e n t s p a s s i n g from the plasma membrane t o the spore w a l l . Method A. c a . X 24,000.  Figures  2-4 L i g h t microscope p i c t u r e s o f t h i c k s e c t i o n s p r e pared by method A and s t a i n e d w i t h T o l u i d i n e b l u e (Appendix C ) . The p i c t u r e s d e p i c t the steps i n the f u s i o n o f h a p l o i d n u c l e i i n the germinat-  ing teliospore.  c a . X 5,040.  I. Figure  £a.  PLATE 2  G e r n e r a l view of a q u i e s c e n t t e l i o s p o r e ( i . e . 0 hr, of g e r m i n a t i o n ) . I n d i c a t e d are m i t o c h o n d r i a (M), e l e c t r o n t r a n s p a r e n t l i p i d bodies ( L ) , and spherosome-like bodies ( S ) . Note the beaked appearance of the d i p l o i d n u c l e u s (dN), the n u c l e o l u s (Nu) and the n u c l e a r envelope (NE). Method B. ca.  X 21,000. Figure  5>b,  P a r t of f i g u r e 5a showing r i d g e s of i n n e r spore w a l l p r o t r u d i n g i n t o p r o t o p l a s t (arrows),  ca. X li2,000.  Figure 6 .  Part of a q u i e s c e n t t e l i o s p o r e ( i . e . 0 h r . of germination) showing the presence of two h a p l o i d nuc l e i (hN). M i t o c h o n d r i a (M) and ER are a l s o p r e sent. Note the s i z e of the m i t o c h o n d r i a .  Method B.  c a . X 29,500.  I.  PLATE 3  F i g u r e 7.  P a r t of a stage-one t e l i o s p o r e (approximately h n r . of h y d r a t i o n ) showing a d i p l o i d nucleus (dN) i n the extremely lobed s t a t e . Note the i n t i m a t e r e l a t i o n s h i p between the l i p i d bodies L and the nucleus d u r i n g t h i s stage. The arrow i n d i c a t e s a p o s s i b l e NE - EH c o n n e c t i o n . A l a r g e p a t c h of f l o c c u l e n t cytoplasm ( f ) i s v i s i b l e . Method A. c a . X 38,500.  F i g u r e 8.  A g e n e r a l view of a stage-one t e l i o s p o r e . The nuc l e o l u s (Nu) i s very conspicuous at one s i d e of the g e n t l y lobed d i p l o i d n u c l e u s . The three w a l l l a y e r s ¥ 1 , W2, and W3 are very d i s t i n c t . Note the f i b r i l l a r nature of the innermost w a l l l a y e r . Two patches of f l o c c u l e n t cytoplasm ( f ) are p r e s e n t . The arrow i n d i c a t e s the p o i n t of f u s i o n between a l a r g e l i p i d body (L) and one of two spherosome-like body ( S ) .  Method C.  c a . X l8,lj.00.  I.  PLATE k  Figure  9.  Part of a stage two t e l i o s p o r e i l l u s t r a t i n g a whorl of endoplasmic r e t i c u l u m (ER) w i t h a swollen c i s t e r na c o n t a i n i n g f l o c c u l e n t m a t e r i a l (arrow). The whorl l i e s i n c l o s e p r o x i m i t y to the d i p l o i d n u c l e us (dN). Note t h a t a f t e r KMnOh f i x a t i o n the n u c l e ar pores (NP) appear simple. Method A. c a . X 39000,  Figure  10.  Part of a stage two t e l i o s p o r e i l l u s t r a t i n g a more advanced stage i n the f o r m a t i o n of the primary hyd r a t i o n v a c u o l e s . A system of s m a l l vacuoles (V), i n t e r c o n n e c t e d by short ER segments, l i e s near the d i p l o i d n u c l e u s (dN). A number of f r e e spherosomel i k e bodies (S) are p r e s e n t . Method A. ca. X 30,000.  Figure  11,  A w e l l formed vacuole (V) l i e s w i t h one end a d j a cent to the d i p l o i d n u c l e u s (dN). Note the vacuo l a r - ER c o n n e c t i o n (arrow). Method A.  ca. X 3lf,500.  Figure  12,  During stage one and two complex system of ER develop i n a s s o c i a t i o n w i t h the d i p l o i d nucleus (dN). The arrow i n d i c a t e s a prominent n u c l e a r envelopeER c o n n e c t i o n . Method A. c a . X 26,000.  I. F i g u r e 13.  PLATE 5  Part of a stage two t e l i o s p o r e i l l u s t r a t i n g s e v e r a l l a y e r s of ER surrounding the d i p l o i d nucleus (dN). Two n u c l e a r pores (NP) and a n u c l e a r envelope-ER connection (arrow) are i n d i c a t e d . Method A.  ca. X 30,000.  Figure lit.  Spherosome-like bodies (s) o f t e n l i e along e l e ments of ER which lead t o the primary h y d r a t i o n  vacuoles ( V ) .  F i g u r e l£.  Method A.  ca. X 28,£00.  General view of a t e l i o s p o r e j u s t p r i o r t o germina t i o n showing the s p h e r i c a l t o ovoid m i t o c h o n d r i a (M) w i t h well-developed p a r a l l e l , p l a t e - l i k e c r i s tae l y i n g i n the l o n g t u d i n a l a x i s of these organelles. Note t h a t the n u c l e a r pores (NP) appear simple a f t e r t h i s method of p r e p a r a t i o n . W e l l developed ER i s v i s i b l e . Method B. c a . X 29,500.  I.  PLATE 6  F i g u r e 16.  General view o f a t e l i o s p o r e j u s t p r i o r to g e r m i n a t i o n . Note the presence o f the l a r g e m i t o c h o n d r i a (M), l i p i d bodies ( L ) , many s m a l l vacuo l e s ( V ) . Patches o f e l e c t r o n dense g r a n u l e s are a s s o c i a t e d w i t h a r e g i o n o f f l o c c u l e n t cytoplasm ( f ) . The p a r i e t a l endoplasmic r e t i c u l u m (ER) tends to s t a c k (arrows). Method A. c a . X 30,000.  Figure 17.  General view o f a t e l i o s p o r e j u s t p r i o r to g e r m i n a t i o n . Note the e c c e n t r i c a l l y p o s i t i o n e d d i p l o i d nucleus (dN) with a w e l l - d e f i n e d nuc l e a r envelope (NE) and n u c l e a r pores (NP). Long segments o f ER are a s s o c i a t e d w i t h the n u c l e u s . Throughout the spore s h o r t ER-elements appear to be g i v i n g r i s e t o s m a l l vacuo l e s by d i l a t i o n . Numerous l i p i d b o d i e s (L) are s t i l l p r e s e n t . The arrows i n d i c a t e a d i v i d i n g mitochondrion and a young m i t o c h o n d r i a l element. Note the elementary s t a t e of the c r i stae i n d i v i d i n g and young m i t o c h o n d r i a . Method A. Ga. X 18,000.  43 BIBLIOGRAPHY A l l e n , J.V., Hess, W.M., and Weber, D.J. 1971. Ultrastruct u r a l I n v e s t i g a t i o n s o f dormant T i l l e t i a c a r i e s t e l i o s p o r e s . Mycologia 63^: 144-156* A l l e n , P.J. fungi.  1965. M e t a b o l i c a s p e c t s Of spore g e r m i n a t i o n i n Ann. Rev. P h y t o p a t n o l . 3 : 3 1 3 - 3 4 1 •  B a r t n i c k i - G a r c i a , S. 1969. C e l l wall differentiation i n phycomycetes. Phytopathology 5 £ : 1065-1071. Bauer, H. 1 9 7 1 . 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Metabolism o f glucomannan p r o t e i n d u r i n g g e r m i n a t i o n o f bean r u s t s p e c i e s . Contr. Boyce Thompson I n s t . 21^: 1 2 3 - 1 3 8 .  PART I I I n i t i a t i o n o f t h e Promycelium and P r o m y c e l i a l  Extension  TABLE OP CONTENTS Page ABSTRACT  k-9  INTRODUCTION  49  MATERIALS AND METHODS  51  OBSERVATIONS  51  Stage Pour:  I n i t i a t i o n o f the Promycelium .......  51  Stage F i v e :  Promycelial Extension  57 65  DISCUSSION I n i t i a t i o n o f the Promycelium  65  Promycelial Extension  69  •  The Spherosomal-vacuolar System ••••••••••••••••••  76  CONCLUSION  33  BIBLIOGRAPHY  86  k9  PART I I I n i t i a t i o n o f t h e Promycelium and  Promycelial  Extension  ABSTRACT I n i t i a t i o n o f the promycelium o f U s t i l a g o h o r d e i i n v o l v e s the l o c a l i z e d d e g r a d a t i o n  o f the i n n e r spore w a l l , t h e l o c a l i z e d  d e p o s i t i o n o f a new w a l l l a y e r , and the s w e l l i n g o f t h e p r o t o plast*  D u r i n g t h i s p e r i o d o f development the endoplasmic  reti-  culum and spherosome-like o r g a n e l l e s ( P t . I ) undergo changes i n distribution*  The s i g n i f i c a n c e o f these changes i s d i s c u s s e d *  Host o f the spore contents f l o w i n t o the promycelium as i t extends*  The s t r u c t u r a l b a s i s f o r t h e e x t e n s i o n o f t h e promy-  c e l i a l w a l l i s unknown*  Mitochondria,  l i p i d b o d i e s , and endo-  plasmic r e t i c u l u m a r e randomly d i s t r i b u t e d and do n o t i n c r e a s e s u b s t a n t i a l l y i n quantity*  Apart from t h e nucleus  the only  o r g a n e l l e s which a p p a r e n t l y change i n number and d i s t r i b u t i o n are those bodies t h a t appear t o be t h e u l t r a s t r u c t u r a l  equiva-  l e n t o f t h e l i g h t m i c r o s c o p i s t s ' spheresomes and v a c u o l e s *  In  d i s c u s s i n g t h e s i g n i f i c a n c e o f these changes i t i s suggested t h a t the spheresomal-vacuolar system i n t h i s fungus i s t h e f u n c t i o n a l e q u i v a l e n t o f t h e animal lysosomal  system*  INTRODUCTION On g e r m i n a t i o n  the t e l i o s p o r e s (probasidlan) of U s t i l a g o  h o r d e i g i v e r i s e t o t u b e - l i k e c e l l s o f l i m i t e d growth known as  50  pro my e e l i a o r m e t a b a s i d i a .  These s t r u c t u r e s are s u p e r f i c i a l l y  s i m i l a r to germ tubes produced by o t h e r nonsexual spores* U s t i l a g o h o r d e i the promycelium i s approximately 2.3 (Range: l.lj.0-2.8 u ) .  In  u wide  Once the spore w a l l has r u p t u r e d the  promycelium extends a t a maximum r a t e o f approximately 0 . 2 5  u  per minute, s l o w i n g down as i t a t t a i n s i t s maximum l e n g t h (Range: 20-30 u ) .  When growth has stopped, the c e l l  divides  twice i n r a p i d s u c c e s s i o n g i v i n g r i s e to a f o u r - c e l l e d promycelium containing the f o u r meiotic products.  There i s v e r y  l i t t l e t e r m i n a l growth once d i v i s i o n has o c c u r r e d . T h i s study i s concerned w i t h those c y t o l o g i c a l  events  which occur d u r i n g spore g e r m i n a t i o n - t h a t i s , d u r i n g promyc e l i u m development.  I n s p i t e o f the f a c t t h a t spores (or  s p o r a n g i a i n some Oomycetes) are n o t o r i o u s l y d i f f i c u l t to p r e pare f o r e l e c t r o n microscopy  p r i o r to g e r m i n a t i o n a number o f  attempts have been made to d e s c r i b e the e a r l y stages o f germ tube i n i t i a t i o n which occur w i t h i n the i n t a c t spore ( s p o r a n g i a l ) w a l l ( E i s n e r e t a l . , 1970;  Hawker and Abbott, 1963b; Hawker,  1966;  Hawker e t a l . , 1970;  Lowery and Sussman, 1961|.; Marchant,  1966;  M i t c h e l l and McKeen, 1970;  Walkinshaw e t a l , , 1 9 6 7 ) .  Although the l i t e r a t u r e r e l a t i n g to germ tube u l t r a s t r u c t u r e i s q u i t e e x t e n s i v e , to the b e s t o f t h e author's knowledge no l i t e r a t u r e i s a v a i l a b l e on the u l t r a s t r u c t u r e o f m e t a b a s i d i a . For the most p a r t germ tubes seem to possess few f e a t u r e s t h a t d i s t i n g u i s h them c y t o l o g i c a l l y from hyphane.  I n most r e s p e c t s  the young extending promycelium o f U s t i l a g o h o r d e i a l s o resemb l e s the a p i c a l hyphal r e g i o n o f the m y c e l i a l mutant d e s c r i b e d  by S t e i n (1970).  51  MATERIALS AND The  METHODS  c u l t u r e s and the methods o f c u l t u r i n g and  sampling  were i d e n t i c a l to those d e s c r i b e d p r e v i o u s l y ( F t . I ) , except t h a t the samples were c o l l e c t e d a f t e r 5 h o u r s , and a f t e r 7 to 9?g hours o f h y d r a t i o n .  During  the l a t t e r p e r i o d ,  30 $ to 9 5 $ o f the spores have begun m e t a b a s i d i a l o f these, 1 5 $ to 5 0 $ have formed the f i r s t lj.0$ have formed the second s e p t a ,  approximately extension;  septum; and 5 $ to  Por example, a f t e r 8 hours  o f h y d r a t i o n the per centage germinauidn«(i, e, the number o f v i s i b l e i n the l i g h t microscope) i s 9 0 $ ,  f p o r e s w i t h promycelia  the per centage o f spores t h a t have formed a t l e a s t one  septum  i s 3 5 $ and the per centage o f spores t h a t have formed three s e p t a i s 2 5 $ (Bech-Hansen, 1 9 6 8 ) , The m a t e r i a l was ing  prepared  to methods A, B and  f o r e l e c t r o n microscopy  C ( F t . I , Table I ) , w i t h the  t h a t none o f the m a t e r i a l was a c e t a t e p r i o r to embedding.  exception  p r e - s t a i n e d i n aqueous u r a n y l F i g u r e lib was  photographed u s i n g  a Z e i s s Fhotomicroscope, from t h i c k s e c t i o n s prepared ing  t o method A and  accord-  accord-  s t a i n e d w i t h 1 $ T o l u i d i n e b l u e i n 1 $ borax.  F i g u r e ll|.b d e p i c t s a l i v i n g c e l l  photographed i n a t h i n l a y e r  o f complete l i q u i d medium between a c o v e r s l i p and a g l a s s slide. OBSERVATIONS STAGE FOUR: The  INITIATION OF THE  first  PROMYCELIUM  c y t o l o g i c a l i n d i c a t i o n t h a t germination  begun i s a change l n the shape o f the p r o t o p l a s t . ly  symmetrical,  The  has previous-  c i r c u l a r or o v o i d c r o s s - s e c t i o n a c q u i r e s  a  52  "beaked" appearance ( P i g s . 1, 3 k&» or l|b).  Closer  observation  r e v e a l s t h a t a v e r y s l i g h t change i n t h e shape o f t h e a c t u a l p r o t o p l a s t i s accentuated b y the presence o f a "cap" o f m a t e r i a l which covers the beaked p o r t i o n o f t h e cytoplasm and p r o t r u d e s i n t o t h e spore w a l l  ( F i g s . 1, 3 and i|.a).  The W a l l . - As d e s c r i b e d  i n p a r t I , the spore w a l l  (W) o f  U s t i l a g o h o r d e i c o n s i s t s o f t h r e e major l a y e r s (Wl, W2, and W3). When t h e beak o f the p r o t o p l a s t f i r s t becomes n o t i c e a b l e , and before  t h e appearance o f the "cap", p e r f o r a t i o n s  l n t h e innermost w a l l l a y e r a t a p o i n t o p p o s i t e 2).  A f t e r preparatory  (p) develop t h e beak ( F i g .  methods A and B, these spaces a r e  u s u a l l y empty, o r o c c a s i o n a l l y they c o n t a i n f a i n t l y material  ( F i g s . 1 and lj.a).  t a i n e l e c t r o n dense f i b r i l s  granular  However, a f t e r method C, they con( F i g . 2).  S h o r t l y a f t e r the f i r s t i n n e r w a l l , the new m a t e r i a l  " p e r f o r a t i o n s " appear i n the (pW) b e g i n s t o accumulate around  the p r o t o p l a s t beak ( F i g . 2 ) .  The new m a t e r i a l i s more e l e c t r o n  t r a n s l u c e n t than i s t h e i n n e r w a l l l a y e r .  When i t i s f i r s t  formed i t i s q u i t e homogeneous b u t , as the l a y e r t h i c k e n s and extends l a t e r a l l y , t h e r e g i o n most p r o x i m a l t o the spore w a l l becomes i n c r e a s i n g l y f i b r i l l a r .  However, a t t h i s p e r i o d o f  development, the o r i e n t a t i o n o f these f i b e r s never approaches the r e g u l a r i t y shown by those o f the i n n e r spore w a l l ( F i g . 6). Consequently, although t h e o l d and new l a y e r s a r e c l o s e l y appressed the boundary between them i s abrupt.  The new w a l l  l a y e r i s a l s o c l o s e l y appressed to the p r o t o p l a s t o f t h e beak, but the boundary here i s o f t e n i n d i s t i n c t  ( F i g s . 1 and 3).  53  Once t h i s p a t t e r n o f development has been i n i t i a t e d , processes i n v o l v e d proceed r a p i d l y and r e g i o n o f p e r f o r a t i o n i n c r e a s e s and  simultaneously.  the  As  extends around the  the  sides  o f the beak, the p r o t o p l a s t seems t o push forward d i s p l a c i n g the p e r f o r a t e d i n n e r spore w a l l l a y e r and expanding i n l e n g t h and  i n w i d t h ( F i g . 1+a).  The  new  w a l l l a y e r thickens  and  ex-  tends l a t e r a l l y a t a r a t e s u f f i c i e n t to ensure t h a t the i n c r e a s i n g s u r f a c e a r e a o f the beak i s encased by i t . As thickness  the  o f the f i b r i l l a r spore w a l l l a y e r i s d i m i n i s h e d  by  the advancing p r o t o p l a s t , the outer e l e c t r o n dense w a l l l a y e r s b e g i n to show s i g n s o f d i s o r g a n i z a t i o n ( F i g . i+a). the spore w a l l r u p t u r e s . was  The  be c o n s i d e r e d  extended beyond the  6  spore i l l u s t r a t e d l n F i g u r e  caught a t t h i s exact moment.  which can now  Finally,  The  beak o f the  protoplast,  as a t r u e promycelium, has  spore w a l l , and  barely  i s e n t i r e l y encased by  a  w e l l - d e f i n e d p o r t i o n o f the newly s y n t h e s i z e d w a l l l a y e r . Between the young promycelium and the o l d spore w a l l the  outer  p a r t o f the newly s y n t h e s i z e d  short  collar.  w a l l m a t e r i a l has  Remaining c e l l d e b r i s has  formed a  sloughed o f f i n t o the medium  accompanied by spore w a l l d e b r i s . Plasma Membrane (PM)  and Paramural B o d i e s . - During  velopment o f t h e protoplasmic appears c r e n u l a t e d  beak, the plasma membrane o f t e n  ( F i g s . 1 and 3).  In F i g u r e s 1 and  r e l a t i v e l y l a r g e areas o f the plasmalemma are separated the spore w a l l . s u r f a c e o f the  de-  3. from  T h i s s e p a r a t i o n l a t e r extends t o the e n t i r e p r o t o p l a s t except f o r the beak r e g i o n .  t h i s l o c a l i z e d area the p r o t o p l a s t and  the new  In  wall layer  are  54 v e r y c l o s e l y apposed; so much so t h a t the plasma membrane boundary between the two intimate contact  i s i n d i s t i n c t i n the r e g i o n o f most  ( F i g s . 1, 2,  3,  and  4-a).  Endoplasmic R e t i c u l u m (ER). - Throughout the p e r i o d , there  pre-germination  Is a c o n s i s t e n t i n c r e a s e i n the amount, and  o f endoplasmic r e t i c u l u m i n the I n t e r i o r o f the s p o r e . elements toward the p e r i p h e r y F i g . 17).  are s h o r t and  scattered  length  ER (Ft. I,  J u s t p r i o r t o , and d u r i n g , the i n i t i a t i o n o f  the  promycelium, t h e r e i s a n o t i c e a b l e i n c r e a s e i n t h e number l e n g t h o f these p e r i p h e r a l elements, p a r t i c u l a r l y i n the p r o x i m a l to the d e v e l o p i n g s e v e r a l ER  beak ( F i g s . 1 and k&).  and region  In F i g u r e  I,  c i s t e r n a e have formed a l o o s e a s s o c i a t i o n j u s t  beneath the i n c i p i e n t metabasidium.  As has been p r e v i o u s l y  noted i n p a r t I , i t i s very d i f f i c u l t to o b t a i n  information  on these membrane systems a f t e r glutaraldehyde-osmium f i x a t i o n because o f the d e n s i t y o f the cytoplasm i n ungerminated spores ( F i g s . 2,  4a,  and  Just before ER  6). the rupture  o f the  spore w a l l , the p e r i p h e r a l  elements undergo a f u r t h e r change i n d i s t r i b u t i o n .  After  potassium permanganate f i x a t i o n a number of spores are observed i n which the s h o r t e r membrane fragments have "disappeared" most o f the o r g a n e l l e s seem t o be  encased, c o l l e c t i v e l y , i n a  continuous "sac" composed o f a s i n g l e continuous ER ( F i g . 3)»  The  sac may  l i e d i r e c t l y beneath and  the plasma membrane, or i t may  be  cisterna  p a r a l l e l to  " p u l l e d away" on one o r more  s i d e s l e a v i n g a f a i r l y l a r g e p o r t i o n o f the the sac and the plasmalemma.  and  cytoplasm between  In t h i s s i t u a t i o n the nucleus  55 and a l l t h e m i t o c h o n d r i a , as w e l l as most o f the l i p i d , u o l e s , and remaining ER, l i e w i t h i n i t . s m a l l v a c u o l e s , spherosome-like of  ER are excluded.  vac-  A few l i p i d b o d i e s ,  o r g a n e l l e s and odd fragments  F i g u r e 8 i l l u s t r a t e s a p o r t i o n o f a de-  v e l o p i n g s a c i n which the I n d i v i d u a l ER elements have not y e t completely f u s e d (arrows).  composing i t  The cytoplasm o u t s i d e  the sac i s s l i g h t l y l e s s dense than t h a t w i t h i n ( F i g . 8).  An  a d d i t i o n a l f e a t u r e o f such formations i s t h a t a t v a r i o u s p o i n t s the membranes are e l a b o r a t e d i n t o more o r l e s s complex knots 3.  (Fig.  arrow).  Spherosome-like  Organelles ( S ) . - Frey-Wyssling et a l .  (1963) f i r s t d e s c r i b e d spherosomes u l t r a s t r u c t u r a l l y i n the h i g h e r p l a n t Zea mays. to  A f t e r permanganate f i x a t i o n these  round  angular b o d i e s a r e c h a r a c t e r i z e d by t h e presence o f a u n i f o r m  semi-dense, g r a n u l a r m a t r i x bounded b y a u n i t membrance, and secondly, by a c o n s t a n t , though p o o r l y d e f i n e d , a s s o c i a t i o n w i t h the endoplasmic  reticulum.  Bodies, r a n g i n g i n diameter  from 0,1 t o 0,6 u and s a t i s f y i n g these c r i t e r i a , have p r e v i o u s l y been noted i n KMnOj^-fixed hordei  teliospores of Ustilago  ( E t , I ) , and have a c c o r d i n g l y been d e s i g n a t e d  "sphero-  some-like," As might be a n t i c i p a t e d from the a s s o c i a t i o n o f the spherosome-like  o r g a n e l l e s w i t h t h e ERjgtheseSbodies#tend  to  l o c a l i z e f o l l o w i n g the r e d i s t r i b u t i o n o f membrane systems t h a t accompanies beak f o r m a t i o n .  C l u s t e r s o f these  spherosome-like  bodies a r e p r e s e n t i n the r e g i o n o f beak development e a r l y i n t h i s stage o f d i f f e r e n t i a t i o n  ( F i g s , 1 and 2 ) . A f t e r the f o r -  £6  mation o f the ER-sac, they come to l i e e i t h e r i n s i d e or o u t s i d e the sac-membrane ( P i g . 3 ) .  They tend to accumulate p e r i -  p h e r a l l y I n the v i c i n i t y o f the two  t r i a n g u l a r areas o f c y t o -  plasm formed when the sac membrane p u l l s away from the plasmalemma ( F i g s . 3 and 8 ) .  The  s m a l l e s t o f the  spherosome-like  bodies are r o u g h l y c i r c u l a r i n c r o s s - s e c t i o n and the membranes tend to be c l e a r l y d e f i n e d although u n i t membrane s t r u c t u r e i s d i f f i c u l t to demonstrate.  The l o n g a x i s o f each o f these o r -  g a n e l l e s a l i g n s i n p a r a l l e l w i t h an ER element, w i t h one o f t h e l o n g e r s i d e s c l o s e l y f o l l o w i n g the c o n t o u r s o f the ER membrane. O f t e n the boundary between the ER and a spherosome-like  organ-  e l l e i s vague - the membranes i n the r e g i o n o f a s s o c i a t i o n b e i n g indistinct.  P r o b a b l y t h i s e f f e c t i s an a r t e f a c t caused by  angle o f s e c t i o n i n g . Where the boundaries  the  between them are  w e l l - d e f i n e d , the membranes are separated from each other a r e l a t i v e l y constant d i s t a n c e o f 5 5 to 80 A  0  by  (Fig. 8).  The most prominent o r g a n e l l e s p r e s e n t i n g l u t a r a l d e h y d e osmium f i x e d t i s s u e are those membrane-bound b o d i e s w i t h e l e c t r o n dense c o n t e n t s . i n t i s s u e prepared  They are p a r t i c u l a r l y  a c c o r d i n g to method C.  spore, the e l e c t r o n dense m a t e r i a l may throughout  very  conspicuous  I n the ungerminated  be e i t h e r s c a t t e r e d  an e l e c t r o n t r a n s p a r e n t m a t r i x i n the f o r m o f dense  entwined s t r a n d s ( F i g . 2 ; P t . I , F i g . 5 ) ,  o r i t may  be  "con-  densed" i n t o a s o l i d core l e a v i n g the r e s t o f the m a t r i x absolutely clear  (Pt. I, F i g . 8 ) .  These are the o n l y o r g a n e l l e s  i n glutaraldehyde-osmium f i x e d t i s s u e which have not been assigned a f u n c t i o n .  They are a l s o the o n l y o r g a n e l l e s whose  57  s i z e range, and p a t t e r n o f d i s t r i b u t i o n can be  s a i d to c o r r e s -  pond to those o f the spherosome-like o r g a n e l l e s d e s c r i b e d i n KMnO^-fixed m a t e r i a l .  W i t h i n the spore these bodies range l n  diameter from O.2I4. to 0 . 8 6 u .  L i k e the spherosome-like b o d i e s ,  t h e i r numbers appear to remain constant  d u r i n g the e a r l y stages  o f h y d r a t i o n and t h e i r d i s t r i b u t i o n i s random.  Too  l i t t l e infor-  mation i s a v a i l a b l e on the d i s t r i b u t i o n o f the endoplasmic culum i n glutaradlyhyde-osmium f i x e d spores  reti-  to v e r i f y t h e i r  asso-  c i a t i o n w i t h the ER-sac, but they do c l u s t e r i n the r e g i o n o f d e v e l o p i n g beak ( F i g . 2 ) .  the  On t h i s b a s i s , i t i s t e n t a t i v e l y  suggested t h a t these o r g a n e l l e s are i d e n t i c a l w i t h  the  spherosome-like o r g a n e l l e s found i n permanganate f i x e d m a t e r i a l . STAGE F I V E :  GERM TUBE EXTENSION  Promyoelial Wall  (pW).  - Oddly enough when the promycelium  f i r s t b u r s t s through the spore w a l l , the new  promyoelial w a l l  which surrounds t h e t i p i s r e l a t i v e l y t h i c k (approximately mu),  and q u i t e compact and f i b r i l l a r  (Fig. 6 ) .  During  30  subse-  quent stages o f e l o n g a t i o n the p r o m y o e l i a l t i p i s almost naked (Fig.  1 0 and 2 0 b ) .  A t r u e w a l l can o n l y be d i s t i n g u i s h e d a t  a d i s t a n c e o f about 2 u behind approximately  1 0 mu  the t i p . At t h i s p o i n t , i t i s  t h i c k but proceeding  basipetally,i t  t h i c k e n s r a p i d l y over a d i s t a n c e o f l e s s than 2 u f r o m the t i p ) to a mature w i d t h o f approximately 33 -  75  ( i . e . 1+ u 6 5 mu  (Range:  mu).  At the apex, the p r o m y o e l i a l w a l l i s p o o r l y d e f i n e d ( F i g . 20b)  and l i t t l e i n f o r m a t i o n has been o b t a i n e d  substructure.  concerning i t s  However, throughout the r e s t o f i t s l e n g t h , i t  i s u n i f o r m l y semi-dense and appears t o be g r a n u l a r or  fibrillar  58  ( F i g s . 18a, 18b, and 2 0 ) .  A f i n e f i b r i l l a r m a t e r i a l extends  from i t s s u r f a c e ( F i g s . 18 and 2 0 ) .  Although i t i s not w e l l  i l l u s t r a t e d i n these p i c t u r e s , t h i s m a t e r i a l i s o f t e n e x t e n s i v e , sometimes a t t a i n i n g l e n g t h s o f almost a micron ( P t . I l l , F i g .  7).  In F i g u r e s l 6 a - c l a r g e pores open from the cytoplasm through the w a l l i n t o the e x t e r n a l medium.  Such pores occur  i n f r e q u e n t l y and have been observed o n l y a f t e r p r e p a r a t i o n by method C.  When t h e y are p r e s e n t , t h e y o c c u r i n l a r g e numbers  a l o n g the l e n g t h o f t h e promycelium.  For example, a t l e a s t  10  pores were v i s i b l e i n a s i n g l e s e c t i o n through the germ tube i l l u s t r a t e d by F i g u r e s l 6 a - c . pores i s approximately 47 mu  The diameter o f these hyphal (Range: 3 8 - 5 3 mu)  at the  s u r f a c e o f the w a l l and narrows to approximately 2 5 mu 23-31 mu)  towards  the e x t e r n a l s u r f a c e .  internal (Range:  They are l i n e d by  plasma membrane. Plasma Membrane (PM). - Throughout the p e r i o d o f metabas i d i a l e x t e n s i o n , the plasma membrane appears t o be u n i f o r m l y smooth and c l o s e l y appressed to the h y p h a l w a l l .  I t Is i n d i s -  t i n c t a t the apex but, i n o u t l i n e , t h e t i p i s remarkably smooth ( F i g s . 10 and 2 0 ) .  No paramural b o d i e s are p r e s e n t .  Endoplasmic R e t i c u l u m (ER) and Ribosomes. - The decrease in  c y t o p l a s m i c d e n s i t y which accompanies  a l l o w s more c r i t i c a l osmium f i x a t i o n .  spore g e r m i n a t i o n  o b s e r v a t i o n o f the ER a f t e r g l u t a r a l d e h y d e -  The average w i d t h o f the u n i t membranes a f t e r  a l l t h r e e p r e p a r a t o r y techniques i s 94.0  A§  (Range: 70-110 A°)  but the width o f t h e inter-membrane space i s q u i t e v a r i a b l e , r a n g i n g from 60 to 160 A  0  a f t e r permanganate and f r o m 60 to  100 A° a f t e r gluteraldehyde-osmium f i x a t i o n .  Single lamellae  59  v a r y i n t o t a l width from 210 to 3 7 5 A . Most o f the endoplasmic r e t i c u l u m flows from the with  spore  the r e s t o f the o r g a n e l l e s , and becomes s c a t t e r e d through-  out the l e n g t h o f the germ tube except i n the extreme apex. I t does not  seem to i n c r e a s e i n q u a n t i t y and  i s very  sparsely  d i s t r i b u t e d , c o n s i s t i n g mainly o f s i n g l e , s h o r t , f l a t t e n e d c i s t e r n a e l y i n g j u s t beneath and p a r a l l e l to the plasma membrane ( P i g s , 5 » 1 8 a and  19).  O c c a s i o n a l l y two o r t h r e e l a -  m e l l a e i n the p e r i p h e r a l cytoplasm w i l l  form stacks  parallel  to the w a l l , and these s t a c k s are o f t e n a s s o c i a t e d w i t h membrane-bound v e s i c l e s (ve)  (Pig. 1 5 ) .  small,  ER elements which  are s i t u a t e d away from the c e l l w a l l are u s u a l l y a s s o c i a t e d w i t h the spherosome-like o r g a n e l l e s the nucleus ( P i g s . 1 7 and 1 9 ) .  ( P i g s . 9 and Ida) o r  Nuclear envelope-ER connec-  t i o n s are r a r e l y seen d u r i n g t h i s  stage.  Ribosomes are v e r y d i s t i n c t i n t i s s u e which has f i x e d i n glutaraldehyde-osmium and ( P i g s . 7 , 9 . 17* and 2 0 a - c ) .  with  been  embedded i n Spurr's p l a s t i c  T h e i r d e n s i t y i s uniform  through-  out the l e n g t h o f the promycelium but tends t o decrease w i t h i n the spore as the germ tube extends.  Figure 7 I l l u s t r a t e s  d e n s i t y g r a d i e n t a c r o s s the neck o f the s p o r e . appear to be the o n l y m a t e r i a l present r e c t l y behind the  c e l l apex.  the  Ribosomes  w i t h i n the 0 . 3 u d i -  Most o f the ribosomes are f r e e ;  however, a s m a l l amount o f rough ER occurs near the envelope ( P i g s . 1 7 and 19 » a r r o w s ) .  nuclear  The elements o f the en-  doplasmic r e t i c u l u m which are a s s o c i a t e d w i t h the plasma membrane ( F i g . 1 5 ) o r w i t h spherosome-like o r g a n e l l e s  ( P i g . 9 ) are  60  smooth. Nucleus  ( N ) . - Normally the nucleus i s i n t h e d i p l o i d  s t a t e when i t migrates  (General I n t r o d u c t i o n ) .  In Figures  1+a and [|.b the spore nucleus l i e s j u s t p o s t e r i o r to the deve l o p i n g beak, " a n t i c i p a t i n g " as i t were, i t s passage i n t o the metabasidium.  U s u a l l y , although n o t always, the nucleus i s one  o f tiie f i r s t o r g a n e l l e s to migrate and i t does so i n a d i s t i n c t i v e and c h a r a c t e r i s t i c manner. The neck by which the promycelium narrowest p a r t o f the c e l l 5 and 7).  l e a v e s the spore i s the  (width = approximately 1 u) ( F i g s .  I n o r d e r t o pass through t h i s narrow r e g i o n the  s p h e r i c a l d i p l o i d n u c l e u s (Average diameter = 2.6 u) e l o n g a t e s , a l t e r i n g i t s shape a p p a r e n t l y spontaneously. l e u s i n F i g u r e 5 i s j u s t emerging celium.  The nuc-  from the neck i n t o the promy-  I t s p o s t e r i o r end i s s t i l l v e r y narrow, w h i l e the  remainder has broadened s t r i p on e i t h e r s i d e .  out l e a v i n g o n l y a narrow c y t o p l a s m i c The width o f the promycelium  (Average  width » 2.3 u) i s not s u f f i c i e n t t o a l l o w the d i p l o i d nucl e u s t o r e t u r n t o i t s s p h e r i c a l shape and i t continues t o r e t a i n an elongate form (approximately 1.5 by 3.5 and 10).  u) ( F i g s . 5  During the passage o f the nucleus up the metabasidium,  the nucleus (Nu), i s always a t t h e extreme p o s t e r i o r ( i . e . towards the spore) and a d i s t i n c t i v e s t r u c t u r e , t h e " c e n t r i o l a r -kine to chore - e q u i v a l e n t t e r i o r end and to one s i d e ing  (CKE)" i s always a t the an-  ( F i g . 5).  Further d e t a i l s  concern-  t h e nucleus and i t s a s s o c i a t e d s t r u c t u r e w i l l be d i s c u s s e d  i n p a r t IV. The nucleus does n o t c o n t i n u e t o move w i t h t h e flow o f  61  cytoplasm and o r g a n e l l e s a l o n g the extending tube.  I t comes  to r e s t i n such a p o s i t i o n t h a t when the c e l l has elongated to  i t s t o t a l l e n g t h the nucleus w i l l  terior half. at  he l o c a t e d i n t h e pos-  In t h e c e l l shown i n Figure 10 the nucleus i s  a p o s i t i o n approximately mid-way along the extending p r o -  mycelium.  S i n c e the d i p l o i d nucleus almost f i l l s  diameter o f the tube, l a r g e r o r g a n e l l e s ( i . e . and spherosome-like  the e n t i r e  mitochondria  b o d i e s ) which have been caught i n t h e  c y t o p l a s m i c stream cause a d i s t o r t i o n i n t h e shape o f the nucleus as they pass by i t on t h e i r way to t h e e x t e n d i n g zone between t h e s t a t i o n a r y nucleus and the apex ( F i g . 1 0 ) . When the nucleus has ceased t o move and when a p i c a l e x t e n s i o n has begun to slow down, o r has stopped, the nucleus undergoes two rounds o f n u c l e a r d i v i s i o n tails will  ( m e i o s i s ) and c e l l d i v i s i o n .  De-  concerning t h e mechanisms i n v o l v e d i n these d i v i s i o n s be d i s c u s s e d i n p a r t s I I I and IV.  The consequence o f  these a c t i v i t i e s i s t h a t a o n e - c e l l e d d i p l o i d promycelium, such as t h a t d e p i c t e d i n F i g u r e s 10 and 20a, i s converted t o a f o u r - c e l l e d promycelium i n which each c e l l c o n t a i n s a s i n g l e h a p l o i d n u c l e u s (hN) ( F i g . 1 8 a ) . Cytoplasmic M i c r o t u b u l e s (Mt). - Cytoplasmic m i c r o t u b u l e s are present i n t h e d e v e l o p i n g promycelium o f U s t i l a g o hordei.  I n F i g u r e 7 a number o f them occur both i n c r o s s -  s e c t i o n and l o n g i t u d i n a l s e c t i o n .  W i t h i n the promycelium they  are o f t e n a s s o c i a t e d w i t h the nucleus and more w i l l be s a i d  about  these s t r u c t u r e s i n p a r t IV. M i t o c h o n d r i a (M). - L i k e t h e n u c l e u s , t h e s p h e r i c a l and o v o i d spore m i t o c h o n d r i a undergo a spontaneous e l o n g a t i o n ass  62  they e n t e r the promycelium randomnly  distributed.  (Pigs 5,  and 7).  They become  L o n g i t u d i n a l s e c t i o n s through the  germ tube r e v e a l many c i r c u l a r and o v a l c r o s s - s e c t i o n s o f these o r g a n e l l e s .  These seem t o cut t a n g e n t i a l l y and i t i s  l i k e l y t h a t they r e p r e s e n t s e c t i o n s through l o n g e r mitochond r i a whose i r r e g u l a r shapes wander back and f o r t h a c r o s s the section.  The maximum l e n g t h measured i n any one s e c t i o n i s  approximately 3,8  u.  I n width they range f r o m 0.2 to 0,5  u.  The numerous l o n g , p l a t e - l i k e c r i s t a e seem to l i e r o u g h l y p a r a l l e l t o the l o n g a x i s o f the mitochondrion and t o extend from one s i d e o f the o r g a n e l l e t o the o t h e r , e f f e c t i v e l y d i v i d i n g i t into  compartments ( P i g , 20),  After preparation  by method C the m a t r i x i s more e l e c t r o n dense than the c y t o plasm, g i v i n g t h e s e o r g a n e l l e s a d i s t i n c t i v e  appearance.  A f t e r glutaraldehyde-osmium f i x a t i o n the o u t e r membrane averages 73 A 90 A °  0  (Range: 61-98  A ) 0  and the i n n e r membrane averages  (Range: 73-109 A ) , 0  Lipid  ( L ) . - L i p i d bodies have been d e s c r i b e d i n p a r t I ,  A few b o d i e s , w i t h about the same s i z e range as those i n the spore, are s c a t t e r e d i n the germ tube  ( P i g s , 10, 18a, and 20a).  Spherosome-like Bodies ( S ) . - Apart from the n u c l e i the spherosome-like b o d i e s are the o n l y o r g a n e l l e s which  undergo  s i g n i f i c a n t changes i n number, appearance, and d i s t r i b u t i o n d u r i n g the development  o f the promycelium.  In F i g u r e  6,  s e v e r a l are seen j u s t p o s t e r i o r to the apex o f the newly formed metabasidium.  In the most a n t e r i o r o f these a s m a l l knot  of  f i n e l y f i b r i l l a r m a t e r i a l l i e s on one s i d e w h i l e the r e s t  of  the m a t r i x i s f i l l e d by d i s p e r s e d f i b r i l s .  Some o f the  spherosrome-like bodies a r e always among the f i r s t o r g a n e l l e s to f l o w i n t o the metabasidium. E a r l y d u r i n g the p e r i o d of e x t e n s i o n l a r g e numbers of these spherosomeM.ike b o d i e s , b o t h l a r g e and s m a l l , a r e d i s t r i b u t e d randomly throughout microscope  the cytoplasm.  In the l i g h t  they appear as conspicuous, r e f r a c t i l e bodies o f  variable size  ( P i g . llj-b) which, i n the l i v i n g organism, move ?  quickly i n a l l directions.  They have been observed  t o move  through d i s t a n c e s a t l e a s t h a l f as l o n g as the promycelium. In t i s s u e s prepared f o r e l e c t r o n microscopy by method C, the l a r g e r ones a l l appear t o be i n the conspicuous s o l i d form  (Pigs 9, 10, ltj., and 20a), and t o be surrounded  core by a  membrane which i s approximately 90 A° wide (Range: 7 l j . - l l l A°j. F i g u r e 9 i l l u s t r a t e s a number of spherosome-like o r g a n e l l e s l y i n g along a short segment o f smooth endoplasmic r e t i c u l u m . At the upper end of the membrane and t o the l e f t  (arrow) i s  a s m a l l body w i t h a tenuous "membrane", a g r a n u l a r semidense m a t r i x and a s m a l l dark core r e g i o n .  T h i s type o f  body i s r e m i n i s c e n t o f the s m a l l e r o r g a n e l l e s w i t h an i n d e f i n i t e membrane p r e v i o u s l y noted i n F i g u r e 8 (Method A ) . T h e i r appearance,  r e l a t i o n s h i p w i t h the ER, and a s s o c i a t i o n  w i t h well-developed  spherosome-like  bodies suggests  that  these smailler bodies are the f o r m a t i v e stages of the l a r g e r . Very l a r g e numbers of what have been designated , spherosome-like b o d i e s a r e present i n glutaraldehyde-osmium p r o m y c e l i a ( F i g . 20a-c). 2.3 u.  fixed  They range i n s i z e from 0.2 t o  The numbers and diameters  of these o r g a n e l l e s a r e  g r e a t e r i n promycelia that have almost a t t a i n e d  their  maximum l e n g t h ( F i g . 20a) tending r a p i d l y  than l n those which are s t i l l  ( F i g . 10).  ex-  P r o g r e s s i n g b a s i p e t a l l y , the con-  t e n t s o f the e l e c t r o n - t r a n s p a r e n t p o r t i o n o f the spherosomel l k e b o d i e s become i n c r e a s i n g l y f i b r i l l a r the base o f the promycelium  ( F i g . 20a).  shown i n F i g u r e 20 two  Toward  large  spherosome-like o r g a n e l l e s appear t o be l n the process of fusing  ( F i g . 20c).  The r e s u l t o f such f u s i o n s i s the  forma-  t i o n o f l a r g e b o d i e s w i t h m u l t i p l e e l e c t r o n dense c o r e s . l a t t e r s t r u c t u r e s are common i n budding emptying  spores ( F i g . 12, V ) .  Two  organelles  promycelia and i n  o t h e r phenomena are appa-  r e n t l y a s s o c i a t e d w i t h aging t i s s u e . rence o f pseudo-myelin  First  f i g u r e s w i t h i n the  ( F i g . 11, arrow).  The  i s the o c c u r -  spherosome-like  Second, when the organism  has  ceased t o extend, what appears to be spherosome-like b o d i e s o c c a s i o n a l l y b e g i n to b l e b v e s i c l e s i n the d i r e c t i o n of the septum ( F i g . 18b)  or the c e l l w a l l  ( F i g . 19).  F i g u r e 18a i l l u s t r a t e s a p o r t i o n o f a f o u r - c e l l e d p e r manganate f i x e d promycelium.  Contrary t o what might be  pected, the number of bodies w i t h t y p i c a l  spherosome-like  s t r u c t u r e has decreased, and the s i z e range remains 0.6  u.  ex-  0.2  to  I n F i g u r e I d a , one o f these b o d i e s has f u s e d with a  v a c u o l e , and appears to be r e l e a s i n g i t s contents (arrow). Vacuoles  ( V ) . - P r o m y c e l i a t h a t have been f i x e d i n g p e r -  manganate c o n t a i n a d i s t i n c t i v e c l a s s o f o r g a n e l l e s which, because  o f t h e i r s i m i l a r i t y to the vacuoles d e s c r i b e d i n  p a r t I , have t e m p o r a r i l y been d e s i g n a t e d by t h e same name (Fig.  18a).  Although not as i r r e g u l a r i n shape as the spore  vacuoles they possess a bounding membrane o f approximately  65  the same width as the t o n o p l a s t (Average width = 96 A )  and  0  o c c a s i o n a l l y c o n t a i n f l o c c u l e n t m a t e r i a l ( F i g . 13)*  The  number o f these bodies i n c r e a s e s w i t h p r o r a y c e l i a l age.  A  v e r y l a r g e vacuole w i t h t h e more i r r e g u l a r shape d e s c r i b e d i n p a r t I develops w i t h i n t h e aging spore as o t h e r c y t o p l a s mic contents move i n t o the promycelium ( F i g . 13)*  In g l u t a -  raldehyde-osmium f i x e d t i s s u e , there i s very l i t t l e f o r the e x i s t e n c e o f unique  evidence  corresponding s t r u c t u r e s .  The  c l o s e s t e q u i v a l e n t i s the l a r g e v a c u o l e - l i k e bodies w i t h f i b r i l l a r contents s i t u a t e d i n the spore and a t t h e base of the f o u r - c e l l e d promycelium i n F i g u r e Hj..  The extent o f the  i n c r e a s i n g v a c u o l a t i o n which o c c u r s a t the base o f the p r o mycelium i s r e a d i l y demonstrated i n l i v i n g m a t e r i a l ( F i g .  Hj.).  DISCUSSION INITIATION OF THE  GERM TUBE  Fungal germ tube w a l l s can be i n i t i a t e d e i t h e r by  exten-  s i o n o f one o f t h e p r e - e x i s t i n g l a y e r s o f t h e spore w a l l , o r w a l l (Hawker, 1966).  by s y n t h e s i s o f a new  U s t i l a g o h o r d e i resemble the l a t t e r case  1963b; Hawker, 1966;  (Hawker and  Hawker e t a l . , 1970;  Walkinshaw e t a l . , 1967).  Marchant,  y» K» 5* and 7)«  1966;  i n f e s t a n s (Eisner  m a t e r i a l i n t h i s smut forms a cap  the d e v e l o p i n g beak and t a p e r s a b r u p t l y to the s i d e s 1#  Abbott,  L i k e the w a l l l a y e r s y n t h e s i z e d  by g e r m i n a t i n g s p o r a n g i a o f Phytophthora e t a l . , 1970), the new  Teliospores of  I t never surrounds  the spore  over  (Figs.  completely  as r e p o r t e d f o r germinating sporangia o f Phytophthora  para-  sltica  (Hemmes, 1969)  (Hawker e t a l . ,  and c o n i d i a o f Cunninghamella elegans  1970).  Although d i f f i c u l t  t o prove because  of the i m p o s s i b i l i t y o f marking t h e extending w a l l l a y e r , the g e n e r a l  impression  i s t h a t growth o f the w a l l i s n o t a p i -  ca p r i o r t o spore w a l l r u p t u r e . i s synthesized and  The f i r s t m a t e r i a l , which  a t the apex ( P i g . 2) r a p i d l y becomes f i b r i l l a r  reasonably r i g i d .  A p p a r e n t l y i t i s then pushed forward,  f o r c i b l y , by t h e expanding p r o t o p l a s t . continues  Meanwhile,  down the s i d e s o f the l e n g t h e n i n g  i s completely encased.  synthesis  beak so that i t  T h i s h y p o t h e s i s would account f o r  the f a c t t h a t when the promycelium f i r s t b u r s t s through the spore w a l l t h e apex i s encased by a w a l l l a y e r which i s a l most 10 mu t h i c k .  Such a w a l l i s n o t again  u n t i l the germ tube has ceased a p i c a l  seen a t the t i p  extension.  Much of the spore's c a p a c i t y f o r s u r v i v a l r e s i d e s i n t h e t h i c k spore w a l l which keeps a l i e n f a c t o r s out, but e q u a l l y w e l l keeps the p r o t o p l a s t i n . p r o t o p l a s t and the s y n t h e s i s  Even w i t h the s w e l l i n g o f t h e  o f a hard  "cap" over the beak,  the a c t o f p e n e t r a t i o n must be d i f f i c u l t . gus  E v i d e n t l y the f u n -  "prepares" i t s path by weakening the innermost spore w a l l  l a y e r over a l o c a l i z e d area before  i t . The c h i t i n o u s  region  does not show signs o f mechanical s t r e s s but l a r g e spaces f i l led lj.a).  by e l e c t r o n dense f i b r i l l a r m a t e r i a l develop ( P i g . 2 and Such formations suggest that t h e w a l l i s weakened enzy-  matically.  F i g u r e 2 i l l u s t r a t e s that t h i s process i s a l r e a d y  well-advanced b e f o r e l a t e r a l extension  the new w a l l l a y e r i s l a i d  down.  The  of the r e g i o n o f p e r f o r a t i o n seems t o p r e -  67  cede e x t e n s t i o n o f the new  w a l l l a y e r , and  i t seems l i k e l y  the l e n g t h o f the r e g i o n o f p e r f o r a t i o n determines the  that  ulti-  mate w i d t h o f the neck r e g i o n . Many o f the previous  s t u d i e s on spore g e r m i n a t i o n have  suggested t h a t the spore w a l l i s r u p t u r e d and Abbott, 1963b; Hawker et a l . , 1970;  mechanically  (Hawker  Hashimoto e t a l . , 1958).  D u r i n g a study o f c o n i d i a l g e r m i n a t i o n i n Fusarium culmorum Marchant (1966) noted t h a t as the spore w a l l begins t o bulge p r i o r to i t s u l t i m a t e r u p t u r e ,  l t becomes d i f f u s e . and on  b a s i s he suggested t h a t enzymatic d e g r a d a t i o n was The  this  involved.  r e s u l t s i n U s t i l a g o h o r d e i support t h i s h y p o t h e s i s .  How-  ever, i t i s u n c e r t a i n whether the a r e a o f d e g r a d a t i o n extends i n t o the o u t e r spore w a l l s . suggestive outer  F i g u r e s lj.a and  6 are perhaps more  o f a mechanical s t r e t c h i n g and r u p t u r i n g o f  e l e c t r o n dense l a y e r s .  The  observations  the  o f Stocks  and  Hess (1970) i n g e r m i n a t i n g F s i l o c y b e b a s i d i o s p o r e s  a l s o sug-  g e s t t h a t both enzymatic d i s s o l u t i o n and  pressure  may  physical  be i n v o l v e d i n r u p t u r i n g the spore w a l l . What i s t h e source o f the enzymes and m a t e r i a l r e s p o n -  s i b l e f o r d e g r a d a t i o n o f the new  layer?  spore w a l l and  The most prominent o r g a n e l l e s  synthesis of  the  i n the beak r e g i o n  are endoplasmic r e t i c u l u m which i s known to be i n v o l v e d i n p r o t e i n s y n t h e s i s and be d i s c u s s e d zymes.  will  l a t e r , are b e l i e v e d to s e q u e s t e r h y d r o l y t i c  To what extent  i s unknown.  spherosome-like bodies which, as  each may  en-  be i n v o l v e d i n which f u n c t i o n  In F i g u r e 2 the f i b r i l l a r  contents o f the  spaces  forming i n the innermost w a l l l a y e r resemble the contents of  the spherosome-like fibrillar  bodies and p a r t i c u l a r l y the more f i n e l y  contents of the l a r g e spherosome-like  t r a t e d i n F i g u r e 6. bodies may  T h i s suggests t h a t the  body  illus-  spherosome-like  be the source of the f i b r i l l a r m a t e r i a l seen i n  the p e r f o r a t i o n s .  In t h e i r e l e g a n t study of hyphal t i p growth  i n Pythium ultimum. Grovej,et a l . ,  (1970) p o s t u l a t e d that  s i c l e s , s i m i l a r i n appearance t o what has been termed  ve-  sphero-  some-like bodies i n t h i s study, are r e s p o n s i b l e f o r w a l l synt h e s i s at the h y p h a l t i p and perhaps zation.  also f o r wall p l a s t i c i -  The h y p o t h e s i s i s p a r t i c u l a r l y a t t r a c t i v e  because,  i n order f o r these v e s i c l e s t o r e l e a s e t h e i r contents o u t s i d e the c e l l ,  they must f i r s t f u s e w i t h the plasma membrane and  hence serve the a d d i t i o n a l f u n c t i o n of g e n e r a t i n g the new membrane n e c e s s a r y t o cover the i n c r e a s i n g s u r f a c e area of the protoplast.  In t h i s r e s p e c t i t must be mentioned that the  ER i n the beak r e g i o n has not been observed  to give r i s e to  a s i g n i f i c a n t number of v e s i c l e s ; no spherosome-like  bodies  have been seen t o f u s e w i t h the plasma membrane, and  the  average w i d t h of the spherosome-like membranes (90 A ) i s 0  c o n s i d e r a b l y l e s s than that of the plasma membrane (121+ A ) . 0  At the beak apex the plasma membrane i s i n d i s t i n c t but does not appear t o be c r e n u l a t e d or t o undergo any n o t i c e a b l e a c tivity;  so, unless the plasma membrane i t s e l f  s y n t h e s i z e s the  enzymes, the manner i n which they pass out of the p r o t o p l a s t remains The  obscure. s i g n i f i c a n c e and frequency of the f o r m a t i o n of an  ER-sac ( F i g . 3) p r i o r t o g e r m i n a t i o n i s unknown.  Presum-  69  a b l y i t may  be a mechanism o f m o b i l i z i n g o r g a n e l l e s  i n an  ap-  p r o p r i a t e p o s i t i o n to pass i n t o the i n c i p i e n t promycelium.  (1967) has d e s c r i b e d the s i m i l a r f o r m a t i o n o f an ER-sac  Moor  p r i o r to the  i n i t i a t i o n o f a bud  romyces c e r e v i s i a e . at one  end  i n vegetative  c e l l s o f Saocha-  A c c o r d i n g to Moor's study the sac i s open  and v e s i c l e 3 are produced from the opening edge  which pass to the  cell wall.  At t h i s p o i n t the w a l l i s p l a s t l -  cized. During various and  Lowry and  s t u d i e s i n f u n g i , B e r l i n e r and D u f f  Sussman  (1965)  (1968) have noted knots o f endoplasmic  r e t i c u l u m s i m i l a r to those which are a s s o c i a t e d w i t h the ER-sac i n Ustilago hordei  and have suggested t h a t they f u n c t i o n  membrane g e n e r a t o r s (Robertson,  1961).  as  However, from t h e i r  appearance i n t h i s fungus they might e q u a l l y w e l l serve as mechanism o f membrane  a  storage.  PROMYOELIAL EXTENSION When the promycelium f i r s t  emerges i t i s encased i n a  r i g i d wall f o r a very b r i e f period.  Evidently before  extension  can o c c u r , the a p i c a l w a l l becomes p l a s t i c i z e d i n some manner and  from t h i s time u n t i l e x t e n s i o n  has  ceased the f i r s t  microns o f the promycelium are surrounded by a t h i n and  two indefi-  n i t e boundary, which g r a d u a l l y merges i n t o the t y p i c a l h y p h a l type w a l l tube.  (Average t h i c k n e s s  O b v i o u s l y the key  extension Elongation  l i e s i n the  = 65 mu)  surrounding the r e s t o f  to e i t h e r hyphal o r  promycelial  s t r u c t u r e and f u n c t i o n o f the  apex.  o f the w a l l o f such t u b e - l i k e c e l l s r e q u i r e s ,  t h a t the t i p remain p l a s t i c , second, t h a t new  the  first,  wall material  be  a c c r e t e d c o n t i n u o u s l y and t h i r d , t h a t new plasma membrane be generated  continuously.  L a s t l y , and most i m p o r t a n t l y , a  mechanism must e x i s t by which these three a c t i v i t i e s a r e controlled  and c o - o r d i n a t e d .  Bracker,  As mentionned p r e v i o u s l y , Grove,  and Morre (1970) have suggested  a system o f a p i c a l  e x t e n s i o n wherein v e s i c l e s c o n t a i n i n g the enzymes and m a t e r i a l s necessary  f o r w a l l p l a s t i c i z a t i o n and s y n t h e s i s would f u s e  w i t h the plasma membrane of the hyphal contents  t o the o u t s i d e .  t i p releasing their  Such a mechanism would, a t once s a -  t i s f y a l l three requirements  f o r hyphal t i p extension.  Sever-  a l other s t u d i e s have i n d i c a t e d an abundance o f v e s i c l e s i n hyphal 1968;  apices  (McClUre e t a l . , 1968; Brenner and C a r r o l l ,  and Hemmes and Hohl, 1969) and the authors have i m p l i -  cated these v e s i c l e s i n t i p e x t e n s i o n . U n f o r t u n a t e l y , i n U s t i l a g o h o r d e i the same problem of determining  the mode o f germ tube i n i t i a t i o n a l s o e x i s t s i n  determining  the mode of germ tube e x t e n s i o n .  t i p i s singularly "uninteresting" Its  The  cytologically  s u r f a c e i s e v i d e n t l y r e l a t i v e l y smooth.  a p i c a l corpuscles cytoplasm  The p r o m y c e l i a l . ( P i g . 20b)I  No lomasomes or  ( B a r t n i c k i - G a r c i a et a l . , 1968) a r e e v i d e n t .  a t the t i p i s a p p a r e n t l y devoid  of endoplasmic  r e t i c u l u m , v e s i c l e s and of other c e l l o r g a n e l l e s w i t h the exc e p t i o n of ribosomes. expect  In s h o r t , the o r g a n e l l e s which one might  t o f i n d a t the apex a r e n o t t h e r e .  Pour p o s s i b i l i t i e s  exist: 1.  The d e n s i t y o f f r e e ribosomes a t the apex  the r e l e v a n t s t r u c t u r e s .  obscures  71  2.  The  preparatory  techniques are not  f i n e d t o demonstrate the r e l e v a n t 3.  No  sufficiently  structures.  a p i c e s have been seen at an a p p r o p r i a t e  example, the promycelium i n Figure 20 may extending a l t h o u g h the w a l l has not y e t The  l a c k o f cytoplasmic  re-  stage; f o r  have stopped thickened.  s t r u c t u r e s at the extreme p r o -  m y c e l i a l apex r e f l e c t s the t r u e l i v i n g s t a t e d u r i n g  ex-  tension. The  problem m e r i t s f u r t h e r study. For the most p a r t , w i t h the e x c e p t i o n  and  o f the appearance  a c t i v i t i e s o f the spherosome-like o r g a n e l l e s , an extending  promycelium o f U s t i l a g o h o r d e i resembles the h y p h a l a p i c a l r e gion.  The mature p r o m y c e l i a l w a l l i s s i m i l a r i n appearance  i n width to the hyphal w a l l ( S t e i n , 1970).  To the b e s t  the author's knowledge t h i s i s t h e f i r s t r e p o r t o f the o f pores i n h y p h a l w a l l s . sent, but when present  and  of presence  The f a c t t h a t they are r a r e l y p r e -  occur i n l a r g e numbers, i n d i c a t e s t h a t  the pores are connected w i t h a p a r t i c u l a r p h y s i o l o g i c a l s t a t e o f the c e l l .  When grown on a r t i f i c i a l medium U s t i l a g o  hordei  s e c r e t e s l a r g e q u a n t i t i e s o f exoenzymes (Bech- Hansen, 1970). P o s s i b l y the pores are i n v o l v e d i n s e c r e t i o n o f exoenzymes, o r o f the mucous coat which i s represented f i b r o u s , m a t e r i a l t h a t covers the promycelium.  c y t o l o g i c a l l y by  external surface o f  the  the  S i m i l a r mucous coats have been r e p o r t e d f o r a  number o f dimorphic f u n g i (O'Hern and Henry, 19j?6; Marchant and  Smith, 1967). As a consequence o f the decrease i n cytoplasmic  density  d u r i n g germination  (Barer and Joseph 1958)  more d e t a i l e d  s t u d i e s can be made o f membranous o r g a n e l l e s .  As i n the  hyphal a p i c a l r e g i o n , d e s c r i b e d by S t e i n )1970), the endoplasmic r e t i c u l u m o f the promycelium i s s p a r s e l y d i s t r i b u t e d and l i e s m a i n l y beneath and p a r a l l e l t o the plasma membrane ( P i g . 18a).  In appearance they are i d e n t i c a l except t h a t a  s m a l l q u a n t i t y o f rough ER which has not been r e p o r t e d i n the h y p h a l a p i c e s , seems to develop i n the  promycelium.  (1966) r e p o r t e d a s i m i l a r i n c r e a s e i n the amount o f  Gorfraan  rough ER a f t e r germination i s commonly accepted  o f spores o f P u l i g o s e p t i c a . I t  t h a t the ER,  which i s sparse or absent  i n r e s t i n g s p o r e s , i n c r e a s e s g r e a t l y d u r i n g the e a r l y stages o f germination crease i n ER  (Bracker, 1967).  In U s t i l a g o h o r d e i the i n -  seems t o be c o n f i n e d to the pre-germinal  stage  of hydration. S t e i n (1970) r e p o r t e d t h a t the o u t e r m i t o c h o n d r i a l membrane was  wider than the inner i n hyphae o f U s t i l a g o h o r d e i .  T h i s has not been s u b s t a n t i a t e d i n t h i s study.  But i n most  other r e s p e c t s , i n c l u d i n g the p a r a l l e l grouping  and  longi-  t u d i n a l o r i e n t a t i o n o f the c r i s t a e , the p r o m y o e l i a l m i t o c h o n d r i a a r e s i m i l a r to the h y p h a l ones.  The  tendency f o r  the c r i s t a e to extend a c r o s s the e n t i r e o r g a n e l l e , e f f e c t i v e l y d i v i d i n g i t i n t o compartments, has f o r Microspor um  canis  (Werner et a l . , 1966)  a l s o been d e s c r i b e d and f o r the t i p  r e g i o n o f young sporangia o f Phycomyces (Peat and Banbury}  1967).  There i s no evidence  t h a t the m i t o c h o n d r i a  o f the  promycelium ever undergo the complex changes which have been r e p o r t e d i n d i f f e r e n t i a t e d r e g i o n s o f the hyphae.  An i n t e r e s t i n g aspect o f the m i g r a t i o n o f o r g a n e l l e s from spore to germ tube  i s the spontaneous a l t e r a t i o n o f shape  which some o f them undergo ( F i g s * 5 and 7)*  Por t h e  nu-  c l e u s such a change i s n e c e s s i t a t e d by the narrow dimens i o n s o f t h e neck and germ tube, but most i f not a l l o f the m i t o c h o n d r i a c o u l d a p p a r e n t l y proceed i n e i t h e r form. Thus, an elongate shape must possess other  physiological  advantages. Once g e r m i n a t i o n has begun i n U s t i l a g o h o r d e i the amount o f endoplasmic  r e t i c u l u m does not i n c r e a s e s i g n i -  f i c a n t l y , nor does the number and s i z e o f l i p i d bodies mitochondria.  and  With the e x c e p t i o n o f the extreme a p i c a l  r e g i o n which c o n t a i n s o n l y ribosomes, are d i s t r i b u t e d randomly throughout  t h e above o r g a n e l l e s  the promycelium thus  e n s u r i n g t h a t , f o l l o w i n g s e p t a t i o n and bud f o r m a t i o n , the s p o r i d i a share e q u a l l y .  The o n l y o r g a n e l l e s which  signi-  f i c a n t l y a l t e r i n number, s i z e and appearance are the spherosome-like  and v a c u o l a r o r g a n e l l e s .  I n the promycelia the number o f "permanganate-type" spherosomal bodies decreases, and t h e i r s i z e range remains the same, while the number and s i z e range o f the raldehyde -osmium- t y p e " b o d i e s both i n c r e a s e . l i g h t microscope  "gluta-  With the  v a c u o l e s can be observed forming a t the  base o f the germ tube as i t extends  ( F i g . H+b).  Organ-  e l l e s which resemble the c l a s s i c a l "vacuole" i n permanganate f i x e d m a t e r i a l are present a f t e r promycelia have been prepared by method A ( F i g . 18a)  but no  "distinctive"  organ-  e l l e s s a t i s f y i n g the n e c e s s a r y c r i t e r i a are o b s e r v a b l y present a f t e r methods B and C. What i s observed a t the base of the promycelium  are l a r g e spherosome-like bodies a p p a r e n t l y i n the  process of f u s i o n ( P i g . 20c).  T h i s suggests that the l a r g e  spherosome-like o r g a n e l l e s and f u s i o n products observed i n glutaraldehyde-osmium f i x e d t i s s u e are i d e n t i c a l t o the "vac u o l e s " of permanganate f i x e d t i s s u e . might expect t o f i n d t e r i a l which might  I f t h i s i s true  one  t r a n s i t i o n a l bodies i n KMnO^ f i x e d  ma-  i l l u s t r a t e the manner i n which the sphero-  some-like b o d i e s g i v e r i s e t o v a c u o l e - l i k e b o d i e s .  At the top  r i g h t o f the promycelium d e p i c t e d i n F i g u r e 18a the three o r g a n e l l e s marked 1. i i ,  and i i i  suggest such a sequence.  The  arrow i n F i g u r e l 8 a a l s o i n d i c a t e s a p o i n t a t which a vacuole i s p o s s i b l y e n g u l f i n g a s m a l l e r spherosome-like body. In p a r t I the "primary h y d r a t i o n v a c u o l e s " seem t o o r i g i nate from d i l a t i o n s of the ER.  Subsequent  e x t e n s i o n of the  primary vacuoles d u r i n g p r o m y o e l i a l e x t e n s i o n undoubtedly occurs through f u s i o n of these w i t h the spherosome-like organelles.  The l a t t e r a l s o seem to be d e r i v e d i n some manner  from the ER.  Thus the mature spore vacuoles i n e v i t a b l y con-  t a i n s e v e r a l e l e c t r o n dense c o r e s ( F i g s . 12 and 13). widths of the ER membranes, the t o n o p l a s t , and.the mal membranes are almost i d e n t i c a l 96 A , 0  and 90 A  0  respectively).  The vacuoles which develop  The  spheroso-  (Average widths = 91+  0  , i n the promycelium are formed  by expansion and f u s i o n of the spherosomal b o d i e s ( F i g s . and 2 0 c ) .  A ,  Formation of v a c u o l e s by the expansion and  20a  fusion  75  of  s m a l l e r elements, has been suggested f o r a l a r g e number o f  f u n g i (Buckley e t a l . , 1966;  Hyde and Walkinshaw, 1 9 6 6 ; Hawker  and Abbott. 1963a; Hawker and A b b o t t , 1963c; Smith and Marchant,  1968). of  Such vaouoles a r e a u t o l y t i c and o f t e n c o n t a i n fragments  other c e l l organelles.  I n U s t i l a g o h o r d e i t h e i r l y t i c na-  t u r e i s a l s o demonstrated through the o b s e r v a t i o n t h a t where t h e i r membranes have been damaged d u r i n g f i x a t i o n the s u r r o u n d i n g cytoplasm i s d i g e s t e d ( F i g . 20a). During f u n g a l spore g e r m i n a t i o n l a r g e v a c u o l e s commonly develop, f i r s t i n the emptying spore case and l a t e r a t t h e base o f t h e extending germ tube o r promycelium Walkinshaw, 1 9 6 6 ; Hawker and Abbott, 1963a).  (Hyde and A similar  effect  has a l s o been noted d u r i n g the e x t e n s i o n o f p o l l e n tubes (Jensen e t a l . , 1968).  That the expansion o f v a c u o l e s a t the  bases o f such t u b e - l i k e c e l l s a c t u a l l y generates the forward motion o f the cytoplasm which r e s u l t s i n a p i c a l e x t e n s i o n has been suggested by B u l l e r  (1933) and Corner ( 1 9 4 8 ) .  v a t i o n s i n U s t i l a g o h o r d e i support t h i s h y p o t h e s i s . mass o f the p r o t o p l a s t does n o t appear t o i n c r e a s e c a n t l y d u r i n g g e r m i n a t i o n and budding. promycelium  The o b s e r F i r s t , the signifi-  Second although the  ceases t o extend a p l c a l l y i n a manner which  indi-  c a t e s t h a t the " p r e s s u r e " f o r forward movement has temporar i l y ceased, t h e subsequent  p r o d u c t i o n o f buds o c c u r s almost  e x p l o s i v e l y i n a matter o f minutes.  T h i s suggests t h a t some  form o f pressure i s generated w i t h i n the tube a f t e r l t has ceased t o extend.  The promycelium i s undergoing e x t e n s i v e v a -  cuolation during t h i s period.  However, a l t h o u g h t h i s hypo-  V6 t h e s i s i s tempting, vacuoles may,  i n some or a l l such systems,  be the consequence, r a t h e r than the cause, of movement (Hyde and .Walkinshaw, 1 9 6 6 ; THE  protoplasmic  Robertson, 1 9 6 8 ) .  SPHEROSOMAL VACUOLAR SYSTEM During t h i s study the term "spherosome-like" hac  applied  somewhat a r b i t r a r i l y t o two  been  groups of o r g a n e l l e s :  group o c c u r r i n g i n KMnO^ f i x e d t i s s u e , and w i t h a q u i t e d i f f e r e n t appearance and  the other  one  group,  s i z e range ( i . e . i n the  promycelium), o c c u r r i n g i n glutaraldehyde-osmium f i x e d t i s s u e . T h i s term has  been a p p l i e d f o r h i s t o r i c a l reasons r a t h e r  from adherence t o any p a r t i c u l a r s c h o o l of thought the  s t r u c t u r e and  f u n c t i o n of these o r g a n e l l e s .  l i g h t microscopists  as spherosomes (Armentrout et a l . , 1 9 6 8 ; Frey-Wyssling  Por many years  named them "spherosomes."  v a r i o u s s t r u c t u r a l and  p l a n t s and  Sorokin  (1963) f i r s t described  to those found i n KMnOj^ f i x e d spores and h o r d e i and  concerning  have r e f e r r e d t o the m o t i l e , s p h e r i c a l , r e -  f r a c t i l e d r o p l e t s which are common i n h i g h e r  1966).  than  and  fungi  Sorokin,  bodies s i m i l a r  promycelia of U s t i l a g o  However, a c c o r d i n g  to  the  f u n c t i o n a l f e a t u r e s which these organ-  e l l e s e x h i b i t at v a r i o u s  stages i n development they might  e q u a l l y w e l l have been c a l l e d microbodies ( F r e d e r i c k et a l . , 1968;  Mollenhauer et a l . , 1966), or cytosomes ( F r e d e r i c k  Newcomb, 1 9 6 9 ) . 1968;  or lysosomes ( M a t i l e , 1 9 6 8 ;  W i l s o n et a l . , 1 9 7 0 ) .  l y reviewed the usage and p l i e d to f u n g i and  Matile* and  W i l s o n et a l . ( 1 9 7 0 ) have  Spichiger recent-  s i g n i g i c a n c e of these terms as  have j u s t l y pointed  out  that what i s  and  apnow  r e q u i r e d are e x h a u s t i v e s t u d i e s on the s t r u c t u r e , d i s t r i b u t i o n ,  77  and b i o c h e m i s t r y o f one or more o f t h e s e b o d i e s i n the same h i g h e r p l a n t o r fungus* The l i t e r a t u r e p e r t a i n i n g t o the f u n c t i o n o f these some-like bodies i s a t best c o n f u s i n g .  sphero-  The c o n f u s i o n a r i s e s  l a r g e l y from the f a c t t h a t they are common to two systems which seem d i a m e t r i c a l l y opposed. merous i n degenerating  On the one hand, they a r e v e r y nu-  systems such as h i g h e r p l a n t c o l e o p -  t i l e s and f r e e c e l l c u l t u r e s (Cronshaw, I96I4.), and aging and dying fungal t i s s u e s  (Wilson e t a l . , 1 9 7 0 ) .  they are a l s o numerous I n p l a n t meristematic ing  tissues  and d i f f e r e n t i a t -  ( F r e d e r i c k et a l . , 1 9 6 3 ) and f u n g a l t i p c e l l s  (Wilson e t a l . , 1 9 7 0 ) * presence  On the o t h e r hand  From t h i s o b s e r v a t i o n the  conspicuous  o f such o r g a n e l l e s i n the spores and p r o m y c e l i a o f  U s t i l a g o h o r d e i i s not s u r p r i s i n g *  Such a system i s not o n l y a  young d i f f e r e n t i a t i n g system b u t , i n another  sense, an aging  systemj B i o c h e m i c a l l y o r i e n t e d s t u d i e s have i n d i c a t e d t h a t  sphero-  some-like b o d i e s i n p l a n t s c o n t a i n p r o t e i n and more s p e c i f i c a l l y h y d r o l y t i c enzymes.  Most o f these s t u d i e s have been  c a r r i e d out i n h i g h e r p l a n t systems ( M a t i l e , 1969)*  Typical  lysosomal h y d r o l a s e s have been d e t e c t e d i n spherosomes o f t o bacco  ( B a l z , 1 9 6 6 ; M a t i l e and S p i c h i g e r , 1 9 6 3 ) and o f corn  (Semadeni, 1 9 6 7 ; M a t i l e , 1 9 6 8 ) . transaminase  M a t i l e ( 1 9 6 8 ) a l s o found a  i n l a r g e r spherosomes o f c o r n and two  r e d u c t a s e s i n t h e s m a l l e r ones.  Although  oxido-  t h e r e have been  fewer s t u d i e s o f t h i s type i n f u n g i s e v e r a l h i s t o c h e r a i c a l s t u d i e s have I n d i c a t e d t h a t f u n g a l spherosomes c o n t a i n a c i d  phosphatases (Armentrout et a l . , 1 9 6 8 ; Buckley et a l . , 1 9 6 8 ) . W i l s o n et a l . (1970) surveyed the Ispherosbmes" of seven d i f f e r e n t f u n g i and  concluded, on a p u r e l y c y t o l o g i e a l b a s i s ,  that the a c t i v i t i e s and  d i s t r i b u t i o n of these o r g a n e l l e s  compatible w i t h t h e i r e q u i v a l e n c e t o animal lysosomes. yeast  c e l l does not  c o n t a i n spherosomes and what has  c l a s s i c a l l y r e f e r r e d t o as t h e " v a c u o l e " has b e i n g the lysosomal e q u i v a l e n t .  been  A number of s t u d i e s have sphero-  some-like bodies o f U s t i l a g o h o r d e i a l s o c o n t a i n l i p i d a l . , 1971;  M a t i l e and  Sorokin,  1966).  Sorokin  P r e y - w y s s l i n g et a l . (1963) and  Semadeni  bodies i n h i g h e r p l a n t s .  Gay  a l . (1968) have a s s o c i a t e d  m o b i l i z a t i o n of l i p i d  and  In a d d i t i o n to smaller  "sphero-  i n Pythium ultimum ( G r o v e e t s a l l , 1 9 7 0 ) ; i t has 5  a l s o t r a n s p o r t the enzymes and  other m a t e r i a l s r e q u i r e d f o r w a l l s y n t h e s i s and w a l l  plasti-  The m u l t i p l i c i t y of f u n c t i o n s i n which f u n g a l  spherosomes are t e n t a t i v e l y i m p l i c a t e d ing;  the  seem t o be i n v o l v e d i n h y p h a l t i p  been suggested that they may  cization.  Buckley  f u n g a l spherosomes w i t h the  to form membranes.  somes" of U s t i l a g o h o r d e i  lipid  Greenwood (196I4.) and  above f u n c t i o n s v e s i c l e s which resemble the  extension  and  (Buckley et a l . , 1966;  (1967) have suggested that spherosomes g i v e r i s e to  et  (Allen  S p i c h i g e r , » 1 9 6 8 ; McKeen, 1 9 7 0 ) ,  more p a r t i c u l a r l y p h o s p h o l i p i d and  The  the appearance of  i n d i c a t e d t h a t i n other f u n g i , bodies s i m i l a r t o the  et  are  i s at f i r s t  disturb-  however, o b v i o u s l y i n organisms as c y t o l o g i c a l l y "simple"  as most f u n g i , some of the o r g a n e l l e s must perform more than one  function.  In U s t i l a g o h o r d e i the s t r u c t u r e and f u n c t i o n of the organ e l l e s which we have termed spherosome-like  bodies and  vacuoles  suggest that these s t r u c t u r e s a c t u a l l y r e p r e s e n t stages i n the d i f f e r e n t i a t i o n and development of a s i n g l e o l a r system.  spherosomal-vacu-  The d i f f e r e n c e s i n the s t r u c t u r e of these  organ-  e l l e s a f t e r v a r i o u s methods of f i x a t i o n , and the changes i n t h e i r appearance and d i s t r i b u t i o n d u r i n g g e r m i n a t i o n can be accommodated by the f o l l o w i n g h y p o t h e s i s . 1.  The  spherosome-like  b o d i e s i n U s t i l a g o h o r d e i con-  t a i n p r o t e i n s and l i p i d s p r o t e i n complex).  (perhaps i n the form of a l i p o -  The p r o t e i n s a r e , a t l e a s t i n p a r t ,  enzymes. 2.  The permanganate-type spherosome-like  meter = 0.11  - o.56  bodies  (Dia-  u) are i d e n t i c a l t o the s m a l l s i z e d  glutaraldehyde-osmium  f i x e d ones (Diameter = 0.21+  - 0.86u).  The vacuoles observed  i n p r o m y c e l i a f i x e d by method A are  the permanganate e q u i v a l e n t of the l a r g e r g l u t a r a i d e h y e osmium f i x e d  3.  spherosome-like b o d i e s  (Diameter more than  E i t h e r the c o n t e n t s of the spherosome-like  change throughout  development or e l s e d i f f e r e n t  are a c t i v a t e d at d i f f e r e n t p o i n t s i n time.  bodies contents  Consequently  these o r g a n e l l e s are i n v o l v e d i n a v a r i e t y of a c t i v i t i e s , some of which may multaneously  be a u t o l y t i c , s y n t h e t i c or b o t h .  Si-  c o n t r o l l e d a l t e r a t i o n i n the c o n t e n t s and  l o c a l i z a t i o n of these bodies have profound d i f f e r e n t i a t i o n of the  organism.  e f f e c t on the  80  i+.  A l t e r a t i o n i n the s t r u c t u r e o f the c y t o l o g i c a l l y ob-  servable  contents can be  c o r r e l a t e d with changes i n enzyme  activity. These f o u r p o i n t s are e l a b o r a t e d As has  i n the, f o l l o w i n g d i s c u s s i o n .  been p r e v i o u s l y i n d i c a t e d , ample evidence e x i s t s  i n the l i t e r a t u r e to support the concept of a l i p i d - p r o t e i n m a t r i x i n spherosome-like bodies (Sorokin and That the  Sorokin,  1966).  "spherosomes" o f U s t i l a g o h o r d e l c o n t a i n l i p i d ,  l e a s t i n some stages o f t h e i r development, i s evident  at  from  the f a c t they o f t e n c o n t a i n p s e u d o m y e l i n - l i k e f i g u r e s ( P i g . Buckley e t a l . (1966) and Gay such f i g u r e s i n the  11).  and Greenwood (1966) d e s c r i b e d  "spherosomes" o f other f u n g i and  inter-  p r e t e d them as a stage i n the m o b i l i z a t i o n o f l i p i d f o r membrane s y n t h e s i s .  F i g u r e s 18b  and 19  i l l u s t r a t e more c l e a r l y  t h a t spherosome-like o r g a n e l l e s i n U s t i l a g o h o r d e i can  evi-  d e n t l y g i v e r i s e to membranes which are spontaneously blebbed off  i n the form o f v e s i c l e s i n the d i r e c t i o n o f the plasma-  lemma.  These membranes appear to have a r e g u l a r  tripartite  s t r u c t u r e and  t h e i r width i s s i m i l a r to the w i d t h o f the  ma membrane.  During a recent  technique was  a p p l i e d to the dormant t e l i o s p o r e s o f the smut  T i l l e t i a caries  study i n which a f r e e z - e t c h  A l l e n e t a l . (1971) d e s c r i b e d  u n i d e n t i f i e d o r g a n e l l e ) which resembles the bodies o f U s t l l a g o h o r d e i . sent  i n these o r g a n e l l e s .  L i p i d was As no  a body ( i . e .  spherosome-like  demonstrated to be  cytochemistry  tempted, the presence o f p r o t e i n has the a p p a r e n t l y  plas-  has  pre-  been a t -  been i n f e r r e d f i r s t  from  h y d r o l y t i c (and t h e r e f o r e enzymatic) a c t i v i t i e s  Si  of  these bodies and  secondly from known r e a c t i o n s of the  dif-  f e r e n t p r e p a r a t o r y techniques on v a r i o u s b i o c h e m i c a l components. The f i n a l i d e n t i f i c a t i o n of the contents of the spherosomel i k e bodies w i l l depend upon the use of a p p r o p r i a t e b i o c h e m i c a l or h i s t o c h e m i c a l t e c h n i q u e s . One  of the problems i n the l i t e r a t u r e on  "spherosomes"  i s that the bodies which are u s u a l l y designated as spherosomes i n permanganate f i x e d m a t e r i a l are q u i t e d i f f e r e n t i n appearance from those so designated i n glutaraldehyde-osmium material.  fixed  U n f o r t u n a t e l y few worker have simutaneously  em-  ployed b o t h t e c h n i q u e s , causing doubt t h a t the c o n c l u s i o n s drawn from d i f f e r e n t elles.  s t u d i e s a c t u a l l y apply t o the same organ-  The f o l l o w i n g f a c t o r s suggest t h a t i n U s t i l a g o h o r d e i  the bodies l a b e l l e d  spherosome-like  a f t e r the d i f f e r e n t p r e -  p a r a t o r y methods are the same: 1.  The appearance of these bodies a f t e r methods A,  and C i s compatible w i t h the expected  B,  e f f e c t s of each  technique on a l i p i d - p r o t e i n c o n t a i n i n g body. 2.  T h e i r numbers, d i s t r i b u t i o n , and  s i z e range i n hy-  d r a t i n g spores i s the same (Pt. I ) . 3.  They c l u s t e r i n an i d e n t i c a l manner t o one s i d e of  the d e v e l o p i n g beak i n germinating I}..  spores.  The a l t e r a t i o n i n number, s i z e , and appearance i n  p r o m y c e l i a i s compatible w i t h the h y p o t h e s i s t h a t the permanganate-type spherosomal body i s i d e n t i c a l w i t h the s m a l l - s i z e d glutaraldehyde-osmium  ones and  that the  permanganate-type vacuoles which develop i n aging p r o -  raycelia  are i d e n t i c a l w i t h the l a r g e - s i z e d g l u t a r a l d e h y d e -  osmium spherosome-like  bodies which a l s o develop i n aging  promycelia. Assuming t h a t these v a r i o u s m a n i f e s t a t i o n s a c t u a l l y r e p r e sent stages In the d i f f e r e n t i a t i o n of a s i n g l e system the a c t i v i t i e s i n which the system has so f a r been i m p l i c a t e d can be summarized, i n the order o f occurrence, as f o l l o w s : 1.  d e g r a d a t i o n o f l i p i d bodies d u r i n g pre-germinal de-  velopment ( P t . I, P i g . 8 . ) 2.  spore w a l l d e g r a d a t i o n and p o s s i b l e s y n t h e s i s o f the ( P i g s . 1 and 2 ) .  new p r o m y c e l i a l W a l l 3.  c y t o p l a s m i c d e g r a d a t i o n o c c u r r i n g o u t s i d e the ER-sac  i n a g e r m i n a t i n g spore  (Pig. 8).  1L.  p r o m y c e l i a l w a l l p l a s t i c i z a t i o n and p o s s i b l y s y n t h e s i s ,  5.  f o r m a t i o n of " v a c u o l e s " by o r g a n e l l e expansion  and/or  fusion. A l l o f these a c t i v i t i e s seem t o i n v o l v e a h y d r o l y t i c component .and on t h i s b a s i s i t I s tempting l a g o h o r d e i the spherosomal-vacuolar somal system.  t o suggest t h a t U s t i -  system i s a l s o the l y s o -  The a c t u a l i d e n t i f i c a t i o n o f these o r g a n e l l e s  as lysosomal e q u i v a l e n t s awaits f u t u r e b i o c h e m i c a l and h i s t o c h e m i c a l evidence.  F u n c t i o n s 1,  2, 3 , and if, mainly i n -  volve the s m a l l e r c l a s s of thse bodies appear as spherosome-like  a f t e r KMnO^ f i x a t i o n ) ; f u n c t i o n 5  i s c o n f i n e d t o the l a r g e r c l a s s v a c u o l e - l i k e a f t e r KMnO^).  ( i . e . those which  ( i . e . those which appear as  An i n t e r e s t i n g o b s e r v a t i o n i s that  d u r i n g s t u d i e s on the lysosomes of r o o t t i p c e l l s o f corn  83  seedlings Matile  (1968) has i d e n t i f i e d two c l a s s e s o f lysosomes  which correspond i n s i z e - range and a c t i v i t i e s to the two c l a s s e s o f spherosome-like bodies d i s c u s s e d i n U s t i l a g o h o r d e i . The heavy lysosomes which c o n t a i n h y d r o l a s e s ,  transaminases,  a r e small spheres 0.1 t o 0.3 u i n diameter,  and oxidoreductases  with membranes resemling  the ER membranes.  The " l i g h t l y s o -  somes" o r "small v a c u o l e s " whioh c o n t a i n h y d r o l a s e s transaminase range i n s i z e from 0,3 The  contents  ior  t o 1.5 u i n diameter.  o f the spherosome-like b o d i e s condense i n t o  dense c o r e s , t h i c k f i b r i l s fine f i b r i l s .  and one  ( i . e . i n pre-germinal  The contents  spores), or  o f the l a r g e "spherosome" p o s t e r -  t o the apex o f the newly formed promycelium i n F i g u r e 6  are o f the f i b r i l l a r  type.  The l a r g e r spherosomes have dense  c o r e s , but t h e y a l s o c o n t a i n a h i g h e r p r o p o r t i o n o f f i b r i l l a r m a t e r i a l than the s m a l l e r ones. (Fig.  20)  and l a r g e vacuoles  I n f u s i n g spherosomal bodies  ( F i g s . 12,  t e n t s are u s u a l l y a l s o p r e s e n t .  13 and 11+)  The c y t o l o g i c a l l y  the confibrillar  s t a t e may r e f l e c t the b i o c h e m i c a l l y s t a t e o f the enzymatic contents•  CONCLUSION I n i t i a t i o n o f p r o m y c e l i a l development i n U s t i l a g o h o r d e i i n v o l v e s the c o n t r o l l e d i n t e g r a t i o n o f t h r e e separate ses : l o c a l i z e d degradation  proces-  o f the spore w a l l , s w e l l i n g o f t h e  p r o t o p l a s t , and s y n t h e s i s o f a new l a y e r o f w a l l m a t e r i a l . F u r t h e r e x t e n s i o n o f the promycelium r e q u i r e s a balanced system o f w a l l p l a s t i c i z a t i o n , w a l l a c c r e t i o n , and plasma membrane  s y n t h e s i s i n the r e g i o n o f the apex.  No c y t o l o g i c a l evidence  has been o b t a i n e d i n t h i s organism t o i n d i c a t e what the s t r u c t u r a l b a s i s f o r t h i s system might During the promycelium  be.  e x t e n s i o n most o f the cytoplasm flows  i n t o the t u b e ; the remaining spore cytoplasm becomes h i g h l y vacuolate.  The endoplasmic r e t i c u l u m , m i t o c h o n d r i a and  lipid  b o d i e s are d i s t r i b u t e d randomnly and do not i n c r e a s e n o t i c e ably i n t o t a l quantity.  One  o f t h e major f u n c t i o n s o f the  ER i n h y d r a t i n g and g e r m i n a t i n g spores seems t o be the f o r mation, d i r e c t l y o r i n d i r e c t l y , o f bodies s a t i s f y i n g c l a s s i c a l d e f i n i t i o n o f the fungus v a c u o l e . g e s t i o n was  the  I n p a r t I , a sug-  made t h a t t h e "primary h y d r a t i o n v a c u o l e " i s formed  d i r e c t l y v i a d i l a t i o n o f the ER intermembrane space.  Subse-  quent  fusion  "vacuole" f o r m a t i o n o c c u r s by t h e expansion and  o f s m a l l e r "spherosome-like" o r g a n e l l e s , which a l s o may  be  d e r i v e d i n some manner from the endoplasmic  Dur-  reticulum.  i n g t h e t r a n s i t i o n from the spherosome-like s t a t e t o the v a c u o l e - l i k e s t a t e these bodies e v i d e n t l y perform a number o f f u n c t i o n s a l l o f which i n v o l v e a h y d r o l y t i c component, and a l l o f which have profound e f f e c t s on the d i f f e r e n t i a t i o n o f the organism.  The  c y t o l o g i c a l evidence i s compatible w i t h the  h y p o t h e s i s t h a t the spherosomal-vacuolar  system l n U s t i l a g o  h o r d e i i s the f u n c t i o n a l e q u i v a l e n t o f the l y s o s o m a l i n animal c e l l s  (DeDuve and Wattiaux, 1966).  system  However, as  some evidence i n d i c a t e s t h a t f u n g a l spherosome-like b o d i e s peform a n a b o l i c f u n c t i o n s ( i . e . w a l l s y n t h e s i s ) as w e l l as c a t a b o l i c the a d o p t i o n o f a more " r e s t r i c t i v e "  nomenclature  may  does not seem to be j u s t i f i e d at p r e s e n t .  II.  PLATE 1  F i g u r e 1.  A g e n e r a l view of a t e l i o s p o r e a t germ tube i n i t i a t i o n . Note the l a r g e p e r f o r a t i o n s (p) i n the inner spore w a l l opposite the protoplasmic beak. The new p r o m y c e l i a l w a l l (pW) has begun t o form over the beak. ER and spherosome-like bodies (S) c l u s t e r i n the beak r e g i o n . The p r o t o p l a s t c o n t a i n s many l a r g e matochondrial and primary v a c u o l e s . Note t h a t the p r o t o p l a s t has p u l l e d away from the inner spore w a l l over most o f the s u r f a c e except i n the beak r e g i o n . Method A. c a . X 25,2000.  F i g u r e 2.  The beak a t the e a r l i e s t stages o f germ tube i n i t i a t i o n . The f i r s t p r o m y c e l i a l w a l l (pW) m a t e r i a l i s present a t the extreme t i p o f the beak. Large p e r f o r a t i o n s (p) w i t h f i b r i l l a r e l e c t r o n dense contents are a l r e a d y present on the inner spore w a l l opposite the beak. Note the c h a r a c t e r i s t i c presence o f the spherosome-like b o d i e s ( S ) . Method C. c a . X 41}.,600.  II.  PLATE 2  F i g u r e 3*  A g e n e r a l view of a t e l i o s p o r e at a s l i g h t l y more advanced stage of promycelium f o r m a t i o n . Note the presence of the continuous ER-sac w i t h i t s a s s o c i ated spherosome-like bodies (S) and knots of endoplasmic r e t i c u l u m (arrows). Most of l i p i d bodies (L) vacuoles (V) and mitochondria l i e i n s i d e of the s a c . Method A, c a . X 19,000.  F i g u r e lj.a.  The beak r e g i o n j u s t p r i o r t o spore w a l l r u p t u r e . Note t h a t the t h i n l a y e r of new w a l l m a t e r i a l (pW) completely surrounds the beak. A l s o note the ext e n s i v e p e r f o r a t i o n s (p) i n the i n n e r s s p o r e w a l l opposite the beak and the i n d i c a t i o n s of s t r e s s i n the outer spore w a l l l a y e r s opposite the beak. P e r i p h e r a l ER elements are p r e s e n t . The d i p l o i d nucleus (dN) has moved i n t o p o s i t i o n behind the beak. Method B. c a . X 22,£00.  F i g u r e l+b.  A t h i o k s e c t i o n prepared by method A and s t a i n e d i n T o l u i d i n e b l u e . Note the c h a r a c t e r i s t i c p o s i t i o n of the n u c l e u s , c a . X b , , 4 0 0 .  F i g u r e 5»  M i g r a t i o n of the d i p l o i d nucleus (dN) and mitochond r i a (M) i n t o the young promycelium. Note the e l ongate forms. A l s o note the c h a r a c t e r i s t i c p o s i t i o n s of the n u c l e a l u s (Nu) and the c e n t r i o l a r k i n e t o c h o r e - e q u i v a l e n t (CKE). The p r o m y c e l i a l w a l l (pW) extends through the neck of the r u p t u r e d spore w a l l but does not completely encase the spore. Method B. c a . X 17,800.  II.  PLATE 3  F i g u r e 6.  Spore w a l l r u p t u r e . A well-formed w a l l l a y e r (pW) i s present about t h e new p r o m y c e l l i a l t i p . Note the "exploded" spore w a l l d e b r i s . S e v e r a l l a r g e spherosome-like bodies (S) c o n t a i n i n g e l e c t r o n dense cores and f i n e f i b r i l l a r m a t e r i a l are present Just b e h i n d the p r o m y c e l i a l apex. L i p i d (L) and the i n n e r spore w a l l p e r f o r a t i o n s (p) are i n d i c a t e d . Method C. ca. X 2 9 , 7 5 0 .  Figure 7 .  M i g r a t i o n i n t o the promycelium. Note the l a r g e spore vacuole (V), the elongated m i t o c h o n d r i a (M) and the presence o f m i c r o t u b u l e s (mt). The prom y c e l i a l w a l l (pW) and a young spherosome-like body are a l s o i n d i c a t e d . Method C. c a . X 3 5 - 5 5 0 .  II. F i g u r e 8.  PLATE k  Part of a g e r m i n a t i n g spore showing the f u s i o n of ER elements t o form the ER-sac and showing the r e l a t i o n s h i p of the sac membranes t o the spherosomel i k e bodies (S) and v a c u o l e s ( V ) . Method A. ca.  X 35,700.  F i g u r e 9.  A s e c t i o n through a promycelium i l l u s t r a t i n g the p o s i t i o n a l r e l a t i o n s h i p between smooth ER and the young spherosome-like b o d i e s (arrow) and the smooth ER and the mature spherosome-like bodies ( S ) .  Method C.  F i g u r e 10.  c a . X 35,7000.  A l o n g i t u d i n a l s e c t i o n through a p a r t of a promyc e l i u m showing a d i p l o i d nucleus (dNf), spherosomel i k e bodies ( S ) , l i p i d bodies ( L ) , and mitochond r i a (M), and a l a r g e number of u n i d e n t i f i e d v e s i c l e s . Method C. c a . X 23,700.  II. Figure 1 1 .  PLATE 5  Large spherosome-like bodies w i t h dense cores and pseudomyelinate f i g u r e s (arrow) i n an aging germi n a t e d spore. Method C. ca. X 29,475.  Figure  12.  Large vacuole (V) c o n t a i n i n g m u l t i p l e e l e c t r o n dense cores and l a r g e q u a n t i t i e s of e l e c t r o n dense f i b r i l l a r m a t e r i a l i n an aging germinated spore. Method C. ca. X 1 5 , 7 5 0 .  Figure  13.  Large spore v a c u o l e (V) c o n t a i n i n g e l e c t r o n dense f i b r i l l a r m a t e r i a l and l a r g e e l e c t r o n t r a n s p a r e n t p a t c h e s . Free spherosome-like b o d i e s (S) are numerous i n the v i c i n i t y of the v a c u o l e . Method A. ca. X 1 9 , 9 5 0 .  F i g u r e ll^ar.  B a s a l c e l l of a f o u r - c e l l e d promycelium showing the presence of a l a r g e vacuole (V) w i t h e l e c t r o n dense f i b r i l l a r c o n t e n t s . Method C. sea. X 1 4 , 0 0 0 .  Figure llfb.  A l i v i n g t w o - c e l l e d promycelium viewed withyphase o p t i c s . Note the presence of the b a s a l vacuoles and the r e f r a c t i l e appearance of the spherosomel i k e bodies, ca. X 2 , 5 0 0 .  II.  PLATE 6  Figure 1 5 .  A l o n g i t u d i n a l s e c t i o n through a promycelium showing stacked ER l y i n g near t o and l n p a r a l l e l w i t h the p r o m y c e l i a l w a l l . V e s i c l e s (ve) are o f t e n l o c a t e d near such ER s t a c k s . Method G. ca. X 4 8 , 0 0 0 .  F i g u r e 16a.  A l o n g i t u d i a l s e c t i o n through a o n e - c e l l e d promyc e l i u m i n which many pores can be seen opening from the p r o t o p l a s t through the c e l l w a l l t o the o u t s i d e . Method C. c a . X 3 2 , 7 5 0 .  F i g u r e 16b.  An e n l a r g e d view o f one of the pores d e p i c t e d i n F i g u r e 16a. Method C. c a . X 5 2 , 4 0 0 .  F i g u r e 16c.  Two other pores from the same promycelium 16a. Method C. c a . X 5 2 , 4 0 0 .  F i g u r e 17.  A h a p l o i d nucleus (hN) w i t h i n the spore a f t e r germi n a t i o n . The arrow i n d i c a t e d the chromatinn u c l e o l a r c o n n e c t i o n . Note t h a t on the s i d e of the c o n n e c t i o n o p p o s i t e the n u c l e o l u s the chromatin strands are c l e a r l y v i s i b l e . A short segment of rough ER l i e s c l o s e t o the n u c l e a r envelope. Method C. c a . X 5 3 , 5 5 0 .  as i n  II.  PLATE 7  Figure  18a.  A g e n e r a l view o f one o f the c e l l s i n a f o u r c e l l e d promycelium. Note the h a p l o i d nucleus (hN) w i t h simple n u c l e a r pores (NP) and the mitochondria.L^JER and l i p i d (L) i s s c a n t . On the r i g h t a spherosome-like body (S) i s b e i n g e n g u l f e d by a l a r g e v a c u o l a r - s t r u c t u r e ( V ) . The bodies on the upper r i g h t l a b e l l e d i , i i , and i i i suggest a p o s s i b l e sequence o f convers i o n o f spherosome-like bodies ( i ) to v a c u o l e l i k e bodies ( i i i ) . V e s i c l e s (ve) are o f t e n conspicuous between the v a c u o l e s (V) o r the spherosome-like bodies (S) and the c e l l w a l l . Method A. c a . X 53,£00.  Figure  18b.  An e n l a r g e d view o f one o f the spherosomel i k e bodies (S) i n F i g u r e Ida which seems t o be g i v i n g r i s e to v e s i c l e s which pass towards the septum. Method A, c a . X 95,200.  Figure  19.  P a r t o f a promycelium showing membrane f o r m a t i o n w i t h i n a spherosome-like body and the b l e b b i n g of v e s i c l e s (ve) from the spherosome-like body towards the c e l l w a l l . Short segments of rough ER (arrow) are present near the h a p l o i d n u c l e u s (hN). ER i s o f t e n a l s o a s s o c i a t e d w i t h the spherosome-like bodies ( i . e . lower r i g h t ) . Method B. c a . X 1+1.000.  II.  PLATE 8  Figure  20a.  A l o n g i t u d i n a l s e c t i o n through a s i n g l e - c e l l e d promycelium showing the appearance and d i s t r i b u t i o n of the mitochondria (M), l i p i d b o d i e s (L) and spherosome-like bodies ( S ) . Method C. ca. X 13,800.  Figure  20b.  The apex of the promycelium d e p i c t e d i n F i g u r e 20a. Note the l a c k of c y t o l o g i c a l l y d i s t i n c t s t r u c t u r e s i n the extreme t i p . Method C. ca. x 32,200.  Figure  20c.  F u s i o n of two spherosome-like b o d i e s t o form a l a r g e v a c u o l a r s t r u c t u r e . Note the two e l e c t r o n dense cores and the l a r g e amount of f i b i l l a r m a t e r i a l i n these o r g a n e l l e s ( s ) . Part of the bounding membranes between the two f u s i n g organe l l e s (s) i s s t i l l p r e s e n t . Method C. ca X 32,200.  S6;  BIBLIOGRAPHY A l l e n , J.V., Hess, W.M., and Weber, D.J. 1 9 7 1 . U l t r a s t r u c t u r a l I n v e s t i g a t i o n o f dormant T i l l e n t i a c a r i e s t e l i o s p o r e s . Mycologia 6 3 : 144-15>6. Armentrout, V.N., Smith, G.C., and Wilson, C L . 1 9 6 8 . Spherosomes and mitochondria i n the l i v i n g f u n g a l c e l l . Am. J . Bot. 5 5 : 1 0 6 2 - 1 0 6 7 . 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M i c r o b i o l .  60* 181-189.  Hemmes, D.E. and Hohl, H.R. 1 9 6 9 . U l t r a s t r u c t u r a l changes i n d i r e c t l y g e r m i n a t i n g s p o r a n g i a o f Phytophthora p a r a s i t i c a .  Am. J . Bot. j>6: 300-313.  Hyde, J.M. and Walkinshaw, C.H. 1966. Ultrastructure of b a s i d i o s p o r e s and mycelium o f L e n z i t e s s a e p l a r l a . J . Bacteriol. ^ 2 : 1218-1227. Jensen, W.A., F i s h e r , D.B., and Ashton, M.E. 1 9 6 8 . Cotton embryogenesis: the p o l l e n cytoplasm. P l a n t a 8 l _ : 206-228. Lowry, R . J . and Sussman, A.S. 1 9 6 4 . U l t r a s t r u c t u r e o f the e r m i n a t i n g Neurospore ascospore. Am. J . Bot. $1: 66 Abstract).  t  Lowry, R.J, and Sussman, A.D, 1968. U l t r a s t r u c t u r a l changes d u r i n g g e r m i n a t i o n o f ascospores o f Neurospore t e t r a sperma. J . gen. M i c r o b i o l . £ l : 4 0 3 - 4 0 9 . Marchant, R. 1966. Pine s t r u c t u r e and spore germination i n Fusarium culmorum. Ann. 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M i k r o b i o l . 60: 340-347. ; S o r o k i n , H.P. and S o r o k i n , S. 1 9 6 6 . The spherosomes o f Campanula p e r s i c l f o l i a L. A l i g h t and e l e c t r o n microscope's t u a y . protoplasms 6 2 : 2 1 6 - 2 3 6 . S t e i n , C.W. 1970. An e l e c t r o n microscope study o f a m y c e l i a l mutant o f U s t i l a g o h o r d e i (Pers.) Lagerh. M.Sc. T h e s i s . U.B.C., Vancouver, UAJN.  Stocks, D . L . j a n d Hess, W.M. 1970. U l t r a s t r u c t u r e o f dormant and germinated b a s i d i o s p o r e s o f a s p e c i e s o f P s i l o c y b e .  Mycologia 62: 176-191.  Walkinshaw, G.H., Hyde, J.M., and Zaridt, J . van 1967. Fine® s t r u c t u r e o f q u i e s c e n t and germinating aecioapores o f Cronartium f u s l f o r m e . J . B a c t e r i o l . 9jyi 2k$-2$k» Werner, H.J., J o l l y , H.w., and Spurlock, B.O. 1966. Electron microscope o b s e r v a t i o n s o f the f i n e s t r u c t u r e o f M i c r o sporum c a n l s • 3 I n v e s t . Dermatol. I+Jb: 130-13U. Werner, H.J. and L l n d b e r g , G.D. 1966 E l e c t r o n microscope o b s e r v a t i o n s o f Helminthosporium v i c t o r i a e . J . gen. M i c r o b i o l , hgx 123=I2TT * W i l s o n , C&L•, S t i e r s , D.L., and Smith, G.G. 1970. lysosomes or spherosomes. Phytopathology 60:  Fungal 216-227.  PART I I I P r o m y c e l i a l S e p t a t i o n and S p o r i d i a l  Formation  TABLE OF CONTENTS Page ABSTRACT  92  INTRODUCTION  92  MATERIALS AND METHODS  9^ 9*1  OBSERVATIONS  9k-  Septation Sporidium Formation  •••••••••  100  DISCUSSION Septation Sporidium Formation  98  100  « e  108  CONCLUSION  110  BIBLIOGRAPHY  112  PART I I I I n i t i a t i o n of the Promycelium and P r o m y c e l i a l  Extension  ABSTRACT In the p r o m y c e l i a complete*  o f U s t l l a g o h o r d e l c r o s s w a l l s are  The p a t t e r n o f s e p t a t i o n resembles t h a t found  i n actinomycetes, i n C o o c i d l o i d e s l m m i t i a , and i n E x i d i a nucleata.  E l a b o r a t e membrane complexes have been a s s o c i -  ated w i t h the i n i t i a t i o n o f the c r o s s w a l l *  Two v a r i a t i o n s  of the normal s e p t a t i o n p a t t e r n are d e s c r i b e d and the r e s u l t s of these v a r i a t i o n s are d i s c u s s e d . The  i n i t i a t i o n of s p o r i d i a involves a l o c a l i z e d p l a s -  t i c i z a t i o n of the p r o m y c e l i a l w a l l f o l l o w e d by  degradation  of the o l d w a l l and subsequent s y n t h e s i s of new w a l l material.  A t present no i n f o r m a t i o n i s a v a i l a b l e t o i n d i c a t e  the manner i n which the mature sporidium the parent  i s separated  from  cell.  INTRODUCTION When the promycelium o f U s t l l a g o h o r d e i has almost a t t a i n e d i t s masimum l e n g t h the d i p l o i d nucleus meiosis.  The f i r s t n u c l e a r d i v i s i o n  undergoes  & e . reduction  s i o n ) i s f o l l o w e d immediately by the f o r m a t i o n septum g i v i n g r i s e t o a t w o - c e l l e d promycelium.  divi-  of a s i n g l e E a c h of  the h a p l o i d n u c l e i a g a i n d i v i d e s (&e. e q u a t i o n a l d i v i s i o n ) and a second round of c e l l d i v i s i o n occurs w i t h the almost  simultaneous  establishment  of two more s e p t a .  Once the  f o u r - c e l l e d s t a t e has been a t t a i n e d each c e l l g i v e s r i s e to  an a d d i t i o n a l daughter c e l l formed by a process  y e a s t - l i k e budding.  of  During p r o d u c t i o n of the bud ( i . e .  s p o r i d i u m or b a s i d i o s p o r e ) the parent c e l l nucleus d i v i d e s a g a i n , m i t o t i c a l l y , one  of the daughter n u c l e i becoming  s i t u a t e d i n the s p o r i d i u m and the other i n the parent T h i s study i s concerned  cell.  w i t h the u l t r a s t r u c t u r a l c y t o p l a s m i c  events which occur d u r i n g s e p t a t i o n and budding, A s p o r i d i u m i s of one 4- and -•  of two mating types  designated  G e n e r a l l y a promycelium g i v e s r i s e t o e q u a l num-  b e r s of each t y p e .  The  i n f e c t i v e dlkaryon i n U s t i l a g o  h o r d e i i s u s u a l l y formed by the f u s i o n of two opposite mating type i n the presence  s p o r i d i a of  of a s u i t a b l e h o s t .  However, among smuts an a l t e r n a t i v e mechanism e x i s t s whereby a d i k a r y o n may Safeeulla,  be e s t a b l i s h e d ( D i c k i n s o n . 1927;  I960),  Duran and  A c y t o p l a s m i c b r i d g e forms d i r e c t l y  tween a p l u s and a minus p r o m y c e l i a l c e l l .  In t h i s  u l a r smut s p e c i e s such b r i d g e s only form between  be-  partic-  immediately  adjacent c e l l s , and are o f t e n r e f e r r e d t o as " k n e e - j o i n t s " . The  apex of the b r i d g e then assumes the p r o p e r t i e s of a  hyphal apex e s t e n d i n g t o produce a b r a n c h . n u c l e i of the c o n f l u e n t c e l l s migrate to  e s t a b l i s h a dikaryon.  The  undivided  i n t o the branch r e g i o n  During t h i s study the  ultrastruc-  t u r a l b a s i s of b r i d g e f o r m a t i o n has been e s t a b l i s h e d .  MATERIALS AND The  materials  METHODS  and techniques are  previously described  ( F t s . I and  i d e n t i o a l t o those  I I ) , the t i s s u e  f i x e d a f t e r 5> t o 7% hours of i n c u b a t i o n . in Figure  10 was  b u f f e r e d at pH served  The  f i x e d f o r 12 hours i n 2.0$  7.2  w i t h 0.01  M« cacodylate  being  promycelium glutaraldehyde  b u f f e r and  d i r e c t l y u s i n g phase c o n t r a s t o p t i c s as  ob-  described  in part I I .  OBSERVATIONS SEPTATION: In U s t i l a g o h o r d e i a simple I n v a g i n a t i o n membrane i n i t i a t e s septum f o r m a t i o n ure  of the  plasma  ( F i g . l a , arrows).  Fig-  l b c l e a r l y demonstrates the u n i t membrane d e l i m i t i n g the  septum i n i t i a l .  Throughout development of the septum t h i s  membrane seems t o be  somewhat narrower (Average w i d t h = 100  than the plasma membrane l i n i n g the l a t e r a l p r o m y c e l i a l or the mature septum (Average w i d t h = 121+ A ) . 0  A ) 0  walls  At the  frontal  edge of the e a r l y i n v a g i n a t i o n the membranes are c l o s e l y appressed  f o r a distance  of $0 t o 100  by an e l e c t r o n t r a n s p a r e n t  mu.  and then are  l a m e l l a Ij. t o 10 mu.  which merges w i t h the l a t e r a l w a l l .  separated  i n diameter  An e l e c t r o n dense amor-  phous substance surrounds the advancing edge from i t s e a r l i est  encept ion  ( F i g . l a , upper r i g h t ) ,  Ingrowth of the septam i n i t i a l  ,  occurs by what i s com-  monly known as " c e n t r i p e t a l i n v a g i n a t i o n " , somewhat a f t e r the f a s h i o n of a c l o s i n g i r i s diaphragm. dense m a t e r i a l continues  Amorphous e l e c t r o n  t o encase the advancing edge  (Fig.3)  and once the membranes have met o f t h i s substance can be (Pig.  l+).  The  and f u s e d c e n t r a l l y remnants  seen l y i n g b e s i d e the f u s e d p l a t e  septa w i t h i n the promycelia  are complete ( i . e . s e p t a l pores are  of U s t i l a g o h o r d e i  absent).  J u s t p r i o r t o , or d u r i n g , p l a t e completion  synthesis  o f the s e p t a l w a l l begins a t the l a t e r a l edges and i s formed in  such a way  as to be continuous w i t h the i n n e r l a y e r s o f  the p r o m y c e l i a l w a l l . The  new  In appearance they are  identical.  w a l l m a t e r i a l i s l a i d down s i m u l t a n e o u s l y  a l l around  the i n s i d e edge o f the i n v a g i n a t e d plasma membrane, thus forming a tube o f w a l l m a t e r i a l i n s i d e the i n v a g i n a t i o n . e l e c t r o n transparent  l a m e l l a w i t h i n the tube i s not  occluded.  During s e p t a l development i t r e t a i n s the constant  diameter  (Ij. - 10 mu)  Once the  first  d e f i n e d by the septum i n i t i a l .  The  p l a t e i s complete t h i s c e n t r a l r e g i o n i s continuous throughout the c r o s s w a l l . any  Subsequent t h i c k e n i n g o f the w a l l a t  p o i n t r e s u l t s from the a c c r e t i o n o f new  material  the plasma membrane on e i t h e r s i d e o f the p l a t e . p l a t e completion,  (130  -  1 5 0 mu).  (with the  synchronously  In c r o s s - s e c t i o n the t o t a l w i d t h across the promy-  e x c e p t i o n o f the t r i a n g u l a r t h i c k e n i n g  (Bracker and B u t l e r , 19631 The  Prom  a t t a i n e d i t s maximum  o f the c r o s s - w a l l i s r e l a t i v e l y constant celium,  ( P i g . ij.).  m a t e r i a l must be d e p o s i t e d  throughout the septum u n t i l i t has thickness  Soon a f t e r  s y n t h e s i s o f the s e p t a l w a l l m a t e r i a l  spreads r a p i d l y along the i n n e r s u r f a c e t h i s p o i n t on new  along  at the extreme l a t e r a l edges.,,  t h i c k n e s s o f t h e m a t e r i a l on e i t h e r s i d e o f the c e n t r a l  96  F i g u r e s 1+, 5 and 6 demonstrate sequen-  lamella i s equal. tially  the t h i c k e n i n g o f the s e p t a l w a l l .  In U s t i l a g o h o r d e i the plasma membrane o f the i n v a g i n a t ing  septum i n i t i a l i s f r e q u e n t l y extended i n t o an e l a b o r a t e  complex ( F i g s . 2,  8,  and 9).  Although s e r i a l s e c t i o n s  an e n t i r e promycelium have not been o b t a i n e d  such complexes (Figs. 2  always seem t o l i e a g a i n s t one o f the l a t e r a l w a l l s and  7)»  septum. and  and there i s a p p a r e n t l y  a t l e a s t one complex per  They a r e v i s i b l e a f t e r both glutaraldehyde-osmium  permanganate f i x a t i o n s ( F i g s . 2,  respectively).  8,  3, and 9, and F i g . 7,  With the l i g h t microscope prominent b o d i e s  o f s i m i l a r s i z e and shape occur  i n a s s o c i a t i o n with  i n g l u t a r a l d e h y d e - f i x e d m a t e r i a l ( F i g . 10). sue  across  septa  In l i v i n g  tis-  conspicuous s t a t i o n a r y r e f r a c t i l e b o d i e s c l e a r l y i n d i c a t e  the p o i n t a t which a septum w i l l l a t e r be v i s i b l e tion, Figs. l i - j ) .  (Introduc-  The u l t i m a t e f a t e o f t h e septum-associ-  a t e d membrane complexes i s unknown but t h e r e i s some evidence t h a t they n o r m a l l y begins to form.  degenerate once the s e p t a l w a l l m a t e r i a l  Not a l l the membrane complexes i n t h e  promycelium are a s s o c i a t e d w i t h t h e septum ( F i g . 7)  and more  w i l l be s a i d about t h e r e l a t i o n s h i p o f these unusual s t r u c t u r e s to other c e l l  o r g a n e l l e s i n p a r t V.  F i g u r e 9 c l e a r l y demonstrates the u n i t s t r u c t u r e o f the membranes composing the complex. age w i d t h o f 96A° (Range:  These membranes have an a v e r -  60 - H+.0 A ° ) .  After  glutaraldehyde-  osmium f i x a t i o n they a r e arranged i n c o n c e n t r i c whorls ( F i g . 2)  o r f o l d s ( F i g s . 8 and 9) but a f t e r KMnOlf-fixation  they  fol-  low a more random, but s t i l l compact, o r i e n t a t i o n ( F i g . 7 ) «  Aside from the complexes no other o r g a n e l l e s are cons p i c u o u s l y a s s o c i a t e d w i t h i n i t i a t i o n or t h i c k e n i n g of the septum*  Spherosome-like b o d i e s and l i p i d b o d i e s o f t e n  occur  i n the v i c i n i t y of the c r o s s - w a l l s but t h e i r occurence does not seem t o be any more f r e q u e n t cytoplasm* lie  Short  than i n other p a r t s of the  segments of endoplasmic r e t i c u l u m sometimes  j u s t beneath and p a r a l l e l w i t h the plasma membrane of a  well-developed  septum i n much the  b o r d e r s on the l a t e r a l w a l l s  same f a s h i o n t h a t the  ER  (Pt* I I ) .  Sometimes the plasma-  lemma i s r e l a t i v e l y smooth ( F i g . 6) and  sometimes i t i s q u i t e  c r e n u l a t e even around mature septa which have presumably a t t a i n e d t h e i r maximum t h i c k n e s s Two  ( F i g s * l£ and  v a r i a t i o n s t o the normal s e p t a t i o n p a t t e r n have  been observed*  The  first,  depicted i n Figure  i n i t e l y abnormal event i n which twin s i d e by  16).  side.  The  l a , is a  def-  septa are l a i d down  second v a r i a t i o n i s i n v o l v e d i n the  d u c t i o n of " k n e e - j o i n t s " .  pro-  T h i s l a t t e r phenomenon i s i n i -  t i a t e d by the p l a s t i c i z a t i o n of the l a t e r a l w a l l over a l o c a l i z e d area on b o t h s i d e s of and The  p r o t o p l a s t s of the two  in, a manner s i m i l a r t o the  As the p r o t o p l a s t s bulge the  p r o m y c e l i a l w a l l i s extended around the outer the i n n e r s u r f a c e where the two together  t o a septum*  c e l l s i n v o l v e d are then "blown  out" as two bulbous e x t e n s i o n s normal budding p r o c e s s .  adjacent  lateral  surface.  expanding r e g i o n s are growing  the s e p t a l w a l l resumes growth u n i d i r e o t i o n a l l y  so continues  t o separate  the two p r o t o p l a s t s  Spherosome-like b o d i e s and  On  ( F i g . 11).  l o n g elements of endoplasmic  and  98  r e t i c u l u m are very numerous and prominent on both  sides  o f the bulbous b r i d g e - i n i t i a l during t h i s e a r l y p e r i o d o f formation.  What then seems to occur might best be  as a metabolic  race between those elements engaged i n syn-  t h e s i z i n g the w a l l s s e p a r a t i n g the two o f elements engaged i n b r e a k i n g  c e l l s and another set  down the p a r t i t i o n  In F i g u r e 12 a p o r t i o n o f the w a l l can s t i l l be the two  described  bulbous c e l l e x t e n s i o n s  while  ( F i g . 12).  seen between  the regions on e i t h e r  s i d e o f i t have become e l e c t r o n t r a n s p a r e n t  and  contain  d e b r i s and v e s i c l e s which seem to be d e r i v e d from the F i n a l l y the degratory  elements pre-dominate and the  t i o n i n g w a l l i n the bulbous r e g i o n i s e l i m i n a t e d the p r o t o p l a s t s to fuse completely  ( F i g . 13).  cell  ER.  parti-  allowing  The  plasma mem-  brane reforms i n some manner about the f r e e end o f the remaining  p o r t i o n o f the septum.  tum  p a r t i a l l y s e p a r a t i n g two  cytoplasmic  bridge.  The  net r e s u l t i s an Incomplete sep-  c e l l s which are now  j o i n e d by  In Figure 13 the nucleus o f one  moved i n t o a p o s i t i o n p r e p a r a t o r y  cell  to e n t e r i n g the b r i d g e  a has  region.  SPORIDIUM FORMATION: Under the l i g h t microscope the f i r s t  sign that a  sporidium  i s about to form i s the appearance o f a b r i g h t r e f r a c t i l e on the s u r f a c e of the promycelium.  spot  A f t e r f i v e to t e n minutes  the w a l l begins to bulge at t h i s p o i n t , and t o extend r a p i d l y . D u r i n g the f i r s t phase o f e l o n g a t i o n the bud  i s narrow, but a f t e r  s e v e r a l m i l l i - m i c r o n s o f growth ( i . e . as measured under the e l e c t r o n microscope i t undergoes a spontaneous b a l l o o n i n g o f the d i a meter to give r i s e to the elongate  o v o i d form o f the mature  sporidium  (Introduction, P i g s . 9 - 15).  The t o t a l development of the  bud from the appearance of the r e f r a c t l i e spot t o the mature sporidium requires 20 t o 1*0 minutes. Under the electron microscope the f i r s t sign that a bud i s about to form i s the disappearance of wall material from a small region of the l a t e r a l promycelial surface.  The  actual disappearance of the wall substance i s preceded by a change i n i t s structure.  The semi-electron  dense, f i b r i l l a r  w a l l thickens and becomes more electron-translucent and apparently homogeneous, with the exception  of the t h i n electron  dense l i n e which has been shown t o separate the wall into two layers.  In Figure 11* a short walless zone i s shown t o be &  bounded by altered wall material which i s , no doubt, i n the process of being degraded, and the protoplast has already begun to bulge i n the d i r e c t i o n of the walless region.  Long  elements of endoplasmic reticulum l y i n g just beneath and p a r a l l e l t o the l a t e r a l wall seem t o end i n the v i c i n i t y of the electron translucent  area.  Figures 15 $ 16, and 17 I l l u s t r a t e sequentially the development of a sporidium.  In Figure 15 a t h i n amorphous  layer of material covers the bud i n i t i a l , and the fractured edges of the o l d promycelial wall are c l e a r l y v i s i b l e .  Be-  neath the new layer of w a l l material a large spherosome-like body i s conspicuously the adjacent  present.  Since the l a t e r a l wall of  c e l l on the other side of the septum also seems  to be thinner than the usual promycelial wall' the c e i l s depicted i n Figure 15 may actually represent bridge-formation  rather than budding.  a stage i n  E a r l y i n t h i s devel-  opmental stage I t i s i m p o s s i b l e to p r e d i c t what course o f d i f f e r e n t i a t i o n the c e l l s  w i l l follow.  As the bud i n c r e a s e s  i n s i z e p a r t o f the p r o t o p l a s t o f the parent c e l l moves i n t o it.  The  p a r e n t a l nucleus d i v i d e s , m i t o t i c a l l y , i n the promy-  celium, i n the bud, o r i n the neck between the two daughter n u c l e i separate i n such a way  and  the  t h a t the parent  and the b a s i d i o s p o r e o b t a i n one nucleus each.  As was  cell noted  d u r i n g the passage o f the d i p l o i d nucleus up the promycelium (Ft.  I I ) the n u c l e o l u s always l i e s a t the p o s t e r i o r end o f  a migrating nucleus.  The mature s p o r i d i u m ( F i g . I d ) , then,  c o n t a i n s a s i n g l e h a p l o i d n u c l e u s , a number o f m l t o c h r o n d l a , l i p i d b o d i e s , and spherosome-like of  endoplasmic  b o d i e s , plus a s m a l l amount  r e t i c u l u m and u n i d e n t i f i e d v e s i c l e s .  When the  bud i s f u l l y formed i t i s cut o f f from the mother c e l l to come an autonomous i n d i v i d u a l . to  be-  No evidence has been o b t a i n e d  i n d i c a t e the manner i n which t h i s s e p a r a t i o n occurs or i n  which the proximal end w a l l o f the s p o r i d i u m i s  completed.  DISCUSSION SEPTATION: The hyphal c e l l s o f most h i g h e r f u n g i m a i n t a i n some s o r t o f c y t o p l a s m i c c o n t i n u i t y w i t h t h e i r neighbours  by v i r t u e  one o r more pores i n the c r o s s - w a l l s s e p a r a t i n g the  of  cells.  In g e n e r a l , the pores o f the ascomycetes are r e p r e s e n t e d by "simple h o l e s " w h i l e the pores o f the basidiomycetes c h a r a c t e r i z e d by e l a b o r a t e s t r u c t u r e s known as the parenthosome" (Moore and McAlear,  1962;  are  "dolipore-  Bracker, 1 9 6 7 ) .  A  major exception t o t h i s r u l e occurs among the heterobasidiomycetes (Bracker, 1 9 6 7 ; E h r l i c h ; et a l . , 1 9 6 8 ) .  In t h i s  group dolipore-septa have been found among the Tremellales (Moore, 1 9 6 5 ; Moore and McAlear, 1962; Wells, 1961+), but simple pores among the Uredinales  (Ehrlich  et a l . , 1 9 6 8 ;  Manocha and Shaw 1 9 6 7 , Moore, 1 9 6 3 , Moore, 1 9 6 5 ) .  It i s  commonly accepted that dolipore-septa do not occur i n the U s t i l a g l n a l e s (Moore, 1 9 6 5 ; Bracker, 1 9 6 7 ; E h r l i c h . et a l . , 1 9 6 8 ) although t o the best of the author's knowledge there i s only one report on smut septa i n existence  (Stein, 1 9 7 0 ) .  Complete septa, such as those observed i n the promycelium of tTstllago hordei, have occasionally been noted i n phycomyoetes, hemiascomycetes, and deuteromycetes (Bracker, 1 9 6 7 ) as w e l l as i n a mycelial mutant of Ustilago hordei (Stein, 1 9 7 0 ) and i n a basidiomycete, and Erke, 1 9 7 1 ) •  Cryptococcus neoformis (Cutler  Septa without pores, are often associated  with s p e c i a l i z e d situations such as sealing off injured or evacuated c e l l s (Wells, I96I4.) and d e l i m i t i n g reproductive structures (Hawker and Oooday, 1 9 6 7 ) .  The f o u r - c e l l e d  metabasidium of Ustilago hordei constitutes a highly s p e c i a l ized system i n which each of the c e l l s contains a d i f f e r e n t set of genetic information (lie. as a r e s u l t of meiosis).  Of  course the cytoplasm of these c e l l s i s derived from a single protoplast, and i t i s unknown how long i t takes f o r the new nucleus t o exert i t s e f f e c t on the i n d i v i d u a l c e l l , but cert a i n l y an influence i s manifest by the time of bridge-format i o n and s p o r i d l a l production.  However, since complete septa  also occur i n a mycelial mutant of t h i s fungus (Stein, 1 9 7 0 )  the p o s s i b i l i t y e x i s t s t h a t such septa may be a c h a r a c t e r i s t i c form i n t h i s  species.  Moore (1965) d e s c r i b e d three p a t t e r n s ' d i v i s i o n i n mycota.  of somatic  cell  The mature septum o f U s t i l a g o h o r d e l  i s of c l a s s i f i c a t i o n s type B, r e p r e s e n t e d v l o l a c e o r u b e r and C o c c l d i o i d e s i m m l t i s .  by Streptomyces I t c o n s i s t s of two  l a y e r s of s e p t a l w a l l ffce. two p l a t e s ) each of which i s continuous w i t h the i n n e r l a t e r a l w a l l and an e l e c t r o n t r a n s parent  l a m e l l a l y i n g between the two p l a t e s ( P i g s . 5. 6,  and 16).  Cross w a l l s of s i m i l a r s t r u c t u r e occur  15  among the  phycomycetes (Akai e t a l . , 1968; Hawker and Gooday, 1967), the ascomycetes (Brenner and C a r r o l l , 1968;  Kreger and V e e h u i s ,  1969;  Moore, 1962), the basidiomycetes (Bracker  1963;  J e r s i l d e t a l . , 1967;  and B u t l e r ,  O'Hern and Henry, 1956;  1961*), and a number o f imperfect human pathogens and R o d r i g e z ,  1968).  Wells,  (Carbonelle  They a l s o occur among the actinomycetes  ( G l o u e r t and Hopwood, 1961; Moore, 1965).  The dimensions of  the mature septa o f U s t i l a g o h o r d e i are almost i d e n t i c a l t o those g i v e n f o r the c r o s s - w a l l s of B h l z o c t o n i a s o l a n ! and B u t l e r , 1963).  In the former the newly-formed c r o s s w a l l  w i d t h i s 6 t o 10 mu ( F i g . 1*), 130 t o 150 mu. ( F i g u r e s 6, of the e l e c t r o n t r a n s p a r e n t 5,  and 6).  the mature c r o s s w a l l w i d t h i s  13, 15, and 16),  and the diameter  l a m e l l a i s I* t o 10 mp ( F i g s . 1*,  In the l a t t e r the r e s p e c t i v e measurements are  7 t o 8 mu, 120 mu, and 10 mu. the  (Bracker  septa which oleave  Wells: (1961+b) has noted t h a t  the hypobasidium o f E x l d l a n u c l e a t e  (a h e t e r o b a s i d i o m y c e t e ) i n t o f o u r h y p o b a s i d i a l have a s i m i l a r s t r u c t u r e .  segments  During septum f o r m a t i o n lemma i n v a g i n a t e s  first,  and  i n t h i s smut fungus the plasmas e p t a l w a l l m a t e r i a l i s not  l a i d down u n t i l the membranes have f u s e d , or have almost fused  across  formation  the centre  of the promycelium.  T h i s mode of  most c l o s e l y resembles t h a t of C o c c i d i o i d e s  immitis  (O'Hern and Henry, 1 9 5 6 ) , c e r t a i n s p e c i e s of Streptomyces (Moore, 1 9 6 5 ) , and  some b a c t e r i a (Chapman, 1 9 5 9 ) , a l l of  which form type B septa a c c o r d i n g All  t o Moore's c l a s s i f i c a t i o n .  of these s p e c i e s a l s o form complete s e p t a .  Moore (1963)  and W e l l s (196U.br) observed a s i m i l a r mechanism of complete septation during t h a l l u s formation p o d o p h y l l i and  Puecinia  d u r i n g hypobasidium segmentation i n E x i d i a  nucleate, respectively. and  i n the a e c i a of  The  fact that a similar structure  developmental p a t t e r n have been observed f o r complete  cross-walls  i n a r u s t :(Moore, 1 9 6 3 ) , a tremellaceous  ( W e l l s , 1963b) and t i o n may other  a smut suggests t h a t t h i s form of  be common among h e t e r o b a s l d i o m y c e t e s .  fungus septa-  In most  cases where s e p t a l development has been observed ( i e .  exception-Hawker and Gooday, 196?)  the  m a t e r i a l keeps pace w i t h the e x t e n s i o n  synthesis  of w a l l  of the plasma mem-  brane so t h a t the septum i n i t i a l , at a l l stages i s a wedge of w a l l m a t e r i a l c l o s e l y surrounded by membrane r a t h e r a flat 1969;  membranous p l a t e  (Brenner and C a r r o l l , 1968;  C o n t i and N a y l o r , 1959;  chant and Smith, 1968; The  Manocha and Shaw, 1967;  Moore, 1962;  advancing l i p of the  than  Campbell, Mar-  W e l l s , 1961+).  septum i n i t i a l  of U s t i l a g o  h o r d e i i s c l o s e l y a s s o c i a t e d w i t h a r i m of e l e c t r o n dense  ( P i g s , 3 and l a ) . A s i m i l a r substance surrounds  material  the advancing edge or the pore r i m of the c r o s s w a l l s o f Ascodemis sphaerospora (Brenner and C a r r o l l , 1968), and S o r d a r i a f i m i c o l a (Purtado, 1971).  In b o t h the l a t t e r  s t u d i e s endoplasmic r e t i c u l u m i s a s s o c i a t e d w i t h septum formation  and/or the e l e c t r o n dense substance.  Hawker and  Gooday (1967) have suggested t h a t the ER surrounding the septum i n i t i a l of R h i z o p u s : a e x u a l i s  (Smith) C a l l e n  gives  r i s e t o v e s i c l e s which c o n t a i n the new w a l l m a t e r i a l , and which r e l e a s e t h i s m a t e r i a l by f u s i n g w i t h the s e p t a l membrane.  Lomasomes may a l s o be i n v o l v e d i n the f o r m a t i o n of (Brenner and C a r r o l l , 1968; Hawker and Gooday,  cross walls  1967).  The source of s e p t a l w a l l m a t e r i a l i n U s t i l a g o  h o r d e ! I s as obscure as the mechanism o f a p i c a l e x t e n s i o n (Pt. I I ) .  As noted b y W e l l s f o r E x i d i a n u c l e a t a  (1961*b),  v e s c i o l e s , endoplasmic r e t i c u l u m , and lomasomes are n o t conspicuously  a s s o c i a t e d w i t h the i n v a g i n a t i n g membrane  or t h i c k e n i n g p l a t e . In U s t i l a g o h o r d e i l a r g e membrane complexes are o f t e n a s s o c i a t e d w i t h the i n v a g i n a t i n g septum i n i t i a l .  What are  now r e q u i r e d are s e r i a l s e c t i o n s through p r o m y c e l i a which are under-goint c e l l d i v i s i o n . prove or disprove  Hopefully  the h y p o t h e s i s t h a t there  or more o f these s t r u c t u r e s present division.  According  t o the c u r r e n t  complex i s an " e x t e n s i o n " brane which l i e s a g a i n s t ( P i g s . 2 and 8).  such s t u d i e s  will  i s always one  i n the r e g i o n o f c e l l data a s e p t a l membrane  of the i n v a g i n a t i n g plasma memone of the l a t e r a l germ tube w a l l s  Whether or not there  i s any r e l a t i o n s h i p  between these complexes and the knots of membrane which form p a r t of the ER-sac i n a g e r m i n a t i n g c e l l at p r e s e n t , b u t the farmer  ( P t . I I ) i s unknown  seems t o be a s s o c i a t e d w i t h the  plasma membrane, and the l a t t e r w i t h the endoplamic r e t i c u l u m . A number of r e s e a r c h e r s have d e s c r i b e d s i m i l a r membrane comp l e x e s among f u n g i .  In the basldiomycetes A r m i l l a r l a melea  ( B e r l i n e r and D u f f ) , Coprlnus micaceous (Edwards, 1969), Coprinus lagopus latum  (Lu, 1965; L u , 1966), and Lycoperdon p e r -  (Mardhant, 1969), the ascomycetes A s p e r g i l l u s f u m i c u l u s  (Edwards, 1969), Neurospora 1968), and Neurospora  tetrasperma  (Lowry and Sussman,  c r a s s a (Kozar and W e i j e r , 1969), and  the i m p e r f e c t f u n g i P a r a c o c c i d i o i d e s l o b o i (Purtado e t a l . 1967) and V e r t l c v j l l i u m d a h l i a e ( G r i f f i t h s , 1970) the r e s p e c t i v e workers have not noted any r e l a t i o n s h i p o f the complexes t o the septum.  The s p e c i f i c a s s o c i a t i o n of s i m i l a r membrane  systems w i t h septa occurs i n the imperfect f u n g i P a r a c o c c i d i o i d e s b r a s i l i e n s i s and Blastomyces  dermatitldis (Carbonelle,  1967, and C a r b o n e l l e and R o d r i g e z , 1968) and i n the b a s i d iomycete  Lenzltes saepiaria  (Hyde and Walkinshaw, 1966).  Membrane systems d e r i v e d from the plasma membrane have l o n g been a s s o c i a t e d w i t h septum f o r m a t i o n among the actinomycetes (Edwards, 1970; E l l a r e t a l . , 1967; G l a u e r t and Hopwood, I960; Imaida and Ogura, 1963) where such s t r u c t u r e s have u s u a l l y been r e f e r r e d t o as "mesosomes" because of the s t r i k i n g  struc-  t u r a l s i m i l a r i t y t o the b a c t e r i a l o r g a n e l l e s of t h a t name. One of the p o s t u l a t e d f u n c t i o n s of the b a c t e r i a l mesosome i s t o a s s i s t i n septum f o r m a t i o n (Rogers, 1970; R y t e r , 1968),  106  The  membrane complex-cross w a l l a s s o c i a t i o n has suggested  a number o f p o s s i b l e f u n c t i o n s f o r these e l a b o r a t e systems: s y n t h e s i s and  of s e p t a l w a l l m a t e r i a l between the c e l l  i t s environment d u r i n g periods  activity  membrane  of i n c r e a s e d  metabolic  ( C a r b o n e l l e , 1967), and i n i t i a t i o n o f c e l l  (Edwards, 1970).  In U s t i l a g o h o r d e l  division  i t i s u n l i k e l y t h a t the  complex a c t s d i r e c t l y i n d e p o s i t i o n of the w a l l  material  s i n c e such systems are most prominent i n the v i c i n i t y o f the  invaginating septal i n i t i a l before  begun.  w a l l t h i c k e n i n g has  However, one of t h e ways i n which a membrane complex  might i n i t i a t e  septum formation  i z e d source of p r e - s y n t h e s i z e d incorporated  would be t o provide  a local-  membrane which would then be  i n t o the developing  membranous p l a t e .  This  h y p o t h e s i s i s supported by the f a c t t h a t the membrane o f t h e septal i n i t i a l  i s continuous w i t h t h a t of the complex and by  the f a c t t h a t the w i d t h o f the i n v a g i n a t i o n membrane  (approxi-  mately 100A°) i s c l o s e r t o the mean width of the u n i t membranes of the complex (96A°) than t o the mean width of the plasmalemma on the s i d e s of the promycelium (121+A ). 0  and D u f f (1965), and Lu (1965 and 1966) have a l s o  Berliner  postulated  t h a t s i m i l a r membrane complexes may a c t as membrane generators i n connection cussion  w i t h other  activities  Further  dis-  of the smut membrane complex, i t s o r i g i n and p o s s i b l e  f u n c t i o n s w i l l be r e s e r v e d The  i n fungi.  u n t i l p a r t V.  l i t e r a t u r e p e r t a i n i n g t o membrane complexes i n  f u n g i has f r e q u e n t l y been confused by the f a i l u r e t o d i s t i n g u i s h these s t r u c t u r e s from a r t e f a c t s produced i n t i s s u e  by  glutaraldehyde f i x a t i o n  Ohal and R o h l i c h , 1966;  1958;  (Fawcett and Susuma,  Palade and Claude, 1 9 U 9 a ;  and Claude, 19U9b; R e v e l et a l . , 1 9 5 8 ) .  The f a c t  Palade that  s t a t i o n a r y b o d i e s of s i m i l a r s i z e t o the membrane complexes can be  observed i n l i v i n g germ tubes at p o i n t s where septa  w i l l subsequently form i n d i c a t e s t h a t such s t r u c t u r e s are not f i x a t i o n a r t e f a c t s .  However, t h i s does not r u l e out  the p o s s i b i l i t y t h a t these membranous whorls are formed by h y p e r a c t i v e membrane s y n t h e s i s which r e s u l t s from growth i n a r i c h medium. b a s i d i a produced  A comparison  should be made w i t h meta-  i n minimal medium or d i s t i l l e d  water.  Fungal membrane complexes have a l s o been confused w i t h pseudo-myelin  f i g u r e s which are f r e q u e n t l y observed i n  vacuoles or lysosomes and I s a a c , 1 9 6 7 ) .  (Smith and Marchant, 1 9 6 8 ;  I n t r a v a c u o l a r pseudomyelinate  Thomas figures  a l s o occur i n U s t i l a g o h o r d e i ( P t . I I ) ; however, the mem¥ brane complexes per se are c l e a r l y not a s s o c i a t e d i n any way w i t h l y s o s o m e - l i k e s t r u c t u r e s . Two  v a r i a t i o n s on the normal s e p t a t i o n p a t t e r n have  been observed.  The f o r m a t i o n of twin septa r e s u l t s i n a  promycelium w i t h f i v e compartments one  of which c o n t a i n s  no nucleus and r a p i d l y becomes v a c u o l a t e d .  Knee-joints  r e s u l t from the p a r t i a l degradation of a mature,  complete  septum and the f o r m a t i o n of a c y t o p l a s m i c b r i d g e between two d e l l s of opposite mating type  and - ) .  These appear  t o be s i m i l a r i n s t r u c t u r e and probably a l s o i n o r i g i n t o the pseudo-septa,  or incomplete  t r a n s v e r s e septa de-  s c r i b e d i n o t h e r basidiomycetes ( E h r l i c h e t a l . , 1963; J e r s i l d e t a l . , 1967; K o l t i n and F l e x e r , 1969).  In p a r t  I I l o n g elements o f endoplasmic r e t i c u l u m and spherosomel i k e bodies were a s s o c i a t e d w i t h w a l l s y n t h e s i s and degradat i o n i n t h e r e g i o n surrounding  the beak d u r i n g  germination.  I n t e r e s t i n g l y , a s i m i l a r a s s o c i a t i o n e x i s t s i n the r e g i o n of the bridge  formation  ( P i g s . 11 and 12).  V e s i c l e s which  seem to be d e r i v e d from the ER accumulate i n the r e g i o n o f degradation  ( F i g . 12) suggesting  involved i n catabolic a c t i v i t i e s  t h a t the ER i s more l i k e l y ( i . e . w a l l degradation)  than i n a n a b o l i c ones ( i . e . w a l l s y n t h e s i s ) . SPORIDIUM FORMATION: B a r t n i c k i - G a r c i a and Lippman (1969) h y p o t h e s i z e d bud  formation  that  i n dimorphic f u n g i might be based on l o c a l -  i z e d resumption o f growth o f the parent  cell,  f o l l o w e d by  a u n i f o r m l y d i s p e r s e d p a t t e r n o f w a l l s y n t h e s i s i n the bud. T h i s would r e s u l t i n s p h e r i c a l , y e a s t - l i k e daughter c e l l s as opposed to a p i c a l hyphal e x t e n s i o n . i n U s t i l a g o h o r d e i a t l e a s t support hypothesis.  The  the f i r s t  observations part o f t h i s  The i n i t i a l t h i c k e n i n g o f the parent  m a t e r i a l which occurs  wall  i n t h i s fungus p r i o r to the produc-  t i o n o f a bud has been observed during budding o f t h e dimorphic f u n g i , P a r a c o c c i d i o i d e s b r a s i l i e n s i s and B l a s tomyces d e r m a t l t i d l s ( C a r b o n e l l e , 1967; C a r b o n e l l e and Rodriguez, 1968), as w e l l as s e v e r a l s p e c i e s o f H i s t o plasma (Edwards e t a l . , 1959).  However, i n U s t i l a g o h o r d e i  the t h i c k e n e d w a l l r e g i o n i s more e l e c t r o n t r a n s l u c e n t than  the mature p r o m y c e l i a l w a l l , w h i l e  i n the human pathogens  the t h i c k e n e d r e g i o n i s more e l e c t r o n dense. homogeneity and  Increased  decreased e l e c t r o n d e n s i t y i n the former  i s presumed t o i n d i c a t e an i n c r e a s e doubtedly the r e f r a c t i l e microscope b e f o r e  in plasticity.  spot which appeas i n the  a sporidium becomes v i s i b l e  t h i s p e r i o d of w a l l p l a s t i c i z a t i o n and the of w a l l  The  Unlight  represents  subsequent p e r i o d  degradation.  In the promycelium of U s t i l a g o h o r d e i , a l o c a l i z e d p o r t i o n of the l a t e r a l w a l l i s e v i d e n t l y completely moved b e f o r e the new  the new  re-  bud w a l l i s formed ( P i g . 11+).  As  m a t e r i a l t h i c k e n s about the neck i t again becomes  s t r u c t u r a l l y continuous w i t h the p r o m y c e l i a l w a l l . mechanism of bud  formation  previously described.  The  This  v a r i e s from the mechanisms bud w a l l i n Saccharomyces  c e r e v i s i a e i s a d i r e c t c o n t i n u a t i o n of the p a r e n t a l w a l l (Marchant and Smith, 1968; Moor, 1967).  cell 1965;  McClary and Bowers,  A s i m i l a r mode of bud  i n i t i a t i o n has  also  been suggested i n s e v e r a l s p e c i e s of Histoplasma (Edwards et a l . , 1959). rupture  Sporobolomyoes-type budding i n v o l v e s  of an outer w a l l l a y e r — t h e bud w a l l b e i n g  extension  of an inner w a l l l a y e r of the parent  1969;  Prusso and W e l l s , 1967).  a new  w a l l l a y e r i s s y n t h e s i z e d beneath the  Smith, 1967)•  new  an  (Calonge,  In Rhodotorula g l u t l n i s o l d and  i n g budding the p a r e n t a l w a l l l a y e r r u p t u r e s , the b e i n g encased by completely  the  material  dur-  bud  (Marchant  Rhodotorula-type budding seems t o be  and the  most s i m i l a r t o s p o r i d i a l f o r m a t i o n Notice  i n Ustilago hordei.  should he made that the y e a s t - l i k e Rhodotorulas  have r e c e n t l y been r e c l a s s i f i e d as a new genus of the h e t e r o b a s i d i o m y c e t e s , the Rhodosporidluro (Banno, 1 9 6 7 ) . Long elements of endoplasmic r e t i c u l u m are prominent beneath the bud i n i t i a l d u r i n g the e a r l i e s t that during knee-joint  formation  observation  v e s i c l e s which are ap-  p a r e n t l y d e r i v e d from the ER are prominent i n the r e g i o n Moor ( 1 9 6 7 ) has suggested t h a t i n Saccharo-  of breakdown.  myces c e r e v i s i a e v e s i c l e s d e r i v e d from the ER c a r r y enzymes which induoe a l o c a l i z e d p l a s t i c i z a t i o n of the w a l l o f the mother c e l l ,  and hence i n i t i a t e bud f o r m a t i o n .  On the other  hand, Marchant and Smith ( 1 9 6 7 ) have a s s o c i a t e d l a r g e amounts of ER and E R - d e r i v e d v e s i c l e s w i t h a c t i v e w a l l during budding.  In U s t i l a g o h o r d e l spherosome-like b o d i e s  are q u i t e prominent once s y n t h e s i s gun  (Fig. 1 5 ) .  synthesis  This observation  t h e s i s previously discussed  of the new w a l l has b e -  helps  t o support the hypo-  i n part I I , that i n t h i s  species  of smut fungus the spherosome-like o r g a n e l l e s f u n c t i o n i n wall  synthesis.  CONOLUSION Septation  i n the promycelium of U s t i l a g o h o r d e l i s  i n i t i a t e d by the " c e n t r i p e t a l i n v a g i n a t i o n " o f membranes which are continuous w i t h the plasma membrane.  The source  of the membranes forming the i n i t i a l membranous p l a t e seems t o be a l a r g e membrane complex.  Further  s t u d i e s are  r e q u i r e d t o t e s t the hypothesis  t h a t at l e a s t one such  complex i s a s s o c i a t e d w i t h every d e v e l o p i n g S e p t a l w a l l m a t e r i a l i s not d e p o s i t e d the  initial.  i n any q u a n t i t y  i n i t i a l p l a t e has been completed.  s e p t a l w a l l m a t e r i a l i s unknown.  septum  The source of the  No pores occur  mature c r o s s w a l l s of the metabasidium. of the r e g u l a r s e p t a t i o n p a t t e r n occur,  until  i n the  Two v a r i a t i o n s one o f which g i v e s  r i s e t o "knee-joints". Budding i s i n i t i a t e d by a s e q u e n t i a l p l a s t i c i z a t i o n and degradation  o f the w a l l o f the parent  cell.  This  stage i s f o l l o w e d by an e x p l o s i v e p r o t r u s i o n of the s p o r i dium accompanied by s y n t h e s i s o f new w a l l m a t e r i a l .  Fur-  t h e r s t u d i e s are r e q u i r e d t o determine the manner i n which the mature sporidium  i s separated  Evidence i s presented  from the promycelium.  which supports the h y p o t h e s i s  t h a t i n U s t i l a g o h o r d e i the endoplasmic r e t i c u l u m f u n c t i o n s i n the break down of mature w a l l m a t e r i a l .  The spherosome-  l i k e o r g a n e l l e s , which may a l s o be i n v o l v e d i n w a l l deg r a d a t i o n and p l a s t i c i z a t i o n , probably  have an a d d i t i o n a l  r o l e i n the b i o s y n t h e s i s o f new w a l l m a t e r i a l .  III.  PLATE 1  Figure l a .  Septum i n i a t i o n . An abnormal s i d e - b y - s i d e s e p t a t i o n . E a c h septum i s formed n o r m a l l y . Three p o i n t s a t which the septum i n i t i a l s can be observed are i n d i c a t e d ( i . e . arrows and i n s e r t ) . E a c h i n i t i a l c o n s i s t s of an i n v a g i n a t i o n of the plasma membrane bounding an e l e c t r o n t r a n s p a r e n t c e n t r a l l a m e l l a and ending i n an amorphous e l e c t r o n dense material. Method C. c a . X 2 6 , 2 0 0 .  Figure l b .  An enlarged view of one side of one of t h e septum i n i t i a l s i n f i g u r e l a . Note the w e l l d e f i n e d plasma membrane (FM), the c e n t r a l l a m e l l a , and the e l e c t r o n dense m a t e r i a l surrounding the f r o n t a l edge. Method C. c a . X 4 5 , 8 5 0 .  Figure  2.  Aiiraore advanced septum i n i t i a l showing a c l e a r c o n t i n u i t y between the plasma membrane d e l i m i t i n g the s e p t a l i n i t i a l and the membranes composing a membrane complex,(mc). The two s i d e s of the septum i n i t i a l are i n d i c a t e d by arrows. Method C. ca. X 3 9 , 3 0 0 .  I I I . PLATE 2 Figure 3 .  A septum i n i t i a l which i s almost complete. The arrow i n d i c a t e s the e l e c t r o n dense f r o n t a l edge m a t e r i a l . Note s e p t a l w a l l m a t e r i a l i s b e g i n n i n g t o be deposited a t the l a t e r a l edges and i s cont i n u o u s w i t h the inner p r o m y c e l i a l w a l l . Lipid b o d i e s ( L ) , m i t o c h o n d r i a (M) and spherosome-like o r g a n e l l e s (S) are i n d i c a t e d . None of these o r g a n e l l e s are s p e c i f i c a l l y a s s o c i a t e d w i t h the formi n g c r o s s - w a l l . Method C. c a . X 3 2 , 7 5 0 .  F i g u r e 1*.  A newly completed c r o s s - w a l l . Note the very t h i n l a y e r of s e p t a l w a l l m a t e r i a l , the c e n t r a l l a m e l l a , and the remains of the e l e c t r o n dense f r o n t a l edge m a t e r i a l (arrow). Method C. c a . X 1+5,850.  Figure  5»  Figure 6 .  A more advanced stage i n s e p t a l w a l l t h i c k e n i n g . The two w a l l p l a t e s ( p i ) and the c e n t r a l l a m e l l a ( c l ) are i n d i c a t e d . Method C. c a . X k$,&50v A mature septum. The two w a l l p l a t e s ( p i ) and the c e n t r a l l a m e l l a ( c l ) are i n d i c a t e d . Method C. c a . X 1*5,850.  9  III. F i g u r e 7.  PLATE 3  A membrane complex (mc) which i a not c l e a r l y ^ a s s o c i a t e d w i t h septum f o r m a t i o n . T h i s complex seems t o be continuous w i t h the ER as w e l l as the plasma membrane. Note the d i s t i n c t i v e f i b r i l s of the mucous coat i n the upper l e f t . Method A. ca. X 35,700.  F i g u r e 8.  A membrane complex (mc) a s s o c i a t e d w i t h the forming septum. Method C. sea. X 35,700.  F i g u r e 9.  An e n l a r g e d view of the membrane complex seen i n F i g u r e 8 showing the connection w i t h the plasma membrane d e l i m i t i n g the septum and showing the s t r u c t u r e of the complex membranes. Method C. ca. X 116,600.  F i g u r e 10.  A l i g h t microscope view of a r e f r a c t i l e membrane complex (mc) a s s o c i a t e d w i t h the f o r m a t i o n of the f i r s t septum. The m a t e r i a l was f i x e d i n 2% g l u t a r a l d e h y d e and photographed u n s t a i n e d w i t h phase o p t i c s , c a . X 3,000.  III.  PLATE  F i g u r e 11.  K n e e - j o i n t f o r m a t i o n . Note the protruberance on e i t h e r side of the septum and the p o o r l y d e f i n e d w a l l s r e g i o n s i n the zone between the two p r o t r u b e r a n c e s . Note the c l u s t e r i n g of ER and spherosome-like b o d i e s (S) beneath the w a l l of the l a r g e r protuberance. A l i p i d body (L) i s a l s o i n d i c a t e d . Method A. c a . X 22,500.  F i g u r e 12.  A more advanced stage i n k n e e - j o i n t f o r m a t i o n . Note the c l u s t e r i n g of ER and shperosome-like b o d i e s (S) i n the b r i d g e r e g i o n , the apparent d e g r a d a t i o n of m a t e r i a l i n the zone between the two bulges which are s t i l l s e p a r a t e . V e s i c l e s (ve) are p r e s e n t i n the r e g i o n of d e g r a d a t i o n and can a l s o be seen i n the adjacent p r o t o p l a s t . Method A. c a . X 15,900.  F i g u r e 13•  A completed k n e e - j o i n t . Note t h a t the plasma membrane has been reformed around the end og the now incomplete septum. The w a l l surrounding the b r i d g e i s s t i l l t h i n n e r than the s e p t a l w a l l . Note the p o s i t i o n of the h a p l o i d nucleus (hN). Method C. c a . X 26,200.  III.  PLATE 5  F i g u r e ll+a.  The e a r l i e s t v i s i b l e I n d i c a t i o n of bud formation [(bracketed r e g i o n ) . A c e n t r a l , w a l l e s s zone i s bounded by a m o d i f i e d zone of p r o m y c e l i a l w a l l . Long ER elements end beneath the m o d i f i e d w a l l zone. Method C. c a . X 5 9 , 5 0 0 .  F i g u r e llj,b.  An e n l a r g e d view of the b r a c k e t e d w a l l r e g i o n i n F i g u r e llj-a. Method C. c a . X 81a,,600.  Figure 1 5 .  A more advanced stage i n bud f o r m a t i o n . Note the appearance of new amorphous w a l l m a t e r i a l c o v e r i n g the now p r o t r u d i n g bud. A spherosome-like body (S) l i e s j u s t behind the bud apex. Method C. c a . X 31,875.  F i g u r e 16.  A developing bud showing the p o s i t i o n of the hapl o i d nucleus (hN). The bud now has a w e l l d e v e l oped w a l l which t h i n s towards the apex. The plasma membrane (PM) i s very d i s t i n c t . Method C. c a . X 31,875.  III.  PLATE 6  F i g u r e 17.  A s p o r i d i u m which has almost a t t a i n e d maximum l e n g t h . Note the narrow neck r e g i o n . Numerous m i t a c h o n d r i a (M) and spherosome-like o r g a n e l l e s (S) are present i n young s p o r i d i a . Method C. ca. X 19,500.  F i g u r e 18.  A mature sporidium. E a c h sporidium c o n t a i n s a s i n g l e h a p l o i d nucleus (hN), l i p i d bodies ( L ) , ER, and numerous u n i d e n t i f i e d v e s c i c l e s ( v c ) . The e l e c t r o n t r a n s p a r e n t zones at e i t h e r end are probably r e g i o n s i n which the spherosomalv a c u o l a r b o d i e s have l y s e d and degraded the surr o u n d i n g cytoplasm. Notetthe w e l l d e f i n e d nuc l e a r envelope (NE). Method B. ca. X 27,600.  112,  BIBLIOGRAPHY A k a i , S., Fukutomi, M., and Kunoh, H. 1968. An e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n of c o n i d i a and hyphae of E r y s i p h e graminls h o r d e i . Mycopathologica et Mycol o g i a A p p u c a t a js5: 2 1 5 - 2 2 2 . Banno, I . 1 9 6 7 . 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Bud f o r m a t i o n i n Sacchar oroyces c e r e v i s i a e and a comparison w i t h the mechanism o r c e l l d i v i s i o n i n other y e a s t s . J . gen. M i c r o b i o l . 53: 161+-169. McClary, D.O. and Bowers, W.D. 1965. The i n t e g r i t y of t h e c e l l w a l l d u r i n g bud f o r m a t i o n i n y e a s t s . Can. J . M i c r o b i o l . 11: 1+1+7-U52. Moor, H. 1967. E n d l p l a s m i c r e t i c u l u m as the i n i t i a t o r of bud f o r m a t i o n i n y e a s t (Sacchromyces c e r e v i s i a e ) . A r c h . M i k r o b i o l . 57: 135-11+57 Moore, R.T. 1962. P i n e s t r u c t u r e of Mycota 1. E l e c t r o m microscopy of the Discomycete Ascodesmis. Nova Hedwigia 5: 263-278. Moore, R.T. 1963. P i n e s t r u c t u r e o f Mycota 10. T h a l l u s formation i n Pucelnla p o d o p h y l l i a e c i a . Mycologia  55: 633-61+2.  ~  Moore, R.T. 1965. The u l t r a s t r u c t u r e o f f u n g a l c e l l s . The F u n g i I eds. 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U n i v e r s i t y of B r i t i s h Columbia, B r i t i s h Columbia, Canada. Thomas, P.L. and I s a a c , P.K. 1 9 6 7 . An e l e c t r o n microscope study of i n t r a v a c u o l a r b o d i e s i n the u r e d i a of wheat stem r u s t and i n hyphae of other f u n g i . Can. J . B o t . 45: 1473-1478. W e l l s , K. 1964a. The b a s i d i a of E x i d i a n u c l e a t a Ultrastructure. Mycologia 5 6 : 3 2 7 - 3 4 1 .  I  W e l l s , K. 1964b. Development.  II  The b a s i d i a of E x i d i a n u c l e a t a Am. J . B o t . 5 1 : 3 6 0 - 3 7 0 .  PART IV Nuclear D i v i s i o n w i t h S p e c i a l Emphasis on M e i o s i s  TABLE OF CONTENTS Page ABSTRACT  118  INTRODUCTION  113  MATERIALS AND METHODS  120  C u l t u r e s and C u l t u r i n g  •  120  L i g h t Microscopy  120  E l e c t r o n Microscopy  123  OBSERVATIONS  123  L i g h t Microscopy E l e c t r o n Microscopy  ••••••••• •  123 •  •••••  DISCUSSION  136  Nuclear Fores r  *  Centriolar-kinetochore-equivalent Cytoplasmic M i c r o t u b u l e s .......••*. Chromosome Number The Model  BIBLIOGRAPHY  127  ••••••••••••••••••••••••  136 137 li+1 11+2 11+7 152  118  PART IV Nuclear D i v i s i o n With S p e c i a l Emphasis on  Meiosis  ABSTRACT A study has been made o f the u l t r a s t r u c t u r e o f the nucleus and a s s o c i a t e d s t r u c t u r e s o f the smut fungus, U s t i l a g o h o r d e l . w i t h s p e c i a l emphasis on the and  s t r u c t u r e o f the n u c l e a r pores, the  position  centriolar-  k i n e l o c h o r e - e q u i v a l e n t , and c y t o p l a s m i c m i c r o t u b u l e s .  Elec-  t r o n microscope o b s e r v a t i o n s o f t h e m e i o t i c and m i t o t i c promyc e l i a l d i v i s i o n s have been compared w i t h l i g h t microscope o b s e r v a t i o n s o f n u c l e a r d i v i s i o n i n both U s t i l a g o h o r d e l Ustilago k i l l e r i .  E v i d e n t l y c e r t a i n aspects o f the m e i o t i c  and m i t o t i c d i v i s i o n s are unusual  and the r e s u l t s have been  i n t e r p r e t e d a c c o r d i n g to Brown and Stack's somatic  and  n u c l e a r d i v i s i o n i n some f u n g i .  (1971) model f o r  The  problem o f chrom-  osome number i s d i s c u s s e d . INTRODUCTION The  t e l i o s p o r e o f U s t i l a g o h o r d e i i s the s e x u a l  and c o n t a i n s the o n l y d i p l o i d nucleus  i n the l i f e  spore,  cycle.  When the spore germinates t h e nucleus u s u a l l y migrates  into  promycelium (metabasidium) i n the d i p l o i d s t a t e ( P t . I I ) , Three c y c l e s of n u c l e a r d i v i s i o n occur i n quick s u c c e s s i o n : the f i r s t  two  d i v i s i o n are the m e i o t i c r e d u c t i o n d i v i s i o n  and the m e i o t i c e q u a t i o n a l d i v i s i o n , r e s p e c t i v e l y , and l a s t i s the m i t o t i c d i v i s i o n which g i v e s r i s e to the  the  first  119  haploid sporidial n u c l e i .  Chromosomes and s p i n d l e s i n the  n u c l e i o f a smut s p e c i e s were f i r s t  d e s c r i b e d by Harper i n  1893, and h i s r e s u l t s have s i n c e been s u b s t a n t i a t e d by other workers.  Most r e s e a r c h e r s  have r e p o r t e d  chromosome number i n smuts i s two. i n preparation concerning  t h a t the h a p l o i d  Because o f the d i f f i c u l t i e s  and i n t e r p r e t a t i o n the s t a t e o f knowledge  the n u c l e a r  c y t o l o g y o f t h e smut f u n g i r e s t s  c i s e l y where i t d i d i n 191+5.  pre-  No photographic r e c o r d e x i s t s ,  whether a t the l i g h t or e l e c t r o n - m i c r o s c o p e  l e v e l and none  o f the d e t a i l s o f n u c l e a r d i v i s i o n i s known.  Contrary  to  the s t a n d - s t i l l i n c y t o l o g i c a l p r o g r e s s ,  genetic  information  has been accumulating r a p i d l y .  various  Ustilago  Recently  s p e c i e s have been used i n s t u d i e s o f recombination  (Holliday,  1961; H o l l i d a y I96I4.; Kozar 1969), o f h a p l o i d i z a t i o n (Day and Jones, 1969), o f g e n e t i c  complementation (Dinoor and Person,  1969) and o f p a r a s i t i s m (Thomas and Person 1965; 1965)*  Halisky,  Furthermore s e v e r a l o f these s t u d i e s i n d i c a t e t h a t  the h a p l o i d chromosome number o f a t l e a s t some smut may be g r e a t e r than two  ( H o l l i d a y , I96I4.; Day and Jones, 1969),  Such s t u d i e s l e a d t o a renewed demand f o r more concerning  species  the n u c l e i o f t h e smut f u n g i .  information,  I n her p r e s i d e n t i a l  address t o the B r i t i s h My c o l o g i c a l S o c i e t y , i n 1939,  Kathleen  Sampson s t a t e d the case most s u c c i n c t l y when she s a i d , ,,, i t would be unwise to become d a z z l e d by the f a s c i n a t i o n s o f smut g e n e t i c s and to f o r g e t how few are the e s t a b l i s h e d f a c t s concerning chromosome behaviour i n t h i s group o f f u n g i . To o b t a i n a c o n v i n c i n g c y t o l o g i c a l picture o f meiosis i n t h i s group i s no easy t a s k ...  120  MATERIALS AND METHODS CULTURES AND  CULTURING  The w i l d - t y p e  s t r a i n s o f U s t i l a g o h o r d e i are the same  as those p r e v i o u s l y d e s c r i b e d light-microscope observations hordei  Included  i n the  photographs, by way o f comparison, a r e  on the n u c l e i o f a m y c e l i a l mutant o f U s t i l a g o  ( S t e i n , 1970)  post-meiotic  i n part I,  ( P i g s , Pl-ij.), and on the m e i o t i c  divisions i n Ustllago k o l l e r i  ( P i g s , Rows G-L),  (Willie)  The m y c e l i a l mutant was prepared and photo-  graphed by Dr. Jean Mayo, and t h e U, k o l l e r i samples D. C. Wighton.  A l l the m a t e r i a l was  i n p a r t I w i t h the e x c e p t i o n  c u l t u r e d as  LIGHT MICROSCOPY (see a l s o Appendix Squash P r e p a r a t i o n s .  by  described  o f t h e m y c e l i a l mutant which  was grown on the s u r f a c e o f agar p l a t e s  parations  and  (Appendix B ) .  C)  - To prepare s t a i n e d squash  pre-  a drop o f medium c o n t a i n i n g the m a t e r i a l was a i r -  d r i e d b r i e f l y on c o v e r - s l i p s , and f i x e d i n one o f the f o l l o w ing  ways: a) f o r one hour i n a c e t i c - a l c o h o l (1:3) t o which  added a few drops o f  was  chloroform.  b) f o r 21*. hours i n B A C - f i x a t i v e A f t e r f i x a t i o n , the m a t e r i a l was  (Lu 1962). s t a i n e d by one o f the  f o l l o w i n g procedures: a) Peulgen ( D a r l i n g t o n and La Cour 1962); U. was h y d r o l y z e d  i n 1 N HC1 a t 60° f o r 10-12  k o l l e r i was h y d r o l y z e d 1 hour.  hordei  minutes: U.  i n 5 N HC1 a t room temperature f o r  The m a t e r i a l was then s t a i n e d f o r 30-I4.O minutes  TABLE I :  Summary of L i g h t Microscope Techniques  Species  £•  K  o  l  l  e  i  >  i  TJ. horde i  Stage  Fixative  Stain  teliospores and sporidia  acetic - alcohol  Feulgen  teliospores  acetic - alcohol  Feulgen Haematoxylin  sporidia spheroplasts and mycelial mutant  acetic - alcohol  Haematoxylin  122 and 8quashed i n 1+5 % a c e t i c  acid,  b) Propiono-haematoxylin A,  1968):  for  10-12  (Henderson and Lu, Method  the m a t e r i a l was h y d r o l y z e d i n 1 N HC1 a t minutes and s t a i n e d f o r 1-2  60°  C  minutes.  A l l photographs were made from f r e s h l y prepared  slides.  S l i d e s were subsequently made permanent w i t h E u p a r o l where desired. The procedures used f o r each c e l l type a r e o u t l i n e d i n Table I . The  c e l l d e p i c t e d i n F i g u r e 33a was f i x e d i n g l u t a r a l d e -  hyde and observed u n s t a i n e d w i t h phase o p t i c s as d e s c r i b e d i n part I I I S e c t i o n s . - The m a t e r i a l (U. h o r d e i ) was prepared a c c o r d i n g to e l e c t r o n microscope sections  (0.25-0.50 u)  ultramicrotome  p r e p a r a t i o n B (Part I ) .  were c u t on a S o r v a l Porter-Blum  Thick  MT-2  and were s t a i n e d w i t h 1 % T o l u i d i n e b l u e i n 1 %  borax. S p h e r o p l a a t s . - S p h e r o p l a s t s were prepared by Mr. M. Holmwood as f o l l o w s :  a m o n o s p o r i d i a l l i n e o f U. h o r d e i was evil,  c u l t u r e d i n m o d i f i e d complete b r o t h c o n t a i n i n g 15 % dextrose and g l u s u l a s e ( E n d o l a b o r a t o r i e s I n c . ) .  A t 22° C 18 - 21\.  hours were r e q u i r e d f o r s p h e r o p l a s t s t o form. was Lu's  The m a t e r i a l  then f i x e d i n a c e t i c - a l o o h o l and s t a i n e d w i t h Henderson and haematoxylin. Microscopy.  - A l l the U. k o l l e r i m a t e r i a l was observed  w i t h a L e i t z 3 V by i+V bellows camera a t f u l l 1  extension.  123  A Z e i s s photomicroscope was  used i n a l l s t u d i e s o f U.  Both m i c r o s c o p e s were equipped w i t h a $\\h mu  hordel.  interference  filter. ELECTRON MICROSCOPY Germinating t e l i o s p o r e s o f U s t i l a g o h o r d e l were prepared for  e l e c t r o n microscopy a c c o r d i n g  described  i n part  to methods A, B and  C as  I. OBSERVATIONS  LIGHT MICROSCOPY D i f f e r e n t stages o f the l i f e  cycle apparently  react  q u i t e d i f f e r e n t l y to the v a r i o u s methods o f s t a i n i n g . s t u d i e s o f the p r o m y c e l i a l  For  divisions, fixation in acetic-  a l c o h o l f o l l o w e d by Feulgen s t a i n i n g ( F i g s . Rows A-Lj_ gave g r e a t e r chromosome c o n t r a s t than d i d haematoxylin ( F i g s . Ml, NI and 0 1 )  w i t h the techniques used.  haematoxylin-; p r e p a r a t i o n s  However, s e v e r a l  of meiosis i n Ustilago  hordei  are i n c l u d e d to show t h a t s i m i l a r c o n f i g u r a t i o n s have been observed ( F i g s . Ml, NI and 0 1 ) . in  On  the o t h e r hand, f i x a t i o n  a c e t i c - a l c o h o l f o l l o w e d by haematoxylin produced much  superior r e s u l t s i n vegetative the m y c e l i a l mutant. s t a i n s the n u c l e o l u s  t i s s u e , p a r t i c u l a r l y with  Haematoxylin, u n l i k e Feulgen, a l s o d e n s e l y (Row  o f the c h r o m a t i n - n u c l e o l a r  P), allowing v i s u a l i z a t i o n  relationship.  L i t t l e i s known about the e a r l y stages o f m e i o t i c  pro-  phase I whioh presumably o c c u r w i t h i n the forming, t h i c k walled  teliospore.  When the nucleus f i r s t  passes i n t o  the  promycelium t h e chromatin i s a l r e a d y h i g h l y c o n t r a c t e d Rows A, B, and G).  (Pigs.  Normally t h i s l a t e prophase chromatin  takes the form o f two elongate d e n s e l y - s t a i n e d  bodies  l y i n g s i d e by s i d e and p a r a l l e l w i t h the l o n g a x i s o f t h e promycelium.  O c c a s i o n a l l y , when the d i p l o i d nucleus i s Just  e n t e r i n g the promycelium, r i n g - l i k e c o n f i g u r a t i o n s ( P i g s . A l , and 0 ) ,  Such c o n f i g u r a t i o n s probably  the e a r l i e s t m e i o t i c celium.  occur represent  stages which can be seen i n the promy-  One c h a r a c t e r i s t i c o f l a t e prophase I n u c l e i i s  t h a t the two chromatin bodies f r e q u e n t l y appear t o be c l o s e l y a s s o c i a t e d a t one o r both ends. Cif, E l , G2, Glj.).  ( P i g s . A2, A3, Alj., B3, Bij.,  A second common f e a t u r e i s t h a t one o r  both b o d i e s may be j o i n e d by a f i n e P e u l g e n - p o s i t i v e to a s m a l l k n o b - l i k e  thread  s t r u c t u r e l y i n g a g a i n s t one o f the  l a t e r a l promycelial walls  ( P i g s . A2, B l , Gif, M).  I n U s t i l a g o h o r d e i and U s t i l a g o k o l l e r i no stage e x i s t s which v i s u a l l y compares with t h e metaphase p l a t e seen a t meiosis I i n higher  p l a n t s and many f u n g i .  The chromatin  bodies c o n t r a c t as n u c l e a r d i v i s i o n approaches ( i . e . compare A2,  B2, and P2; a l s o G2 and H2).  minimum d i p l o i d l e n g t h  As they a t t a i n t h e i r  (approximately 1.6 u) they b e g i n  to r o t a t e and come t o l i e a t an angle t o the l o n g i t u d i n a l a x i s o f the metabasidium ( P i g . E 2 ) .  Following  t h i s there i s  a poorly defined  stage i n which l i t t l e d e t a i l i s d i s c e r n -  ible  These l a s t two stages a r e i n f r e q u e n t l y  (Pig. E3).  seen and a r e probably s h o r t .  When the chromatin bodies  again become d i s t i n c t they l i e i n a w e l l - d e f i n e d  anaphase  125  c o n f i g u r a t i o n ( P i g s . Elf.,  H3-4.,  I I , and  01).  Viewed w i t h  p l a n a r o p t i c s the Peulgen s t a i n i n g r e a c t i o n i s c o n s i d e r a b l y l e s s i n t e n s e , s u p p o r t i n g the common assumption t i o n d i v i s i o n has occured  t h a t reduc-  compare H 2 and H 3 ) ,  (i.e.  The  daughter h a p l o i d n u c l e i p u l l a p a r t r a p i d l y and i n so d o i n g the two  chromatin bodies w i t h i n each mucleus continue the  c o n t r a c t i o n which was  i n i t i a t e d i n the d i p l o i d s t a t e .  The  minimum l e n g t h a t t a i n e d by the chromatin b o d i e s o f h a p l o i d nuclei i s  1.0  u  (Pigs.  and  P2,  Jl-2).  O c c a s i o n a l l y the  p a i r o f anaphase chromatin b o d i e s i n each o f the s e p a r a t i n g n u c l e i appear to be j o i n e d a t the "poleward" end and 1 2 ) .  (Pigs. ELL,  I n F i g u r e s PI and 1 3 a chromatin t a i l i s l a g g i n g  behind each o f t h e chromatin p a i r s and p r o j e c t i n g i n the d i r e c t i o n o f i t s homologue. conspicuous knob.  The t a i l s each t e r m i n a t e i n a  During the anaphase m i g r a t i o n the  two  chromatin bodies g e n e r a l l y l i e p a r a l l e w i t h the l o n g i t u d i n a l promycelial axis. Good l i g h t microscope p i c t u r e s were r a r e l y o b t a i n e d of  t h e second and t h i r d p r o m y c e l i a l d i v i s i o n s , which are  e s s e n t i a l l y mitotic i n nature.  Amazingly  e x c e p t i o n o f the reduced l e n g t h o f the two  enough, with the chromatin b o d i e s ,  both these d i v i s i o n s p r o g r e s s i n a manner e s s e n t i a l l y i c a l w i t h the r e d u c t i o n d i v i s i o n .  ident-  The m e i o t i c e q u a t i o n a l  d i v i s i o n i s r e p r e s e n t e d here by F i g u r e s F 4 . and L (note the arrow marking t h e f i r s t sections  (M3-4.,  N3-I4.,  and  septum) and the s e t o f s e r i a l  03-4-).  The f i r s t p o s t - m e i o t i c  d i v i s i o n i s r e p r e s e n t e d b y F i g u r e J 3 - 4 where the nucleus  126  lies  i n the neck o f the forming bud.  and I I I c o n f i g u r a t i o n s ( F i g . J3-U-)  a  r  Decondensed prophase I I e  infrequent, suggesting  e i t h e r t h a t chromatin decondensation between d i v i s i o n s i s " o p t i o n a l " o r that i t occurs r a p i d l y . III  the condensed  During prophase  I I and  chromatin bodies r a r e l y l i e p a r a l l e l t o the  l o n g p r o m y c e l i a l a x i s ; u s u a l l y they a r e angled s t e e p l y a c r o s s it  ( F i g s . Fq. and N3-lj.).  Little  d e t a i l i s d i s c e r n i b l e i n the  t i n y anaphase I I and I I I n u c l e i b u t , i n g e n e r a l , they  resemble  anaphase I c o n f i g u r a t i o n ( F i g . L ) . By way o f comparison  a few photographs  o f somatic m i t o t i c  d i v i s i o n s have been i n c l u d e d (Rows P, Z and R ) . F i g u r e s Ql-lj. and Rl-2 r e p r e s e n t s p o r l d i a l d i v i s i o n s .  Again very  l i t t l e d e t a i l can be seen i n h a p l o i d s p o r l d i a l n u c l e i .  Where  d i v i s i o n - l i k e f i g u r e s do o c c u r they a r e l o c a t e d e i t h e r i n the p a r e n t a l c e l l o r , more o f t e n , i n the neck between the parent c e l l and the bud ( R l - 2 ) . (Ql-2  Two p r o p h a s e - l i k e c o n f i g u r a t i o n s  and Q3-I4.) and an anaphase (Rl-2) a r e shown.  three c e l l s ,the two chromatin b o d i e s a r e c l e a r l y S e v e r a l attempts have been made to produce  In a l l visible.  sporldial  sphero-  p l a s t s o f U s t i l a g o h o r d e i s i n c e removal o f the w a l l a f f o r d s c o n s i d e r a b l e advantages  f o r f i x i n g , s t a i n i n g and adequately  squashing the p r e p a r a t i o n s .  U n f o r t u n a t e l y most o f the n u c l e i  seen thus f a r i n s p h e r o p l a s t c u l t u r e s seem to be l n a r e s t i n g state.  R3 and Rl+ a r e photographs  t a i n i n g prophase-like f i g u r e s . chromatin bodies All"  o f two s p h e r o p l a s t s con-  Again there a r e two d i s t i n c t  (Length = 1.0 u ) .  p r e p a r a t i o n s o f the m y c e l i a l mutant were made w i t h  127  haematoxylin*  The n u c l e i and chromatin bodies o f the  m y c e l i a l mutant are e x c e p t i o n a l l y l a r g e , b e i n g comparable to those o f the m e i o t i c d i p l o i d s t a t e *  At present the  p l o i d y o f t h e m y c e l i a l mutant i s unknown* e x i s t s t h a t i t may be a t r u e d i p l o i d communication)*  The p o s s i b i l i t y  ( J . Mayo, p e r s o n a l  I n any case t h e l a r g e s i z e o f t h e n u c l e i  does allow c l e a r o b s e r v a t i o n o f the two chromatin b o d i e s . Haematoxylin has the advantage  o f s t a i n i n g the n u c l e o l u s  as w e l l as t h e chromatin; i n row P the conspicuous n u c l e o l u s , which l i e s a t the end o f t h e n u c l e u s , i s i n d i c a t e d by arrows. One o r both o f the chromatin masses a r e a t t a c h e d to the nucleolus.  F i g u r e s P I , P2, and P3 are c o n s i d e r e d to be i n  l a t e m i t o t i c prophase.  A number o f anomalous f i g u r e s  like  those shown i n F i g u r e Plj. o c c u r , and these have been i n t e r p r e t e d as e a r l y somatic anaphase; the daughter  chromatin  bodies have begun t o separate but the n u c l e o l u s which i s persistent, i s yet undivided. ELECTRON MICROSCOPY U l t r a s t r u c t u r e o f the Nucleus and A s s o c i a t e d S t r u c t u r e s . The o b s e r v a t i o n s concerning the u l t r a s t r u c t u r e and a c t i v i t i e s o f the n u c l e u s and i t s a s s o c i a t e d s t r u c t u r e s a r e based on glutaraldehyde-osmium otherwise s t a t e d .  f i x e d m a t e r i a l (Methods B and C) u n l e s s  Potassium permanganate l e a c h e s out and  d e s t r o y s both n u c l e i c a c i d s and m i c r o t u b u l a r s t r u c t u r e s which m a i n l y compose t h e n u c l e a r apparatus. F i g u r e 5 i l l u s t r a t e s a h a p l o i d , e a r l y prophase  I I nucleus  128  i n a promycelium prepared by method C,  The n u c l e a r  envelope i s r a r e l y d i s t i n c t a f t e r  glutaraldehyde-osmium  f i x a t i o n but when measurementsjare  p o s s i b l e the average  double membrane w i d t h i s 3 0 0 A  0  (Range 210-1+00 Ao) w i t h  each o f the u n i t membranes averaging 83 A  0  (Range £ 0 - 1 2 0  A ). 0  A f t e r KMnOj^ - f i x a t i o n the r e s p e c t i v e measurements are 27£ A  0  (Range 220-31+0 A°) and 80 A . 0  I n s p i t e o f the  tenuous n a t u r e o f the n u c l e a r membranes a f t e r g l u t a r aldehyde-osmium f i x a t i o n t h e nucleus i s never d i f f i c u l t to d i s t i n g u i s h .  F i r s t , the o u t e r s u r f a c e of the envelope  i s o f t e n accentuated by a conspicuous attachment o f r i b osomes ( F i g . £ ) ,  Second, the nucleoplasm i s l e s s  dense  than the cytoplasm ( F i g s . 7 , ll+, and 28). The n u c l e a r envelope o f U s t l l a g o h o r d e l i s f r e q u e n t l y i n t e r r u p t e d by n u c l e a r pores.  In m a t e r i a l prepared by meth-  ods A and B, these pores appear as simple " h o l e s " , a l l o w i n g d i r e c t c o n t i n u i t y between the nucleoplasm and cytoplasm. However, a f t e r glutaraldehyde-osmium f i x a t i o n and embedding i n Spurr's p l a s t i c , i t i s c l e a r t h a t the pores a r e o c c l u d e d by e l e c t r o n - d e n s e m a t e r i a l .  In cross-section, t h i s material  i s r a t h e r amorphous ( F i g . 2 3 ) ,  but i n face-view the c i r c u l a r  pore r e g i o n , which has an average diameter o f 1 , 0 6 0 A° (Range: 9 5 0 1 1 0 0 A ° ) , i s h i g h l y s t r u c t u r e d  (Fig. 6a-c).  A r i n g o f g r a n u l a r or m i c r o t u b u l a r elements l i e s  just  inside  the pore p e r i p h e r y and i s a p p a r e n t l y embedded i n the e l e c t r o n dense substance.  One or two l a r g e g r a n u l e s (or m i c r o t u b u l e s )  l i e i n the c e n t r e (Average diameter 1 9 0 - 2 U + AO),  Fine  129  f i b r i l l a r m a t e r i a l (Average diameter = 1+2 A )  radiates  0  f r o m the c e n t r a l element i n a l l d i r e c t i o n s towards the pore periphery.  Pores of t h i s d e s c r i p t i o n are most prominent  i n n u c l e i which are c l o s e t o d i v i s i o n ( P i g . 23 and P t . V, Pig.  3&)  and c y t o p l a s m i c microtubules  are i n e v i t a b l y  present  near such pores ( P i g . 6a-to,  i n the p e r i p h e r a l cytoplasm  and  23). The n u c l e o l u s i s u s u a l l y the most conspicuous s t r u c t u r e i n the nucleoplasm ( P i g s . f>,  20,  22,  2 3 , 2l+,  27-9,  and  31).  In shape i t i s s p h e r i c a l t o o v o i d ; i t s average diameter i s 1.06 it  u i n b o t h d i p l o i d and h a p l o i d n u c l e i .  In s t r u c t u r e  c o n s i s t s of l o o s e l y o r g a n i z e d , e l e c t r o n - d e n s e ,  granular  m a t e r i a l i n t e r s p e r s e d by patches of l e s s dense nucleoplasm ( P i g s . 2 2 and 2 3 ) .  One  i s d i v i d e d i n t o two  separate  g r a n u l a r zone ( P i g . 5,  o f t e n has  23,  the impression  s e c t i o n s by a t h i n ,  and 2 7 ,  i s always c l o s e l y appressed on one of the n u c l e a r envelope. (Pts.  I I and  i o r end  that It  arrows).  less-  The  nucleolus  s i d e to the i n n e r s u r f a c e  As has been n o t e d p r e v i o u s l y  111$, the n u c l e o l u s always l i e s at the  of the m i g r a t i n g nucleus  • jfehe promycelium or i n t o the bud,  poster-  ( i . e . during migration  up  or d u r i n g anaphase r e p u l -  sion) . At the a n t e r i o r end  of the m i g r a t i n g nucleus  is  another c y t o l o g i c a l l y conspicuous body which w i l l be r e f e r r e d to  i n t h i s paper as the  (CKE).  ( P i g s . 7 and 2 2 ) .  extra-nuclear  centriolar-4cenetochore-equivalent, The  CKE  Is, s t r i c t l y  ( i . e . the main body of the CKE  speaking,  lies  outside  130  the n u c l e a r e n v e l o p e ) ; however, i t s s t r u c t u r e and are i n t i m a t e l y a s s o c i a t e d w i t h n u c l e a r events. n u c l e i are s l i g h t l y elongate has  ceased,  23),  (Pig. 7 ) .  activities  Migrating  When the  migration  the n u c l e i round-up i n s o f a r as i s p o s s i b l e ( P i g .  and the CKE  assume a p o s i t i o n c l o s e l y appressed  outer s u r f a c e o f the n u c l e a r envelope and d i r e c t l y to the n u c l e o l u s .  t o the  opposite  U s u a l l y i n n o n - d i v i d i n g n u c l e i , the  CKE  l i e s i n a c y t o p l a s m i c w e l l p r o t r u d i n g i n t o the nucleoplasm. During prophase I , t h i s w e l l i s shallow 23),  (Pig. 3 ,  22,  and  but i t i n c r e a s e s l n depth d u r i n g prophase I I and I I I  ( F i g . 7* and 2 0 ) o f the CKE.  corresponding w i t h an i n c r e a s e i n the  size  The w e l l seems to be a r e l a t i v e l y s t a b l e p a r t of  prophase n u c l e a r s t r u c t u r e whether or not the CKE l i e s w i t h i n it.  F i g u r e s 17a-b  and l 8 a - b , i l l u s t r a t e r e s p e c t i v e l y a  n u c l e a r w e l l without U s u a l l y the CKE cytoplasm  a CKE and a n u c l e a r w e l l w i t h a  i s surrounded by a h a l o o f low d e n s i t y  from which o t h e r cytoplasmic o r g a n e l l e s i n c l u -  d i n g ribosomes are excluded.  The  remains I n t a c t ( F i g . 3 and 1 1 ) .  adjacent n u c l e a r membrane On i t s i n s i d e s u r f a c e i s  a cap o f e l e c t r o n dense nucleoplasmic  m a t e r i a l ( i . e . chromatin)  which i s a s s o c i a t e d with the presence o f t h e kinetochore-equivalent 7,  CKE.  the chromatin  (Figs. 7,  9,  18b,  centriolar-  and 2 0 ) .  cap i s c l e a r l y b i p a r t i t e .  In F i g u r e  Notably,  this  i s the o n l y p o i n t on the n u c l e a r envelope t o which the chromatin  can be shown to a t t a c h .  S t r u c t u r e s resembling the CKE have never been i n ungerminated spores but whether t h i s i s due  observed  to the a c t u a l  131' absence of She  body or t o the f a c t t h a t i t i s masked by  d e n s i t y of the cytoplasm i s unknown.  the  A single centriolar-  M n e t b c h o r e - e q u i v a l e n t i s l o c a t e d a t the a n t e r i o r of  the  d i p l o i d n u c l e u s when i t f i r s t b e g i n s t o migrate up the promy-— I I , P i g . 5)  c e l i u m . #Ft. oval,  In the  c r o s s - s e c t i o n , the CKE  ( i . e. p a r t i c u l a r l y i n prophase I ) , c i r c u l a r ,  s l i g h t l y t r i a n g u l a r except when d i v i d i n g .  37© by 3 7 0 mu by  (Figg 9 ) .  equivalent  I I I , i t i n c r e a s e s to as much as  This increase i s r e a d i l y  observed  c o n s i s t s of a t a n g l e 0  an amorphous e l e c t r o n - d e n s e and  of f i n e f i b r i l s w i t h  (Range: 3 : 8 - ^ 9 A ) 0  matrix  embedded i n  (Figs § - 1 3 ) *  core t h a t f o l l o w s the contours  of the body on the s i d e opposite  9 and  an  In prophase  I I I , i t o f t e n develops what appears to be a dense  elongated face  the  S t r u c t u a l l y , the c e n t r l o l a r - ^ k e n e t o c h o r e -  average diameter of 28.1* A  12).  are two  of the  outer  t o the n u c l e u s  O c c a s i o n a l l y , as i n F i g u r e s 1 0 and 1 1 ,  d i s t i n c t zones i n the CKE,  s i d e and a r e g i o n i n which the f i b r i l s  on the  other.  T h i s e f f e c t may  passes through, and In p r o m y c e l i a  are  (Figs.  zone  "spun-out"  be produced when the s e c t i o n  i s i n the same plane  as the dense  core/  of U s t i l a g o h o r d e i , each prophase I, I I ,  I I I nucleus i s associated with a s i n g l e small  k i n e t o c h o r e - e q u i v a l e n t .tsuObviously  centriolar-  f o r t h i s t o be  c e n t r i o l a j ? - k i n e t ©chore-equivalent must r e p l i c a t e nuclear d i v i s i o n .  sur-  there  a dense f i b r i l l a r  on one  and  ( P i g . 8)  comparing F i g u r e s 8 and 9 which are a t approximately  same m a g n i f i c a t i o n .  II  or  I t s maximum  s i a e i n prophase I i s approximately 1 6 5 by 9 5 mu but d u r i n g prophase I I and  is  O c c a s i o n a l l y elongate  so  the  once p e r  fibrillar  structures  —  132  have been observed l y i n g p a r a l l e l w i t h the n u c l e a r envelope ( F i g s . I4.-I6) and these have been i n t e r p r e t e d as d i v i d i n g centriolar-klnetochore-equivalents. fibrils, CEE,  I n F i g u r e 15?, f i n e  s i m i l a r i n s t r u c t u r e to those c o n s t i t u t i n g the  seem to connect the d i v i d i n g CKE t o the n u o l e a r en-  velope. During l a t e prophase,  c y t o p l a s m i c m i c r o t u b u l e s develop  i n a s s o c i a t i o n w i t h the nucleus and the CKE. an average diameter o f 2 4 4 A  0  (Range:  The t u b u l e s have  2 0 0 - 2 5 0 A ° ) w i t h an  e l e c t r o n t r a n s p a r e n t c e n t r a l core (Average diameter = 1 0 2 A ) . 0  For the most p a r t these m i c r o t u b u l e s l i e i n the l o n g i t u d i n a l a x i s o f the promycelium o r a t a s l i g h t angle to i t ( F i g s . 1 9 , 2 0 and 23 )  5  19 and 2 0 ) .  and r a d i a t e from the v i c i n i t y o f the CKE ( F i g s . The t u b u l e s do n o t o r i g i n a t e d i r e c t l y from the  CKE i t s e l f but from the g l o b u l a r e l e c t r o n - d e n s e s t r u c t u r e s (mtoc) i n the v i c i n i t y o f the CKE ( F i g s . 2 1 ) .  Other  t u b u l e s end i n s i m i l a r e l e c t r o n dense s t r u c t u r e s  micro-  (Fig.2 0 ) .  Nuclear D i v i s i o n . - F i g u r e s 2 2 t o 3 3 a r e arranged s e q u e n t i a l l y to demonstrate  the main stages o f the promy-  c e l i a l d i v i s i o n s a c c o r d i n g t o the author's  interpretation.  F i g u r e s 2 2 t o 26 i l l u s t r a t e m e i o s i s I ( i . e. r e d u c t i o n d i v i s i o n ) and F i g u r e s 2 7 t o 3 3 * m e i o s i s I I ( i . e. e q u a t i o n a l d i vision).  The sequence o f 2 7 to 3 3 i s a oomposite o f observa-  t i o n s made from d i v i s i o n I I and I I I n u c l e i .  These d i v i s i o n s  are e s s e n t i a l l y i d e n t i c a l i n appearance w i t h the e x c e p t i o n t h a t when d i v i s i o n I I I I o c c u r s i n the parent c e l l t h e n u l e a r a x i s ( i . e . an Imaginary l i n e drawn through the two p o l e s )  133,  l i e s at an angle of about 45° to the longitudinal promycelial axis while the nuclear axis of d i v i s i o n II l i e s roughly a l l e l to the longitudinal c e l l a x i s .  par-  Unless the nuclear  axis can be determined, septa counted, or a bud i s v i s i b l e i t i s impossible to d i s t i n g u i s h d i v i s i o n II and III n u c l e i . The d i p l o i d fusion nucleus i n Figure 2 2 i s about 4 . 2 5 u by 1.70  p.  The arrow indicates the d i r e c t i o n of the promy-  c e l i a l apex.  At the posterior end of the nucleus l i e s the  prominent nucleolus and d i r e c t l y opposite to the nucleolus and about one-third of the nuclear length behind the anterior nuclear t i p i s the centriolar-kinetochore-equivalent. Between the nucleolus and the CKE i s a band of nucleoplasm of increased density ( i . e. chromatin) about 0.7 This nucliolar-ehromatin-JC'KE  configuration i s c h a r a c t e r i s t i c  of the migrating prophase I nucleus. schematically i n Figure 35a*  p wide and 1.3 p long.  It i s represented  When the nucleus has  ceased  Its forward motion i t shortens and rounds-up insofar as i s possible.  In Figure 23 the CKE has s h i f t e d i t s p o s i t i o n  p o s t e r i o r l y with respect to the nucleus so that i t now  lies  midway along the nuclear length and at the same time the nucleolus<shas begun to move from a posterior to a l a t e r a l position. feating.  The impression i s that the entire nucleus i s rDeThe nucleolar-CKE distance i s shortened to 0.6  Such late prophase I n u c l e i are associated with l y i n g p a r a l l e l with the length of the nucleus  microtubules  ( i . e. with the  promycelial a x i s ) , on the side occupied by the CKE. 23 i s represented acheraatically i n Figure 3 5 b .  p.  Figure  No  data are a v a i l a b l e on the d i v i s i o n of the  kinetochore-equivalent  at r e d u c t i o n d i v i s i o n .  centriolar-  In the next  c h a r a c t e r i s t i c stage t h a t i s observed the nucleus and  elongates  c o n s t r i c t s i n the c e n t e r , becoming dumbbell shaped ( F i g . In F i g u r e 23 the two bulbous ends of the nucleus  21+).  v i r t u a l l y separated. the CKE's i s v i s i b l e nucleus. 35>d.  The n u c l e o l u s has  one  of  on the r i g h t s i d e of the lower daughter  F i g u r e 2l\ i s r e p r e s e n t e d  As the two  d i v i d e d and  have  diagrammatically  i n Figure  daughter n u c l e i p u l l apart the n u c l e a r mem-  brane undergoes a very s h o r t p e r i o d of p a r t i a l breakdown ( F i g u r e s 2$ and 26). e q u i v a l e n t has  In F i g u r e 26  the c e n t r i o l a r - k i n e t o c h o r e -  moved from a l a t e r a l to an a p i c a l p o s i t i o n .  Note the condensation i n F i g u r e s 2$ and 26.  of the c h r o m a t i n - n u c l e o l a r  material  A f t e r KMnO^ f i x a t i o n the n u c l e i c a c i d  c o n t a i n i n g m a t e r i a l ( I . e. n u c l e o l u s and chromatin) i s r e p r e s e n t e d by e l e c t r o n - t r a n s p a r e n t patches ( F i g s . 2$  and  33)•  in  F i g u r e 7 I l l u s t r a t e s a h a p l o i d daughter nucleus  repulsion.  The n u c l e a r envelope has been r e c o n s t i t u t e d and  the c e n t r i p l a r - T - k i n e t o c h o r e - e q u i v a l e n t i s i n the l e a d p o s i t i o n . IhYiFigure 27 diameter - 1.8 prophase I I .  u) has The  d i r e c t l y opposite CKE  the h a p l o i d daughter nucleus  CKE  ( Average  come to r e s t and has presumably  entered  l i e s i n a characteristic postion  the n u c l e o l u s . A V  line.drawn  through the  .  and b i s e c t i n g the n u c l e o l u s would c r o s s the l o n g i t u d i n a l  p r o m y c e l i a l a x i s almost p e r p e n d i c u l a r l y . a l a t e prophase I I nucleus  F i g u r e £8  j u s t p r i o r t o CKE  illustrates  division.  s e c t i o n has passed d i r e c t l y through the centre of the  The nucleus  135  i n the same plasne as the chromatin which i s condensed clearly visible. CKE  One  chromatin body d i r e c t l y  and  j o i n s the  to the n u c l e o l u s , and the second c o l l s v e r t i c a l l y upwards  i n F i g u r e 28  (arrows).  Both o f the chromatin bodies  are  j o i n e d d i r e c t l y to the CKE by a p a i r o f f i b r i l s which have an average diameter o f 8 0 A 28 i s r e p r e s e n t e d  78-83 A ) .  (Range:  0  Figure  0  s c h e m a t i c a l l y l n F i g u r e 35®.  Shortly after  t h i s stage the c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t moves out o f the n u c l e a r w e l l , elongates p r e v i o u s l y ( F i g s , 29  35f).  and  and d i v i d e s as  described  In F i g u r e 3 0 the daughter  CKE'a are m i g r a t i n g around the n u c l e a r envelope which i s o u t l i n e d by the presence o f n u c l e a r pores, and has  a l r e a d y begun to elongate F i g u r e 30  celiaJl axis. Figure 35g.  nucleus  i n the d i r e c t i o n o f the promy-  i s represented  F i g u r e 31 shows the two  a d i v i s i o n I I I nucleus  the  schematically i n  CKE's a t the p o l e s  of  ( i . e. note the p o s i t i o n o f the  n u c l e a r a x i s w i t h r e s p e c * t o the p r o m y c e l i a l a x i s ) . n u c l e o l u s l i e s i n a c h a r a c t e r i s t i c p o s i t i o n to one the n u c l e a r e x i s and midway between the p o l e s  The side of  (Fig. 3 5 M .  As i n M e i o s i s I the n u c l e a r envelope p a r t i a l l y breaks down and  the daughter n u c l e i move a p a r t  In F i g u r e 32 microtubules daughter n u c l e u s .  ( F i g s . 32 and  can be seen p a s s i n g i n t o the open  Presumably the m i c r o t u b u l e s  represent  p a r t o f the d i v i s i o n s p i n d l e which i n i n a d e q u a t e l y by the techniques  35k).  employed.  preserved  Under the l i g h t microscope  u s i n g phase c o n t r a s t o p t i c s , t h i s s p i n d l e appears i n glutaraldehyde  f i x e d m a t e r i a l as a t h i n dark l i n e i n the  136  nuclear axis  (Pig. 3 3 b ) . DISCUSSION  NUCLEAR PORES Since C a l l a n and Torolin ( 1 9 5 0 ) f i r s t d i s c o v e r e d "pores" o r " h o l e s " i n the n u c l e a r envelope o f amphibian oocyte n u c l e i i t has become i n c r e a s i n g l y c l e a r t h a t these r e g i o n s are not j u s t simple "spaces" a l l o w i n g the f r e e  dif-  f u s i o n o f substances between the nucleus and cytoplasm (Feldherr, 1 9 6 5 ) . are  When p r o p e r l y prepared the n u c l e a r pores  f i l l e d w i t h a v a r i e t y o f s t r u c t u r a l components embedded  i n an e l e c t r o n opaque substance.  These pores seem to be  u b i q u i t o u s among e u k a r y o t i c organisms of  and a l a r g e volume  l i t e r a t u r e p e r t a i n i n g to t h e i r chemical and s t r u c t u r a l  p r o p e r t i e s now e x i s t s  (Abelson and Smith 1970; prank and  Scheer, 1970; Koshiba e t a l . ,  1970; Yoo and Hayley,  1967).  There a r e many r e p o r t s o f n u c l e a r pores i n f u n g i (Bracker, I967) but i n most cases they appear as simple " h o l e s " i n the  n u c l e a r envelope.  I n U s t i l a g o h o r d e i p r e p a r a t o r y methods  A and B (Pt I ) e v i d e n t l y d e s t r o y the complex pore which i s so prominent  apparatus  i n t i s s u e f i x e d i n glutaraldehyde-osmium  and embedded i n Spurr's p l a s t i c  (Pigs. 6a-c).  The observ-  a t i o n s on the n u c l e a r pores o f t h i s fungus agree w i t h the f i n d i n g s i n o t h e r p l a n t s and animals i n r e s p e c t o f the p r e sence, appearance,  and s i z e o f the pore r e g i o n s , p e r i p h e r a l  g r a n u l e s , c e n t r a l g r a n u l e s , and r a d i a t i n g f i l a m e n t s .  A complex  pore s t r u c t u r e o f t h i s type has a l s o been d e s c r i b e d i n Copr i n u s lagopus (Lu, 1965) and Ascobolus s t e r c o r a r i u s  (Wells, 1970).  137  In  U s t i l a g o h o r d e l there i a an i n c r e a s e i n the number and  prominence of n u c l e a r pores as t h e n u c l e i approach d i v i s i o n . GENTRIOLAR-KENETOCHORE -EQUIVALENT Although t r u e c e n t r i o l e s have been found i n phycomycetes w i t h a m o t i l e ; s t a g e i n the l i f e  cycle  (Bracker 1967)  they  do not occur among da oomycetes and b a s i d i o m y c e t e s . Y e t . when a p p r o p r i a t e l y s t a i n e d t i s s u e ' i s observed i n t h e l i g h t microscope  t i n y s p h e r i c a l , e l o n g a t e , or r e c t a n g u l a r b o d i e s  f r e q u e n t l y l i e a t the p o l e s of d i v i d i n g f u n g a l n u c l e i and i f these b o d i e s are observed a t v a r i o u s stages of the d i v i s i o n c y c l e they behave i n a " c e n t r i o l a r " manner. the e l e c t r o n microscope sist  Under  these c e n t r i o l e - l i k e s t r u c t u r e s  con-  of s e m i - e l e c t r o n dense, u s u a l l y amorphous, m a t e r i a l .  During d i v i s i o n they l i e a t the p o l e s of the s p i n d l e t u b u l e s and are the hub of the a s t r a l r a y s .  micro-  Wells (1970)  has r e c e n t l y reviewed the l i t e r a t u r e p e r t a i n i n g t o these b o d i e s and n u c l e a r d i v i s i o n i n the Ascomycetes: n o t a b l e , among these s t u d i e s are those of Beckett and W i l s o n (1968) *  n  Podospora a n s e r i n a , Robinow and Marak (1966) i n Sacchar--  omyces c e r e v i s i a e , Schrantz Wells  (1967) i n P u s t u l a r i a c u p u l a r l s ,  (1970) i n Ascobolus s t e r c o r a r l u s and Z i c k l e r (1970)  i n two s p e c i e s of Ascobolus and two s p e c i e s of Podospora. Probably because of the d i f f i c u l t y  of f i x i n g  f u n g i w i t h a p p r o p r i a t e E . M. techniques smium) l i t t l e  basidiomycetous  ( i . e. g l u t a r a l d e h y d e  i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g the  " c e n t r i o l a r - e q u i v a l e n t " i n these f u n g i .  G i r b a r d t (1968),  138  Lu  (1965  and 1967b), and Motta (1967  described  and 1969)  have  s t r u c t u r e s which appear to be the " c e n t r i o l a r -  e q u i v a l e n t " i n P o l y s t i o t u s v e r s i c o l o r , Coprinus 1agopus, and A r m i l l a r i a m e l l e a r e s p e c t i v e l y . In U s t i l a g o h o r d e i the body which l i e s at the poles the d i v i s i o n s p i n d l e has been termed the equivalent cussed.  The  shape and and  (CKE)  centriolar-kinetochore-  f o r reasons which w i l l be subsequently d i s -  CKE  o f U s t i l a g o h o r d e i c l o s e l y resembles i n  s i z e the  " c e n t r i o l a r - e q u i v a l e n t " i n Coprinus lagopus  A r m i l l a r i a melleaj  t h a t o f P o l y s t i c t u s v e r s i c o l o r seems  to be s l i g h t l y d i f f e r e n t .  In b o t h U s t i l a g o h o r d e i  Coprinus lagopus (Lu, 1967b) t h i s body has structure. 28.4. CKE  The diameter o f t h e f i b r i l s  A°) and  the  probable P e u l g e n - p o s i t i v e  should  be  d i v i s i o n and f i r s t present,  (Berkson, 1970;  sub-  the  Why  the  CKE meidtic  m i t o t i c d i v i s i o n i s , at  phenomenon i s widespread among f u n g i  Lu, 1967a; O l i v e , 1965;  Z i c k l e r , 1970).  nature o f  prophase o f the second  post-meiotic  unknown; but the  a fibrillar  centriolar-kinetochore-  composed p a r t l y o f DNA.  increase i n s i z e during  and  (Average diameter =  i n t h i s smut fungus suggest t h a t the  e q u i v a l e n t may  of  Singleton,  1953;  As i n most o t h e r basidiomycetes and  cetes s t u d i e d u l t r a s t r u e t u r a l l y , the  ascomy-  "centriolar-kinetochore-  e q u i v a l e n t " o f U s t i l a g o h o r d e i i s c l o s e l y a s s o c i a t e d with the outer  s u r f a c e o f the n u c l e a r  envelope at a l l times,  and  except d u r i n g the very b r i e f p e r i o d o f d i v i s i o n i t s e l f , i t l i e s w i t h i n a cytoplasmic ( G i r b a r d t , 1968; 1966;  Wells, 1970;  Lu, 1965;  well protruding Motta, 1969;  Z i c k l e r , 1970).  i n t o the  Robinow and  nucleus Marak,  139  In U s t i l a g o an o r g a n e l l e - f r e e zone surrounds the CKE.  This  i s a l s o the case i n P o l y s t i c t u s v e r s i c o l o r  1968)  and Ascobolus  s t e r o o r a r i u s (Wells, 1970)  (Girbardt,  and,  as p o i n t e d out  by W e l l s i s r e m i n i s c e n t o f the robosome-free zone which f r e q u e n t l y surrounds t r u e c e n t r i o l e s . The l a r g e v a r i e t y o f names which have been a p p l i e d to the bodies t h a t a c t as c e n t r i o l a r e q u i v a l e n t s d u r i n g f u n g a l nuclear d i v i s i o n i s at best confusing. r e c e n t l y reviewed the terminology to ascomycetes.  The most commonly used terms are  Z i c k l e r , 1970)  Marak, 1966; Pickett-Heaps  (1970) has  as i t has been a p p l i e d  (Lu, 1967a), "centrosome" (Lu, 1965; 1968;  Wells  and  Beckett and  W e l l s , 1970).  Wilson,  s  " c e n t r i o l a r plaque"  "centriole"  (Robinow and  However, as p o i n t e d out  by  (1969c) i n an a r t i c l e on the e v o l u t i o n o f the  m i t o t i c apparatus,  the use o f terms t h a t suggest  true  c e n t r i o l e s when a p p l i e d to these f u n g a l s t r u c t u r e s may misleading.  R e c e n t l y G i r b a r d t (1968) has  be  c o i n e d the phrase  " k i n e t o c h o r e - e q u i v a l e n t " as a more a p p r o p r i a t e term i n Polystictus versicolor. to be d i r e c t l y attaohed  In t h i s fungus the chromatin  to t h i s body which I s a p p a r e n t l y  i n v o l v e d i n d i r e c t i n g independent motions o f both nucleus  as a whole, and  appears  the chromatin  within.  the  The  "chromosomes" o f P o l y s t i c t u s do not a c t as independent e n t i t i e s but pass through the d i v i s i o n c y c l e as i f they  are  c o l l e c t i v e l y a t t a c h e d to t h i s s t r u c t u r e which a c t s as a common k i n e to chore. T h i s i s not a new  Hence the term  concept.  "kinetochore-equivalent;!'  I t has l o n g been observed  with  11+0  the l i g h t microscope t h a t l n many f u n g i the attached by a f i n e f i l a m e n t to  chromatin Is  a s m a l l knob at the edge  o f the nucleus i n r e s t i n g n u c l e i and  throughout some or a l l  stages i n n u c l e a r d i v i s i o n , e i t h e r m e i o t i c  (Berkson  B r i t t o n , 1969;  O l i v e , 19U9)  or m i t o t i c  M i t c h e l l and  McKeen, 1970).  1968;  Berkson, 1970;  1967a, Marks, 1965;  Lu,  D u r i n g d i v i s i o n , as i n P o l y s t i c t u s , the many o f these f u n g i do not but  form a c l a s s i c a l metaphase p l a t e  a centriolar-equivalent.  "chromosomes" o f U s t i l a g o h o r d e i Peulgen-positive  up  03-1+).  B l , Gl+, and  perhaps d u r i n g  Ml)  and  the l a t e r a l  CKE  small  promycelial  this association  Presumably the knob i s e i t h e r the  j u s t beneath the  meiotic  persists  d i v i s i o n ( F i g s . M3-1+, N3-1+, and  t o c h o r e - e q u i v a l e n t i t s e l f o r the lies  The  are a l s o a t t a c h e d t o a  knob l y i n g a g a i n s t  ( F i g s . A2,  to and  d i v i d i n g knob to  j o i n e d ; the knob a c t i n g as both a c o l l e c t i v e  k i n e t o c h o r e and  wall  (Girbardt,  "chromosomes" o f  behave as i f c o n t r o l l e d s o l e l y by the  which they are  and  centriolar-kine-  condensed chromatin cap  (Fig. 7),  that  T h i s a s s o c i a t i o n has  observed u l t r a s t r u c t u r a l l y a t telophase ( F i g . 7 ) , prophase (Fig.  22),  and  l a t e prophase ( F i g . 28), but  not y e t  at  metaphase. The  p o s s i b i l i t y t h a t a s i n g l e s t r u c t u r e may  f u l f i l the f u n c t i o n s not  o f b o t h c e n t r i o l e and k i n e t o c h o r e i s  as unusual as i t may  structures  at once  at f i r s t  appear.  F i r s t both  are known to g i v e r i s e d i r e c t l y or i n d i r e c t l y  to m i c r o t u b u l e s .  Second, some evidence e x i s t s t h a t  during  spermatogenesis i n v i v i p a r i d s n a i l s centromeres are  trans-  formed d i r e c t l y i n t o c e n t r i o l e s ( P o l l i s t e r and  Pollister,  been  1943).  T h i r d , the " k i n e t o c h o r e - e q u i v a l e n t " i n U s t i l a g o  h o r d e i resembles  very c l o s e l y i n s t r u c t u r e the f k i n e t o c h o r e "  of some animal c e l l s  ( B r i n k l e y , 1966) s i n c e i t i s composed  of an e l e c t r o n dense a x i a l f i l a m e n t ( F i g s . 9, 12, and 1^) surrounded hy f i n e f i b r i l l a r  material.  CYTOPLASMIC MICROTUBULES In s i z e and appearance the m i c r o t u b u l e s of U s t i l a g o h o r d e i are s i m i l a r t o those of h i g h e r p l a n t s (Newcombe, 1968). The p o s i t i o n and l a t e prophase  development of these aggregations  are r e m i n i s c e n t of the n u c l e a r - a s s o c i a t e d "prophase  bands"  of the c y t o p l a s m i c m i c r o t u b u l e s which h e r a l d the onset of n u c l e a r d i v i s i o n i n many p l a n t s (Burgess and N o r t h c o t e , 1967; Burgess, 1970a and b, Pickett#Heaps,  1969a and b ) .  They may serve as " d i r e c t i o n markers" which predetermine the d i r e c t i o n of the m i t o t i c s p i n d l e  (Burgess, 1970a)  or they may be a r e s e r v o i r of p r e s y n t h e s i z e d m i c r o t u b u l e s which move i n t a c t  i n t o the s p i n d l e  (PIckett-Heaps, 1969a).  Most of the c y t o p l a s m i c m i c r o t u b u l e s of U s t i l a g o do not r a d i a t e d i r e c t l y from the CKE ( P i g s . 19 and 20) but seem t o terminate i n g l o b u l a r , e l e c t r o n - d e n s e r e g i o n s of cytoplasm ( P i g . 21).  Similar densities  (fatbe) o f t e n occur  at the, d i s t a l i n d s of other t u b u l e s ( P i g . 20). Recent evidence suggests that these dense r e g i o n s o f cytoplasm which occur i n a s s o c i a t i o n w i t h m i c r o t u b u l e s i n many plants  (Burgess, 1970b) and animals  ( T i l n e y and Goddard,  1970) are the a c t u a l s i t e s of m i c r o t u b u l e s s y n t h e s i s ( i . e. microtubule-organizing  centres).  U|2  CHROMOSOME NUMBER: When Harper ( 1 8 9 8 ) f i r s t d e s c r i b e d  chromosomes and  s p i n d l e s i n n u c l e i o f U s t i l a g o aoabiosae. he s t a t e d t h a t the chromosome number was e i g h t to t e n f o r t h i s With t h e e x c e p t i o n  o f Dickinson  species.  ( 1 9 3 1 ) , however, subsequent  c y t o l o g i c a l s t u d i e s among the U s t i l a g i n a l e s have not supported t h i s h i g h  chromosome count.  (193U) and H i r s c h h o r n  Kharbush ( 1 9 2 7 ) , Wang,  (191+5) whose i n v e s t i g a t i o n s c o l l e c -  t i v e l y d e a l t w i t h f i v e d i f f e r e n t genera i n c l u d i n g Ustllago species  eleven  concluded t h a t the h a p l o i d number i s two.  These chromosome counts were made m a i n l y a t m e i o s i s , but i n f i v e s p e c i e s a t m i t o s i s as w e l l . been confirmed by Rawitscher  T h i s count has a l s o  ( 1 9 2 2 ) , Wang (1914-3), Das (191+9),  and Person and Wighton (I96I4.).  Our o b s e r v a t i o n s  i n Ustilago  h o r d e i and U s t i l a g o k o l l e r i i n d i c a t e t h a t two elongate chromatin bodies are present  i n m e i o t i c and m i t o t i c prophase  n u c l e i up t o , and i n c l u d i n g d i v i s i o n , and t h a t d u r i n g  ana-  phase two chromatin bodies c o n s t i t u t e each daughter complement.  However, c o n s i d e r i n g  cytology,  current i n v e s t i g a t i o n s o f fungal  the presence o f two chromatin bodies does not  n e c e s s a r i l y i n d i c a t e that n = 2J Undoubtedly i n t h e n a j o r i t y o f f u n g i , m e i o s i s occurs classically  ( O l i v e , 1 9 6 5 ; Lu, 1 9 6 5 ; Lu, 19.67a and b;  S i n g l e t o n , 1 9 5 3 ; Westergaard and von W e t t s t e i n , The  chromosomes pass through the l e p t o t e n e ,  1965).  zygotene,  pachytene, d i p l o t e n e , and d i a k i n e s i s prophase stages,  line  up on a t y p i c a l metaphase p l a t e , p u l l a p a r t on a normal  s p i n d l e and regroup i n t o daughter h a p l o i d n u c l e i at telophase.  However, i n U s t i l a g o h o r d e i , and s e v e r a l other  fungi^ m e i o s i s a p p a r e n t l y does not conform to t h i s a l pattern.  tradition-  In these s p e c i e s m e i o s i s and m i t o s i s l o o k i d e n t -  i c a l except t h a t the q u a n t i t y of prophase chromatin i s g r e a t er  i n the former than i n the l a t t e r .  Furthermore, the  d i v i s i o n f i g u r e s i n these s p e c i e s s t r o n g l y resemble the d i v i s i o n f i g u r e s i n the many f u n g i (ascomycetes and b a s i d iomycetes) which demonstrate a normal m e i o t i c p a t t e r n but an abnormal m i t o t i c one.  Robinow and Caten (1969) have  r e c e n t l y reviewed the l i t e r a t u r e p e r t a i n i n g to t h i s  latter  c l a s s of f u n g i which i n c l u d e s such w e l l - s t u d i e d s p e c i e s as Aspergillus nldulans  (Robinow and Caten, 1969), S c h i z o p h y l l u m  commune ( B a k e r s p i e g e l , 1959), Neurospora c r a s s a  (Namboodiri  and Lowry, 1967), and Fusarium oxysporum (Robinow,  personal  communication). These a p p a r e n t l y unusual d i v i s i o n f i g u r e s a l l have the f o l l o w i n g c h a r a c t e r i s t i c s i n common when observed i n the l i g h t microscope : 1. The chromatin takes the form of two elongate chromosome-like bodies l y i n g p a r a l l e l w i t h or at a s l i g h t angle t o the l o n g i t u d i n a l c e l l a x i s i n prophase anaphase  nuclei,  and telophase n u c l e i .  2. At a l l observable stages, each of the two chromosome—Like bodies i s j o i n e d by a f i n e to  filament  a k n o b - l i k e s t r u c t u r e which seems to precede the  nucleus.  This knob-like structure d i v i d e s , gives  m rise  t o the d i v i s i o n s p i n d l e , and g e n e r a l l y behaves  i n a manner s u g g e s t i n g t h a t i t i s the  centriolar-  equlvalent. S i g n i f i c a n t l y , i n a l l cases where meiosis i s normal but  mit-  o s i s i s n o t , the m e i o t i c complement c o n s i s t s of more than two  chromosome p a i r s .  Por example, i n A s p e r g i l l u s n i d u l a n s  e i g h t b i v a l e n t s can be demonstrated  c y t o l o g i c a l l y at meiosis  ( E l l i o t , I960) and t h i s number agrees w i t h the number of l i n k a g e groups e s t a b l i s h e d g e n e t i c a l l y  (Kafer>  1958)*  emphasize the p o i n t even more d r a m a t i c a l l y , somatic  To  nuclei  i n a d i p l o i d v a r i e t y of A. n i d u l a n s which must have s i x t e e n chromosomes, only two  chromatin b o d i e s are observable  and  these l o o k j u s t l i k e those of the h a p l o i d (Robinow and  1968).  C l e a r l y the presence  of two  Caten,  chromatin bodies i n t h i s  type of nucleus i s not a s u f f i c i e n t r e a s o n t o conclude  that  n = 21 That unusual n u c l e a r c o n f i g u r a t i o n s occur i n many fungi i s a f a c t . i s t o determine sibilities  The problem t h a t aofifronts the s i g n i f i c a n c e  cytologists  of t h i s f a c t .  Two  pos-  e x i s t : e i t h e r these c o n f i g u r a t i o n s r e f l e c t mere-  the inadequacy  of a v a i l a b l e c y t o l o g i c a l techniques as a p p l i e d  to f u n g a l n u c l e i or e l s e they r e s u l t from genuine i a l variation.  behavor-  U h f o r t u n a t e l y , f u n g a l n u c l e i are f o r the  most p a r t very s m a l l ; they are u s u a l l y c o n s t r a i n e d i n very narrow tubes w i t h t h i c k wa11s  .that;defy squashing,  and the chromatin f r e q u e n t l y f a i l s t o s t a i n w i t h the u s u a l chromosome s t a i n s  (11 e, Feulgen, haematoxylin,  green p y r o n i n e ) .  These f a c t o r s have, very  methyl-  justifiably,  l e d f u n g a l c y t o l o g i s t s t o be h i g h l y s k e p t i c a l of these unusual c o n f i g u r a t i o n s . t h a t the  However, r e c e n t evidence suggests  i n t e r p r e t a t i o n a l problem may  have another b a s i s :  1. Among f u n g i i n which unusual chromosomal c o n f i g u r a t i o n s are  observed these c o n f i g u r a t i o n s  c o n s i s t e n t l y and  they have t h e i r own  occur  equally  consistent c h a r a c t e r i s t i c s . 2. These c o n f i g u r a t i o n s arethe same i n b o t h material  (Pigs.  m a t e r i a l and  M3-U, N3-J4,  and  03-Aj) and  The  squashed  t h e r e f o r e p r o b a b l y do not r e s u l t from  squashing the m a t e r i a l i n a c o n f i n e d 3.  sectioned  yeast,  space.  Saccharomyoes c e r v i s i a e , has  at l e a s t  eighteen  l i n k a g e groups and presumably, t h e r e f o r e ,  eighteen  chromosomes.  Protoplasts  of yeast  r e a d i l y produced, e l i m i n a t i n g the problem of w a l l and y e t no amount of squashing w i l l r e s o l v e the two anaphase. hordei not  are the  further  chromatin b o d i e s commonly seen at  A similar situation exists i n Ustilago  ( F i g s . R3,  Rlj).  Therefore  the problem does  seem,to be e n t i r e l y on of c o n s t r a i n t .  1|. As has been d i s c u s s e d p r e v i o u s l y ,  considerable,  l i g h t microscope evidence i n d i c a t e s that i n these f u n g i the two  elongate chromosomal bodies are a t t a c h e d  anently  t o the  still  " c e n t r i o l a r equivalent".  inconclusive, e l e c t r o n microscopic  perm-  Although observations  i n S c h i z o p h y l l u m commune ( G i r b a r d t , 1968), S a c c h a r o myoes pombe (Robinow, p e r s o n a l  communication) and  Ustilago hordei hypothesis.  ( P i g s . 7, 22, and 28) support  this  C l e a r l y , i f i t i s true that the "chromo-  somes" a r e permanently a t t a c h e d kinetochore-equivalent  to the c e n t r i o l a r -  d i v i s i o n c o u l d not c o n c e i v a b l y  occur i n the c l a s s i c a l f a s h i o n . I f the u s u a l chromosome c o n f i g u r a t i o n i n s p e c i e s  like  A s p e r g i l l u s n i d u l a n s , Neurospora c r a s s a , Schizophyllum and Fusarium oxysporum are not due to t e c h n i c a l then one i s f o r c e d to conclude t h a t the v i s i b l e a t m e i o s i s become  commune,  inadequacies  chromosomes  l i n k e d together  i n some manner  i n two groups i n somatic n u c l e i . The  c l a s s of f u n g i which demonstrate the double  chromatin-body c o n f i g u r a t i o n a t meiosis i s q u i t e s m a l l to date. heterobasidiomycetes  as w e l l as m i t o s i s  Included i n the c l a s s are c e r t a i n  such as the smuts U s t i l a g o h o r d e i and  U s t i l a g o k o l l e r i , the r u s t s P u e c i n i a l o b a t a (Berkson, and Coleosporlum vernonia heterobasidiomycete  1961).  ( O l i v e , 19U9)  Ceratobasidium  and the c e r a t o b a s l d i a c e o u s  practlcolum  (Saksena,  A l s o i n c l u d e d are s e v e r a l s p e c i e s of the horaobasidio-  mycete Marasmius (Duncan and MacDonald, 1965)  and the a s c o -  mycete Saccharomyces c e r e v i s i a e (Robinow, p e r s o n a l ication). way  1970),  At present  commun-  there does not seem to be any c o n c l u s i v e  i n which to determine c y t o l o g i c a l l y the t r u e chromosome  number of these f u n g i , p a r t i c u l a r l y i n s p e c i e s l i k e U s t i l a g o h o r d e i and U s t i l a g o k o l l e r i where the e a r l y m e i o t i c prophase stages have not been seen.  247 Recent g e n t l e s t u d i e s i n d i c a t e that some U s t l l a g o s p e c i e s may have more than two l i n k a g e groups. (I96I4)  Holliday  has p r e s e n t e d evidence f o r the e x i s t e n c e  of f i v e  chromosomal arms i n U s t i l a g o maydis and Day and J o n e s ( 1 9 6 9 ) have concluded from h a p l o i d i z a t i o n s t u d i e s that  Ustllago  v i o l a c e a has at l e a s t t e n chromosome p e r genome. a t e l y the c y t o l o g i c a l data i n these s p e c i e s able f o r comparison w i t h U s t i l a g o h o r d e i .  Unfortun-  i s not a v a i l In s p i t e of the  a n a l y s i s of a r e l a t i v e l y l a r g e volume of r e c o m b i n a t i o n data a r i s i n g out of many d i f f e r e n t crosses  i n Ustilago  only one l i n k a g e group has been found (Person, supporting  the s u g g e s t i o n  hordei  unpublished),  t h a t U s t i l a g o h o r d e i may have  a low chromosome numbers. I n t e r e s t i n g l y only one centromere has been mapped i n b o t h U s t i l a g o h o r d e i and U s t i l a g o v i o l a c a e . T h i s i s i n agreement w i t h the p o s s i b i l i t y that the "chromosomes" share a common  kinetochore.  THE MODEL Brown and Stack a l t e r n a t i v e theory  (1971)  have r e c e n t l y formulated  an  of somatic d i v i s i o n on f u n g i , ( s p e c i f i c a l l y ]  A s p e r g i l l u s mldulans ), s t a r t i n g from the assumption that the  double chromatin body c o n f i g u r a t i o n r e f l e c t s the a c t u a l  chromosomal behaviour p a t t e r n . Figure  3U w i t h the k i n d p e r m i s s i o n  designed to s a t i f y two 1.  T h e i r model, d e p i c t e d i n of the a u t h o r s , i s  criteria:  I t must i n c o r p o r a t e  of the unusual n u c l e a r  the c h a r a c t e r i s t i c f e a t u r e s c o n f i g u r a t i o n s as they have  been observed i n the l i g h t and e l e c t r o n microscope:  DIAGRAM I (FIGURE 3k) Brown and Stack's Model f o r Somatic Nuclear D i v i s i o n i n Some Fungi  Model f o r Meiosis i n U s t i l a g o h o r d e i  SEPTUM  i ) the chromosomes are j o i n e d end-to-end i n two  groups,  i i ) the two  chromatin  bodies are permanently  j o i n e d to a common k i n e t o c h o r e - e q u i v a l e n t , i i i ) the k i n e t o c h o r e  a l s o a c t s as the  centriolar-  e q u i v a l e n t and g i v e s r i s e to the s p i n d l e , i v ) at d i v i s i o n the chromosomes do not form a metaphase p l a t e but remain s t r e t c h e d out i n two lines. v) the chromosomes r e a c h t h e i r maximum degree o f contraction at telophase. 2.  The model must account f o r t h e f a c t t h a t d u r i n g  somatic  d i v i s i o n i n the hyphae o f A s p e r g i l l u s n i d u l a n s  s i s t e r chromatid  s e g r e g a t i o n and n u c l e a r m i g r a t i o n  are non-random (Rosenberger and K e s s e l , 1968), most i n t r i g u i n g experiment Rosenberger and found t h e i r r e s u l t s were compatible chromatids c o n t a i n i n g DNA segregate F i g u r e 35  Kessel  w i t h t h e view t h a t  s t r a n d s o f the same age  as a u n i t d u r i n g m i t o s i s .  depicts schematically meiosis i n U s t i l a g o hordei  as i t would be model.  In t h e i r  i n t e r p r e t e d a c c o r d i n g to Brown and  Stack's  The o r i g i n a l model has been m o d i f i e d o n l y i n s o f a r  as necessary  to make i t compatible  with a meiotic  situation  and w i t h the d e t a i l s o f s t r u c t u r e and f u n c t i o n i n the t i c u l a r fungus.  For example F i g u r e 35 takes i n t o account  both n u c l e a r r o t a t i o n and the f a c t t h a t at l e a s t one chromatin  par-  bodies o f U s t i l a g o h o r d e i d i r e c t l y l i n k s  o f the  the  centriolar-kinetochore-equivalent  t o the nucleolus.  How-  ever n e i t h e r o f these s i t u a t i o n s i s l i k e l y t o be unique t o Ustilago. nuclear  Nuclear r o t a t i o n i s a well-known concomitant of ( A i s t and Wilson, 1 9 6 8 ) and  d i v i s i o n of many f u n g i  (Girbardt, 1968)  evidence i n b o t h S c h i z o p h y l l u m commune and  Saccharomyce3 pombe (Robinow, p e r s o n a l  communication)  suggests t h a t the c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t and nucleolus The  are j o i n e d v i a a t l e a s t one of the chromatin b o d i e s  diagrammatic r e p r e s e n t a t i o n  by F i g u r e s  ( F i g . 35>) i s supported  2 2 and 3 3 a which d e p i c t the major stages i n the  d i v i s i o n process.  At present  the author c o n s i d e r s the  data i n U s t i l a g o h o r d e i t o be more compatible w i t h Brown and  Stack's model f o r f u n g a l n u c l e a r  c l a s s i c a l pattern.  d i v i s i o n t h a t w i t h the  But a l l stages have not been seen i n  s u f f i c i e n t d e t a i l and the m a t e r i a l i s c e r t a i n l y  subject  to r e i n t e r p r e t a t i o n . AC KNOWLEDGEMENT The  o r i g i n a l model f o r somatic n u c l e a r d i v i s i o n i n  A s p e r g i l l u s n i d u l a n s was conceived  by D r . R. M. Brown  ( U n i v e r s i t y of Texas a t A u s t i n , U. S. A.) and was based on the p r e v i o u s indebted  work of many o t h e r s .  The author i s  t o Dr. Brown f o r h i s k i n d p e r m i s s i o n  of t h i s model i n the p r e p a r a t i o n  t o make use  of t h i s t h e s i s .  a l s o due t o Dr. C. Robinow ( U n i v e r s i t y of Western Canada) whose k i n d l y c r i t i c i s m s , i n f o r m a t i o n ,  Thanks i s Ontario,  and advice  have c o n t r i b u t e d g r e a t l y t o the ideas expressed i n p a r t TV.  IV.  PLATE 1  M e i o t i c d i v i s i o n s f o l l o w i n g t e l i o s p o r e germination i n U s t i l a g o hordei. Feulgen s t a i n e d . Phase o p t i c s . F i g u r e s A l - lj., B l - I;,  C l . Mid-prophase I .  Figures C 2 I ; ,  E l . Late prophase I .  Figure  E2,  DI - l|,  Late prophase I .  F i g u r e s E3 — lj., F I — 3« Figure Note:  F l * . Metaphase  Anaphase  I.  II.  i n plates 1 - 3 (a) the l o n g i t u d i n a l axes of the promycel i a are h o r i z o n t a l . (b) one s c a l e d i v i s i o n r e p r e s e n t s 1 mics* ron. ( T o t a l = 10 u.) c a . X 5,000.  2  1  *>  4  •  3  J I «•» ^  4  —•  ^»  or*  •i)  IV.  PLATE 2  M e i o t i c and m i t o t i c d i v i s i o n s succeeding t e l i o s p o r e germination i n U s t i l a g o k o l l e r i . Feulgen s t a i n e d . Planar o p t i c s . Figures Gl - 4 .  Mid-prophase I .  F i g u r e s HI - 2 .  Late prophase I .  F i g u r e s H 3 - 4 , I I - 4 , J l - 2 , K. Figure  L.  Figure  J3 - 4 .  Note:  Anaphase  IIX  I.  (arrow i n d i c a t e s f i r s t  Mitosis I I I .  c a . X 5,000.  Anaphase  septum).  IM  ' i i i i i i I  IV.  PLATE 3  Rows M, N, and 0 . M e i o t i c d i v i s i o n s succeeding t e l i o s p o r e germination i n U s t i l a g o h o r d e i . Planar o p t i c s . Group a: Haematoxylin. F i g u r e M l . Mid-prophase Figure N l .  Late prophase I  F i g u r e 0 1 . Anaphase I Group b :  s e r i a l s e c t i o n s s t a i n e d i n T. b l u e .  F i g u r e s M3 - Lt, N3 - l\ and 03 -  4.  Row P. M e i o t i c m y c e l i a l d i v i s i o n s Planar o p t i c s . F i g u r e s PI - 3 Figure  Metaphase I I . (arrows i n d i c a t e n u c l e o l u s ) .  Mid-prophase.  P i i . Metaphase.  Rows Q, and R. M i t o t i c s p o r i d i a l and s p h e r o p l a s t Planar opt i c s. Figure Q l - 2 .  Prophase.  F i g u r e Q3 -  Metaphase.  k»  F i g u r e R l - 2.  Anaphase.  F i g u r e R3 - k»  Metaphase.  Note:  ca. X 5 , 0 0 0 .  division.  IV.  PLATE 1+  F i g u r e $,  A g e n e r a l view of a h a p l o i d nucleus (hN) showing the n u c l e o l u s (Nu) i n i t s c h a r a c t e r i s t i c p o s i t i o n t o one side of the nucleus and a g a i n s t the n u c l e a r envelope (NE). The arrow indicates: a t h i n e l e c t r o n t r a n s p a r e n t zone a p p a r e n t l y d i v i d i n g the nuc l e o l u s i n t o two. Note the attachment of ribosomes t o the outer surface of the n u c l e a r envelope. Method G. c a . X 1+0,600.  Figure 6 a .  Nuclear pores i n c r o s s - s e c t i o n . Note the microt u b u l e i n the upper l e f t . Method C. ca. X 3 7 , 6 5 0 .  f  Figure 6 b .  A l o n g i t u d i n a l s e c t i o n through a promycelium showi n g the p o s i t i o n of the n u c l e a r pore r e g i o n d e p i c t ed i n F i g u r e 6 a . Method C. c a . X 1+0,600.  Figure 6 c .  An e n l a r g e d view of two of the n u c l e a r pores from figure 6 a . Note the c e n t r a l granules and the f i n e f i l a m e n t s r a d i a t i n g from the c e n t r a l granule t o a r i n g of p e r i p h e r a l g r a n u l e s . Method C. ca. X 95,200. Note:  the b l a c k s c a l e r e p r e s e n t s only 0 . 1  u.  IV.  PLATE 5  Figure  7.  A m i g r a t i n g h a p l o i d nucleus (hN). Note the elongate form of the nucleus and the c h a r a c t e r i s t i c p o s i t i o n of the c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t (CKE) at the narrow end. The chromatin cap l y i n g on the i n ner s i d e of the n u c l e a r envelope (NE) opposite the CKE i s c l e a r l y b i p a r t i t e . Method C. c a . X 1*7,600.  Figure  8.  The c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t a t prophase I. Note the s m a l l s i z e of the CKE and i t s p o s i t i o n w i t h i n a shallow cytoplasmic w e l l . The n u c l e a r envelope (NE) appears t o be i n t a c t . T h i s i s an e n l a r g e d view of t h e CKE seen i n F i g u r e 22. Method B. c a . X 60,1+00.  Figure  9.  The c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t at prophase II. Note the s i z e of the CKE i n comparison w i t h the CKE i n F i g u r e 8 . A l s o note the narrow e l e c t r o n dense zone w i t h i n the CKE and the chromatin cap. Method C. c a . X 6 0 , 7 0 0 .  Figure  10.  The CKE at prophase I I . Note the f i b r i l l a r ance. Method C. c a . X 129,900.  appear-  Figure 1 1 .  The CKE a t prophase I I . Note the f i b r i l l a r appearance and the two zones of v a r y i n g e l e c t r o n d e n s i t y . Method C. c a . X 90,000.  12.  The CKE at prophase I I . Note the f i b r i l l a r appearance of the CKE and the t h i n e l e c t r o n dense zone, w i t h i n the CKE. Method C. ca. X 188,250.  Figure  Figure 13•  The CKE at prophase I I . Note the surrounding l e s s e l e c t r o n dense h a l o . Method C. c a . X 126,900. Note: 0.1 u  The b l a c k s c a l e on F i g u r e s only.  8 t o 13  represents  IV.  PLATE 6  Figure  11+.  The d i v i s i o n of the c e n t r i o l a r - k i n e t o c h o r e e q u i v a l e n t (CKE) d u r i n g the t h i r d p r o m y o e l i a l n u c l e a r d i v i s i o n which gives r i s e t o the f i r s t s p o r l d i a l nucleus. Note t h a t the CKE has l e f t the n u c l e a r w e l l and i s now elongate and p a r a l l e l w i t h the n u c l e a r envelope. Method C. ca. X 13,000.  Figure  15.  An e n l a r g e d view of the elongate CKE i n F i g u r e 11+. Note the f i b r i l l a r nature of the CKE, the n u c l e a r envelope (NE) and the f i n e f i b r i l s pass i n g between the CKE and the n u c l e a r e n v e l o p e . Method C. ca. X 1 2 7 , 5 0 0 .  Figure  16.  An elongate CKE which has been i n t e r p r e t e d as a stage i n C K E - d i v i s i o n . Note the f i b r i l l a r nature of the CKE. Method C. ca. 1 2 7 , 5 0 0 .  Figure  17a.  A s e c t i o n through the n u c l e a r r e g i o n showing an e n l a r g e d n u c l e a r pore which may be a permanent CKE-well. Method C. ca. X 5 3 , 5 5 0 .  Figure  17b.  An e n l a r g e d view of the w e l l - r e g i o n seen i n F i g ure 1 7 a . Method C. c a . X 158,600.  Figure l 8 a .  A s e c t i o n through the h a p l o i d nucleus showing the CKE w i t h i n the w e l l . Method C. ca. X 2 9 , 7 5 0 .  Figure l8b.  An enlarged view of the CKE w i t h i n the n u c l e a r w e l l seen i n F i g u r e l 8 a . Note the appearance of the chromatin cap i n c r o s s - s e c t i o n . Method C. ca. X 1 5 9 , 0 0 0 . Note: the b l a c k s c a l e on F i g u r e s 17b and represents 0 . 1 u only.  l8b  IV.  PLATE 7  F i g u r e 19.  Microtubules (mt) which o f t e n o r i g i n a t e from the v i c i n i t y o f the c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t (CKE) appear to end i n dense amorphous s t r u c t u r e s resembling the m i c r o t u b u l e - o r g a n i z i n g centres (mtoc) o f h i g h e r animals and p l a n t s . Note t h a t t h e CKE l i e s i n i t s c h a r a c t e r i s t i c p o s i t i o n on the s i d e o f the h a p l o i d nucleus (hN) d i r e c t l y o p p o s i t e the nuc l e o l u s (Nu). Method C. c a . X 36,000.  F i g u r e 20.  Microtubules (mt) r a d i a t i n g from the v i c i n i t y o f the c e n t r i o l a r - k i n e t o c h o r e - e q u l v a l e n t (CKE).  Method C. c a . X 51,250.  F i g u r e 21.  M i c r o t u b u l e s (mt) ending i n the v i c i n i t y o f t h e c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t (CKE) do not terminate a t the CKE i t s e l f but r a t h e r end i n e l e c t r o n dense bodies (mtoc) surrounding t h e CKE. Method C, c a . X 96,200.  F i g u r e 22.  D i p l o i d prophase I nucleus (dN) l y i n g i n the metab a s i d i u m . The arrow i n d i c a t e s the d i r e c t i o n o f the p r o m y c e l i a l apex. Note the p o s t e r i o r p o s i t i o n o f t h e n u c l e o l u s and the p o s i t i o n o f t h e c e n t r i o l a r kinetochore - e q u i v a l e n t (CKE) on the s i d e o f t h e nucleus o p p o s i t e the n u c l e o l u s . The CKE l i e s i n a shallow cytoplasmic w e l l approximately o n e - t h i r d of the n u c l e a r l e n g t h b e h i n d the n u c l e a r apex. A band o f e l e c t r o n dense chromatin connects the nuc l e o l u s to the n u c l e a r envelope j u s t beneath the  CKE.  Method B.  c a . X 31+,000.  2?  IV.  PLATE 8  Figure 2 3 ,  D i p l o i d nucleus (dN) i n l a t e prophase I . The CKE has moved t o a p o s i t i o n midway along the n u c l e a r l e n g t h and the n u c l e o l u s (Nu) has s h i f t e d l a t e r a l l y t o the s i d e opposite the CKE. The n u c l e a r envelope has become v e r y i n d i s t i n c t and i s mainly o u t l i n e d by the p o s i t i o n of the n u c l e a r pores (NP). M i c r o t u b u l e s (mt) l i e p a r a l l e l with the l o n g i t u d i n a l c e l l a x i s on the CKE-side of the n u c l e u s . Method 0 . ca. X 51,100.  F i g u r e 2I4,  E a r l y anaphase I . Note the dumb-bell shaped nuc l e a r p r o f i l e , and the a p p a r e n t l y i n t a c t n u c l e a r envelope. A n u c l e o l u s (Nu) i s d i s t i n c t i n each h a p l o i d nucleus (hN). One of the CKE's i s v i s i b l e on the r i g h t s i d e of the lower n u c l e u s . Method C. c a . X 1 9 , 1 2 5 .  Figure 2 5 .  E a r l y anaphase I suggesting a p e r i o d of p a r t i a l breakdown of the n u c l e a r envelope (NE). Note that the e l e c t r o n - t r a n s p a r e n t r e g i o n s r e p r e s e n t n u c l e i c a c i d c o n t a i n i n g m a t e r i a l . Method A. ca. X 2 0 , 7 0 0 0 .  F i g u r e 26.  Telophase I n u c l e u s . The CKE has moved t o the l e a d i n g end of the m i g r a t i n g n u c l e u s . Note the d e n s i t y of the chromatin and p a r t i a l breakdown of the n u c l e a r envelope on the r i g h t s i d e of the figure. Method C. c a . X 3 5 , 7 0 0 .  IV.  PLATE 9  Figure  27.  E a r l y prophase I I n u c l e u s . (hN). Note the charact e r i s t i c p o s i t i o n s i s o f the CKE and the n u c l e o l u s (nu). Arrow I n d i c a t e s a t h i n e l e c t r o n t r a n s p a r e n t zone a p p a r e n t l y d i v i d i n g the n u c l e o l u s i n two. Method C. ca. X 1 0 , 9 5 0 .  Figure  28.  Late prphase I I nucleus (hN). M i c r o t u b u l e - l i k e s t r a u c t u r e s seem t o be r a d i a t i n g from the enlarged CKE. One very condensed chromatin body h o i n s the CKE d i r e c t l y t o the n u c l e o l u s (Nu) and a second chromatin body i s v i s i b l e c o i l i n g upwards i n t h e f i g u r e . The two arrows i n d i c a t e the p o s i t i o n s of the chromatin b o d i e s . Each chromatin body i s j o i n e d d i r e c t l y to the CKE by at l e a s t one p a i r of chromatin s t r a n d s . Method C. c a . X 1+7,600. 1  Figure  29.  CKE-replication during nuclear d i v i s i o n I I . The n u c l e o l u s (Nu) a l s o seems t o be b i p a r t i t e . Method C. X c a . 1+7,600.  Figure  30.  M i g r a t i o n of the daughter CKE's d u r i n g d i v i s i o n I I . The n u c l e a r r e g i o n i s demarcated by the p o s i t i o n of the n u c l e a r pores (NP). Note t h a t the nucleus i s becoming e l o n g a t e . Method c. c a . X 1+0,650.  IV. PLATE 10 Figure  31.  The metaphse or e a r l y anaphase e q u i v a l e n t at d i v i s i o n I I I . The two enlarged GKE's d i e at the p o l e s of the n u c l e u s . Note t h a t the n u c l e a r a x i s l i e s at an angle of about 1+5° t o the p r o m y c e l i a l a x i s . The n u c l e o l u s (Nu) l i e s i n a c h a r a c t e r i s t i c p o s i t i o n t o one s i d e of the n u c l e a r a x i s and midway between the p o l e s . Method c. ca. X. 1+2,000.  Figure  32.  Anaphase I I . M i c r o t u b u l e s (mt) p e n e t r a t e the daught e r n u c l e u s on the s i d e o p p o s i t e the lead end during n u c l e a r m i g r a t i o n . The n u c l e a r envelope i s broken on the s i d e through which the m i c r o t u b u l e s pass i n t o the n u c l e u s . Method c. c a . X 32,7£0.  Figure  33a,  Anaphase I I . Note the incomplete n u c l e a r envelope Part of the f i r s t septum i s v i s i b l e i n the upper r i g h t . Method A. X c a . 17,700.  Figure  33b,  A l i g h t microscope view of anaphase I I i n m a t e r i a l f i x e d i n GA and viewed w i t h phase o p t i c s . The ' second round of n u c l e a r d i v i s i o n i s not always synchronous. The s i n g l e arrow i n d i c a t e s a nucleus i n prophase I I ; the double arrow i n d i c a t e s a nuc l e u s i n anaphase I I . The t h i n dense l i n e i n the l o n g i t u d i n a l a x i s of the d i v i d i n g nucleus r e p r e sents the s p i n d l e , c a . X 2,800.  152 BIBLIOGRAPHY Abelson, H.T. and Smith, G.H. 1 9 7 0 . Nuclear p o r e s : the pore-annulus r e l a t i o n s h i p i n t h i n s e c t i o n . J. Ultrastruet.. 30: 558-588. A i s t , J.R. and Wilson, C.L. 1 9 6 8 . I n t e r p r e t a t i o n o f n u c l e a r d i v i s i o n figures- i n v e g e t a t i v e hyphae o f f u n g i . Phytopathol. 5 8 : 876-877. B a k e r s p i e g e l , A. 1959. 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O l i v e , L.S. 1949. Karyogamy and m e i o s i s i n the r u s t C o l e o sporium v e r n o n l a e . Am. J . B o t . 3 6 : 4 I - 5 I 4 .  155  O l i v e , L.S. 1 9 6 5 . Nuclear behaviour d u r i n g m e i o s i s . In The Fungi I . An Advanced T r e a t i s e , ed. Ainsworth, G.G., and Sussman, A.S., Academic P r e s s , N.Y. pp. 1 4 3 - 1 6 1 . Person, 0 . and Wighton, D. I96I4.. The chromosomes o f U s t i l a g o . Can. J . Genet. C y t o l . 6,: 21)2 ( A b s t r a c t ) Pickett-Heaps, J.D. 1 9 6 9 a . Preprophase microtubules and storaatal d i f f e r e n t i a t i o n i n Commelina cyanes. Aust. J . B i o l . S c i . 2 2 : 375-391. P i c k e t t - H e a p s , J.D. 1969b. Preprophase m i c r o t u b u l a r bands i n some abnormal m i t o t i c c e l l s o f wheat. J . C e l l S c i . It: 3 9 7 - 4 2 0 . P i c k e t t - H e a p s , J.D. 1 9 6 9 c . 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Saksena, H.K. 1961. Ceratobasidium  i l : 717-72TT  Nuclear phenomena i n the basidium o f p r a t l c o l u m ( K o l i l a ) O l i v e . Can. J . Bot.  Sampson, K. 1 9 3 9 . L i f e c y c l e s o f smut f u n g i . Mycol. Soc. 23_: 1 - 2 3 .  Trans.  Brit.  S c h r a n t z , J.P. 1 9 6 7 . Presence d'un a s t e r cours des mitoses de l a s q u e e t de l a f o r m a t i o n des ascospores chez l ' A s comycete P u s t u l a r i a c u p u l a r i s ( 1 . ) Fuck. C. R. Acad. S c i . P a r i s , S e r . D. 2 6 4 : 127C-1277. 1  S i n g l e t o n , J.R. 1 9 5 3 . Chromosome morphology and the chromatin c y c l e i n the ascus o f Neurospora c r a s s a . Am. J . Bot. |t0: 124-14.3.  Stein,~C.¥. 1 9 7 0 * An e l e c t r o n microscope study Of a m y c e l i a l mutant o f U s t i l a g o h o r d e i (Pers.) Lagerh. M.Sc- T h e s i s , U.B.C., Vancouver, CUT! Thomas," P.L. and Person, C. virulence i n Ustilago.  1 9 6 5 . G e n e t i c c o n t r o l o f lowCan. J . Genet. C y t o l . 7 : 5 8 3 - 5 8 8 .  T i l n e y , L.G. and GOddard, J . 1 9 7 0 . N u c l e a t i n g s i t e s f o r the assembly o f c y t o p l a s m i c m i c r o t u b u l e s i n the ectodermal c e l l s o f A r b a c l a p u n c t u l a t a . J . C e l l B i o l . I j ^ : 561+-575* Wang, C.S. 1 9 4 3 . S t u d i e s on the c y t o l o g y o f U s t i l a g o c r a m e r l ! Phytopathology 3 3 : 1 1 2 2 - 1 1 3 3 . Wang, D.T. 193U- C o n t r i b u t i o n a 1 » e t u d e des U s t i l a g i n e e s ( C y t o l O g i e du p a r a s i t e e t p a t h o l o g i e de l a c e l l u l e h o t e ) . Le B o t a n i s t s 2h: 5 3 9 - 6 7 0 . W e l l s , K. 1 9 7 0 . L i g h t and e l e c t r o n microscope s t u d i e s o f Ascobolus s t e r c o a r i u s I Nuclear d l v i i i o n s i n the a s c u s . Mycologia 62: 7 6 1 - 7 V U . Westergaard, M. and von W e t t s t e i n , D. 1 9 6 5 . 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Chromosome 30: 287-304.  PART V The P o s i t i o n a l R e g u l a t i o n of C e l l and N u c l e a r  Division  TABLE OF CONTENTS Page ABSTRACT  158  INTRODUCTION  158  MATERIALS AND METHODS  159  OBSERVATIONS  160  DISCUSSION  161  BIBLIOGRAPHY  IJk  PART V The  P o s i t i o n a l Regulation  of C e l l and Nuclear D i v i s i o n  ABSTRACT Elaborate  membrane complexes have p r e v i o u s l y been  a s s o c i a t e d w i t h septum i n i t i a t i o n  in Ustllago hordei.  E v i d e n t l y s i m i l a r complexes are formed i n the v i c i n i t y of the nucleus d u r i n g m e i o t i c  prophases I and I I .  The  h y p o t h e s i s i s presented that the complexes are p a r t of a system governing the p o s i t i o n a l . r e g u l a t i o n of c e l l and nuclear  division  i n the promycelium.  A review i s presented  of the l i t e r a t u r e p e r t a i n i n g t o f u n g a l membrane complexes, and t h e i r r e l a t i o n s h i p t o mesosomes of b a c t e r i a and the G o l g i apparatus of higher  p l a n t s and animals.  INTRODUCTION In p a r t I I I membrane complexes were d e s c r i b e d i n a s s o c i a t i o n w i t h the septum i n i t i a l and  of U s t i l a g o h o r d e i  the suggestion  was made that these membranous s t r u c -  t u r e s are i n v o l v e d  i n the i n i t a t i o n of c r o s s w a l l forma-  tion.  The observations  presented.in  t h i s paper i n d i c a t e  t h a t the membrane complexes form from the plasma membrane i n a s s o c i a t i o n w i t h prophase n u c l e i .  Their  subsequent  a c t i v i t i e s are compatible with the h y p o t h e s i s that they are an important p a r t of the mechanism which  regulates  the p o s i t i o n i n g of c e l l and n u c l e a r d i v i s i o n with r e s p e c t to  each o t h e r . In h i g h e r p l a n t s the c e l l w a l l I d e l i m i t i n g two daughter  c e l l s forms as a d i r e c t t e l o p h a s i c c o n t i n u a t i o n of the nuc l e a r d i v i s i o n which gave r i s e t o the two new n u c l e i , - t h e m i c r o t u b u l a r s p i n d l e apparatus reorganized t o p a r t i c i p a t e p l a t e *(Neweombe, 1969).  of the parent nucleus  being  i n the f o r m a t i o n of the c e l l  The m i c r o t u b u l a r apparatus  asso-  c i a t e d with many f u n g a l n u c l e i i s of very l i m i t e d d u r a t i o n and disappears completely have s e p a r a t e d .  as soon as the daughter n u c l e i  In septate f u n g i c r o s s w a l l s  subsequently  form, u s u a l l y v i a c e n t r i p e t a l i n v a g i n a t i o n of the l a t e r a l w a l l and plasma membrane.  M i c r o t u b u l e s do not seem t o be  i n v o l v e d and t h e r e are no obvious connections between t h i s process and n u c l e a r d i v i s i o n .  Yet some method of e n s u r i n g  that the septum separates the daughter n u c l e i must e x i s t . Such a mechanism i s p a r t i c u l a r l y  important  in a  metabasi-  dium, l i k e t h a t of U s t i l a g o h o r d e i where f a i l u r e t o maint a i n a one-to-one r e l a t i o n s h i p between the nucleus c e l l would g r e a t l y decrease  and the  the m e i o t i c e f f i c i e n c y .  MATERIALS AND METHODS The m a t e r i a l s and techniques used are the same as those p r e v i o u s l y d e s c r i b e d ( P t . I ) .  Methods A, B, and  C, f o r p r e p a r i n g t i s s u e f o r e l e c t r o n microscopy l i n e d i n p a r t I , Table I .  are out-  160  OBSERVATIONS Membrane complexes, s i m i l a r  i n appearance t o those  a s s o c i a t e d w i t h septum i n i t i a t i o n  (Pt. I l l ) ,  a l s o occur  contiguous t o the n u c l e i of U s t l l a g o h o r d e i . shows a c r o s s - s e c t i o n through a very prominent  Figure 1 complex  which seems t o be i n d i r e c t c o n n e c t i o n w i t h the n u c l e a r envelope.  The nucleus t o which t h i s complex i s a t t a c h e d  i s a d i p l o i d nucleus which f i x e d w h i l e m i g r a t i n g i n t o the promycelium.  Figure 2 i l l u s t r a t e s a sequential section  through the same c e l l and about first.  0.2 u removed from the  In the promycelium, whomever s i m i l a r membranous  s t r u c t u r e s are observed  i n a s s o c i a t i o n w i t h the u n d i v i d e d  f u s i o n n u c l e u s , they are found i n a c h a r a c t e r i s t i c  position.  The complex i s always pressed c l o s e t o the p r o m y c e l i a l w a l l at the p o s t e r i o r end of the nucleus  ( i . e . the end c l o s e s t  t o the spore: F i g s . 3a and 1*), and consequently i s adjacent t o the n u c l e o l u s ( F i g . k U ) .  It also l i e s  nucleus o p p o s i t e t o the s i n g l e lent  (Fig. U ) .  A  on the s i d e of the  centriolar-kinetochore-equiva-  group of v e s i c l e s of v a r i o u s dimensions i s  i n e v i t a b l y l o c a t e d i n the v i c i n i t y of the complex and a l s o at the p o s t e r i o r end of the nucleus  ( F i g s . 3a, 3b, and 1*).  These v e s i c l e s may be a s s o c i a t e d w i t h m i c r o t u b u l e s . F i g u r e 5> i s i n t e r p r e t e d as a s e c t i o n through a dumbb e l l shaped nucleus which i s i n the process of r e d u c t i o n d i v i s i o n and which has probably proceeded s t a t e e q u i v a l e n t t o e a r l y anaphase I .  to a physiological  Presumably the hap-  l o i d chromosome complements and the daughter n u c l e o l i have  161  separated  (Ft. VI).  A membrane complex appears i n c r o s s -  s e c t i o n i n the middle of the c o n s t r i c t i n g r e g i o n between the daughter n u c l e i .  A t a s l i g h t l y l a t e r stage when the daughter  n u o l e i o f the f i r s t m e i o t i c  d i v i s i o n have v i r t u a l l y  separated  the membrane complex w i t h i t s accompanying v e s i c l e s i s again v i s i b l e i n the i n t e r n u c l e a r zone ( F i g . 6) Although membranous systems have not y e t been observed i n close proximity  t o metaphase  I I o r anaphase I I n u o l e i  they do o c c u r i n the p e r i n u c l e a r r e g i o n d u r i n g prophase I I ( F i g s . 7a and 7b),  and prophase I I I ( F i g . 8).  Figure  7a  i l l u s t r a t e s the r a r e case i n which two separate complexes seem to be a s s o c i a t e d w i t h a s i n g l e  nucleus.  The o r i g i n o f the membrane complexes i s s t i l l  uncertain.  However, i n s e v e r a l oases where n u o l e i have been observed c l o s e t o the plasmalemma, the plasma membrane i n v a g i n a t e s d i r e c t i o n o f the n u c l e u s . not  i n the  The i n v a g i n a t i n g membrane does  t r a v e l i n a s t r a i g h t l i n e but c o i l s on i t s e l f i n double  oonoentric  layers  ( F i g s . 9a and 9 b ) .  I t seems reasonable  t h a t c o n t i n u a t i o n of t h i s k i n d o f a c t i v i t y would give r i s e t o s t r u c t u r e s resembling membrane complexes.  DISCUSSION Membranous s t r u c t u r e s , s i m i l a r i n appearance t o the membrane complexes o f U s t i l a g o h o r d e i , have been observed i n a t l e a s t a dozen d i f f e r e n t f u n g i  (Ft. I l l ) including  phycomycetes, b a s i d i o m y c e t e s , ascomycetes, and imperfect forms (Table I ) . The complexes seem t o be a s s o c i a t e d w i t h  162  s e p t a i n two human pathogens, and Blastomyces  dermatitidis  Paracoccidioides b r a s i l i e n s i s ( C a r b o n e l l e , 1967; Carbonelle  and Rodrigez, 1 9 6 8 ) and i n two basidiomycetes L e n z l t e s (Hyde and Walkinshaw, 1 9 6 6 ) and U s t i l a g o h o r d e i  aaepiaria (Pt.  I I I ) . I n both o f t h e preceding basidiomycetes, mem-  brane complexes a l s o occur i n p r o x i m i t y to the n u c l e i ; a s i m i l a r r e l a t i o n s h i p has been noted i n P a r a c o c c i d i o d e s (Purtado e t a l , , 1967)  loboi  Lu, 1966),  and Coprinus lagopus  (Lu, 1965;  To date, the o n l y s t u d i e s which have c o n s i d e r e d  t h i s a s s o c i a t i o n i n any d e t a i l a r e those i n Coprinus. c o r d i n g t o L u (1965  and 1966)  Ac-  a s i n g l e membrane complex  forms i n the b a s i d i u m o f Coprinus logopus d u r i n g prophase I of meiosis.  At t h i s time the complex which i s 0.9 t o 1.0  u  i n diameter l i e s c l o s e to the d i p l o i d f u s i o n nucleus and g i v e s r i s e to l a r g e numbers o f v e s i c l e s . p l e x d e s c r i b e d i n t h i s paper resembles  The U s t i l a g o com-  the Coprinus complex  both i n time and p l a c e o f o r i g i n and i n i t s a s s o c i a t i o n with vesicles  ( P i g s . 3a, 3D, and i+).  In U s t i l a g o h o r d e l i t has  not been proven t h a t the complex a c t u a l l y produces icles.  the ves-  However, a t metaphase I the Coprinus complex p r o i f e r -  a t e s r a p i d l y to almost f i v e times i t s prophase s i z e w h i l e that o f U s t i l a g o remains unchanged.  Whether t h i s i s due to  a d i f f e r e n c e i n f u n c t i o n o r perhaps o n l y i n the degree o f f u n c t i o n i s unknown. C o n s i d e r i n g the r e l a t i v e l y wide d i s t r i b u t i o n o f t h i s type o f membranous body among f u n g i , one wonders why so l i t t l e a t t e n t i o n has been p a i d to i t .  There a r e probably three  b a s i c reasons f o r t h i s n e g l e c t .  F i r s t , as d i s c u s s e d i n  p a r t I I I , there has been a g e n e r a l tendency t o avoid  these  bo di es because of the p o s s i b i l i t y t h a t they may simply be f i x a t i o n a r t e f a c t s or e l s e t h a t they may r e s u l t from the a u t o l y t i c degradation  of other  "normal" o r g a n e l l e s .  dence has p r e v i o u s l y been presented n e i t h e r of these p o s s i b i l i t i e s Ustilago hordei.  which i n d i c a t e d t h a t  i s l i k e l y t o be the case i n  Second, s t r e s s f u l c o n d i t i o n s are known t o  cause the r o l l i n g up of ER-elements i n h i g h e r p l a n t s et  Evi-  a l . , 1961+) and animals (Fawcett  and Susuma, 1958).  p o s s i b i l i t y should be examined c r i t i c a l l y because most of the t i s s u e prepared has been a r t i f i c i a l l y  cultured.  plasmic r e t i c u l u m a l s o occur  (Whaley This  i n fungal studies  f o r e l e c t r o n microscopy  However whorls of endo-  i n some cases  during  apparently  normal development; t h i s i s p a r t i c u l a r l y t r u e i n embryonic tissues  (Fawcett  and Susuma, 1958; Robertson, 1961).  U s t i l a g o hordei a f u r t h e r point t o consider  In  i s that the  complex seems t o a r i s e from a r o l l i n g up of the plasma membrane and not of the endoplasmic r e t i c u l u m . that l i t t l e  A third  reason  information i s a v a i l a b l e p e r t a i n i n g t o fungal  membrane complexes i s t h a t no one, w i t h the e x c e p t i o n (1965)» appears t o have obtained  sufficient  of Lu  information  i n d i c a t i n g t h a t these unusual s t r u c t u r e s might perform some physiological function.  I submit t h a t membrane complexes  i n U s t i l a g o h o r d e i do serve a d e f i n i t e and important  func-  t i o n , — n a m e l y the p o s i t i o n a l r e g u l a t i o n of c e l l and n u c l e a r divisions.  The suggest 1.  observations  i n the promycelia  o f t h i s smut fungus  the f o l l o w i n g h y p o t h e s i s : During  l a t e m e i o t i c prophase there i s a l o c a l i z e d  p r o l i f e r a t i o n of the plasma membrane opposite the b a s a l p o r t i o n of the d i p l o i d 2.  nucleus.  The p r o l i f e r a t i n g membrane i n v a g i n a t e s and c o i l s on  itself  (Pigs-*; 9a and 9b) t o f i v e r i s e t o a r e a s o n a b l y  l a r g e s t r u c t u r e c o n s i s t i n g of c o n c e n t r i c u n i t membrane layers 3.  ( P i g s . 3 a , 3b and 4 ) .  The membrane complex, i n some way, e s t a b l i s h e s f i r m  connection w i t h the n u c l e u s .  The nature  of t h i s contact  i s unknown.  P o s s i b l y the n u c l e a r envelope i s i n v o l v e d  i n formation  of the complex b u t t h e r e i s no d i r e c t  evidence  for this.  In F i g u r e 1 the u n i t membrane of  the complex, and the n u c l e a r envelope seem t o be i n direct 4.  connection.  The n u c l e a r r o t a t i o n d e s c r i b e d i n p a r t IV which  b r i n g s the c e n t r i o l a r - k i n e t o c h o r e - e q u i v a l e n t and the n u c l e o l u s i n t o a medial p o s i t i o n w i t h r e s p e c t t o the l e n g t h of the d e p l o i d nucleus  a l s o c e n t r e s the mem-  brane complex. 5>.  During r e d u c t i o n d i v i s i o n the daughter h a p l o i d  n u c l e i separate  and the membrane complex, h a v i n g now  l o s t c o n t a c t w i t h the n u c l e a r envelope, middle of the l e n g t h e n i n g and  6).  remains i n the  i n t e r n u c l e a r zone ( F i g s . £  6. The f r e e membrane complex then begins to " u n r o l l " p r o v i d i n g the membrane to form the i n i t i a l s e p t a l p l a t e (Pt. I l l ,  P i g s . 2 and 5).  ?• Having completed i t s f u n c t i o n the membrane complex is  destroyed.  8. Hew complexes form i n a s s o c i a t i o n w i t h the prophase I I n u c l e i and the c y c l e begins  again.  T h i s t e n t a t i v e h y p o t h e s i s w i l l demand c o n s i d e r a b l e  testing.  One o f the immediate requirements i s to o b t a i n s e r i a l t i o n s s i n c e there closeness  i s no other way to a c c u r a t e l y  sec-  judge the  o f the complex-nucleus r e l a t i o n s h i p o r the one-  to-one-to-one r e l a t i o n s h i p o f the nucleus, p l e x , and septum.  membrance com-  Should t h i s h y p o t h e s i s prove c o r r e c t  there  i s no reason to assume t h a t the same mechanism i s n e c e s s a r i l y a c t i v e d u r i n g somatic m i t o t i c d i v i s i o n s although l i m i t e d e v i dence i n d i c a t e s t h a t i t may be. evolved  P o s s i b l y t h i s mechanism has  as a s p e c i a l i z e d a d a p t a t i o n  to the p e c u l i a r problems  o f undergoing m e i o s i s i n a metabasidium. Other authors have proposed d i f f e r e n t f u n c t i o n s which such membranous s t r u c t u r e s might f u l f i l l  i n f u n g i and the  complexes i n U s t i l a g o h o r d e i may c a r r y o u t one o r more o f these i n a d d i t i o n t o t h a t o f p o s i t i o n i n g .  I n Coprinus  1 ago pus. Lu (1965) has p o s t u l a t e d t h a t s i n c e the m e i o t i c d i v i s i o n s occur i n r a p i d s u c c e s s i o n  the membrane complex,  which p r o l i f e r a t e s enormously at metaphase I , might be the generator o f a d d i t i o n a l nuclear  envelope.  T h i s does not  seem to be t h e case i n U s t i l a g o s i n c e the complex-associated  166  v e s i c l e s have not been observed t o f u s e w i t h the n u c l e a r membrane, and most of the v e s i c l e s remain zone f o l l o w i n g n u c l e a r d i v i s i o n bility  (Pig. 6).  i n the  internuclear  A second  possi-  i s t h a t the complex a c t s as an anchor which b r i n g s  the nucleus t o a s t a t i o n a r y p o s i t i o n p r i o r t o d i v i s i o n . p a r t IV i t was  In  suggested t h a t Brown and Stack's theory (1971)  of f u n g a l n u c l e a r d i v i s i o n i s at present the most  likely  model t o account f o r the phenomena which occur i n U s t i l a g o hordei during meiosis.  T h i s theory i s p a r t l y based  e a r l i e r work by Rosenberger  on the  and K e s s e l (1968) which showed  non-random s i s t e r chromatid s e g r e g a t i o n and n u c l e a r m i g r a t i o n i n the hyphae of A s p e r g i l l u s n i d u l a n s .  I n t e r e s t i n g l y the  l a t t e r authors p o s t u l a t e d t h a t i n order t o o b t a i n the r e s u l t s which they d i d the o l d e s t of the s e g r e g a t i n g u n i t s should be anchored t o some s t a t i o n a r y p a r t of the c e l l u l a r membrane system.The U s t i l a g o complex resembles system of b a c t e r i a  s t r i k i n g l y the mesosomal  (Rogers, 1970; R y t e r , 1968)  mycetes (Edwards, 1970;  and Pitz-James, I960).  and  actino-  Structurally  b o t h the complex and the mesosome c o n s i s t of c o i l e d membranes which are d e r i v e d from the plasma membrane.  They b o t h occur  i n a s s o c i a t i o n w i t h the n u c l e a r m a t e r i a l and w i t h the forming septa; consequently they have b o t h been i m p l i c a t e d i n maint a i n i n g c e r t a i n s t r u c t u r a l and f u n c t i o n a l r e l a t i o n s h i p s be4tween c e l l and n u c l e a r d i v i s i o n . between the two  The  obvious  similarity  s t r u c t u r e s has prompted some workers t o r e f e r  t o these s t r u c t u r e s as " f u n g a l mesosomes" (Edwards,  1969;  Kozar and W e i j e r , to  imply  1969).  some s o r t of e v o l u t i o n a r y s i g n i f i c a n c e which  not e x i s t at a l l . reason  T h i s i s hazardous because i t tends  According  t o the c u r r e n t data there i s  t o assume t h a t the undeniable  systems i s not by way logy.  may  resemblance of the  of i d e n t i t y but r a t h e r by way  two  of ana-  F i r s t the c u r r e n t model of the b a c t e r i a l mesosome  i n d i c a t e s t h a t i t i s composed of t u b u l a r or v e s i c u l a r membranes (Ryter, 1968)  although  membrane theory s t i l l  exist  some proponents of the l a m e l l a r  (Highton,  problem i n b a c t e r i a l c y t o l o g y has f i x a t i o n procedures r e s u l t  different  R y t e r , 1968), and  same s i t u a t i o n e x i s t s i n U s t i l a g o h o r d e l  Obviously  and  b a c t e r i a , and there i s  t h a t the l a m e l l a r conformation  actinomycetes.  probably  occurs Ryter,  Second, w h i l e b o t h systems are  r i v e d from the plasma membrane they are probably i n the same manner (Imaeda and  Ogura, 1963;  Rogers, 1970).  c e l l u l a r f u n c t i o n s such as c e l l u l a r  (Ryter, 1968), w a l l s e c r e t i o n (Rogers, 1970) s e c r e t i o n (Beaton, 1968). out any  de-  not formed  T h i r d , the b a c t e r i a l mesosome c a r r i e s out a v a r i e t y of important  the  ultrastructural  among gram-negative b a c t e r i a (Kakefuda e t a l . , 1967; 1968)  one  ( F i g s . 1, 3*> a n d l l O ) .  i s t h a t most of the  s t u d i e s have d e s c r i b e d gram-positive good evidence  the  i n a d i f f e r e n t appearance of the  i s s u e i s not yet c l o s e d .  A further complication  Of course  l o n g been t h a t  mesosomes (Burdett and Rogers, 1970; must admit t h a t the  1970).  other  respiration and exoenzyrae  Whether the f u n g a l complex c a r r i e s  of these a d d i t i o n a l f u n c t i o n s w i l l remain unknown  u n t i l the a p p r o p r i a t e b i o c h e m i c a l  and/or h i s t o c h e m i c a l  infor-  168)  mation i s a v a i l a b l e .  However, the f i r s t  two p o s s i b i l i t i e s  seem t o be u n l i k e l y . Lu  (1965 and 1966) has noted t h a t f u n g a l  membrane  complexes a l s o resemble the G o l g i apparatus i n some aspects of s t r u c t u r e and f u n c t i o n , and has hence r e f e r r e d t o these s t r u c t u r e s as G o l g i .  Undoubtedly the membrane complex formed  i n Coprinus lagopus d u r i n g  meiotic  prophase I bears a g r e a t e r  resemblance t o a G o l g i than does the complex i n U s t i l a g o h o r d e i i n i t s s t r u c t u r e , i t s production  of v e s i c l e s , and i t s  probable f u n c t i o n a l s i g n i f i c a n c e as a nuclear  membrane gen-  e r a t o r . ' The s t r u c t u r e of the f u n g a l membrane complex i s p a r t i c u l a r l y reminiscent c e r t a i n types of algae and Gawlik, 1970).  of the G o l g i apparatus d e s c r i b e d i n (Bouck, 1965; Brown, 1969; M i l l i n g t o n  Another f e a t u r e which the two systems  share i n common i s a tendency t o be s i t u a t e d i n the p e r i nuclear Morre  region.  Beams and K e s s e l  (1968) and Mollenhauer and  (1966) have reviewed the v a r i o u s f u n c t i o n s of the  Golgi apparatus—the l a t t e r with p a r t i c u l a r reference t o plants.  N o t a b l y , f u n c t i o n s which G o l g i serve i n animals,  h i g h e r p l a n t s , and some f u n g i are a l s o f u n c t i o n s which have been suggested f o r f u n g a l membrane complexes, f o r example enzyme s e c r e t i o n and  transformation  1966).  ( G r i f f i t h s , 1970) and membrane  generation  ( B e r l i n e r and D u f f , 1965; Lu, 1965 and  In f u r t h e r support of the h y p o t h e s i s t h a t  membrane complexes are the G o l g i - e q u i v a l e n t  fungal  i s the observa-  t i o n t h a t membrane complexes are u s u a l l y not observed i n f u n g i which possess the u s u a l p l a n t dictyosomes (Table I I ) .  TABLE I Membrane Complexes and Golgi i n Fungi Membrane Complexes  Golgi  Phycomycetes: Peronaspora parasitica  Phycomycetes: Chou (1970)  Albugo C a n d i d a Nowakowskiella prorusa Peronospora P tTca Peronospora hurTca Phytophthora erythroseptica i^nytopntnora mrestans"" Phytopnthora parasitica Pythium debTryanum Pytnium u l t imum  Berlin & Bowen (196!+) Chambers et a l . (1967) Davison (1968)  a r a 3 l  m a n a  Peyton & Bowen (1963) Chapman & V u j i c i c (1965) Ehrlich & E h r l i c h (1966) t!  II  II  Hawker (1963) Grove et a l . (1967)  Saprolegna ferax Heath & Greenwood (1971) Ascoraycetes: Neurospora crassa Neurospora tetrasperma  Neobulgar l a pur a  Kozar & Weijer (1969) Lowry & Sussman  Moore & McAlear (1963)  (1968)  Basidiomycetes: A r m i l l a r i a mellea Coprinus lagopus Lenzites saepiaria Lycoperdon perlatum Blastomyces derma tl^HTd is Parac occ idiocies hrasiliensis Paracoccidiodes loboi V e r t i c i l l i u m dahliae  Berliner & Duff (1965) Lu (1965 & 1966)  Hyde & Walkinshaw (1966) Marchant (1969) Carbonelle & Rodrigez (1968) ibid Furtado et a l . (1967)  Griffiths  (1970)  Puccinia podophylli  Moore (1963)  17©  At present t o o l i t t l e  information i s available  ing f u n g a l membrane complexes t o attempt  t o I d e n t i f y them  w i t h e i t h e r the mesosomal system or the G o l g i Consequently we should perhaps  concern-  apparatus.  r e t a i n the simple term " f u n g a l  membrane complex" f o r these s t r u c t u r e s and so a v o i d a nomenc l a t u r e which might r e s u l t  i n misleading implications.  t a i n l y the complexes resemble  Cer-  mesosomes and some types of  G o l g i i n c e r t a i n aspects of s t r u c t u r e and f u n c t i o n . the ontogeny of the three systems i s d i f f e r e n t , a separate e v o l u t i o n a r y o r i g i n .  However,  indicating  What seems most l i k e l y t o  be the case i s t h a t c o i l e d membrane systems of t h i s  type  have o p t i m a l p r o p e r t i e s f o r c e r t a i n k i n d s of p h y s i o l o g i c a l f u n c t i o n s r e l a t i n g t o s e c r e t i o n , and membrane g e n e r a t i o n and s t o r a g e , and t h a t these p r o p e r t i e s have been ^ u t i l i z e d " i n dependently  i n d i f f e r e n t c l a s s e s of organisms.  V. Figure 1  A l a r g e membrane complex (mc) i n d i r e c t c o n n e c t i o n w i t h the n u c l e a r envelope of a prophase I nucleus  (dN).  Figure 2  PLATE 1  Method A.  c a . X 21}.,750.  A c o n s e c u t i v e s e c t i o n about 1000 A removed from f i g u r e 1 showing the p o s i t i o n of the whole nucleus w i t h r e s p e c t t o the membrane complex. Method A. 0  ca. X 13,500v  V.  PLATE 2  F i g u r e 3a.  A prophase I nucleus (dN) i n the promycelium showi n g the p o s t e r i o r p o s i t i o n o f the membrane complex (mc) and the p o s i t i o n a l r e l a t i o n s h i p between the complex and the v e s i c l e s ( v e ) . The CKE l i e s i n a p o s i t i o n opposite the complex. The n u c l e a r bounda r y i s demarcated by n u c l e a r pores (NP). Method A. c a . X 37,200.  F i g u r e 3b  An enlarged view of the membrane complex and v e s i c l e r e g i o n seen i n f i g u r e 3a. Method A. c a . X 7h»k- 0, Q  F i g u r e 4,  A prophase I nucleus (dN) showing the c h a r a c t e r i s t i c p o s i t i o n s of the membrane complex (mc) v e s i c l e s (ve) and n u c l e o l u s (Nu). The arrow i n d i c a t e s one o f the c h r o m a t i n - n u c l e o l a r c o n n e c t i o n s . Method C. c a . X 47,500.  F i g u r e 5.  D i v i s i o n I nucleus i n the dumb-bell stage. The nuc l e u s i s the same as t h a t shown i n p a r t IV f i g u r e 24. Note the p o s i t i o n of the membrane complex (mc). Method c. c a . X 18,000.  V.  PLATE 3  F i g u r e 6.  As the nucleus (N) elongates, c o n s t r i c t s c e n t r a l l y and d i v i d e s the membrane comples (mc) comes t o l i e i n a c e n t r a l p o s i t i o n between the two s e p a r a t i n g daughter n u c l e i . A l s o note the v e s i c l e s i n the i n t e r n u c l e a r zone. Method B. c a . X 18,700.  F i g u r e 7a.  A h a p l o i d nucleus a s s o c i a t e d w i t h two membrane complexes (mc's). The membrane complex on the lower l e f t i s a s s o c i a t e d w i t h v e s i c l e s . Method C. c a .  X 30,000. F i g u r e 7b.  An enlarged view of the membrane complex (mc) and v e s i c l e s seen i n f i g u r e 7a. Method C. c a .  X 61;,400. F i g u r e 8.  A h a p l o i d nucleus showing the i n t i m a t e r e l a t i o n s h i p between the membranes of the complex and the n u c l e a r  envelope.  Method B.  c a . X 60,600.  V.  PLATE k  F i g u r e 9a.  Formation of a membrane complex i n v a g i n a t i o n of the plasma membrane. Method B. c a . X 31,000.  F i g u r e 9b.  An e n l a r g e d view of the forming complex seen i n F i g u r e 9a. Method B. c a . X 81,200.  F i g u r e 10.  A l o n g i t u d i a l s e c t i o n i l l u s t r a t i n g : t h e attachment between a membrane complex and the plasma membrane bounding a t h i c k e n i n g septum. Method B. c a . X 37,000.  171  BIBLIOGRAPHY Beams. H.W. and K e s s e l , R.G. s t r u c t u r e and f u n c t i o n .  1968. The Golgie a p p a r a t u s : I n t . Rev. C y t o l . 2 ^ : 2 0 9 - 1 7 6 .  Beaton, C.D. 1968. An e l e c t r o n microscope study o f the mesosomes o f a p e n i c i l l l n a s e - p r o d u c i n g Staphyloco ecus. J . gen. M i c r o b i o l . £ 0 : 37-1+ * 2  B e r l i n , J.D, and Bowen, C.C. 1961+. The m o s t - p a r a s i t e i n t e r f a c e o f Albugo C a n d i d a on Raphanus s a t i v u s . Am. J . Bot. ^ 1 : I4j4.5-lj.52. B e r l i n e r , M.D. and Duff, R.H. 1 9 6 5 . U l t r a s t r u c t u r e o f A r m i l l a r i a m e l l e a hyphae. Can. J . B o t . b^: 171-172. Bouck, G.B. 1 9 6 5 . Fine s t r u c t u r e and o r g a n e l l e a s s o c i a tions i n algae. J . C e l l B i o l . 26_: 5 2 3 - 5 3 7 . Brown, R.M. 1969. Observations on t h e r e l a t i o n s h i p o f t h e G o l g i apparatus t o w a l l f o r m a t i o n i n a marine Chrysophycean a l g a , P l e u r o c h r y s i s s c h e r f f e l i i Pringsheim. J . C e l l B i o l . Ifcl; 1 0 9 - 1 2 3 . Brown, R.M. and Stack, S.M. 1971. (unpublished)•  P e r s o n a l communication  B u r d e t t , I.D.J, and Rogers, H.J. 1 9 7 0 . M o d i f i c a t i o n o f the appearance o f mesosomes i n s e c t i o n s o f B a c i l l u s l i c h e n i forrais a c c o r d i n g to the f i x a t i o n procedures. J . U l t r a s t r u c t . Res. ^ 0 : 354-367. C a r b o n e l l e , L.M. 1 9 6 7 . C e l l w a l l changes during the budd i n g process o f P a r a c o c c i d i o i d e s b r a s i l i e n s l s and Blastomyces d e r m a t i t i d i s . J . B a c t e r i o l . 91+: 213-223. C a r b o n e l l e , L.M. and Rodrigez, J . 1 9 6 8 . M y c e l i a l phase o f P a r a c o c c i d i o d e s b r a s i l e n s i s and Blastomyces d e r m a t i t i d i s : an e l e c t r o n microscope study. J . B a c t e r i o l . ~ 9 6 : 533-51+3. Chambers, T.C., Markus, K., and Willoughby, L.G. 1 9 6 7 . The f i n e s t r u c t u r e of t h e mature zoosporangium o f Nowakowskiell a profusa. J . gen. M i c r o b i o l , l+j>. 135-11+1. :  Chapman, J.A. and V u j i c i c , R. 1 9 6 5 . The f i n e s t r u c t u r e o f s p o r a n g i a o f Phytophthora e r y t h r o s e p t i c a Pehtyb. J . gen. M i c r o b i o l . 1+1.: 2 7 5 - 2 9 o T Chou, O.K. 1 9 7 0 . An e l e c t r o n microscope study o f host p e n e t r a t i o n and e a r l y stages o f haustorium f o r m a t i o n o f Peronospora p a r a s i t i c a ( F r . ) T u l . on cabbage c o t y l e dons. Ann. Bot. 3k: 189-201+.  172 Davison, E.M. 1968. Cytochemistry and u l t r a s t r u c t u r e o f hyphae and h a u s t o r i a of Pieronospora p a r a s i t i c a . Ann.  B o t . 3 2 : 613-621.  Edwards, M. 1969. Mesosome-like s t r u c t u r e s i n b l u e - g r e e n algae and lower e u k a r y o t i c forms. B i o p h y s . J . 9} PA 176 (Abstract). Edwards, R.P. 1970. E l e c t r o n microscope i l l u s t r a t i o n s o f d i v i s i o n i n Mycobacterium l e p r a e . J . Med. M i c r o b i o . 3: 493-498. ~ ; E n r l i e b , M.A. and E h r l i c h , H.G. 1966. U l t r a s t r u c t u r e o f the hyphae and h a u s t o r i a o f Phytophthora i n f e s t a n s and hyphae: o f Phytopbthora p a r a s i t i c a l Can. J. Bot. 4 4 f i l l 9 5 - 1 5 0 4 * Fawcett, D.W. and Susuma, I . 1958. Observations on the c y t o p l a s m i c membranes o f t e s t i c u l a r c e l l s , examined by phase c o n t r a s t and e l e c t r o n microscopy. J . Biophys. C y t o l . ly: 1 3 5 - l U l * F i t z - J a m e s , P.C. I960. P a r t i c i p a t i o n of the c y t o p l a s m i c membrane «iin the growth and spore formation o f b a c i l l i . J . B i o p h y s . Biochem. C y t o l . B : 5 0 7 - 5 2 8 . Furtado, J.S., B r i t o , T. de, and F r e y m u l l e r , E . 1967* ture and r e p r o d u c t i o n of P a r a c o c c i d i o i d e s l o b o i . Mycologia 5 9 : 2 8 6 - 2 9 4 .  Struc-  G r i f f i t h s , D.A. 1970. The f i n e s t r u c t u r e o f V e r t i c i l l i u m d a h l i a e Kleb c o l o n i z i n g c e l l o p h a n e . Can. J. M i c r o b i o l . 17: 7 9 - 8 1 . Grove, S.N., Morre, D.J., and B r a c k e r , C E . 1967. The G o l g i apparatus as a s i t e o f endomembrane d i f f e r e n t i a t i o n i n Pythium ultimum. Am. J . B o t . 5 4 : 6 3 8 ( A b s t r a c t ) . Hawker, L.E. 1963. F i n e s t r u c t u r e o f Pythium debaryanum Hesse and i t s probable s i g n i f i c a n c e l — N a T . 197: 618-619. Heath, T.B. and Greenwood, A.D. "1971. U l t r a s t r u c t u r a l o b s e r v a t i o n s On the kinetosome, and G o l g i b o d i e s d u r i n g the a s e x u a l l i f e c y c l e of S a p r o l e g n i a . Z e i t . Z e l l f o r s c h .  112: 371-389.  ~  Hlghton, P.J, 1970. An e l e c t r o n microscope study o f the s t r u c t u r e of mesosomal membranes i n B a c i l l u s l l c b e n l f o r m l s . J . U l t r a s t r u c t . Res. 31,: 2 4 7 - 2 5 9 . Hyde, J.M. and Walkinshaw, C H . 1966, Ultrastructure,of b a s l d i o s p o r e s and mycelium o f L e n z i t e s s a e p i a r i a . J. B a c t e r i o l . 92: 1218-1227.  Imaeda, T. and Ogura, M. 1963. Formation o f i n t r a c y t o p l a s m i c membrane systems o f Mycobacteria r e l a t e d to c e l l d i v i s i o n ; '  J . B a c t e r i o l . 8 5 : 15'0-163.  Kakefuda, T., Holden, J.T., and Utech, N.M. 1967. Ultrastruct u r e o f tbe membrane system i n L a c t o b a c i l l u s plantarum. J . B a c t e r i o l . 9 3 : 1*72-1*82. Kozar, F . and W e i j e r , J . 1 9 6 9 . E l e c t r o n dense s t r u c t u r e s i n Neurospora c r a s s a . Can. J . Genet. C y t o l . 1 1 : 613-616. Lowry, R.J. and Sussman, A.S. 1 9 6 8 . U l t r a s t r u c t u r a l changes d u r i n g g e r m i n a t i o n o f a s c o s p o r e s . J . gen. M i c r o b i o l . 5 1 : 1*03-4*09. Lu, B.C. 1 9 6 5 . F i n e s t r u c t u r e i n f r u i t i n g b o d i e s o f Coprinus, w i t h s p e c i a l emphasis on chromosome s t r u c t u r e s . Ph.D. T h e s i s , The U n i v e r s i t y o f A l b e r t a , Edmonton, A l b e r t a , CAN. Lu, B.C. 1 9 6 6 . G o l g i apparatus o f the b a s i d i o m y c e t e Coprinus lagopus . J . B a c t e r i o l . 92: I831-183I*. Marchant, R. 1 9 6 9 . The f i n e s t r u c t u r e and development o f the f r u c t i f i c a t i o n o f Lycoperdon perlaturn. T r a n s . B r i t . M y c o l . Soc. 5 3 : 6 3 - 6 8 . M i l l i n g t o n , W.F. and Gawlik, A. 1 9 7 0 . U l t r a s t r u c t u r e and i n i t i a t i o n o f w a l l p a t t e r n i n Pediaetrum boryanum. Am. J . B o t . 5 7 : 5 5 2 - 5 6 1 . " Mollenhauer, H.H. and Morre, D.J. 1 9 6 6 . G o l g i apparatus and p l a n t s e c r e t i o n . Ann. Rev. P I . P h y s i o l . I J : 27-1*6. Moore, R.T. 1 9 6 3 . F i n e s t r u c t u r e o f mycota. X I Occurrence o f the G o l g i dictyosome i n the heterobasidlomycete P u c c i nia p o d o p h y l l i . J. Bacteriol. 86: 866-871. — Moore, R.T. and McAlear, J.H. 1 9 6 3 . F i n e s t r u c t u r e o f mycota !*• The occurrence o f the G o l g i dictyosome i n the fungus Neobulgaria pura. J . C e l l B i o l . 1 6 : 131-11*1. Newcombe, E.H. 1 9 6 9 . P l a n t M i c r o t u b u l e s . Ann. Rev. P I . Physiol. 2 0 : 253-287. Peyton, G.A. and Bowen, C G . 1 9 6 3 . The h o s t - p a r a s i t e i n t e r f a c e o f Peronospora manshurica on G l y c i n e max. Am. J . Bot. 5 0 : 7 8 V - 7 9 9 . Robertson, J.D. 1 9 6 1 . New u n i t membrane o r g a n e l l e o f Schwann ce11. In B i o p h y s i o l o g l c a l and P h a r m a c o l o g i c a l A c t i o n s , e d . Shane, A.M., Am. ASSOC. AOV. o r s c i .  m Rogers, H.J. 1970. B a c t e r i a - l growth and the c e l l envelope. B a c t e r i o l . Rev. 3J+: 19l*-21l*. Rosenberger, R.P. and K e s s e l , M. 1968. Non-random s i s t e r chromatid s e g r e g a t i o n and n u c l e a r m i g r a t i o n i n hyphae A s p e r g i l l u s n i d u l a n s . J . B a c t e r i o l . 96: 1208-1213. o  f  Ryter, A. 1968. A s s o c i a t i o n o f the nucleus and the membrane of b a c t e r i a : a m o r p h o l o g i c a l study. B a c t e r i o l . Rev.  32: 39-51*.  Whaley, W.G., Kephart, J.B., and Mollenhauer, H.H. 1961*. The dynamics o f c y t o p l a s m i c membranes d u r i n g development. In C e l l u l a r Membranes i n Development, e d . Locke, M., H#H2 lol!aj..j. Acaaemic .rress, i n c . ppT 135-157. ?  175  GENERAL CONCLUSION "The  t h i n g s t h a t are not y e t done." ( I s a i a h , 1+6.10)  A g e n e r a l i z e d c y t o l o g i c a l study o f an e n t i r e stage i n the l i f e c y o l e o f an organism has c e r t a i n advantages and c e r t a i n disadvantages; however, i n approaohing an organism as l i t t l e known as U s t i l a g o h o r d e i , t h i s step i s both nec e s s a r y and v a l u a b l e . miscellaneous  stages  Casual  observation of organelles at  i n the l i f e c y c l e o f an organism r e -  v e a l s the remarkable v a r i e t y o f f a s c i n a t i n g s t r u c t u r e s t o be found.  Unfortunately,  t h i s approach does l i t t l e t o e s -  t a b l i s h the e q u a l l y i n t e r e s t i n g and perhaps more r e l e v a n t i n t e r r e l a t i o n s between o r g a n e l l e s and o r g a n e l l e systems. In most cases, as i n U s t l l a g o h o r d e i , the e s s e n t i a l organ e l l e s are always p r e s e n t . contents,  I t i s the changes i n the s i z e ,  d i s t r i b u t i o n and i n t e r r e l a t i o n s h i p s which d i r e c t  the course o f d i f f e r e n t i a t i o n i n the organism. n e r a l i z e d study enoourages the o b s e r v a t i o n its  e n t i r e t y , thus a l l o w i n g one to peroeive  C l e a r l y a ge*  o f development i n e x a c t l y which  o r g a n e l l e s and systems may be important a t c e r t a i n developmental s t a g e s .  Perhaps the most important r e s u l t s o f such a  study are, f i r s t ,  the f o r m u l a t i o n o f hypotheses  concerning  e x a o t l y what i s important and when and where i t i s important; and,  second, the f o r m u l a t i o n o f methods by which these hypo-  theses might be t e s t e d .  176  T h i s study i n d i c a t e s the f e a s i b i l i t y o f s t u d y i n g p r e germinal mlcroanatomieal  changes i n h y d r a t i n g spores.  Con-  s i d e r i n g t h a t many o f the fundamental events which l e a d up to metabasidium f o r m a t i o n b e g i n l o n g b e f o r e germination i t s e l f , the p a u c i t y o f i n f o r m a t i o n i n t h i s developmental i s astounding]  What i s now  r e q u i r e d are e x t e n s i v e s t u d i e s on  a v a r i e t y o f spores, coupled w i t h the a p p r o p r i a t e and h i s t o c h e m i c a l t e c h n i q u e s .  and  biochemical  I n U s t i l a g o h o r d e i . two  p o r t a n t aspects to study are the composition c u l e n t cytoplasm"  stage  o f the  im-  "floc-  the development o f the r e s p i r a t o r y  p a t t e r n as r e f l e c t e d i n the p e c u l i a r i t i e s o f the mitochond r i a l population.  Another g e n e r a l area d e s e r v i n g o f f u r t h e r  study i s t h a t o f p r o m y c e l i a l e x t e n s i o n . I n p a r t I , the evidence  suggests  t h a t the n u c l e a r enve-  l o p e g i v e s r i s e to the endoplasmic r e t i c u l u m , and the endop l a s m i c r e t i c u l u m i t s e l f g i v e s r i s e to the "primary  hydra-  t i o n " vacuoles by d i l a t i o n o f the intermembranous space. More study i s r e q u i r e d to determine the exact manner i n which t h i s happens; i n f o r m a t i o n from glutaraldehyde-osmium f i x e d m a t e r i a l i s necessary.  C e r t a i n l y one o f the next steps i n  p u r s u i n g t h i s q u e s t i o n should be the a p p l i c a t i o n o f the Gomori r e a c t i o n (or some e q u i v a l e n t technique) t o determine whether the primary  vacuoles have acid-phosphate  activity,  ( i . e. are l y t i c i n n a t u r e ) . Vacuoles chanisms.  i n U s t i l a g o h o r d e i are formed by s e v e r a l  I n the metabasidium, spherosome-like  bodies  meseem  to g i v e r i s e It© v a c u o l a r s t r u c t u r e s by d i l a t i o n and/or f u sion.  T h i s would imply t h a t they a r e e q u i v a l e n t i n f u n c t i o n  to the animal lysosome. c e r t a i n l y without somal a c t i v i t y  However, t h i s  cannot be decided f o r  appropriate histochemlcal tests f o r l y s o -  ( i . e . the Gomori r e a c t i o n ) .  The  evidence i n  U s t i l a g o h o r d e i suggests t h a t these o r g a n e l l e s may  be i n -  v o l v e d i n other f u n c t i o n s as w e l l as l y t i c ones; thus, even i f the spherosomal bodies s h o u l d prove to have lysosomal a c t i v i t i e s , the author f e e l s t h a t one would be w e l l a d v i s e d not t o conclude t h a t t h a t i s t h e i r o n l y f u n c t i o n . Much y e t remains to be l e a r n e d about the n u c l e i o f smut fungi.  To date, t h e evidence suggests t h a t the m e i o t i c d i v i s  i o n f i g u r e s are most compatible w i t h Brown and Stack's model f o r somatic n u c l e a r d i v i s i o n i n some f u n g i .  However, more"  i n f o r m a t i o n i s r e q u i r e d on a l l stages o f n u c l e a r d i v i s i o n p a r t i c u l a r l y a t the e l e c t r o n microscope  l e v e l , and  s e c t i o n i n g through e n t i r e n u c l e i i s r e q u i s i t e .  serial  Studies of  the somatic s p o r i d i a l n u c l e a r d i v i s i o n s should a l s o be made. One  also suspects that the techniques c o u l d be improved s u f -  f i c i e n t l y to demonstrate microtubules more c l e a r l y and  so  e s t a b l i s h t h e i r r e l a t i o n s h i p to the d i v i d i n g n u c l e u s .  Some  e f f o r t should be made t o determine  the e x t e n t to which t h i s  mechanism o f d i v i s i o n occurs among f u n g i and any g e n e t i c imp l i c a t i o n s should be i n v e s t i g a t e d There now  ( e s p e c i a l l y at m e i o s i s ) .  seems to be l i t t l e doubt t h a t membrane com-  p l e x e s are common among f u n g i , and i t seems  unreasonable  t h a t these complexes should not be s t u d i e d more s e r i o u s l y . In U s t i l a g o h o r d e i . these complexes appear to p l a y some p a r t  i n e s t a b l i s h i n g the p o s i t i o n a l r e l a t i o n s h i p between a septum and the p r o c e e d i n g n u c l e a r d i v i s i o n .  Serial sectioning i s  r e q u i r e d t o e s t a b l i s h the one-to-one-to-one r e l a t i o n s h i p  be-  tween membrane complex, n u c l e u s , and  cell  d i v i s i o n mutants may  septum.  S t u d i e s on  prove u s e f u l i n d e t e r m i n i n g the  signi-  f i c a n c e of the complex. The disadvantage i n a g e n e r a l i z e d study of t h i s i s t h a t although a m u l t i t u d e of hypotheses  suggest them-  s e l v e s , few d e f i n i t e c o n c l u s i o n s can be drawn. merely p o i n t s the way. and hypotheses  type  Such a study  The author hopes that the o b s e r v a t i o n s  presented a r e s u f f i c i e n t l y well-documented  and  o f s u f f i c i e n t i n t e r e s t to encourage o t h e r s , as w e l l t o pursue answers to these  problems.  APPENDIX A C u l t u r e Medium (A)  COMPLETE BROTH Vogel's s o l u t i o n ( d i l u t e ) D i s t i l l e d water Tryptophane Casein hydrolysate Yeast e x t r a c t ( D i f c o ) Sucrose or Dextrose Vitamin solution 1.  Note:  2. (B)  V i t a m i n s o l u t i o n t o be added a f t e r a u t o claving. To make complete p l a t e s add 20 gm. B a c t o agar b e f o r e a u t o c l a v i n g .  VOGEL'S SOLUTION ( c o n c e n t r a t e ) Nao c i t r a t e . 2H2O KH^PO. anhydrous NHi NO-J anhydrous  MgSOh^. 7H 0 2  CaClg . 2 H 0 Trace element s o l u t i o n D i s t i l l e d water Chloroform 2  Note:  (C)  20 m l . 11. £0 mg. 5 gm. 55 gnu (20 gm. ( (10 gm. 10 m l .  1. 2. 3.  123 gm. 2$0 gm. 100 gm.  10 gm.  5 5 750 2  gm. ml. ml. ml.  Add chemicals s u c c e s s i v e l y w i t h s t i r r i n g . S t o r e a t room temperature. D i l u t e 50-fold w i t h d i s t i l l e d water b e f o r e use.  VITAMIN SOLUTION Thiamin Riboflavin Pyridoxin C a l c i u m pantothenate Benzoic a c i d Nicotinic acid Choline chloride Inositol F o l i c acid D i s t i l l e d water t o a t o t a l o f  Note:  1. 2.  100 mg. £0 rag. 50 mg;. 200 mg.. £0 mg. 200 mg. 200 mg. 1*00 mg. £0 mg. 1 1.  S t o r e a t 1*°C. Use 10 m l . o f v i t a m i n s o l u t i o n p e r l i t r e o f s t e r i l e medium.  (D)  TRACE ELEMENT SOLUTION C i t r i c acid . 1H 0 ZnSOj. . 7H2O FpfNliJpSO). . 6HpO 2  CUSOK  .  5R20  MnSOJT , lHpO  H3POR anhydrous • 2H 0  NS2M0O1,  2  Chloroform H 0 distilled 2  Note:  1.  Store at room temperature.  5 gm* $ gm. 1 gmi 0.25 gm. 0.05 gm.  O.OS^gBU  0.05 gnu  1 ml. 95 ml.  APPENDIX B Preparation o f M a t e r i a l f o r E l e c t r o n Microscopy i  NOTE: 1.  Tbe e n t i r e procedure i s c a r r i e d out a t room temperature.  C o l l e c t i o n . - R e s t i n g spores are o b t a i n e d b y s p l i t t i n g open  the k e r n e l s o f smutted heads and shaking the spores i n t o a t e s t tube c o n t a i n i n g a few m i l l i - l i t r e s o f d i s t i l l e d water.  These  are shaken v i o l e n t l y f o r s e v e r a l seconds t o wet the spores, and are c e n t r i f u g e d immediately a t low speeds f o r 1-2 minutes on a P h i l l i p s Drucker Combination c e n t r i f u g e L-708,  The water  i s decanted o f f and the spores resuspended i n the d e s i r e d fixative.  The time i n which the r e s t i n g spores are i n water  must be kept t o a minimum i n order t o a v o i d the p o s s i b i l i t y of a c t i v a t i o n , Por spores which have been h y d r a t e d h a l f - a n - h o u r or more i n b r o t h , a s u i t a b l e a l i q u o t o f the spore suspension ( t h i s depends on' the c o n c e n t r a t i o n o f s p o r e s , - u s u a l l y 10-20 m l . i s s u f f i c i e n t ) i s c e n t r i f u g e d down.  The b r o t h i s decanted o f f ,  and the p e l l e t washed once i n water  (before  KMnO^-fixation)  or i n the a p p r o p r i a t e b u f f e r , f o r 1-2 minutes.  After  repel-  l e t i n g , the l i q u i d i s poured o f f and the m a t e r i a l resuspended i n the d e s i r e d 2.  fixative.  F i x a t i o n . - The two f i x a t i o n procedures used a r e : 1)  1.5$ KMnO^ (aqueous) - Potassium permanganate i s d i s s o l v e d i n d i s t i l l e d water o v e r - n i g h t and f i l t e r e d b e f o r e u s e . F i x a t i o n time i s 10 - 20 minutes.  2)  2.0$ G l u t a r a l d e h y d e - 1 p a r t w i t h 34 p a r t s 0.01 M cacodylate b u f f e r  ( S a b a t i n i e t a l . , 1961j B r a c k e r  and Grove, p e r s o n a l communication).  The pH o f the  b u f f e r i s a d j u s t e d t o 7.0-7.2 with HC1 b e f o r e u s e . F i x a t i o n time i s 12-16 hours (the longer time i s r e q u i r e d f o r the r e s t i n g s p o r e s ) . When u s i n g glutaraldehyde the f i x a t i v e i s important Ustilago hordei.  as a f i x a t i v e the o s m o l a r i t y o f  i n o b t a i n i n g optimal r e s u l t s f o r  The o s m o l a r i t y o f the growth medium, the growth  medium p l u s the m a t e r i a l , and the f i n a l 2% g l u t a r a l d e h y d e s o l u t i o n were determined on an Advance Osmometer (Model 3W). The  r e s u l t s (Table I ) i n d i c a t e d t h a t the osmolaritjres o f a l l  three a r e w i t h i n £ 0 m i l l i - o s m o l e s o f each  other.  I t i s a d v i s a b l e t o c a r r y out the f i r s t hour o f f i x a t i o n under vacuum.  T h i s i s p a r t i c u l a r l y t r u e o f r e s t i n g spores  which tend t o f l o a t . throughout 3.  The m a t e r i a l i s a g i t a t e d  continuously  fixation.  Washing. - The f i x e d m a t e r i a l i s washed f o r a minimum o f  lh hour i n water o r b u f f e r (whichever i s a p p r o p r i a t e )  changing  the washing f l u i d 6-10 times.  recovered  The m a t e r i a l i s always  by c e n t r i f u g a t i o n . I*.  P o s t - f i x a t i o n . - Only t h e g l u t a r a l d e h y d e - f i x e d m a t e r i a l i s  p o s t - f i x e d i n OsO^.  OsO^ i s prepared  by mixing equal p a r t s o f  e i t h e r 2% or" \\% OsO^ i n d i s t i l l e d water with the a p p r o p r i a t e buffer.  P o s t - f i x a t i o n time i s 3 t o 3^g h o u r s .  5>.  Washing. - P o s t - f i x e d m a t e r i a l i s washed as i n step 3«  6.  U r a n y l acetate s t a i n i n g . - Glutaraldehyde-osmium f i x e d  m a t e r i a l m a y b e p r e ^ a t a i n e d w i t h 0.3$ U r ( A e ) water f o r 2-J* h o u r s .  2  in distilled  Note t h i s step i s OPTIONAL and i s  183  u s u a l l y n o t d e s i r a b l e f o r germinated  material (pt. I I ) . I f  t h i s s t e p i s used the m a t e r i a l should be rewahed as i n steps 3 and 5 . 7.  Agar embedding. - A t t h i s stage, the spores a r e c o l l e c t e d  on a m i l l i p o r e f i l t e r .  The m a t e r i a l i s coated on the f r e e s i d e  by dropping 2 $ water agar is  (I4.7  0  C) onto the s u r f a c e .  The f i l t e r  s t r i p p e d o f f onoe the agar has s o l i d i f i e d and the under  s u r f a c e i s s i m i l a r i l y coated.  The p e l l e t i s c u t i n t o s m a l l  p i e c e s f o r embedding. 8.  A l c o h o l d e h y d r a t i o n . - A l l m a t e r i a l ( K M n O ^ f i x e d and GA-Os  f i x e d ) i s dehydrated 50$,  through a standard e t h a n o l s e r i e s :  30$,  7 0 $ , 8 5 $ , 9 5 $ , 1 0 0 $ . The agar-embedded t i s s u e i s passed  through  the f i r s t  5 s o l u t i o n s i n approximately  1 hour, and  g i v e n 3 changes i n a b s o l u t e a l c o h o l o f 3 0 minutes each ( T o t a l = I3g h r . ) . NOTE:  9.  Propylene  I n the t e x t , 3 b a s i c p r e p a r a t o r y methods are d i s t i n g u i s h e d , noted A, B and C. Method A i n v o l v e s KMnOi, f i x a t i o n . Methods B and ;C i n v o l v e GA-Os f i x a t i o n and remain the same u n t i l the end o f the a l c o h o l d e h y d r a t i o n . B and C a r e d i s t i n g u i s h e d on the b a s i s o f the embedding p l a s t i c . Methods A and B a r e comp l e t e d by f o l l o w i n g steps 9 and 1 0 ; method C by s t e p 1 1 o n l y .  oxide d e h y d r a t i o n . - Propylene  oxide i s added  drop-by-drop to the f i n a l a b s o l u t e e t h a n o l change u n t i l the s o l u t i o n i s h a l f - a n d - h a l f (Time = 3g h o u r ) . are made i n Propylene 10.  oxide  Three changes  (Time = 1 h o u r ) .  Embedding i n Epon 8 1 2 . - The m a t e r i a l i s i n f i l t r a t e d by  Epon 8 1 2 (Ladd o r S h e l l O i l ) ; ( L u f t , 1 9 6 1 ; 7A:3B).  Plastic  minus a c c e l e r a t o r i s added drop-wise to the b l a s t propylene oxide  change over a p e r i o d o f 1 ^ - 2 hours t o a f i n a l h a l f - a n d - h a l f  solution.  The m a t e r i a l i s l e f t o v e r n i g h t , uncovered and f r e s h  p l a s t i o i s made ( i n c l u d i n g the a c c e l e r a t o r , 1.5#-DMP 30).  The  f r e s h p l a s t i o i s changed 3 times (3g hour each) b e f o r e embedding. The Epon 812  i s hardened by p o l y m e r i z i n g the b l o c k s 12 hours  at  28-32  37° C and  11.  hours a t  60°  C.  Embedding i n Spurr's media. - The m a t e r i a l i s i n f i l t r a t e d  by Spurr's  standard embedding medium ( F o l y s c i e n c e s  (Spurr, 1969;  Standard medium A f i r m ) .  Incorporated)  I t i s brought to the  p l a s t i c through immersion i s f i v e standard s o l u t i o n s o f i n c r e a s ing 1  p l a s t i c concentration: : 1, 2 : 1, 3 : 1.  Spurr's  : ETOH = 1  6-7 12.  h r . a t 70°  em-  C.  cut on a S o r v a l l Porter-Blum o f diamond k n i v e s  - S i l v e r to grey s e c t i o n s are  MT-2  ultramicrotome  using glass  (Dupont) k n i v e s , and are p i c k e d up on  carbon-  The m a t e r i a l i s p o s t - s t a i n e d i n a s a t u r a t e d s o l u -  t i o n o f u r a n y l a c e t a t e i n $0% (Reynolds,  (^ h r . each) and  Harden Spurr's by p o l y m e r i z i n g the b l o c k s  Post-embedding technique.  coated g r i d s .  : 2,  I t i s l e f t i n the l a s t s o l u t i o n o v e r n i g h t ;  then i s put through 3 changes o f p l a s t i c bedded i n b l o c k s .  : 3, 1  1963).  ethanol f o l l o w e d by l e a d c i t r a t e  A f t e r methods A and B the s t a i n i n g times  are 30-lj.O minutes and  20-30  minutes r e s p e c t i v e l y ; while  after  method G the times are 3-k- hours (37° C) and 30-ij.O minutes respectively.  185  TABLE I Osmolarity o f B r o t h , B r o t h p l u s M a t e r i a l , and 2 $ Glutaraldehyde Material  Measurement (milli-osmoles)  Complete b r o t h B r o t h p l u s 0 h r . spores B r o t h p l u s 7 h r . spores 2% G.A.  NOTE:  (cacodylate b u f f e r )  290.5 289.8 291.0 312 5 312 5 3Hi'.5 313 3 315 3 320 5 336 0 335.5 339.5  Measurements were made on an Advance Instrument Osmometer (Model 3W).  BIBLIOGRAPHY L u f t , J.H. 1961. Improvements i n epoxy r e s i n embedding methods. J . Biophys. Bioohem. C y t o l . £ : I+.09-I|.llij.Reynolds, E.S. 1963* The use of l e a d c i t r a t e at h i g h pH as an e l e c t r o n opaque s t a i n i n e l e c t r o n microscopy. J. Cell B i o l . 1 2 : 208-212. S a b a t i n i , D.D., Bensoh, K.G. and B a r r n e t t , R.J. 1963. Cytoc h e m i s t r y and e l e c t r o n mioroscopy - p r e s e r v a t i o n o f c e l l s u l a r u l t r a s t r u c t u r e and enzyme a c t i v i t y by aldehyde f i x a tion/ J . C e l l B i o l . 12.: 19-58. S p u r r , A.R. 1969. A low v i s c o s i t y epoxy r e s i n embedding medium f o r e l e c t r o n microscopy. J . U l t r a s t r u c t . Res. 26: 31-43.  186  APPENDIX C L i g h t Microscope F i x i n g and  S t a i n i n g Procedures  SQUASH PREPARATIONS In a l l cases a drop of medium c o n t a i n i n g air-dried  the m a t e r i a l i s  b r i e f l y on c o v e r s l i p s b e f o r e b e i n g t r e a t e d by one  of  the f o l l o w i n g methods, a)  (Acetic-alcohol)-Feulgen  Acetic-alcohol fixative:  1 pt. g | a c i a l a c e t i c acid with  Mix:  3 pt. absolute a l c o h o l . Add:  a few  drops of c h l o r o f o r m to  every 10 ml. Feulgen S t a i n  (Darlington  and  of above s o l u t i o n .  La Cour, 1 9 6 2 ) :  Pour 2 0 0  cc.  b o i l i n g d i s t i l l e d water over 1 gram of B a s i c F u c h s i n shake.  Cool to 50°C; f i l t e r  i n t o brown or darkened  b o t t l e w i t h ground g l a s s stopper and (Hydrochloric  acid).  Cool to 25°C and  anhydrous sodium b i s u l f i t e (K2S2O4).  metabisulfite  i t w i l l decolourize. add  0 . 5 - 1 . 0 gm  S O 2 water:  Mix:  gm.  A f t e r 21+ h r . properly  of d i s t i l l e d water w i t h 1 gm.  1.  Wash two  2.  Fix in acetic-alcohol - 1  3.  Wash s i x times i n d i s t i l l e d  1*..  Hydrolyse i n 1 N HC1  times i n d i s t i l l e d  gm.  potassium  shake, and  and  HC1  either 1  I f i t does not d e c o l o r i z e  tassium m e t a b i s u l p h i t e Method:  add  (NaHSOj^) or 1-3  charcoal,  stock  2 0 cc. I N  Keep i n the dark.  of A c t i v a t e d  2 0 0 ml.  add  and  filter. po-  1 0 cc. 1 N H C 1 .  H 2 O - 5 min.  each.  hr. H0 2  - 5 min.  @ 60° C. - 8 min.  each.  or i n  18?  $ N HC1 @ 22° C. - 1 h r . 5.  Wash 2 times i n tap water - 5 min. each.  6.  Wash f o u r times i n d i s t i l l e d  7.  Feulgen - 1-2 h r .  8.  Rinse twice i n SO2 water - 5> min. each.  9.  Wash 6 £imes i n d i s t i l l e d  10.  water - £ min. each  water - 5 min. each  S t o r e c o l d u n t i l use.  To squash the c o v e r s l i p i s i n v e r t e d  ( i . e . m a t e r i a l down) on  a g l a s s s l i d e on which a drop o f h&% a c c e t i c a c i d has been p l a c e d , and p r e s s u r e a p p l i e d .  A l l m a t e r i a l was photographed  from f r e s h l y prepared s l i d e s .  A f t e r o b s e r v a t i o n the s l i d e i s  f l o a t e d o f f i n a b s o l u t e a l c o h o l and remounted b)  i n Euparol.  BAC-Propi onic-haemat o x y l i n  BAC-fixative  (Lu, 1962);  Mix:  9 pt. n-butyl alcohol, 6 p t . g l a c i a l a c e t i c a c i d , and 2 - 3 pt.  Use: Propionic-haematoxylin  10$ aqueous chromic a c i d .  fresh.  (Henderson and Lu, 1968): Stock s o l u t i o n s :  A) 2$ haematoxylin in 50$ propiacid. B) 0 . 5 $ i r o n  alum  in 50$ propiacid. Mix:  e q u a l volumes  o f A and B. I f  s t o c k s o l u t i o n s a r e f r e s h mix t o g e t h e r one day b e f o r e use. Aged stock s o l u t i o n s  (i.e. 3  mon. or more) can be mixed and used immediately.  Method:  1.  Wash two timed  2.  F i x i n B A C - f i x a t i v e @ room temperature - 2l± h r .  3.  Wash s i x times at  in distilled  water - £ min. each.  i n d i s t i l l e d water ( l a s t change  60° C.) - 5> min.  each.  i+.  Hydrolyze  5.  Wash two times i n tap water - 5 min. each.  6.  Wash f o u r times  7.  D r a i n c o v e r s l i p and apply a few drops of staim macerating  8.  i n 1 N HC1 @ 60° C. - 12 min.  i n d i s t i l l e d water - 5 min. each.  the t i s s u e w i t h an i r o n  needle.  I n v e r t onto a g l a s s s l i d e and a p p l y  M a t e r i a l i s photographed from f r e s h l y prepared  pressure.  s l i d e s and i s  made permanent as d e s c r i b e d i n procedure a. c)  (Acetic-alcohol)-(Propiono-haematoxylin) P r e p a r a t i o n of the f i x a t i v e  ( i . e . a c e t i c - a l c o h o l ) i s des-  c r i b e d i n p a r t a and p r e p a r a t i o n of the, s t a i n i n p a r t b. Method:  The method i s the same as that d e s c r i b e d i n p a r t b s u b s t i t u t i n g a c e t i c - a l c o h o l f o r B A C - f i x a t i v e i n step 2, and s h o r t e n i n g the f i x a t i o n time t o 1 h r .  SECTIONS Fixative:  Method A, B, or C as d e s c r i b e d f o r e l e c t r o n m i c r o s copy (Appendix B ) .  Toluidine blue: Method:  1.  Mix 1 gm. T o l u i d i n e blue i n 100 ml. 1$ borax.  T h i c k s e c t i o n s ( 0 . 2 5 - 0.50 u) a r e picked up w i t h a s m a l l copper loop  (1-2 mm d i a m e t e r ) , and t r a n s -  f e r r e d t o a drop of water on a c l e a n g l a s s  slide.  2.  Evaporate water by h e a t i n g g e n t l y over a flame.  3.  P l a c e a drop o f s t a i n d i r e c t l y on s e c t i o n s .  1+.  Heat u n t i l the edge of the drop t u r n y e l l o w .  5.  Wash the s t a i n o f f i n tap water.  6.  Dehydrate 1 min. ihir.i70# e t h a n o l f o l l o w e d by 1-2 min. i n a b s o l u t e a l c o h o l .  7.  Pass s l i d e through x y l e n e f o r 1 min.  8.  Mount i n immersion o i l and s e a l w i t h n a i l  polish.  BIBLIOGRAPHY D a r l i n g t o n , C D . and La Cour, L.P. 1962. The H a n d l i n g of Chromosomes. George A l l e n and Unwin L t d . , London. Henderson, S.A. and Lu, B . C 1968. The use of haematoxylin f o r squash p r e p a r a t i o n s of chromosomes. S t a i n Technology U3 : 233-236. Lu, B.C. 1962. A new f i x a t i v e and improved p r o p i on o-c a m i n e squash technique f o r s t a i n i n g fungus n u c l e i . Can. J .  Bat. kO : 8I4.3-847.  

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