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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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1971. N In presenting th i s thes i s in p a r t i a l f u l f i lmen t of the requirements for an advanced degree at the Un iver s i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extens ive copying of th i s thes i s f o r scho la r l y purposes may be granted by the Head of my Department or by h i s representat ives . It is understood that copying or pub l i ca t i on o f th i s thes i s fo r f i nanc i a l gain sha l l not be allowed without my wr i t ten permiss ion. Depa rtment The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada For out of olde feldes, as men seytb, Cometh a l t h i s newe corn from yer to yere, And out of olde books, i n good feytb, Cometh a l t h i s newe science that men l e r e . The Parlement of Foules Georrrey Chaucer i ABSTRACT A comparative l i g h t and electron microscope technique has been used to study the c y t o l o g i c a l changes accompanying t e l i o -spore ( i . e . probasidium) germination i n Ustilago hordei (Pers.) Lagerh. Special emphasis has been placed on determining the u l t r a s t r u c t u r a l events involved i n karyokinesls, e s p e c i a l l y meiosis, and cytokinesis. The thesis i s divided into f i v e parts, of which the f i r s t i s concerned with pre-germinal d i f f e r e n t i a t i o n . The great i n -crease i n microanatomical complexity which occurs during the pre-germinal stages i s due l a r g e l y to an increase i n the amount of endoplasmic reticulum (ER) and to the formation of "primary hydration vacuoles." Evidently the nuclear envelope gives r i s e to the new ER which i n turn d i l a t e s to form the vacuoles. This i s accompanied by an increase i n mitochondrial size and the de-velopment of patches of patches of "flocculent cytoplasm." Part II concerns the i n i t i a t i o n and subsequent extension of the metabasidium ( i . e . promycelium)• I n i t i a t i o n involves t h e l l o c a l i z e d degradation of the inner spore wall, and deposi-t i o n of new wall material. The ER and spherosome-like bodies seem to be associated with these a c t i v i t i e s . Once spore wall rupture has occurred the s t r u c t u r a l basis of promycelial exten-sion i s unknown but 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 organelles appear to 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. i i Septation, knee-joint formation, and budding are discussed i n part I I I . Elaborate membrane complexes are associated with cross wall i n i t i a t i o n . A membranous plate i s completed across the c e l l before septal wall thickening begins. The i n i t i a t i o n of s p o r i d i a ( i . e . basidiospores) 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 promyoelial wall followed by degradation of the o l d wall and subsequent synthesis of new wall material. Bridge-formation r e s u l t s when two adjacent c e l l s give r i s e to bud-like processes which grow together and subsequently fuse to produce a protoplasmic bridge. The structure and a c t i v i t i e s of the metabasidial n u c l e i and t h e i r associated structures are discussed i n part IV, Both meiosis and mitosis are unusual i n that the two chromatin bodies apparently remain attached to the centriolar-kinetochore-equivalent and at l e a s t one of the chromatin bodies i n attached to the nucleolus throughout the d i v i s i o n cycle. The r e s u l t s are compatible with Brown and Stack's (1971) model f o r somatic nuclear d i v i s i o n i n some fungi. Membrane complexes, resembling those which I n i t i a t e septa, form i n association with prophase nu c l e i and maintain a s p e c i f i c r e l a t i o n with the nucleus throughout d i v i s i o n . In part V the suggestion i s made that these complexes form part of a mechanism c o n t r o l l i n g the p o s i t i o n a l relationships of nuclear and c e l l d i v i s i o n s i n the promycelium. i i i TABLE OP CONTENTS Page ABSTRACT i TABLE OP CONTENTS t i i i LIST OF TABLES i v LIST OF DIAGRAMS iv" LIST OP FIGURES v LEGEND OF SYMBOLS v i i ACKNOWLEDGEMENT v i i i GENERAL INTRODUCTION 1 PART I: Pregermination Development i n Hydrating Teliospores li+ PART I I : I n i t i a t i o n of the Promycelium and Promycelium Extension I4.8 PART I I I : Promyoelial Septation and S p o r i d i a l Formation 91 PART IV: Nuclear D i v i s i o n with Special Emphasis on Meiosis 117 PART V: The Po s i t i o n a l Regulation of C e l l and Nuclear D i v i s i o n 157 GENERAL CONCLUSION 175 APPENDIX A 179 APPENDIX B 181 APPENDIX C 186 i v LIST OF TABLES Page PART I : TABLE I - Summary of Electron Microscope Techniques • • • • 19 PART IV: TABLE I - Summary of Light Microscope Techniques •••••••••••• 1 2 1 PART V: TABLE I - Membrane Complexes and Golgi i n Fungi 169 APPENDIX: TABLE I - Osmolality of Broth, Broth plus Material, and 2% Glutaraldehyde , 185 LIST OF DIAGRAMS Page GENERAL INTRODUCTION: DIAGRAM I - L i f e Cycle of Ustilago hordel ..... 3 PART IV: 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 II (FIGURE 35) -Model f o r Meiosis i n Ustilago hordel ....... 11+9 V LIST OP FIGURES INTRODUCTION: Figure 1 . Figures 2 - l i . PART I : Figure 1 . Figures 2 -Figures 5 - 6 . Figures 7 - 8 . Figures 9 - 1 2 . Figure 1 3 • Figure 111. Figures 15 - 1 7 . PART I I : Figures 1 -Figure 5 . Figure 6 . FIGURE 7 . Figures 8 - 9 . Figures 11 - l l | . . Figure 1 5 . Figure 1 6 . Figure 1 7 . Figure 1 8 . Figure 1 9 . A time lapse study of teliospore germina-t i o n . One-, two-, and three-celled state ( l i g h t microscopy). Quiescent t e l i o s p o r e . Karyogamy (li g h t microscopy). Quiescent t e l i o s p o r e . Stage one te l i o s p o r e . Stage two te l i o s p o r e . Stage two te l i o s p o r e . ER-NE relationship, Stage two te l i o s p o r e . ER-vacuolar r e l a -t ionship. Stage three t e l i o s p o r e . F u l l y activated. Promycelium i n i t i a t i o n . Nuclear and mitochondrial migration. Spore wall rupture and promycelium emer-gence . Migration of organelles. Spherosome-like bodies. Spherosome and vacuoles i n aging t i s s u e . ER stacks i n the promycelium. Pores i n the promyoelial wall, Haploid nucleus. The c e l l of a f o u r - c e l l e d promycelium, Spherosomal membrane formation. Figure 20. PART I I I : Figures 1 - 6 . Figures 7 - 10. Figures 11 - 13. Figures Ui - 18. PART IV: Plate 1. Plate 2. Plate 3. Figure $, Figure 6. Figure 7. Figures 8 - 1 3 . Figures U4. - 16. Figures 17 - 18. Figures 19 - 21. Figures 22 - 26. Figures 27 - 33. PART V: Figures 1 - 6 . Figures 7 - 8 . Figure 9. Figure 10. The s i n g l e - c e l l e d promycelium. Septation. Membrane complexes. Bridge formation. Sporidium formation. Meiosis i n TJ. hordei ( l i g h t microscopy). Meiosis i n U. k o l l e r i ( l i g h t microscopy) Meiosis and mitosis i n U. horde! ( l i g h t microscopy). General view of a haploid nucleus. Nuclear pores. Migrating haploid nucleus. The CKE. Replication of the CKE. The nuclear w e l l and the CKE. Microtubules. Meiosis I. Meiosis II and post-meiotic m i t o s i s . Membrane complexes and the prophase I nucleus. Membrane complexes and the prophase II nucleus. Membrane complex formation. Membrane complex septum attachment. v i i LEGEND OP SYMBOLS ch — chromatin CKE - centriolar-kinetochore-equivalent c l - c e n t r a l lamella (septum) ER — endoplasmic reticulum f - f l o c c u l e n t cytoplasm L — l i p i d body M — mitochondrion mc — membrane complex rat - microtubule mtoc — microtubule organizing centre N — nucleus (dN - d i p l o i d nucleus, hN - haploid nucleus) NE - nuclear envelope NP - nuclear pore Nu — nucleolus P - perforations P i - plate (septum) PM — plasma membrane pW - promyoelial wall S — spherosome-like body V — vacuole ve - v e s i c l e W — spore wall (Wl - outer wall layer) (W2 - middle wall layer) (W3 - inner wall layer) Magnifications - The black scale represents 0.5 u °n electron micrographs and 1.0 u on l i g h t microscope pictures unless otherwise indicated. v i i l ACKNOWLEDGEMENT I particularly wish to thank my supervisor, Professor Clayton Person for his encouragement of independent research and for his guidance and valuable suggestions i n the pre-paration of this thesis. Thanks are also due to Dr. T. Bisalputra and other members of my thesis committee for i n -valuable discussions concerning the preparation of the ma-nuscript. 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.) for their helpful criticism and advice i n the preparation of part IV. I would also like to express my appreciation to Mr. L. I i . Veto for his expert teaching, technical assistance and advice i n electron microsoopy and photography. To Margaret Shand and Carole Stanley, many thanks for providing assistance of many kinds and for your moral support in times of " c r i s i s . " The smooth running of the "smut" lab i s largely 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 lab" for their constant encouragement and helpful discussions. Lastly, and most importantly, I wish to thank my hus-band, Dave, whose encouragement, help, understanding, and i n f i n i t e patience enabled me to complete this investigation and manuscript. 1 GENERAL INTRODUCTION The h i s t o r i c a l development of sci e n t i f i c interest in the smut fungi centres on one of Man's most pressing problems, - the World's food supply. Many of these tiny plant pathogens which constitute the order Ustilaginales (subclass Hetero-basidiomycetidae) are parasitic on cereal grains* The crop diseases which they cause greatly reduce not only crop y i e l d but also quality; as v i t a l agents of crop damage they are sec-ond only to rusts (Uredinales). Since cereals constitute app-roximately one-quarter of the total food consumed by Man, the economic importance of this group of fungi i s clear (Fischer and Holton, 1958; Christensen, 1963)» n o t sur-prisingly, much research has been devoted to i t s control. During the last 20 to 30 years this pursuit has led to the opening up of new and complex realms of study bent upon elucidating the physiological and genetical interrelationships between host and parasite (Flor, 191+2 and 1951+J Halisky, 1965; Person, 1959). Such studies, while of potential economic value, hold a theoretical fascination of their own. In addition smut fungi, particularly members of the genus Ustllago. have found favour as purely genetic tools being used i n studies of muta-tion (Hood, 1968), recombination (Holliday, 1961 and I96I4.; Kozar, 1969)* haploidization (Day and Jones, 1969)* and gene-t i c complementation (Dinoor and Person, 1969). The species most extensively used are Ustllago mavdis. Ustilago violaceae. 2 and Ustllago hordel. Considering the importance of the smuts both economically and as a research t o o l , and considering the energies which have been devoted to c e r t a i n aspects of smut physiology and genetics, amazingly l i t t l e oytologioal information i s available concerning any one species. Fischer and Holton have reviewed the cytology of the smuts up to 1957* Almost a l l knowledge of the structure, and l i f e cycle of these organisms has been ob-tained by l i g h t miorosoopy; i n general very l i t t l e i s known about the ultrastructure 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 ' point of view, i s the paucity of information concerning the nuclei of these fungi, e s p e c i a l l y at meiosis. The major purpose of the work presented i n t h i s thesis was to obtain information on ohromosome number, nuclear structure, and mechanism of d i v i s i o n during the meiotic and post-meiotic stages i n Ustilago hordei (Fers.) Lagerh. A comparative l i g h t and eleotron microscope teohnique has been employed f o r t h i s purpose. In Ustllago hordel, as i n other smuts, the meiotic process i s i n e x t r i c a b l y associated with other c e l l u l a r events pertaining to the germination of the sexual spore - the teliospore. The scope of the work was, therefore, broadened to include a o y t o l o g i c a l analysis of terliospore a c t i v a t i o n and germination, of germ tube extension, and of basidiospore formation. A detailed description of the variations i n the l i f e cycles of d i f f e r e n t species of the U s t i l a g i n a l e s oan be found i n The  Biology and Control of the Smut Fungi (Fischer and Holton, 1957 )»• For convenience, the l i f e cycle of Ustilago hordei i s pre-3 DIAGRAM I L i f e cyle of Ustilago hordel A schematic representation of the l i f e cycle of Ustilago hordei. Haploid nuclei 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 of i t s l i f e cycle. The pa-r a s i t i c stage i s init i a t e d only after the fusion of two com-patible haploid cells (sporidia) of different mating type (+ and -) to form a dikaryon. The dikaryotic state i s maintained u n t i l the parasitic stage culminates with the fusion i n pairs, of compatible haploid nuclei and subsequent production of d i -ploid teliospores. On teliospore germination, the diploid fu-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 to bud i n yeast-like fashion to form a clone of haploid cells (basidiospores or sporidia). There are two clones of each mating-type per promycelium* The term fteliospores" i s commonly applied to the sexual spores of rusts and smuts; however, notice should be taken that these structures are now more properly referred to as "pro-basidia". Germinating teliospores give rise to specialized tub-like cells of limited growth which are commonly called "pro-mycella (Hawker, 1966), In keeping with the new terminology the term "promycelium" should be replaced by "metabasidium". Throughout this thesis these terms w i l l be used interchangeably. The most complete study to date on teliospore germination and basidiospore production in Uatilago hordei is that of D, T, Wang (193if). In her studies Masig observed most of the funda-mental events which characterize this stage. The resting spore contains a single large nucleus which undergoes division shortly after the metabasidium is formed. In the particular strain ob-served by Wang this f i r s t nuclear division usually takes place INTRODUCTION. PLATE 1 Figures l a - 0. S e r i a l photographs of teliospores of Ustilago hordei germinating i n a thin layer of complete brotn between a glass s l i d e and a cover s l i p . 1 a and 1 b represent re s t i n g spores and spores a f t e r one hour of hydration respectively. F i g -ures l c - 0 were taken at half-hour i n t e r v a l s (Total = 6^ g n r . ) . The arrow i n l i indicates the f i r s t septumj the arrows i n l j indicate the second and t h i r d septa. 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 spor-dia 6an be detected i n l j , the t h i r d sporidia i n 11 and the fourth sporidia i n In. The pro-mycelium on the right i s undergoing Knee-joint formation, ca, X 1,000. Note: each scale d i v i s i o n represents one mi-cron ( i . e . t o t a l scale length i s 10 microns). 5 within the spore, one of the daughter n u c l e i subsequently migrat-ing into the promycelium, but she noted that sometimes the s i n -gle nucleus w i l l f i r s t migrate into the germ tube and then d i -vide. The l a t t e r i s almost always the case i n the s t r a i n of Ustilago hordel studied i n thi s t h e s i s . The two daughter n u c l e i subsequently divide again, either simultaneously or indepen-dently. According to Wang, three septa are l a i d down se-parating the four n u c l e i . In the stock culture used i n t h i s thesis the three septa are not l a i d down together. The f i r s t septum i s l a i d down immediately a f t e r the f i r s t nuclear d i v i s i o n d i v i d i n g the promycelium i n two. The other two septa are f o r -med immediately following the second nuclear d i v i s i o n , one on either side of the f i r s t . The promycelium i t s e l f contains three of the chambers, the fourth chamber l y i n g i n the spore. A l -most immediately each c e l l gives r i s e to a sporidium, and each of the nuc l e i divides again, one of the daughter n u c l e i pass-ing into the bud, the other remaining i n the parent c e l l . As an alternative to s p o r i d i a l production Wang observed that two of the chambers sometimes anastamose v i a a bridge which by-passes a septum. The bridge then extends a p i c a l l y to form a branch. The two undivided n u c l e i of the joined c e l l s pass into the branch to i n i t i a t e a dikaryon. These events which were pre-v i o u s l y observed by Wang and which constitute the major portion of t h i s thesis are summarized photographically i n the i n t r o -ductory Figures la-o and 2 - l i . In addition to her observations on the nucleus Wang also studied the "cytome" which i s now r e -ferred to as the mitochondria, the "ergastome" consisting of 6 osmiophilic l i p i d bodies, and formation of vacuoles i n the germinating spore. Variations on the pattern of teliospore germination and basidlospore production in 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 ultrastructural information i s available pertaining to the heterobasidiomycetes i n general, and to the Ustilaginales i n particular. Several workers have used scanning electron microscopy (Hille and Brandos, 1956) or surface replica techniques (Khanna et a l . , 1966) to ob-serve the surface features of smut teliospores, Kukkonen and Vaissalo (I96I4.) made a very preliminary attempt to investigate teliospore formation in Anthracoldea aapersa, Freeze-etching has revealed some of the fine structural features of the resting 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 sporidia o f Patilago hordei (Hlgham, personal communication). The cy-tology of mycelial mutant of Ustilago hordei has been studied i n some detail by Stein (1970). Fullerton (1970) has investiga-ted several aspects of the intracellular hyphae of eleven species of smut i n their corresponding hosts. Among other hetero-basidiomycetes studies include those on basldial development i n the tremellaceous fungus Exidia nucleata (Wells, 196i|.a and 1964b), budding i n Tremella mesenterica (Bandoni and Bisalputra, 1971)* and those on uredospore germination (Ehrlich and Ehrlich, 1969; Manocha and Shaw, 1967; Sussman et a l , , 1969* Williams and Leglngham, 1962j.) and various aspects of the host-parasite 7 relationship among rusts (Bracker, 1967; Ehrlich and Ehrlich, 1963; Van Dyke and Hooker, 1969)* Some information i s also available concerning the Rhodotorulas (Marchant and Smith, 1967) which have now been reclassified as heterobasidiomycetes (Banno, 1967). Fungal ultrastructure has recently been reviewed by Bracker (1967); i n many respects the cytology of the hetero-basidiomycetes i s very similar to that of other fungi. The literature pertaining to the metabolism (Allen, 1965) and cytology (Bracker, 1967) of fungal spores i s now quite vol-uminous. Excellent reviews of many aspects of 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 cytological investigations have been extensive, almost a l l studies have considered only quiesoent spores and spores i n whloh germination has occurred. Throughout this study the term "germination" w i l l be applied to the formation of the metabasidium. L i t t l e information i s available concerning the very important events which occur i n the activating pre-germinal spore and which lead up to the process of germination i t s e l f . An attempt i s made i n this study to determine the sequential morphological changes which take place i n the t e l i o -spore and metabasidium before and after germination. Such studies give r i s e to certain interesting conclusions concer-ning the changing numbers and distribution of organelles. Pa r t i -cular attention has been paid to the nature and origins of the INTRODUCTION. PLATE 2 Figure 2. The one-celled state as seen with phase optics i n a l i v i n g promycelium growing in a t h i n layer of com-plete broth between a glass s l i d e and a cover s l i p . The large d i p l o i d nucleus (dN) and a "spherosome-l i k e body" i s indicated, ca. X 4,600. Figure 3. The two-celled state as seen with phase optics in a promycelium f i x e d i n 2$ glutaraldehyde in 0.01 M ca-codylate guffer. The f i r s t septum (arrow) and one of the haploid n u c l e i (hN) are indicated. Note the condensed state of the nuclear chromatin, ca. X 4,600. Figure 1).. The f o u r - c e l l e d state as seen with phase optics in a l i v i n g promycelium grown as in figure 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 associated with the f i r s t septum. Three of the four haploid n u c l e i are ob-vious, ca. X lj.,600. Note: each scale d i v i s i o n represents one micron, ( i . e . t o t a l scale length i s 10 microns). 1 • • • • 1 1 ' 1 • 1 8 endoplasmic reticulum and the vacuoles i n the germinating sys-tem, and to the possible functions of the "spherosome-like bodies" (S) which are r e a d i l y observable i n the l i g h t miorosoope (Pig. 2). Several excellent reviews of meiosis and mitosis i n fungi are available (Burnett, 1968; Olive, 1953; Olive, 1965; Robinow and Bakerspiegel, 1965). At best the study of fungal nuclei i s d i f f i c u l t and with the exception of yeast n u c l e i , those of the smuts are perhaps the most d i f f i c u l t to observe and i n t e r -pret. The i n i t i a l problem i s the small size of the n u c l e i (1.5-2.5 u) • This i s compounded by the p r a o t i o a l l y indestruc-t i b l e fungal wall, and the general unresponsiveness of the protoplast to the usual methods of f i x i n g and s t a i n i n g . Further* more, the e a r l y meiotic stages occur within the forming thick-walled teliospore which i s usually embedded i n dead host tissue - material whioh i s impossible to squash, and d i f f i c u l t to section. Chromosomes and spindles were f i r s t described i n the nuclei of a species of Ustllago by Harper ;(1898). Rawitscher (1922) subsequently confirmed the presenoe of a spindle i n d i v i d i n g nuolei and described i t s intranulear nature. The teliospore i s the sexual spore, and presumably represents the only d i p l o i d c e l l i n the l i f e c y c l e . I t i s commonly assumed that meiosis oocurs during the f i r s t two (or three) nuclear d i v i s i o n s a f t e r the teliospore germinates, but the d e t a i l s of these nuclear d i -visions are so unclear that there i s s t i l l some doubt as to which represents the reduction division,(Sampson, 1939; Hirsohhorn, 9 1 9 4 5 ) . Wang (1934) and Sampson (1939) suggest that the f i r s t d i v i s i o n i s reductional. This has been supported gen e t i c a l l y and i s now commonly assumed to be the case; but Hirschhorn ( 1 9 4 5 ), Das ( 1 9 4 9 ) , and even Fischer and Holton (1957) support the idea that the second d i v i s i o n i s the reductional one. Kharbush ( 1 9 2 7 ) , Wang (1934), and Hirschhorn (1945) c o l l e c t i v e l y studied f i v e d i f f e r e n t genera including eleven species of U s t i - lago. From t h e i r chromosome counts at meiosis and mitosis they concluded that n = 2 . With the exception of Harper (1898) and Dickson (1931) 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, 1 9 6 4 ) . To the best of the author's knowledge t h i s i s the 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 of nuclear a c t i v i t y i n a smut fungus. Investigation of the metabasidial c e l l and nuclear d i v i s i o n s indicates that c e r t a i n i n t e r e s t i n g relationships e x i s t between the two. Special consideration has been given to the problems of chromosome number and mechanism of nuclear d i v i s i o n i n Ustilago hordei, and the data have been reanalysed i n the l i g h t of the electron microscope observations and the current trends i n fungal cytology. BIBLIOGRAPHY Ainsworth, G.G. and Sussman, A,S, (ed.) 1 9 6 5 . The Fungi; An  Advanced T r e a t i s e . I I . Academic Press, N.Y. 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 investigations of dormant T i l l e t l a caries t e l i o -spores, Mycologia 6 ^ : l i | i i - l 5 6 , A l l e n , P.J, 1 9 6 5 , Metabolic aspects of spore germination i n fu n g i . Ann, Rev. Phytopathol. 2L: 313-311-2 • Bandoni, R.J, and Bisalputra, A,A, 1 9 7 1 . Budding and f i n e structure of Tremella mesenterioa haplonts. Can. J. Bot, 2 7 - 3 0 . Banno, I. 1 9 6 7 , Studies on the sexuality o f Rhodotorula. J . Gen. Appl. Microbiol, 1^: 1 6 7 - 1 9 6 , Bracker, C.E, 1 9 6 7 . Ultrastructure of fungi. Ann, Rev, Phy-topathol. £ : 314.3-37^. Burnett, J.H, 1 9 6 8 . Nuclear d i v i s i o n . Fundamentals of Myco- logy, Edward Arnold (Publishers) Ltd, London, pp, 3 o 0 -Chrlstensen, J , J , 1 9 6 3 , Corn smut caused by Ustilago maydis Monograph # 2 , Am, Pathol, Soc, Das, MIC, 19U-9. Morphology and cytology of Entyloma micro- sporum (Unger) Schroet, and Urooystis anemones (Pers.) Wint, on Ranunculus repens L~. Ind, Phytopathol, 2,: 1 0 8 -Day^ sAijW. , and Jones, J.E. 1 9 6 9 . Sexual and parasexual ana-l y s i s of Ustilago violaceae. Genet, Res, 1J±.: 1 9 5 - 2 2 1 . Dickson, S, 1 9 3 1 . Experiments on the physiology and genetics of the smut f u n g i . C u l t u r a l characters. Pt. I I , The e f f e c t of c e r t a i n external conditions on t h e i r segrega-t i o n , Proc. Roy, Soc, B, 108 : 3 9 5 - 4 3 2 , Dinoor, A,, and Person, C, 1969. Genetic complementation l n Ustilago hordel. 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 , Electron microscopy o f the host-parasite r e l a t i o n s h i p s i n stem rust 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 Puccini a graminis. Can, J , Bot, lj/7_: 2 0 6 l - 2 0 6 l j . . Fischer, G.W,, and Holton, C.S, 1 9 5 7 . Biology and Control of  the Smut Fungi. The Ronald Press Co., N.Y, 1 2 7 . 11 Flor, H.H. 1942* Inheritance of pathogenicity of Melampsora  l i n i . Phytopathology 3 2 : 653 - 6 6 9 . Flor, H.H. 1954* Host-parasite interaction in flax rust - i t s genetics and other implications. Phytopathology h 5 : 680-8 5 . ' ' ' . Fullerton, R.A. 1 9 7 0 . An electron microscope study of the intracellular hyphae of some smut fungi (Ustilaginales). Aust. J. Bot. l B : 2 8 5 - 2 9 2 . Halisky, P.M. 1965* Physiologic specialization and genetics of the smut fungi. III. Bot. Rev. 3 1 : 1 1 4 - 1 5 0 . Harper, R.A. 1 8 9 6 . Nuclear phenomena in the smuts. Trans. Wise. Acad. S c i . Arts Lett. 5 : 4 7 6 - 4 9 8 . Hawker, L.E. 1 9 6 6 . Germination: morphological and anatomical changes. The Fungus Spore, ed. Madelin, M.F. Butter-wprtbs, 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 versateliospores 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 unter-suchung der sporenoberflache einiger Us111ago-Arten. Phyto-pathol. Z. 2 9 : 104-109. Hirschhorn, E. 1 9 4 5 . Several smut fungi. Mycologia 3 7 : 2 1 7 -2 3 5 . Holliday, R. 1 9 6 l . The genetics of Ustilago maydis. Genet. Res. 2 : 2 0 4 ^ 2 3 0 . Holliday, R. 1964. The induction of mitotic, recombination by mitomycin C in Ustilago and Saccharomyces. Genetics, Princeton 5 0 : 3 ^ 3 - 3 3 5 . Hood, CH. 1 9 6 8 . U.V. induced leth a l i t y and mutation in syn-chronized cultures of Ustilago hordei sporidia. Mut. Res. 6 : 391 - 4 0 0 . ' Khanna, A., Payak, M.M., and Mahta, S.C. 1 9 6 6 . Teliospore morphology of some smut fungi. I Electron microscopy. Mycologia 5 8 : 5 6 2 - ^ 6 9 . Kharbueb, S.S. 1927* Contribution a 1'etude des phenomenes sexuels chez les Ustilaginee's. Ann. S c i . Mat. Bot. 9 : 285-297. "" Kozar, F. 1969. Mitotic recombination in biochemical mutants of Ustilago hordei. Can. J. Genet. Cytol. 1 1 : 961 -966. 12 Kukkonen, I * and Valssalo, T . 1 9 6 I L . An eleotron mioroaoope study on spore formation i n a smut. Ann, Bot* Penn. 1 ; 236-249. " Madelin, M.P, 1966, The Fungus Spore, Colston Papers No, 18, Butterworths, London. Manocha, M.S., and Shaw. 1967. Electron microscopy of uredo-spores of Melampsora l i n i and of rust-infected f l a x . Can. J. Bot. 2j5: 1575-1582. Merchant, R., and Smith, D .6. 1967* Wall structure and bud formation i n Rbodotorula g l u t i n i s . Arch. Mikrobiol. 58* 2 4 8 - 2 5 6 . — 3 — Olive, L.S. 1953* The structure and behaviour of fungus nuc-l e i . Bot. Rev. 19: ^ 3 9 - 5 8 6 . Olive, L.S. 1965. Nuclear behaviour during meiosis. The Fungi I The Fungal C o l l . ed. Ainsworth, G.C. and~Suss-~ man, A.s. Academic Press, N.Y. pp. 143 -161. Person^ CO. 1959. Gene-for-gene relationships i n host-para-s i t e systems. Can. J . Bot. 373 &101-1130. Person, C and Wighton, D. 1964. The chromosomes of Ustilago. Can. J. Bot. 6_: 242 (Abstr.). Rawitacner, F. 1922. Beitrage zur kenntnis des Ustilagineen. I I . Z e i t . fur Bot. 273-296. Robinow, C F . and Bakerspigel, A. 1965. Somatic n u c l e i and forms of mitosis i n fungi. The Fungi I The Fungal C e l l , ed. Ainsworth, G.C. and Sussman, A . S . Academic press, N.Y. pp. 119-139. Sampson, K. 1939. L i f e cycles of smut fungi. Trans. B r i t . Mycol. Soc. £2: 1 - 2 3 . Stein, C,W. 197p. An electron microscope study of a mycelial mutant of Ustilago hordei ( P e r s i ) • M.Sc. The s i s , Univer-s i t y of 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: Their Dogga mancy and Germination. Harper and Row, Publishers, 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 studies of cold-dormant and ger-minating uredospores of Puecinia graminls var. t r i t i c i . Can. J. Bot. 1^7} 2073-2077^ Van Dyke, C G . and Hooker, A.L. 1969. Ultrastructure of host and parasite i n interactions of Zea maya with Puecinia so sogfeli. Phytopathology 59: 19itfZT%'GT~' 13 Wang, C.S, 194-3. Studies on the cytology of Ustilago crameri. Phytopathology j£: 1 1 2 2 - 1 1 3 3 . Wang, D.T. 1934* Contribution a l»etude des Ustilaginees (Cytologic du parasite et pathologie de l a c e l l u l e hote). Le Botaniste 26: 539-670. Wells, K. 1964a. The basidi a of Ex l d l a nucleata I. U l t r a -structure, Mycologia £6: 3 2 7 - 3 4 1 . Wells, K. 1964b. The basidi a of E x i d i a nucleata I I . Develop-ment. Am. J . Bot. £ 1 : 360-370. Williams, P.G. and Ledingham, G.A, 1964* Fine structure of wheat stem rust uredospores. Can. J . Bot. l±2: l 5 0 3-lf> 0 8 . PART I Pregermination Development i n Hydrating Teliospores TABLE OP CONTENTS Page ABSTRACT . 15 INTRODUCTION 15 MATERIALS AND METHODS . 16 Cultures and Culturing 16 Sample Taking •• 17 Preparation f o r Electron Microscopy • 1 8 OBSERVATIONS 20 Preparatory Methods • 20 Resting Spores • • • ••• 22 Stage One 26 Stage Two • 27 Stage Threes F u l l y Activated Spore 28 DISCUSSION 30 The Spore Wall 30 The Protoplast •• ••• 32 CONCLUSION IP-BIBLIOGRAPHY k-3 15 PART I Pregermination Development i n Hydrating Telloapores ABSTRACT An attempt has been made to determine the sequence of cyto-l o g i c a l changes occurring during pregermination imbibition of Ustilago hordel t e l i o s p o r e s . The f i r s t detectable changes are an increase i n the amount of endoplasmic reticulum, the develop-ment of patches of "flocculent cytoplasm", and a peculiar amoe-boid a c t i v i t y of the nucleus. Shortly a f t e r , vaouoles begin to appear i n the c e n t r a l regions of the protoplast. The mitochon-d r i a increase i n size throughout the pregermination period but show no evidence of d i v i s i o n u n t i l shortly before germination. L i p i d does not seem to decrease s u b s t a n t i a l l y . Hypotheses are put forward to account f o r the orgin of the endoplasmic r e t i -culum and of "primary vacuoles", INTRODUCTION In a recent review of fungal u l t r a s t r u c t u r e , Bracker (196?) states the need f o r further information concerning the cytology • » of spore germination, and p a r t i c u l a r l y the need f o r studies i n material which i s amenable to s p e c i f i c structure-function ana-l y s i s . With t h i s i n mind an attempt has been made to study, l n some d e t a i l , the sequential c y t o l o g i c a l changes occurring dur-ing the pre-germination development of imbibing teliospores of the smut fungus, Ustllago hordei» 16 Hopefully more information w i l l be forthcoming. Most fine struoture studies of the germination of fungus spores have i n -cluded only observations of spores in the dormant or quiescent state, and spores during and after germination. Very l i t t l e attention has been paid to the important events which lead up to germination i t s e l f (Corfman, 1966; Hyde and Walkinshaw, 1 9 6 6 ) . The teliosporea of Ustilago hordei belong to the category of resting spores which Allen ( 1965) refers to as "environ-mental", that i s , they w i l l resume development immediately when the environment permits. In this case only the presence of free water i s required for germinations to proceed. The teliospore i s also the sexual spore. It contains the only diploid nucleus in the entire l i f e cycle (Intro. Diagram I ) . At present very l i t t l e i s known about the ultrastructure of spores among the Ustilaginales. Kukkonen and Vaissalo (1961+) have studied teliospore formation in Anthracoldea as- peraa, and several workers have investiggted the surface fea-tures of mature spores (Hille and Brandes, 1956). Recently, freeze-etching has been used to 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 in these studies were the product of crosses between the two 17 wild-type haploid mating strains I j J and E3, isolated by Thomas ( 1 9 6 U ) * A l l were produced in the f i e l d . None of the samples had been stored longer than twelve months, Culturing, - It i s known that when spore samples are germ-inated 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 in a shake culture consisting of a modified oomplete Vogel's broth prepared ac-cording to 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 con-centration, the faster is the germination rate. The i n i t i a l spore concentrations employed were not controlled. During the early studies the f i r s t promycelia were detected at approxi-mately five hours after the i n i t i a t i o n of hydration. In sub-sequent 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 half hours. However, the sequence of stages seen in a l l cases ap-peared to be indentical. Only the period of time spent in each stage was longer in the former case. Each of the events des-cribed i s , therefore, a composite of a number of observations, each observation being made ln the context of the Individual experiment• During the early studies pre-germination samples were tak-en at o, h, 2h» and £ hours; in later studies at 0, h$ 1, and 2 hours. Each sample consisted of 3 milli 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 in the preparation procedure the spores were recovered by centrifugation• The material was f i x -ed far electron microscopy, at room temperature, according to one of the following procedures: 1. 1.5/6 KMnOj^  (aqueous) for 20 minutes. 2. 2.0$ glutaraldehyde ln 0.01 M cacodylate buffer at pH 7.0 or 7-2 for 12 to 16 hours (the longer time was required by the resting spores), followed by washing in buffer and post-fixation in 1.0 or 2.0$ OsO^ ln the same buffer for 3-3% hours. The material was then washed in either d i s t i l l e d water (pro-cedure 1) or buffer (procedure 2). Glutataldehyde-osmium f i x -ed spores were subsequently stained in 0.5$ aqueous uranyl acetate for 2-4 hours. After pelleting in 2% water-agar the spares were dyhydrated through a standard ethanol series. The material was then either passed through a standard propylene oxide series and embedded in Epon 812, or was directly embedded in Spurr's plastic (Spurr, 1969). The three basic preparatory methods are outlined in Table I. Sections were cut our on a Sorvail Porter-Blum MT-2 u l t r a -microtome using glass or diamond knives, and were post-stained in a saturated solution of uranyl acetate in 70$ ethanol follow-ed by lead citrate (Reynolds, 1963). A l l sections were viewed with an Hitachi HS-75 microscope operating at 5 0 KV. TABLE I Summary of Electron Microscope Technique Method Fixation B l . f $ KMnOlj. aqueous 2.0$ buffered g l u -teraldehyde 2.0% buffered g l u -teraldehyde Post Fixation 1-2$ buffered OsC^ 1-2$ buffered OsO^ Dehydration Ethanol-pro-pylene oxide Ethanol-pro-pylene oxide Ethanol Embedding Epon Epon Spurr's H 20 LIGHT MICROSCOPY (see a l s o Appendix C) Thick sections (0*25 u) were made of r e s t i n g spores and spores a f t e r one hour of hydration, which had been prepared according t o electron microscope method A (KMnO^). They were stained with 1.0$ Toluidine blue i n 1,0$ eorax. A Zeiss photo-microscope, equipped with 51+6 mn interference f i l t e r , was used i n a l l studies, OBSERVATIONS PREPARATORY METHODS Methods A, B, and C each provide 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 properties of the l i v i n g organism. This i s a strong argument f o r the necessity of employing more than one technique before attempting to V i s u a l i z e what the l i v i n g state of the c e l l might have been. Unless otherwise stated a l l the events here described have been seen with a l l three proce-dures . Method A appears to produce the highest l e v e l of a r t e f a c t . As has often been noted, permanganate causes both nucleic a c i d and l i p i d to leach out; the chromatin, nucleolus, ribsomes, and l i p i d bodies being conspicuously absent. D i s t o r t i o n of the c y t o l o g i c a l contents also occurs. The protoplast and many of the organelles appear to shrink i n t o t a l s i z e ; the i n t r e -e l s ter n a l spaces of the ER and nuclear envelope, and the i n t e r -membranous space of the mitochondria are usually expanded. No doubt, the grotesque shapes of the mitochondria seen i n the 21 dormant spores i n Figure 1 are artefaots caused by KMn% f i x a -t i o n * Such d i s t o r t i o n s never occur i n glutaraldehyde-osmium f i x -ed material* Nevertheless, permanganate f i x a t i o n consistently gives good membrane contrast and provides s u f f i c i e n t morphological information* I t also allows r a p i d and even penetration o f the embedding p l a s t i c * As has been noted by a number of authors (Sussman, et a l * , 1969)* t h i s l a t t e r property i s of considerable importance i n dealing with thick-walled spores* Because of these aspects, much of the information contained i n t h i s report has come from tissu e prepared according to method A* Glutaraldehyde-osmium f i x a t i o n tends to preserve the c e l l s i n a more natural state, but the a p p l i c a t i o n i s more problematic* Both pH and osmolarity appear to be important factors (Appendix B, Table I ) * Method B, i n which material i s embedded i n Epon 312, appears to r e s u l t i n the leaching out of some protein-aceous material, causing an increase 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 with t h i s method as suggested by Sussman et a l * (1969), i s improper penetration o f the p l a s t i c (Figs* 6 and 1 5 ) . Low v i s c o s i t y Spurr's embedding p l a s t i c retains a greater proportion of the ground substance and i n f i l t r a t e s the tissue evenly* However, there i s a general tendency towards negative s t a i n i n g of the membranes* This problem i s unique to 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 physiological i n -a c t i v i t y of the membranes or by masking of the membranes by dense cytoplasm (Walkinshaw et a l * , 1967; Weiss, 1963) i s un-known* 22 RESTING SPORES Spore Wall (W). - Considering that a r e s t i n g spore has a t o t a l diameter of 8 - 1 0 u, the spore wall, which i s approx-imately 1 u t h i c k (Ranges 0 . 6 3 - 1 .2i j . u), i s i t s most pro-minent constituent. I t i s composed of three d i s t i n c t layers (Pigs. 1 to 8 ) . The outermost layer (Wl), which i s the t h i n -nest (Average width = 80 rau), i s of medium electron density and of constant width around the entire surface. Its exter-na l face i s s l i g h t l y i r r e g u l a r but there i s no c h a r a c t e r i s t i c pattern of organization. This layer does not seem to be v i s i b l e a f t e r preparation with method B. In Figure 8 , patches of amorphous electron dense material can be seen adhering to the outer surface. The middle layer (W2) of the spore wall i s exceedingly electron dense and s t r u c t u r a l l y amorphous. The thickness of t h i s layer i s very v a r i a b l e ; on one side of the spore i t may be l i t t l e wider than the outermost layer (Range: 0 . 1 2 - 0 . 1 6 u), while on the opposite side i t may be as t h i c k as 0 . 6 2 - 0 . 7 0 u. Usually the boundary between the middle layer and the inner-most layer (W3) i s abrupt. This t h i r d layer i s the l e a s t dense of the three, and also the most structured. I t consists of f i b r i l s oriented p a r a l l e l to the circumference of the spore and embedded i n a homogeneous matrix. After gluteraldehyde-os-mium f i x a t i o n , a s taining gradient apparently exists i n t h i s l a y e r and the density of f i b e r s decreases towards the protoplast. These f i b r i l s are most d i s t i n c t with methods A and C. This layer i s O .33 to 0 . 5 3 u t h i c k . Its i n t e r n a l surface i s uneven and ridges p e r i o d i c a l l y project f o r distances of approximately 23 60 mu into the protoplast (Figs. $b and 1 7 ) . Plasma Membrane (PM).- When teliospores are f i x e d i n KMnOj^  the protoplast p u l l s away from the spore wall 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 with g l u t -araldehyde and osmium, the plasmalemma i s r a r e l y seen at t h i s stage. The average unit membrane thickness 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 with methods B and C indicate that i n the l i v i n g c e l l i t i s probably clo s e l y applied to the spore wall except where "paramural bodies" are present. In the r e s t i n g spore, small quantities of amorphous mater-i a l are sometimes situated between the plasma membrane and the spore wall (Figs. 1 , 1 1 , and 17). These simple "paramural bo-d i e s " do not appear to be membrane bound and show none of the complex tubular or vescicular forms commonly associated with such structures (Marchant and Rabards, 1 9 6 8 ) . In addition, where the plasma membrane has pulled away from the wa l l , d i s -t i n c t f i b e r s with an average diameter of 22 A 0 are v i s i b l e be-tween the protoplast and the spore wall (Figs, l a , l b , and 1 5 ) . Endoplasmic Reticulum (ER) and Ribosomes. — I n res t i n g spores, the ER i s sparse. Usually a series of short c i s t e r n a l elements l i e just beneath, and p a r a l l e l with, the plasma mem-brane, while i n the central regions a few short fragments of indeterminate morphology are scattered at random. Although the amount of data on glutaraldehyde-osmium f i x e d tissue i s l i m i t e d , i n t h i s respect, the ER membranes appear to be smooth. 2k The ribosomes o f U. hordei measure 100 to 15>0 A 0 i n d i a -meter. In the r e s t i n g protoplast these are very densely packed. Most, i f not a l l , of them appear to be f r e e . Nucleus (N). - Di p l o i d r e s t i n g n u c l e i (dN) are positioned c e n t r a l l y i n the spore and appear to be spherical to ovoid i n shape. Often the nucleus appears to be beaked on one sid e . The diameter ranges from 2.0 to 2.7 u (Figs. 1 and 5) . Occa-s i o n a l l y a r e s t i n g spore has two hapoid n u c l e i (hN), each having a diameter ranging from 1 .5 to 1.8 u (F i g . 6). Light microscope observations on r e s t i n g spores and spores a f t e r One hour o f hydration indicate that nuclear fusion can occur during hydration (Figs. 2, 3 and !{.), but no Information i s avai 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 the r e s t i n g spore the nuclear envelope (NE) tends to be poorly defined. This lack of d e f i n i t i o n may be due to i n -adequate f i x a t i o n , or to a r e a l difference i n the physiologi-c a l state of the membrane, or to both. Only a h i n t o f the f a m i l i a r double membrane i s observed l n Figure 5 and few nuclear pores can be seen. In glutaraldehyde-osmium f i x e d t i s s u e the nucleolus (Nu), appears to be uniformly granular and electron dense. In agreement with the r e s u l t s obtained from hyphal n u c l e i (Stein, 1970) the nucleolar diameter mea-sures approximately 1 u (Range 0.93 - 1»19 u) In both hap-l o i d and d i p l o i d n u c l e i . Mitochondria (M). - The mitochondria generally are located randomly i n the r e s t i n g teliospore although there may be a tendency to aggregate i n the more cen t r a l regions (F i g . 5)» 25 At t h i s stage t h e i r cross-sectional shape i s ovoid to round. No evidence has been found f o r the presence of elongate mito-chondria. As was previously mentioned, the distorted shapes observed a f t e r permanaganate f i x a t i o n are believed to be a r t e -f a c t u a l . The average maximum length of the r e s t i n g mitochon-d r i a as determined after,glutaraldehyde-osmium f i x a t i o n , i s 0.38 u (Range : 0.2° - 0.60 f i ) . The c r i s t a e , which are f a i r l y well-developed, are p l a t e - l i k e , and tend to be arranged i n p a r a l l e l arrays which may or may not l i e i n the long axis of the mitochondrion. L i p i d Bodies (L). - The r e s t i n g teliospore contains a large number of l i p i d bodies randomly scattered throughout the protoplast. After permanganate f i x a t i o n they appear as con-spicuous electron transparent bodies which sometimes contain traces of semi-electron dense material ( P i g . l ) . They are bounded by an electron-dense l i n e about liO A° thick, which cannot be shown to have unit membrane structure even at high magnifications. No analgous membrane-like structure i s pre-sent a f t e r preparatory methods B and C. After glutaraldehyde-osmium f i x a t i o n the l i p i d bodies are usually s l i g h t l y electron dense and homogeneous. In the spore these bodies range i n size from O.J. to 0 .5 p. Spherosome-like Bodies (S). After permanganate f i x a t i o n semi-dense, membrane-bound bodies are often observed .which range i n size from 0.1 to 0.6 u (diameter). They are usually associated with fragments of endoplasmic reticulum. Evidence i s presented i n part II that these bodies are i d e n t i c a l with 26 those membrane-bound bodies which contain very electron-dense material a f t e r glutaraldehyde-osmium f i x a t i o n (Pigs. 5 and 7)« In spores these l a t t e r bodies range i n size from 0.2 to 1.9 u. STAGE ONE Approximately one-half to one hour aft e r the beginning of hydration the nucleus of the teliospore undergoes a r a d i c a l change i n shape. It becomes lobed. The extent of t h i s condi-t i o n varies from a mild d i s t o r t i o n (Pig. 8) to an extreme state i n which the nucleus i s deeply indented and sends put long attenuated arms that bulge in t o nuclear lobes (Pig. 7 ) » This extreme state has only 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 fi x e d material i s l i m i t e d . When the nucleus i s i n the lobed condition l i p i d bodies appear to become c l o s e l y associated with the nuclear envelope. Figure 7 shows a l i p i d body, within a protoplasmic arm, exten-ding deep int o a nuclear c l e f t . At t h i s time l i p i d bodies are also occasionally seen i n close association with the mem-bren^-bound bodies containing electron dense material (S). In Figure 8 a l i p i d body appears to be fused with two such organelles (Pig. 8, arrow). Another early c y t o l o g i c a l change found to occur i n the ac t i v a t i n g teliospore i s an increase i n the amount of smooth ER i n the inner regions of the protoplast, p a r t i c u l a r l y sur-rounding and p a r a l l e l with the nuclear envelope. The ER appears to be connected with the nuclear envelope at some points (Pig. 7» arrow). 27 Shortly af t e r hydration begins, well-developed, electron transparent zones develop i n the cytoplasm (Pigs. 7 and 8). These zones, referred to as "flocculent cytoplasm" ( f ) , are r e l a t i v e l y large and appear to have structure. Occasionally, a f t e r permanganate f i x a t i o n , large semieleotron dense granules (Average diameter = 0.l8 u) are located i n , or around the edges of, the f l o c c u l e n t areas (Pig. 16). Similar regions have also been reported i n aging hyphae of U. hordei, but only a f t e r glutaraldehyde-osmium f i x a t i o n (Stein, 1970)* STAGE TWO At approximately h a l f way through the pregermination i n -t e r v a l , complex who Els of ER develop i n the cytoplasm ({Pig. 9). A l l the information pertaining to these whorls has been derived from permanganate-fixed material; the density of the protoplast a f t e r glutaraldehyde-osmium f i x a t i o n renders any observation 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 proximity to the nucleus i s f o r t u i t o u s , or whether the proximity 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 with flo c c u l e n t contents appear i n the cytoplasm (Pigs. 10 and 11). They are bounded by a unit,imembrane, the tonoplast, which has an average t h i c k -ness of 96 A 0 (Range: 80-120 A 0 ) . After 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 project outwards sharply, expecially at points of contact with what appears to be !.ER (Pig. 11, arrow). In Figure 10 several vacuoles are apparently interconnected and are p a r t i a l l y surrounded by a complex* system 28 of ER, One end of these early vacuolar systems always l i e s i n the v i c i n i t y of the nucleus i n the observed material (Pigs, 10 and 1 1 ) . Spherosome-like bodies are often present i n the v i c i n i t y of developing vacuolar complexes. At t h i s stage they do not always appear to be associated with ER (Pig. 10<)', Well-deve-loped spherosomes commonly occur, as w e l l , aligned along por-tions of the ER which appear to be associated with developing vacuoles (Pig, ll).). As the spores approach germination a large proportion of the n u c l e i are associated with long ER cisternae (Pig. 1 7 ) . Sometimes a nucleus i s almost completely surrounded by sever-a l layers of cisternae which l i e p a r a l l e l with the nuclear envelope ( F i g . 1 3 ) * In Figures 12 and 13 two such cisternae c l e a r l y connect with the nuclear envelope (Arrows). FULLY ACTIVATED SPORE (STAGE THREE) Figures 15* 1 6 , and 17 i l l u s t r a t e the maximum state of d i f f e r e n t i a t i o n that the spore attains before the metabasidium begins to form. Certain of i t s components remain e s s e n t i a l l y unchanged from the dormant state (Figs. 1 , 5 and 6 ) . Although the spore increases s l i g h t l y i n volume during imbibition, the spore wall does not show any noticeable change i n thickness, nor i s there any evidence of stretching. Simple paramural bodies are s t i l l present (Fig. 1 7 ) . Apparently the number, s i z e , and p o s i t i o n of both the l i p i d bodies, and the sphero-some-like organelles remain constant. However, a comparison of Figure 1 with Figure 17 (method A) or of Figures 5 and 6 2 9 with Figure 15 (method B) indicates a much greater degree of c y t d l o g i c a l complexity i n the f u l l y activated spores• This increase i n complexity has been achieved v i a the stages a l -ready discussed. The sum t o t a l of these changes, plus the f i n a l steps i n the preparation ,for germination, can be sum-marized as follows; Endoplasmic Reticulum. - In the f u l l y activated spore, both the t o t a l quantity, and the length of i n d i v i d u a l cisternae that can be traced i n any one section increase considerably, p a r t i c u l a r l y i n the perinuclear region (Fig 17). Rather l a t e i n the pregermination period a further change occurs i n the d i s t r i b u t i o n of ER. The cisternae tend to stack (Fig. 16, arrows). Vacuoles. - Vacuoles are not present i n dormant spores. By the time jthe hydrated spore reaches the stage of germination i t contains at least one large vacuole plus a variable number of smaller vacuoles. In Figure 17 there are a number of pai r s of these s n a i l vacuoles joined together by what appears to be short ER segments. Huelei. - The nucleus which i s once again more regular i n shape has become e c c e n t r i c a l l y situated i n the spore (Figs. 15 and 17). In Figure 17 the double nature of the nuclear en-velope i s c l e a r l y v i s i b l e . Several figures also indicate the presence of many simple nuclear pores (UP) (Figs. 9, 10, 11, 13, 15* and 17). Further d e t a i l s concerning the meiotic nucleus w i l l bsadiscussed i n part IV. Mitochondria. - Figure 6 and Figure 15 i l l u s t r a t e material 30 prepared by method B and are at the same magnification, A com-parison o f these two f i g u r e s c l e a r l y shows the dramatic increase i n the s i z e o f the mitochondria during hydration. The average maximum length o f the mitochondria i n f u l l y activated spores, a f t e r glutaraldehyde-osmium f i x a t i o n , i s 0.75 u (Range: 0.7 -1 .2 u), a fig u r e which i s almost twice that o f the r e s t i n g mitochondria (0.3d u ) . A comparison o f Figure 5 with Figure 15 indicates that there i s no s i g n i f i c a n t change i n the shape of mature mitochondria, or i n the number, length, and arrange-ment of c r i s t a e . The number of mitochondria does not appear to increase s i g n i f i c a n t l y throughout pregermination development. Only i n the very l a t e s t stages depicted i s there evidence that the mitochondria are beginning to d i v i d e . In Figure 17 the arrows indicate a mitochondrion apparently i n the process of con-s t r i c t i o n , and a double membrane-bound organelle which i s i n -terpreted as a cross-section through an immature mitochondrion. The most conspicuous feature of these d i v i d i n g and immature organelles i s the poor development of the c r i s t a e . Flocculent Cytoplasm. - The patches of "flocculent cyto-plasm 1 1 which develop very early i n the hydration process are s t i l l present ( F i g . 16). They do not seem to be l o c a l i z e d i n any p a r t i c u l a r area of the spore. DISCUSSION THE SPORE WALL Teliospores have very complex walls - a f a c t which i s pro-31 bably important i n t h e i r a b i l i t y to survive (Allen, 1 9 6 5 ) . No doubt t h i s same complexity, compounded by an astonishing t h i c k -ness, i s responsible f o r the lack of c y t o l o g i o a l information concerning the development and germination of these spores. In agreement with previous studies on smut fungi (Graham, I960; Hess and Weber, 1970) the teliospore wall of Ustilago hordei consists of three major layer. Graham (I960) also reported an intermediate cementing layer i n T i l l e t i a contraversa Kuhn. The amorphous electron dense material adhering to the outer surface may be the collapsed mucus-like j e l l y which surrounds the developing spore i n i t i a l s (Kukkonen and Vaissalo, 1961+). In agreement with the studies of H i l l e and Brandes (1956) the teliospore of Ustllago hordei appears to be exceptional among smuts i n possessing a smooth outer surface. A l l non-motile fungal spores possess at l e a s t one e l e c -tron dense layer, and t h i s layer i s never the most proximal to the cytoplasm. Its dense nature i s due to the presence of melanin which i s postulated to give resistance to r a d i a t i o n damage and to enzyme l y s i s (Bartnicki-Garcia, 1969). The innermost wall layer of Ustilago hordei i s the only f i b r i l l a r l a yer. This i s i n agreement with biochemical studies i n T i l l e t i a contraversa which show that c h i t i n i s predominant i n the inner layers but i s lacking i n the /outer (Graham, I960). The density of the " c h i t i n " f i b r i l s decreases i n the v i c i n i t y of the protoplast. A s i m i l a r gradient has been noted i n the inner w a l l layers of Neurospora ascospores (Lowry and Sussman, 196I4.). The ridgeswhich project i n t o the protoplast are s i m i l a r 32 to those demonstrated by freeze-etching i n vegetative fungal c e l l s (Bauer, 1 9 7 0 ; Hess, 1968), and conidia (Campbell, 1 9 6 9 b ; Sassen et a l . , 1 9 6 7 ) * THE PROTOPLAST The plasma membrane o f Ustilago hordei appears to be wider (approximately 1 2 0 A 0 ) than that usually reported for fungi ( 6 0 - 8 0 Ao), Corfman ( 1 9 6 6 ) , however, gives s i m i l a r dimensions f o r the spores of a myxomycete. Unlike the plasma membrane o f the hyphae of Ustilago (Stein, 1 9 7 0 ) that o f un-germinated spores i s r e l a t i v e l y smooth, A crenulate plasma membrane i s often associated with active growth (Marchant et a l , , 1 9 6 7 ) or with aging (Hawker, 1965) i n fungi. During the development o f conidia 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 convoluted during the period of wall f o r -mation, becomes smooth i n the mature spore (Campbell, 1 9 6 9 a ) . The tendency f o r the plasma membrane to p u l l away from the spore wall a f t e r permangante f i x a t i o n i s probably a r t e f a c t u a l . Usually paramural bodies are not found i n mature spores (Marchant and Robards, 1 9 6 8 ) , The amorphous structures that are sometimes seen l y i n g between the plasma membrane and spore wall (Figs. 1 and 1 7 ) do not appear to be eithe r tubular or vesicular and i t i s questionable whether they a c t u a l l y con-s t i t u t e paramural bodies. However, the p o s s i b i l i t y e x i s t s that these simple inclusions may be r e s i d u a l breakdown pro-ducts of paramural bodies which were active during spore w a l l deposition (Campbell, 1 9 6 9 a ; C a r r o l l , 1 9 6 9 ; Wilsenach and Kessel, 1 9 6 5 ) * The nature of the f i b e r s which connect the 3 3 protoplast with the spore wall i s unknown, but presumably t h e i r function i s to maintain close adherence of these two parts o f the spore during prolonged storage and dessication, G r i f f i t h s (1971) has described s i m i l a r f i b e r s i n the b a s i -diospores of Panaeolus compaaaulatua (L,) Fr, The paucity of endoplasmic reticulum and the p a r i e t a l p o s i t i o n of the ER are features which are common to a number of r e s t i n g spores (Buckley et a l , , 1 9 6 6 ; E h r l i c h and E h r l i c h , 1969), The short p a r i e t a l fragments are postulated to be the remanent of an ER-net which was active i n the thickening of the immature spore w a l l (Reeves, 1 9 6 7 ) , Kukkonen and Vaissalo (1961+) claim that ER i s pressed against the mucous walls of the sporogenous hyphae shortly after spore w a l l formation begins i n a smut fungus, Anthracoidea aspersa. An increase i n the amount o f endoplasmic reticulum during germination appears to be almost ubiquitous among fungi (Bracker. 1 9 6 7 ; f o r exception see Sussman et a l . , 1 9 6 9 ) and i s also c h a r a c t e r i s t i c of the dormant seed of higher plants. This increase r e f l e c t s a generalized acceleration of the me-t a b o l i c rate which accompanies a c t i v a t i o n and germination (Allen, 1 9 6 5 ) . With Ustilago hordel one of the f i r s t i n d i -cations o f increased metabolic a c t i v i t y i n the spore i s the appearance of short ER elements i n the i n t e r i o r , p a r t i c u l a r l y i n the v i c i n i t y of, and p a r a l l e l with the nuclear envelope. During the pregermination period t h i s nuclear-associated ER continues to develop, and may form several p a r a l l e l layers of cisternae almost completely surrounding the nucleus ( F i g . 1 3 ) , Several other reports of s i m i l a r nuclear-ER associations e x i s t i n the l i t e r a t u r e of fungi (Buckley et a l . , 1966; Gay and Greenwood, 1966; Peat and Banbury, 1967; Wells, 1 9 6 4 a ; and Wells, 1965) and higher plants (Esau and G i l l , 1971). Figures 12 and 13 indicate that during the period of maximal develop-ment of t h i s association, clear connections exist between the ER and the nuclear envelope. Such connections are w e l l -documented i n fungal hyphae although r a r e l y i n spores (Namboodiri, 1 9 6 6 ) . The o r i g i n of the abundant ER which forms i n spores dur-ing the f i r s t few hours of hydration appears to have pre-viously 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 to originate from the nuclear envelope. In view of the f a c t that i n Ustilago  hordei the f i r s t ER to be formed l i e s i n the v i c i n i t y of the nuclear envelope, and that nuclear envelope-ER connections can be demonstrated, t h i s would appear to be probable. Abundant evidence indicates that 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 hly active c e l l s such as eggs, the nuclear 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 (Wischnitzer, 1970). As germination becomes imminent, smooth entoplasmic r e t i c u -lum tends to form stacks i n the cytoplasm - p a r t i c u l a r l y i n a p a r i e t a l p o s i t i o n (Fig. 16), Some authors have suggested that these stacks of membranes, frequently seen i n fungi, r e -semble simple Golgi (Campbell, 1969b). However, the r e s u l t s of t h i s work tend to support the view of Wells (1964a and 1964b) that since these stacks are not always associated with the nucleus or with v e s i c l e production, they cannot j u s t i f i -3 5 ably be referr e d to as Golgi. Such aggregations o f ER are not r e s t r i c t e d to fungi, and t h e i r possible 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 (1971) review the l i t e r a t u r e on t h i s stacking phenomenon i n plant and animal c e l l s , with p a r t i c u l a r emphasis on the evidence l i n k i n g i t with either an active or a passive state. In most fungi the development of stacks of smooth membranes appears to be associated with highly active states such as ascosporeawall formation ( C a r r o l l , 1 9 6 9 ) , and nuclear d i v i s i o n i n mature b a s l d i a (Manocha, 1 9 6 5 ; Wells, 1961ib). In Ustilago hordei they form just p r i o r to the f i r s t u l t r a s t r u c t u r a l signs that germination has begun. Vacuoles are not present i n most r e s t i n g or dormant spores (with the exception of the powdery mildews) but are formed during the processes of a c t i v a t i o n and germination (Hyde and Walkinshaw, 1 9 6 6 ; Hawker et a l . , 1 9 7 0 ; Hawker and Abbott, 1 9 6 3 ; Niederpruem and Wessels, 1 9 6 9 ; Voelz and Niederpruep, 1961+). Such vacuolation appears to be a necessary adjunct o f hydration and germination - so much so that i f the vacuoles are damaged, or prevented from expansion, the spore ceases to germinate ( M i t c h e l l and McKeen, 1 9 7 0 ) . In spite o f the obvious importance of the development of these organ-e l l e s i n the spore no previous attempt has been made to determine t h e i r origin J Several theories e x i s t to account f o r the o r i g i n s o f plant vacuoles. Vacuoles may arise v i a d i l a t i o n of the intramem-branous cisternae of either the endoplasmic reticulum (Buvat, 1 9 6 1 ) or the Golgi apparatus (Marinos, 1 9 6 3 ) . Prom her studies 36 on the merlstems of the higher plant Anthoceros. Manton (1962) proposed that vacuoles always aris e from pre-existing vacuoles. B e l l and Muhlethaler (I96I4-) concluded that they could a r i s e from the degeneration of mitochondria and p l a s t i d s . Matile and Spichiger (1968) have more rec e n t l y proposed that vacuoles are formed by the development and coalescence of lyaosome-llke bodies. The l a s t four suggestions can be eliminated i n the con-s i d e r a t i o n o f vaouole development i n the ungerminated spore of Ustilago hordei because of the following f a c t s : 1 . No organelles which can be i d e n t i f i e d as Golgi are present i n U. hordei. 2. Resting spores of th i s fungus possess no vacuoles. 3 . Evidence f o r mitochondrial degeneration has been ob-served i n agirghyphae but never i n hydrating spores. To the contrary, the mitochondria during pregerminatlon development are seen to increase i n siz e and number ( i . e. only i n the f i n a l pregerminatlon stages), ij.. Those membrane-bound dense bodies which, during and after germination, appear to act as lysosomal equivalents (Ft. I I ) , do not coalesce or change i n number or siz e throughout pregerminal development. At about the time when vacuoles begin to appear i n some spores, "Whorls" of endoplasmic reticulum occur i n other spores. Such whorls have not been seen i n spores which could be shown to possess a vacuole. Often, a whorl has one or more i n f l a t e d cisternae (Fig. 9 , arrow), the contents of which are 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 the f l o c c u l e n t 37 contents detectable In developing vacuoles. The evidence, therefore, seems to be most consistent with the hypothesis of Buvat. I t i s suggested that i n Ustilago hordei the "primary vacuoles. 1 1 which are responsible f o r the i n i t i a l swelling of the 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 the cisternae i n whorls of ER. Figures 9 , 1 0 , and 1 1 are representative of the postulated developmental sequence. As germination approaches, t h i s process seems to become more generalized, so that, as seen i n Figure 1 7 , small fragments o f ER throughout the spore appear to give r i s e to s i m i l a r d i l a t i o n s . The hypothesis i s further supported by the f a c t that the tonoplast membrane (Average width = 96 A°) i s the same width as the u n i t mem-branes of the ER (Average width = 9ij. A ° ) . Robinson, Park and McClure (1969) have presented evidence that vacuoles, Induced i n the hyphae of Aspergillus niger by the vacuolation f a c t o r , also form by expansion o f the ER lumen. What i s now required i s the ap p l i c a t i o n of the appropriate histochemical tests 1 . e. for acid phosphatase) to determine f i r s t whether vacuoles show lysosomal a c t i v i t y and second whether they are i n f a c t de-ri v e d from the ER. Similar hypotheses have recently been suggested f o r the o r i g i n and development of vacuoles i n the meristematic regions of the shoot and adventitious roots of Glechoma hedeaoa L. (Bowes, 1 9 6 5 ) . and i n the root meristems of Lupinus albua (Mesquita, 1 9 6 9 ) . Mesquita was able to show that the rough ER, which gives r i s e to the vacuoles, i s continuous with the nuclear envelope, and that there i s d i r e c t communication be-tween the intravacuolar space and the perinuclear c i s t e r n a . Such a d i r e c t connection has not been observed i n Ustilago  hordei but i t has been mentioned that the whorls of ER are i usually i n close proximity with the nuclear envelope, and that one end of the f i r s t primary vacuoles i s usually also very near the nucleus. A detailed discussion of the meiotic nucleus of U s t i - lago hordei i s presented i n part IV, but a few points, which perhaps have s p e c i a l relevance to the germination process, deserve mention here. Normally, the teliospores of smut fungi are considered to contain a single d i p l o i d nucleus. To the best of the author's knowledge th i s i s the f i r s t study to suggest that t h i s i s not always the case. The f a c t that the mature spore may sometimes have two haploid n u c l e i (Pigs. |2, 3, h* and 6) suggests that the synchronization of karyogamy with spore formation i s loose. It i s unkown whether such spores, i n which karyogamy has evidently been delayed, are capable of undergoing germination and meiosis. A second unusual feature i s the apparent p l a s t i c i t y of nu-clear shape which develops very early i n hydration. Corfman (1966) reported a s i m i l a r occurrence shortly a f t e r the be-ginning of hydration i n the spore of a myxomycete, Fuligo  septica (L.) Weber, and suggested that i t s function was to maximize the p o t e n t i a l f o r nucleocytoplasmic i n t e r a c t i o n . In Ustilago hordei the lobed state seems to be associated with the movement of the nucleus from a c e n t r a l to a p e r i -pheral p o s i t i o n i n the spre. This suggests that i t may be 39 a manifestation of true nuclear amoeboid motion. Whether the apparent association of l i p i d bodies with the lobate nucleus i s f o r t u i t o u s , or functional i s unknown. L i p i d bodies are also associated with the n u c l e i of some phycomycetes during gamete formation (Blondel and Turian, I960), The observations reported here on the development of the mitochondrial population during germination seem to be at var-iance with the commonly accepted views. In a l l cases so f a r reported the quiescent mitochondria are much larger than those seen a f t e r germination (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-ted that the decrease i n mitochondrial s i z e i s often accom-panied by an increase l n the t o t a l number, suggesting that the large mitochondria divide to form smaller ones. During teliospore germination i n Ustilago hordei the exact opposite seems to occur. The mitochondria, which are o r i g i n a l l y quite small, double t h e i r average maximum length during hydration. The population does not increase noticeably i n number and f i -gures which have been interpreted as d i v i s i o n f igures (Fig, 1 7 , arrows) occur only p r i o r to germination. During the l a s t stage discussed here, and i n subsequent stages, very few such figures occur at any one time, and the average s i z e of the mitochondria l n the population, i n general, continues to i n -crease u n t i l w e l l into metabasidia extension. I t would be i n -t e r e s t i n g to know whether th i s unusual behaviour r e f l e c t s some difference i n the developing r e s p i r a t o r y pattern of UstJilago hordei t e l i o s p o r e s . On the other hand i t may simpler? i i O r e s u l t from the f a c t that the protoplast of the teliospore does not a c t u a l l y increase greatly i n mass during the f o r -mation of the promycelium. The r e s t i n g mitochondria of t h i s fungus also seem to be unique i n the f a c t that t h e i r c r i s t a e are well-developed and r e t a i n the p a r a l l e l grouping previously reported f o r the hyphae (Stein, 1-970). Li p i d i s probably the most common storage material i n fungus spores, p a r t i c u l a r l y i n the spores of rusts and smuts which germinate at the expense of t h e i r pwn reserves (Allen, 1965). A decrease i n the number of l i p i d or o i l bodies during germination i s almost ubiquitous among fungi (Hawker et a l . , 1970 Manocha and Shaw, 1967; Walkinshaw et a l . , 1967; Williams and Ledingham, I96I1; McKeen, 1970). In t h i s respect Ustilago hordei also appears to be unique, since no decrease i n the number or size of the l i p i d bodies i s detectable. However, the occasional association of l i p i d bodies with the mem-brane- bound, lysosome-like organelles (Pig. 8) may be i n d i -cative that some u t i l i z a t i o n does occur. It seems l i k e l y that, because the spores were; germinated i n a comparatively r i c h medium, they may be less dependent on endogenous reserves. Flocculent, or foamy, cytoplasm occurs i n the spores of several fungi, and various funtions have been attributed to i t . Campbell (1969a) suggests that i t represents dextran accumulations; Hyde and Walkinshaw (1966) and Voelz and Niederpruem (I961i) suggest that they are regions i n which glycogen, or some other storage compound, has been leached out. Sussman et a l . (1969) postulate that such regions serve kl the function of vacuoles. The observation that i n Ustilago  hordei such regions are often associated with clusters of large granules (Pig. 16) supports the idea that " f l o c c u -l e n t " cytoplasm r e s u l t s from the leaching out of some com-ponent. No histochemical i d e n t i f i c a t i o n was attempted; the size and appearance of the granules are compatible with suggestions that they could be glycogen, dextran, or perhaps glucan. Wynn and Gajdusek (1968) have shown that during the germination of uredospores of the bean rust, Uromyces phaseolj, a portion of the endogenous reserves are transferred to a temporary res e r v o i r of polysaccharide (probably glucan), which i s u t i l i z e d i n the rapid elongation of germ tubes. With the electron microscope, t h i s storage form i s said to be repre-sented by p a r t i c l e s which are s i m i l a r i n apperance to glycogen. CONCLUSION During the period of pregerm|nation hydration, the t e l i o -spore of Ustilago hordei undergoes a number of u l t r a s t r u c t u r a l changes commonly associated with germination; the amount of endoplasmic reticulum increases, temporary storage material accumulates, and vacuoles form. However, the fungus appears to be unusual i n the f a c t that the mitochondria do not decrease i n size or increase n o t a b l y i n number, and the f a c t that the number and size of l i p i d bodies does not decrease. The f e a s i -b i l i t y of sequential pregermination studies i s c l e a r . The importance of s i m i l a r studies i n other types of fungus spores i s made manifest by the f a c t that many, i f not most, of the 42 metabolic changes which r e s u l t i n germination occur within the spore long before the event i t s e l f . Any attempt to corre-l a t e such metabolic changes with post-germinal c y t o l o g i c a l ob-servations i s of l i m i t e d value, and may be misleading. In the author's opinion the previous "mystery" concerning the o r i g i n of the endoplasmic reticulum and the "primary hydration va-cuoles" i s indefensible. I t has, t e n t a t i v e l y , been suggested that the ER i s derived i n some manner from the nuclear en-velope, and that the primary vacuoles form by d i l a t i o n of ER cisternae. These suggestions are c e r t a i n l y stimulating, as well as being of c r i t i c a l importance to any understanding o f fungal spore germination, and are worthy of f u r t h e r study. I. PLATE 1 General view of a quiescent teliospore ( i . e . 0 hr. of germination), showing the three wall layers (Wl, W2, and W3), the plasma membrane (PM), and the structured d i s t r i b u t i o n of l i p i d bodies (L), spherosieme-like bodies (S), mitochondria (M), endoplasmic reticulum (ER) and the d i p l o i d fusion nucleus (dN). Method A. ca. X 24,400. Part of figur e 1 a showing the re 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 the f i n e filaments passing from the plasma membrane to the spore w a l l . Method A. ca. X 24,000. Figures 2-4 Light microscope pictures of thick sections pre-pared by method A and stained with Toluidine blue (Appendix C). The pictures depict the steps i n the fusion of haploid n u c l e i i n the germinat-ing tel i o s p o r e . ca. X 5,040. Figure l a . Figure l b . I. PLATE 2 Figure £a. Figure 5>b, Figure 6 . Gerneral view of a quiescent teliospore ( i . e . 0 hr, of germination). Indicated are mitochondria (M), electron transparent l i p i d bodies (L), and sphero-some-like bodies (S). Note the beaked appearance of the d i p l o i d nucleus (dN), the nucleolus (Nu) and the nuclear envelope (NE). Method B. ca. X 21,000. Part of figure 5a showing ridges of inner spore wall protruding into protoplast (arrows), ca. X li2,000. Part of a quiescent teliospore ( i . e . 0 hr. of ger-mination) showing the presence of two haploid nu-c l e i (hN). Mitochondria (M) and ER are also pre-sent. Note the size of the mitochondria. Method B. ca. X 29,500. I. PLATE 3 Figure 7. Part of a stage-one teliospore (approximately h nr. of hydration) showing a d i p l o i d nucleus (dN) i n the extremely lobed state. Note the intimate r e l a t i o n -ship between the l i p i d bodies L and the nucleus during t h i s stage. The arrow indicates a possible NE - EH connection. A large patch of f l o c c u l e n t cytoplasm (f) i s v i s i b l e . Method A. ca. X 38,500. Figure 8. A general view of a stage-one teliospore. The nu-cleolus (Nu) i s very conspicuous at one side of the gently lobed d i p l o i d nucleus. The three wall layers ¥ 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 wall layer. Two patches of floc c u l e n t cytoplasm (f) are present. The arrow i n -dicates the point of fusion between a large l i p i d body (L) and one of two spherosome-like body (S). Method C. ca. X l8,lj.00. I. PLATE k Figure 9. Figure 10. Figure 11, Figure 12, Part of a stage two teliospore i l l u s t r a t i n g a whorl of endoplasmic reticulum (ER) with a swollen c i s t e r -na containing flo c c u l e n t material (arrow). The whorl l i e s i n close proximity to the d i p l o i d nucle-us (dN). Note that a f t e r KMnOh f i x a t i o n the nucle-ar pores (NP) appear simple. Method A. ca. X 39000, Part of a stage two teliospore i l l u s t r a t i n g a more advanced stage i n the formation of the primary hy-dration vacuoles. A system of small vacuoles (V), interconnected by short ER segments, l i e s near the d i p l o i d nucleus (dN). A number of free spherosome-l i k e bodies (S) are present. Method A. ca. X 30,000. A well formed vacuole (V) l i e s with one end adja-cent to the d i p l o i d nucleus (dN). Note the vacu-olar - ER connection (arrow). Method A. ca. X 3lf ,500. During stage one and two complex system of ER de-velop i n association with the d i p l o i d nucleus (dN). The arrow indicates a prominent nuclear envelope-ER connection. Method A. ca. X 26,000. I. PLATE 5 Figure 13. Part of a stage two teliospore i l l u s t r a t i n g several layers of ER surrounding the d i p l o i d nucleus (dN). Two nuclear pores (NP) and a nuclear envelope-ER connection (arrow) are indicated. Method A. ca. X 30,000. Figure l i t . Spherosome-like bodies (s) often l i e along e l e -ments of ER which lead to the primary hydration vacuoles (V). Method A. ca. X 28,£00. Figure l£. General view of a teliospore just p r i o r to germin-ation showing the spherical to ovoid mitochondria (M) with 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 longtudinal axis of these organ-e l l e s . Note that the nuclear pores (NP) appear simple a f t e r t h i s method of preparation. Well de-veloped ER i s v i s i b l e . Method B. ca. X 29,500. I. PLATE 6 Figure 16. General view of a teliospore just p r i o r to ger-mination. Note the presence of the large mito-chondria (M), l i p i d bodies (L), many small vacu-oles (V). Patches of electron dense granules are associated with a region o f flocculent cyto-plasm ( f ) . The p a r i e t a l endoplasmic reticulum (ER) tends to stack (arrows). Method A. ca. X 30,000. Figure 17. General view of a teliospore just p r i o r to ger-mination. Note the e c c e n t r i c a l l y positioned d i p l o i d nucleus (dN) with a well-defined nu-clear envelope (NE) and nuclear pores (NP). Long segments of ER are associated with the nucleus. Throughout the spore short ER-ele-ments appear to be giving r i s e to small vacu-oles by d i l a t i o n . Numerous l i p i d bodies (L) are s t i l l present. The arrows indicate a d i -viding mitochondrion and a young mitochondrial element. Note the elementary state of the c r i -stae i n d i v i d i n g and young mitochondria. Method A. Ga. X 18,000. 43 BIBLIOGRAPHY Al l e n , J.V., Hess, W.M., and Weber, D.J. 1971. 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Electron microscope study of the o r i g i n and development of the vacuoles i n r o o t - t i p c e l l s of '{>"i?. Lupinus albus L. J . U l t r a s t r u c t . Res. 2 6 : 2 4 2 - 2 5 0 . M i t c h e l l , N.L. and McKeen, W.E. 1 9 7 0 . Light and electron microscope studies on the conidium and germ tube of Sphaerotheca macularis. Can. J . M i c r o b i o l . Ibj 2 7 3 - 2 8 0 . Namboodiri, A.N. 1 9 6 6 . Electron microscopic studies on the conidia and hyphae of Neurospora crassa. Caryologia 19: - 1 1 7 - 1 3 3 . ~ Niederpruem, D.J. and Wessels, J.G.H. 1 9 6 9 . C y t o d i f f e r e n t i a -t i o n and morphogenesis i n Schizophyllum commune. Bac*-t e r i o l . Rev. 3 3 : 5 0 5 - 5 3 5 . 1+6 Peat, A* and Banbury, G.H. 1967. Ultr a s t r u c t u r e , protoplasmic streaming, growth and tropisms of Pbycomyces sporangio-sphDres. I General introduction. iL.Tne u l t r a s t r u c t u r e of the growing zone. New Pbytol. 66: 475-4*8ij,• Reeves, P. 1 9 6 7 . The fi n e structure of ascospore formation i n Pyronema domesticum. Mycologia 5 9 : 1 0 1 8 - 1 0 3 3 . Reynolds, E.S. 1 9 6 3 . The use of lead c i t r a t e at high pH aa an .electron opaque s t a i n i n electron micro a copy. J . C e l l B i o l . 1 7 : 2 0 8 - 2 1 2 . Robinson, P.M., Park, D. and McClure, W.K. 1 9 6 9 . Observations on induced vacuoles i n fungi. Trans. B r i t . Mycol. Soc. 52: 1*1*7-450. Sassen, M.M.A., Remsen, C C . and Hess, W.M. 1 9 6 7 . Pine s t r u c -ture of Penecllllum conidiospores. Protoplasms 61+: 7 5 - 8 8 . Spurr, 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 electron microscopy. J . U l t r a s t r u c t . Res. 26: 31-43. Stein, G.W. 1970. An electron microscope study of a mycelial mutant of Ustilago hordei (Pera.) Lagerh. M.Sc. Thesis. U n i v e r s i t y or i s r i T i s n Columbia, B r i t i s h Columbia, Canada. Susaman, A.a., Lowry, R.J., Diirkee, T.L. and Maheahware R. 19o9. U l t r a s t r u c t u r a l studies of cold-dormant and ger-minating uredospores of Puccinia gramlnla var. t r i t i c i . Can. J . Bot. | 7 : 2 0 7 3 - 2 0 7 7 " ~ ~ ~ Thomas, P.L. 1 9 6 5 . Virulaenee l n Ustilago hordel (Pera.) Lagerh. M.Sc. Thesia, University or Alberta, Alberta Canada. Voela, H. and Niederpruem, D.J. 1 9 6 4 . Pine structure of basidiospores of Schizophyllum commune. J . B a c t e r i o l . 8 8 : 1 5 4 9 - 1 5 0 2 . ~ Walkinshaw, CH., Hyde, J.M., and Zahdt, J. Van 1967* Pine structure of quiescent and geminating aeciospores of Cronartium fuslforme. J . B a c t e r i o l . 9 ^ : 245-254* wella, K. 1 9 6 4 a . The basidia of Ex i d l a nucleate. I U l t r a -structure. Mycologia 5 6 : 3 2 7 - 3 4 1 . Wells, K. 1 9 6 4 b . The basidia of Exid i a nucleata. II Develop-ment. Am. J. Bot. 5 1 : 36O-37TT; Wells, K. 1 9 6 5 . 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PART II I n i t i a t i o n of the Promycelium and Promycelial Extension TABLE OP CONTENTS Page ABSTRACT k-9 INTRODUCTION 4 9 MATERIALS AND METHODS 5 1 OBSERVATIONS 5 1 Stage Pour: I n i t i a t i o n of the Promycelium ....... 5 1 Stage F i v e : Promycelial Extension 57 DISCUSSION 65 I n i t i a t i o n of 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 of the Promycelium and Promycelial Extension ABSTRACT I n i t i a t i o n of the promycelium of Ustilago hordei involves the l o c a l i z e d degradation of the inner spore w a l l , the l o c a l i z e d deposition of a new wall l a y e r , and the swelling of the proto-p l a s t * During t h i s period of development the endoplasmic r e t i -culum and spherosome-like organelles (Pt.I) undergo changes i n d i s t r i b u t i o n * The sig n i f i c a n c e of these changes i s discussed* Host of the spore contents flow i n t o the promycelium as i t extends* The s t r u c t u r a l basis f o r the extension of the promy-c e l i a l w a l l i s unknown* Mitochondria, l i p i d bodies, and endo-plasmic reticulum are randomly di s t r i b u t e d and do not increase s u b s t a n t i a l l y i n quantity* Apart from the nucleus the only organelles which apparently change i n number and d i s t r i b u t i o n are those bodies that appear to be the u l t r a s t r u c t u r a l equiva-l e n t of the l i g h t microscopists' spheresomes and vacuoles* In discussing the si g n i f i c a n c e of these changes i t i s suggested that the spheresomal-vacuolar system i n t h i s fungus i s the fun c t i o n a l equivalent of the animal lysosomal system* INTRODUCTION On germination the teliospores (probasidlan) of Ustilago  hordei give r i s e to tube-like c e l l s of lim i t e d growth known as 5 0 pro my e e l i a or metabasidia. These structures 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 other nonsexual spores* In Ustilago hordei the promycelium i s approximately 2.3 u wide (Range: l.lj.0-2.8 u ) . Once the spore wall has ruptured the promycelium extends at a maximum rate of approximately 0 . 25 u per minute, slowing down as i t attains i t s maximum length (Range: 20 -30 u). When growth has stopped, the c e l l divides twice i n rapid succession giving r i s e to a f o u r - c e l l e d promy-celium containing the four meiotic products. There i s very l i t t l e terminal growth once d i v i s i o n has occurred. This study i s concerned with those c y t o l o g i c a l events which occur during spore germination - that i s , during promy-celium development. In s p i t e of the f a c t that spores (or sporangia i n some Oomycetes) are notoriously d i f f i c u l t to pre-pare for electron microscopy p r i o r to germination a number of attempts have been made to describe the early stages of germ tube i n i t i a t i o n which occur within the i n t a c t spore (sporangial) wall (Eisner et a l . , 1970; Hawker and Abbott, 1963b; Hawker, 1966; Hawker et a l . , 1970; Lowery and Sussman, 1961|.; Marchant, 1966; M i t c h e l l and McKeen, 1970; Walkinshaw et al , ,1967) . Although the l i t e r a t u r e r e l a t i n g to germ tube ultr a s t r u c t u r e i s quite extensive, to the best of the 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 of metabasidia. For the most part germ tubes seem to possess few features that 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. In most respects the young extending promycelium of Ustilago hordei also resem-bles the a p i c a l hyphal region o f the mycelial mutant described by Stein (1970). 5 1 MATERIALS AND METHODS The cultures and the methods of c u l t u r i n g and sampling were i d e n t i c a l to those described previously (Ft. I ) , except that the samples were co l l e c t e d a f t e r 5 hours, and a f t e r 7 to 9?g hours of hydration. During the l a t t e r period, approximately 30 $ to 95$ of the spores have begun metabasidial extension; of these, 1 5 $ to 5 0 $ have formed the f i r s t septum; and 5 $ to lj.0$ have formed the second septa, Por example, a f t e r 8 hours of hydration the per centage germinauidn«(i, e, the number of fpores with promycelia v i s i b l e i n the l i g h t microscope) i s 9 0 $ , the per centage of spores that have formed at l e a s t one septum i s 3 5 $ and the per centage of spores that have formed three septa i s 2 5 $ (Bech-Hansen, 1 9 6 8 ) , The material was prepared f o r electron microscopy accord-ing to methods A, B and C (Ft. I, Table I ) , with the exception that none of the material was pre-stained i n aqueous uranyl acetate p r i o r to embedding. Figure lib was photographed using a Zeiss Fhotomicroscope, from t h i c k sections prepared accord-ing to method A and stained with 1 $ Toluidine blue i n 1 $ borax. Figure ll|.b depicts a l i v i n g c e l l photographed i n a t h i n layer of complete l i q u i d medium between a cov e r s l i p and a glass s l i d e . OBSERVATIONS STAGE FOUR: INITIATION OF THE PROMYCELIUM The f i r s t c y t o l o g i c a l i n d i c a t i o n that germination has begun i s a change l n the shape of the protoplast. The previous-l y symmetrical, c i r c u l a r or ovoid cross-section acquires a 5 2 "beaked" appearance (Pigs. 1, 3 k&» or l|b). Closer observation reveals that a very s l i g h t change i n the shape of the actual protoplast i s accentuated by the presence o f a "cap" of material which covers the beaked portion o f the cytoplasm and protrudes into the spore wall (Figs. 1, 3 and i|.a). The Wall. - As described i n part I, the spore wall (W) of Ustilago hordei consists o f three major layers (Wl, W2, and W3). When the beak of the protoplast f i r s t becomes noticeable, and before the appearance of the "cap", perforations (p) develop l n the innermost wall layer at a point opposite the beak (Fig. 2). A f t e r preparatory methods A and B, these spaces are usually empty, or occasionally they contain f a i n t l y granular material (Figs. 1 and lj.a). However, afte r method C, they con-t a i n electron dense f i b r i l s ( F i g . 2). Shortly a f t e r the f i r s t "perforations" appear i n the inner w a l l , the new material (pW) begins to accumulate around the protoplast beak (Fig. 2). The new material i s more electron translucent than i s the inner w a l l l a y e r . When i t i s f i r s t formed i t i s quite homogeneous but, as the layer thickens and extends l a t e r a l l y , the region most proximal to the spore wall becomes increasingly f i b r i l l a r . However, at t h i s period of development, the or i e n t a t i o n of 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 inner spore w a l l ( F i g . 6). Consequently, although the old and new layers are cl 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 also c l o s e l y appressed to the protoplast of the beak, but the boundary here i s often i n d i s t i n c t (Figs. 1 and 3). 53 Once t h i s pattern of development has been i n i t i a t e d , the processes involved proceed r a p i d l y and simultaneously. As the region of perforation increases and extends around the sides of the beak, the protoplast seems to push forward di s p l a c i n g the perforated inner spore w a l l layer and expanding i n length and i n width ( F i g . 1+a). The new w a l l layer thickens and ex-tends l a t e r a l l y at a rate s u f f i c i e n t to ensure that the i n -creasing surface area of the beak i s encased by i t . As the thickness of the f i b r i l l a r spore w a l l l a y e r i s diminished by the advancing protoplast, the outer e l e c t r o n dense wall layers begin to show signs of disorganization (Fig. i+a). F i n a l l y , the spore w a l l ruptures. The spore i l l u s t r a t e d l n Figure 6 was caught at t h i s exact moment. The beak of the protoplast, which can now be considered as a true promycelium, has barely extended beyond the spore wa l l , and i s e n t i r e l y encased by a well-defined portion of the newly synthesized w a l l l a y e r . Between the young promycelium and the o l d spore wall the outer part of the newly synthesized wall material has formed a short c o l l a r . Remaining c e l l debris has sloughed o f f into the medium accompanied by spore wall debris. Plasma Membrane (PM) and Paramural Bodies. - During de-velopment of the protoplasmic beak, the plasma membrane often appears crenulated (Figs. 1 and 3). In Figures 1 and 3. r e l a t i v e l y large areas of the plasmalemma are separated from the spore w a l l . This separation l a t e r extends to the e n t i r e surface of the protoplast except for the beak region. In t h i s l o c a l i z e d area the protoplast and the new w a l l layer are 54 very c l o s e l y apposed; so much so that the plasma membrane boundary between the two i s i n d i s t i n c t i n the region o f most intimate contact (Figs. 1, 2, 3, and 4-a). Endoplasmic Reticulum (ER). - Throughout the pre-germination period, there Is a consistent increase i n the amount, and length of endoplasmic reticulum i n the I n t e r i o r o f the spore. ER elements toward the periphery are short and scattered (Ft. I, F i g . 17). Just p r i o r to, and during, the i n i t i a t i o n o f the promycelium, there i s a noticeable increase i n the number and length of these peripheral elements, p a r t i c u l a r l y i n the region proximal to the developing beak (Figs. 1 and k&). In Figure I, several ER cisternae have formed a loose association just beneath the i n c i p i e n t metabasidium. As has been previously noted i n part I, i t i s very d i f f i c u l t to obtain information on these membrane systems a f t e r glutaraldehyde-osmium f i x a t i o n because of the density of the cytoplasm i n ungerminated spores (Figs. 2, 4a, and 6). Just before the rupture of the spore wall, the peripheral ER elements undergo a further change i n d i s t r i b u t i o n . A f t e r potassium permanganate f i x a t i o n a number of spores are observed i n which the shorter membrane fragments have "disappeared" and most of the organelles seem to be encased, c o l l e c t i v e l y , i n a continuous "sac" composed of a single continuous ER c i s t e r n a (Fig. 3)» The sac may l i e d i r e c t l y beneath and p a r a l l e l to the plasma membrane, or i t may be "pulled away" on one o r more sides leaving a f a i r l y large portion o f the cytoplasm between the sac and the plasmalemma. In t h i s s i t u a t i o n the nucleus 55 and a l l the mitochondria, as well as most of the l i p i d , vac-uoles, and remaining ER, l i e within i t . A few l i p i d bodies, small vacuoles, spherosome-like organelles and odd fragments of ER are excluded. Figure 8 i l l u s t r a t e s a portion of a de-veloping sac i n which the Individual ER elements composing i t have not yet completely fused (arrows). The cytoplasm outside the sac i s s l i g h t l y l e s s dense than that within ( F i g . 8). An add i t i o n a l feature of such formations i s that at various points the membranes are elaborated into more or less complex knots (Fig. 3. arrow). Spherosome-like Organelles (S). - Frey-Wyssling et a l . (1963) f i r s t described spherosomes u l t r a s t r u c t u r a l l y i n the higher plant Zea mays. After permanganate f i x a t i o n these round to angular bodies are characterized by the presence of a uniform semi-dense, granular matrix bounded by a unit membrance, and secondly, by a constant, though poorly defined, association with the endoplasmic reticulum. Bodies, ranging i n diameter from 0,1 to 0,6 u and s a t i s f y i n g these c r i t e r i a , have pre-viously been noted i n KMnOj^-fixed teliospores of Ustilago  hordei (Et, I ) , and have accordingly been designated "sphero-some-like," As might be anticipated from the association of the spherosome-like organelles with the ERjgtheseSbodies#tend to l o c a l i z e following the r e d i s t r i b u t i o n of membrane systems that accompanies beak formation. Clusters of these spherosome-like bodies are present i n the region of beak development early i n t h i s stage of d i f f e r e n t i a t i o n (Figs, 1 and 2). After the f o r -£ 6 mation of the ER-sac, they come to l i e either inside or out-side the sac-membrane (Pig. 3 ) . They tend to accumulate p e r i -pherally In the v i c i n i t y of the two triangular areas of cyto-plasm formed when the sac membrane p u l l s away from the plasma-lemma (Figs. 3 and 8 ) . The smallest of the spherosome-like bodies are roughly c i r c u l a r i n cross-section and the membranes tend to be c l e a r l y defined although unit membrane structure i s d i f f i c u l t to demonstrate. The long axis of each of these or-ganelles aligns i n p a r a l l e l with an ER element, with one of the longer sides c l o s e l y following the contours of the ER membrane. Often the boundary between the ER and a spherosome-like organ-e l l e i s vague - the membranes i n the region of association being i n d i s t i n c t . Probably t h i s e f f e c t i s an artefact caused by the angle of sectioning. Where the boundaries between them are well-defined, the membranes are separated from each other by a r e l a t i v e l y constant distance of 5 5 to 80 A 0 ( F i g . 8 ) . The most prominent organelles present i n glutaraldehyde-osmium f i x e d tissue are those membrane-bound bodies with very electron dense contents. They are p a r t i c u l a r l y conspicuous i n tissue prepared according to method C. In the ungerminated spore, the electron dense material may be either scattered throughout an electron transparent matrix i n the form of dense entwined strands ( F i g . 2 ; Pt. I, F i g . 5 ) , or i t may be "con-densed" into a s o l i d core leaving the r e s t of the matrix absolutely clear (Pt. I, F i g . 8 ) . These are the only organelles i n glutaraldehyde-osmium f i x e d t i s s u e which have not been assigned a function. They are also the only organelles whose 57 size range, and pattern of d i s t r i b u t i o n can be said to corres-pond to those of the spherosome-like organelles described i n KMnO^-fixed material. Within the spore these bodies range l n diameter from O.2I4. to 0 . 8 6 u. Like the spherosome-like bodies, t h e i r numbers appear to remain constant during the early stages of hydration 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 i n f o r -mation i s available on the d i s t r i b u t i o n of the endoplasmic r e t i -culum i n glutaradlyhyde-osmium f i x e d spores to v e r i f y t h e i r asso-c i a t i o n with the ER-sac, but they do c l u s t e r i n the region of the developing beak (F i g . 2 ) . On t h i s basis, i t i s t e n t a t i v e l y suggested that these organelles are i d e n t i c a l with the spherosome-like organelles found i n permanganate f i x e d material. STAGE FIVE: GERM TUBE EXTENSION Promyoelial Wall (pW). - Oddly enough when the promycelium f i r s t bursts through the spore w a l l , the new promyoelial wall which surrounds the t i p i s r e l a t i v e l y thick (approximately 30 mu), and quite compact and f i b r i l l a r (Fig. 6 ) . During subse-quent stages of elongation the promyoelial t i p i s almost naked (Fig. 10 and 2 0 b ) . A true w a l l can only be distinguished at a distance of about 2 u behind the t i p . At t h i s point, i t i s approximately 10 mu t h i c k but proceeding b a s i p e t a l l y , i t thickens r a p i d l y over a distance of l e s s than 2 u ( i . e . 1+ u from the t i p ) to a mature width of approximately 6 5 mu (Range: 33 - 7 5 mu). At the apex, the promyoelial wall i s poorly defined (Fig. 20b) and l i t t l e information has been obtained concerning i t s substructure. However, throughout the r e s t of i t s length, i t i s uniformly semi-dense and appears to be granular or f i b r i l l a r 58 (Figs. 18a, 18b, and 20). A f i n e f i b r i l l a r material extends from i t s surface (Figs. 18 and 20). Although i t i s not well i l l u s t r a t e d i n these pictures, t h i s material i s often extensive, sometimes at t a i n i n g lengths of almost a micron (Pt. I l l , F i g . 7). In Figures l6a-c large pores open from the cytoplasm through the w a l l into the external medium. Such pores occur infrequently and have been observed only a f t e r preparation by method C. When they are present, they occur i n large numbers along the length o f the promycelium. For example, at l e a s t 10 pores were v i s i b l e i n a single section through the germ tube i l l u s t r a t e d by Figures l 6 a - c . The diameter of these hyphal pores i s approximately 47 mu (Range: 3 8 - 5 3 mu) at the i n t e r n a l surface of the wall and narrows to approximately 2 5 mu (Range: 23-31 mu) towards the external surface. They are l i n e d by plasma membrane. Plasma Membrane (PM). - Throughout the period of metaba-s i d i a l extension, the plasma membrane appears to be uniformly smooth and c l o s e l y appressed to the hyphal w a l l . I t Is i n d i s -t i n c t at the apex but, i n o u t l i n e , the t i p i s remarkably smooth (Figs. 10 and 20). No paramural bodies are present. Endoplasmic Reticulum (ER) and Ribosomes. - The decrease i n cytoplasmic density which accompanies spore germination allows more c r i t i c a l observation of the ER a f t e r glutaraldehyde-osmium f i x a t i o n . The average width o f the unit membranes a f t e r a l l three preparatory techniques i s 94.0 A§ (Range: 70-110 A°) but the width of the inter-membrane space i s quite v a r i a b l e , ranging from 60 to 160 A 0 a f t e r permanganate and from 60 to 100 A° a f t e r gluteraldehyde-osmium f i x a t i o n . Single lamellae 59 vary i n t o t a l width from 210 to 375 A . Most of the endoplasmic reticulum flows from the spore with the rest of the organelles, and becomes scattered through-out the length of the germ tube except i n the extreme apex. It does not seem to increase i n quantity and i s very sparsely d i s t r i b u t e d , consisting mainly of single, short, flattened cisternae l y i n g just beneath and p a r a l l e l to the plasma mem-brane (Pigs, 5 » 1 8 a and 19). Occasionally two or three l a -mellae i n the peripheral cytoplasm w i l l form stacks p a r a l l e l to the w a l l , and these stacks are often associated with small, membrane-bound v e s i c l e s (ve) (Pig. 1 5 ) . ER elements which are situated away from the c e l l w a l l are usually associated with the spherosome-like organelles (Pigs. 9 and Ida) or with the nucleus (Pigs. 17 and 1 9 ) . Nuclear envelope-ER connec-tions are r a r e l y seen during t h i s stage. Ribosomes are very d i s t i n c t i n tissue which has been f i x e d i n glutaraldehyde-osmium and embedded i n Spurr's p l a s t i c (Pigs. 7 , 9 . 17* and 2 0 a-c). Their density i s uniform through-out the length of the promycelium but tends to decrease within the spore as the germ tube extends. Figure 7 I l l u s t r a t e s the density gradient across the neck of the spore. Ribosomes appear to be the only material present within the 0 . 3 u d i -r e c t l y behind the c e l l apex. Most of the ribosomes are f r e e ; however, a small amount of rough ER occurs near the nuclear envelope (Pigs. 17 and 19 » arrows). The elements of the en-doplasmic reticulum which are associated with the plasma mem-brane (Fig. 1 5 ) or with spherosome-like organelles (Pig. 9 ) are 60 smooth. Nucleus (N). - Normally the nucleus i s i n the d i p l o i d state when i t migrates (General Introduction). In Figures 1+a and [|.b the spore nucleus l i e s just posterior to the dev-eloping beak, "an t i c i p a t i n g " as i t were, i t s passage into the metabasidium. Usually, although not always, the nucleus i s one of tiie f i r s t organelles 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 leaves the spore i s the narrowest part of the c e l l (width = approximately 1 u) (Figs. 5 and 7). In order to pass through t h i s narrow region the spherical d i p l o i d nucleus (Average diameter = 2.6 u) elon-gates, a l t e r i n g i t s shape apparently spontaneously. The nuc-leus i n Figure 5 i s just emerging from the neck into the promy-celium. Its posterior end i s s t i l l very narrow, while the remainder has broadened out leaving only a narrow cytoplasmic s t r i p on either side. The width of the promycelium (Average width » 2.3 u) i s not s u f f i c i e n t to allow the d i p l o i d nuc-leus to return to i t s spherical shape and i t continues to r e -t a i n an elongate form (approximately 1.5 by 3.5 u) (Figs. 5 and 10). During the passage of the nucleus up the metabasidium, the nucleus (Nu), i s always at the extreme posterior ( i . e . towards the spore) and a d i s t i n c t i v e structure, the "cent-r i o l a r -kine to chore -equivalent (CKE)" i s always at the an-t e r i o r end and to one side (Fig. 5). Further d e t a i l s concern-ing the nucleus and i t s associated structure w i l l be discussed i n part IV. The nucleus does not continue to move with the flow of 61 cytoplasm and organelles along the extending tube. I t comes to r e s t i n such a position that when the c e l l has elongated to i t s t o t a l length the nucleus w i l l he located i n the pos-t e r i o r h a l f . In the c e l l shown i n Figure 10 the nucleus i s at a po s i t i o n approximately mid-way along the extending pro-mycelium. Since the d i p l o i d nucleus almost f i l l s the enti r e diameter of the tube, larger organelles ( i . e . mitochondria and spherosome-like bodies) which have been caught i n the cytoplasmic stream cause a d i s t o r t i o n i n the shape of the nucleus as they pass by i t on t h e i r way to the extending zone between the stationary nucleus and the apex (Fig. 10). When the nucleus has ceased to move and when a p i c a l extension has begun to slow down, or has stopped, the nucleus undergoes two rounds of nuclear d i v i s i o n (meiosis) and c e l l d i v i s i o n . De-t a i l s concerning the mechanisms involved i n these d i v i s i o n s w i l l be discussed i n parts I I I and IV. The consequence o f these a c t i v i t i e s i s that a one-celled d i p l o i d promycelium, such as that depicted i n Figures 10 and 20a, i s converted to a fo u r - c e l l e d promycelium i n which each c e l l contains a single haploid nucleus (hN) (Fig. 18a). Cytoplasmic Microtubules (Mt). - Cytoplasmic micro-tubules are present i n the developing promycelium o f Ustilago  hordei. In Figure 7 a number of them occur both i n cross-section and lo n g i t u d i n a l section. Within the promycelium they are often associated with the nucleus and more w i l l be said about these structures i n part IV. Mitochondria (M). - Like the nucleus, the spherical and ovoid spore mitochondria undergo a spontaneous elongation ass 62 they enter the promycelium (Pigs 5, and 7). They become randomnly d i s t r i b u t e d . Longitudinal sections through the germ tube reveal many c i r c u l a r and oval cross-sections of these organelles. These seem to cut tangentially and i t i s l i k e l y that they represent sections through longer mitochon-d r i a whose i r r e g u l a r shapes wander back and f o r t h across the section. The maximum length measured i n any one section i s approximately 3,8 u. In width they range from 0.2 to 0,5 u. The numerous long, p l a t e - l i k e c r i s t a e seem to l i e roughly p a r a l l e l to the long axis of the mitochondrion and to extend from one side of the organelle to the other, e f f e c t i v e l y d i v i d i n g i t into compartments (Pig, 20), After preparation by method C the matrix i s more electron dense than the cyto-plasm, giving these organelles a d i s t i n c t i v e appearance. After glutaraldehyde-osmium f i x a t i o n the outer membrane av-erages 73 A 0 (Range: 61-98 A 0 ) and the inner membrane averages 90 A° (Range: 73-109 A 0 ) , L i p i d (L). - L i p i d bodies have been described i n part I, A few bodies, with about the same size range as those i n the spore, are scattered i n the germ tube (Pigs, 10, 18a, and 20a). Spherosome-like Bodies (S). - Apart from the n u c l e i the spherosome-like bodies are the only organelles 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 during the development of the promycelium. In Figure 6, several are seen just posterior to the apex of the newly formed metabasidium. In the most anterior o f these a small knot of f i n e l y f i b r i l l a r material l i e s on one side while the rest of the matrix i s f i l l e d by dispersed f i b r i l s . Some o f the spherosrome-like bodies are always among the f i r s t organelles to flow into the metabasidium. Early during the period of extension large numbers of these spherosomeM.ike bodies, both large and small, are d i s -tributed randomly throughout the cytoplasm. In the l i g h t microscope they appear as conspicuous, r e f r a c t i l e bodies of variable size (Pig. llj-b) which, ? i n the l i v i n g organism, move quickly i n a l l d i r e c t i o n s . They have been observed to move through distances at least h a l f as long as the promycelium. In tissues prepared f o r electron microscopy by method C, the larger ones a l l appear to be i n the conspicuous s o l i d core form (Pigs 9, 10, ltj., and 20a), and to be surrounded by a membrane which i s approximately 90 A° wide (Range: 7lj. - l l l A°j. Figure 9 i l l u s t r a t e s a number of spherosome-like organelles l y i n g along a short segment of smooth endoplasmic reticulum. At the upper end of the membrane and to the l e f t (arrow) i s a small body with a tenuous "membrane", a granular semi-dense matrix and a small dark core region. This type of body i s reminiscent of the smaller organelles with an inde-f i n i t e membrane previously noted i n Figure 8 (Method A). Their appearance, r e l a t i o n s h i p with the ER, and association with well-developed spherosome-like bodies suggests that these smailler bodies are the formative stages of the larger. Very large numbers of what have been designated , sphero-some-like bodies are present i n glutaraldehyde-osmium fixed promycelia (Fig. 20a-c). They range i n size from 0.2 to 2.3 u. The numbers and diameters of these organelles are greater i n promycelia that have almost attained t h e i r maximum length (Fig. 20a) than l n those which are s t i l l ex-tending r a p i d l y ( F i g . 10). Progressing b a s i p e t a l l y , the con-tents of the electron-transparent portion of the spherosome-l l k e bodies become increasingly f i b r i l l a r (Fig. 20a). Toward the base of the promycelium shown i n Figure 20 two large spherosome-like organelles appear to be l n the process of fusing (Fig. 20c). The r e s u l t of such fusions i s the forma-t i o n of large bodies with multiple electron dense cores. The l a t t e r structures are common i n budding promycelia and i n emptying spores ( F i g . 12, V). Two other phenomena are appa-r e n t l y associated with aging t i s s u e . F i r s t i s the occur-rence of pseudo-myelin figures within the spherosome-like organelles ( F i g . 11, arrow). Second, when the organism has ceased to extend, what appears to be spherosome-like bodies occasionally begin to bleb v e s i c l e s i n the d i r e c t i o n of the septum (Fig. 18b) or the c e l l wall (Fig. 19). Figure 18a i l l u s t r a t e s a portion of a f o u r - c e l l e d per-manganate f i x e d promycelium. Contrary to what might be ex-pected, the number of bodies with t y p i c a l spherosome-like structure has decreased, and the si z e range remains 0.2 to 0.6 u. In Figure Ida, one of these bodies has fused with a vacuole, and appears to be r e l e a s i n g i t s contents (arrow). Vacuoles (V). - Promycelia that have been f i x e d ingper-manganate contain a d i s t i n c t i v e class of organelles which, because of t h e i r s i m i l a r i t y to the vacuoles described i n part I, have temporarily been designated by the 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 of approximately 65 the same width as the tonoplast (Average width = 96 A 0) and occasionally contain f l o c c u l e n t material (Fig. 13)* The number of these bodies increases with proraycelial age. A very large vacuole with the more i r r e g u l a r shape described i n part I develops within the aging spore as other cytoplas-mic contents move into the promycelium (Fig. 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 evidence f o r the existence of unique corresponding structures. The closest equivalent i s the large vacuole-like bodies with f i b r i l l a r contents situated i n the spore and at the base of the f o u r - c e l l e d promycelium i n Figure Hj.. The extent of the increasing vacuolation which occurs at the base of the pro-mycelium i s r e a d i l y demonstrated i n l i v i n g material (Fig. Hj.). DISCUSSION INITIATION OF THE GERM TUBE Fungal germ tube walls can be i n i t i a t e d either by exten-sion of one of the pre-existing layers of the spore w a l l , or by synthesis of a new w a l l (Hawker, 1966). Teliospores of Ustilago hordei resemble the l a t t e r case (Hawker and Abbott, 1963b; Hawker, 1966; Hawker et a l . , 1970; Marchant, 1966; Walkinshaw et a l . , 1967). Like the wall layer synthesized by germinating sporangia of Phytophthora infestans (Eisner et a l . , 1970), the new material i n t h i s smut forms a cap over the developing beak and tapers abruptly to the sides (Figs. 1# y» K» 5* and 7) « I t never surrounds the spore completely as reported f o r germinating sporangia of Phytophthora para-s l t i c a (Hemmes, 1969) and conidia of Cunninghamella elegans (Hawker et a l . , 1970). Although d i f f i c u l t to prove because of the i m p o s s i b i l i t y of marking the extending wall layer, the general impression i s that growth of the wall i s not a p i -ca p r i o r to spore wall rupture. The f i r s t material, which i s synthesized at the apex (Pig. 2) r a p i d l y becomes f i b r i l l a r and reasonably r i g i d . Apparently i t i s then pushed forward, f o r c i b l y , by the expanding protoplast. Meanwhile, synthesis continues down the sides of the lengthening beak so that i t i s completely encased. This hypothesis would account f o r the f a c t that when the promycelium f i r s t bursts through the spore wall the apex i s encased by a wall layer which i s a l -most 10 mu thick. Such a wall i s not again seen at the t i p u n t i l the germ tube has ceased a p i c a l extension. Much of the spore's capacity f o r s u r v i v a l resides i n the thi c k spore wall which keeps a l i e n factors out, but equally well keeps the protoplast i n . Even with the swelling of the protoplast and the synthesis of a hard "cap" over the beak, the act of penetration must be d i f f i c u l t . Evidently the fun-gus "prepares" i t s path by weakening the innermost spore wall layer over a l o c a l i z e d area before i t . The chitinous region does not show signs of mechanical stress but large spaces f i l led by electron dense f i b r i l l a r material develop (Pig. 2 and lj.a). Such formations suggest that the wal l i s weakened enzy-matically. Figure 2 i l l u s t r a t e s that t h i s process i s already well-advanced before the new wall layer i s l a i d down. The l a t e r a l extension of the region of perforation seems to pre-67 cede extenstion of the new wall l a y e r , and i t seems l i k e l y that the length of the region of perforation determines the u l t i -mate width of the neck region. Many of the previous studies on spore germination have suggested that the spore wall i s ruptured mechanically (Hawker and Abbott, 1963b; Hawker et a l . , 1970; Hashimoto et a l . , 1958). During a study of c o n i d i a l germination i n Fusarium culmorum Marchant (1966) noted that as the spore wall begins to bulge p r i o r to i t s ultimate rupture, l t becomes d i f f u s e . and on t h i s basis he suggested that enzymatic degradation was involved. The r e s u l t s i n Ustilago hordei support t h i s hypothesis. How-ever, i t i s uncertain whether the area of degradation extends int o the outer spore walls. Figures lj.a and 6 are perhaps more suggestive of a mechanical stretching and rupturing of the outer electron dense la y e r s . The observations o f Stocks and Hess (1970) i n germinating Fsilocybe basidiospores also sug-gest that both enzymatic d i s s o l u t i o n and physical pressure may be involved i n rupturing the spore w a l l . What i s the source of the enzymes and material respon-s i b l e f o r degradation of the spore wall and synthesis of the new layer? The most prominent organelles i n the beak region are endoplasmic reticulum which i s known to be involved i n protein synthesis and spherosome-like bodies which, as w i l l be discussed l a t e r , are believed to sequester hy d r o l y t i c en-zymes. To what extent each may be involved i n which function i s unknown. In Figure 2 the f i b r i l l a r contents of the spaces forming i n the innermost wall layer resemble the contents of the spherosome-like bodies and p a r t i c u l a r l y the more f i n e l y f i b r i l l a r contents of the large spherosome-like body i l l u s -trated i n Figure 6. This suggests that the spherosome-like bodies may be the source of the f i b r i l l a r material seen i n the perforations. In t h e i r elegant study of hyphal t i p growth i n Pythium ultimum. Grovej,et a l . , (1970) postulated that ve-s i c l e s , s i m i l a r i n appearance to what has been termed sphero-some-like bodies i n t h i s study, are responsible f o r wall syn-thesis at the hyphal t i p and perhaps also f o r wall p l a s t i c i -zation. The hypothesis i s p a r t i c u l a r l y a t t r a c t i v e because, i n order f o r these vesi c l e s to release t h e i r contents outside the c e l l , they must f i r s t fuse with the plasma membrane and hence serve the addi t i o n a l function of generating the new mem-brane necessary to cover the increasing surface area of the protoplast. In t h i s respect i t must be mentioned that the ER i n the beak region 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 to fuse with the plasma membrane, and the average width of the spherosome-like membranes (90 A 0) i s considerably less 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 to be crenulated or to undergo any noticeable ac-t i v i t y ; so, unless the plasma membrane i t s e l f synthesizes the enzymes, the manner i n which they pass out of the protoplast remains obscure. The si g n i f i c a n c e and frequency of the formation of an ER-sac ( F i g . 3) p r i o r to germination i s unknown. Presum-69 ably i t may be a mechanism of mobilizing organelles i n an ap-propriate position to pass into the i n c i p i e n t promycelium. Moor (1967) has described the s i m i l a r formation of an ER-sac p r i o r to the i n i t i a t i o n of a bud i n vegetative c e l l s of Saocha- romyces cerevisiae. According to Moor's study the sac i s open at one end and v e s i c l e 3 are produced from the opening edge which pass to the c e l l w a l l . At t h i s point the wall i s p l a s t l -c i z e d . During various studies i n fungi, Berliner and Duff (1965) and Lowry and Sussman (1968) have noted knots of endoplasmic reticulum s i m i l a r to those which are associated with the ER-sac i n Ustilago hordei and have suggested that they function as membrane generators (Robertson, 1961). However, from t h e i r appearance i n t h i s fungus they might equally well serve as a mechanism of membrane 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 occur, the a p i c a l wall 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 extension has ceased the f i r s t two microns of the promycelium are surrounded by a t h i n and i n d e f i -n i t e boundary, which gradually merges into the t y p i c a l hyphal-type wall (Average thickness = 65 mu) surrounding the rest of the tube. Obviously the key to either hyphal or promycelial extension l i e s i n the structure and function of the apex. Elongation of the wall of such tube-like c e l l s requires, f i r s t , that the t i p remain p l a s t i c , second, that new wall material be accreted continuously and t h i r d , that new plasma membrane be generated continuously. Lastly, and most importantly, a mechanism must exist by which these three a c t i v i t i e s are con-t r o l l e d and co-ordinated. As mentionned previously, Grove, Bracker, and Morre (1970) have suggested a system of a p i c a l extension wherein vesicles containing the enzymes and materials necessary f o r wall p l a s t i c i z a t i o n and synthesis would fuse with the plasma membrane of the hyphal t i p releasing t h e i r contents to the outside. Such a mechanism would, at once sa-t i s f y a l l three requirements f o r hyphal t i p extension. Sever-a l other studies have indicated an abundance of vesicl e s i n hyphal apices (McClUre et a l . , 1968; Brenner and C a r r o l l , 1968; 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 extension. Unfortunately, i n Ustilago hordei the same problem of determining the mode of germ tube i n i t i a t i o n also exists i n determining the mode of germ tube extension. The promycelial. t i p i s s i n g u l a r l y "uninteresting" c y t o l o g i c a l l y (Pig. 20b)I Its surface i s evidently r e l a t i v e l y smooth. No lomasomes or a p i c a l corpuscles (Bartnicki-Garcia et a l . , 1968) are evident. The cytoplasm at the t i p i s apparently devoid of endoplasmic reticulum, ves i c l e s and of other c e l l organelles with the ex-ception of ribosomes. In short, the organelles which one might expect to f i n d at the apex are not there. Pour p o s s i b i l i t i e s e x i s t : 1. The density of free ribosomes at the apex obscures the relevant structures. 71 2. The preparatory techniques are not s u f f i c i e n t l y r e -fi n e d to demonstrate the relevant structures. 3. No apices have been seen at an appropriate stage; for example, the promycelium i n Figure 20 may have stopped extending although the w a l l has not yet thickened. The lack of cytoplasmic structures at the extreme pro-mycelial apex r e f l e c t s the true l i v i n g state during ex-tension. The problem merits further study. For the most part, with the exception o f the appearance and a c t i v i t i e s of the spherosome-like organelles, an extending promycelium of Ustilago hordei resembles the hyphal a p i c a l r e -gion. The mature promycelial wall i s s i m i l a r i n appearance and i n width to the hyphal w a l l (Stein, 1970). To the best of the author's knowledge t h i s i s the f i r s t report of the presence of pores i n hyphal w a l l s . The f a c t that they are r a r e l y pre-sent, but when present occur i n large numbers, indicates that the pores are connected with a p a r t i c u l a r p h y s i o l o g i c a l state of the c e l l . When grown on a r t i f i c i a l medium Ustilago hordei secretes large quantities of exoenzymes (Bech- Hansen, 1970). Possibly the pores are involved i n secretion of exoenzymes, or of the mucous coat which i s represented c y t o l o g i c a l l y by the fibrous, material that covers the external surface of the promycelium. Similar mucous coats have been reported f o r a number of dimorphic fungi (O'Hern and Henry, 19j?6; Marchant and Smith, 1967). As a consequence of the decrease i n cytoplasmic density during germination (Barer and Joseph 1958) more detailed studies can be made of membranous organelles. As i n the hyphal a p i c a l region, described by Stein )1970), the endo-plasmic reticulum of the promycelium i s sparsely d i s t r i b u t e d and l i e s mainly beneath and p a r a l l e l to the plasma membrane (Pig. 18a). In appearance they are i d e n t i c a l except that a small quantity of rough ER which has not been reported i n the hyphal apices, seems to develop i n the promycelium. Gorfraan (1966) reported a s i m i l a r increase i n the amount of rough ER after germination of spores of Puligo septica. I t i s commonly accepted that the ER, which i s sparse or absent i n r e s t i n g spores, increases greatly during the early stages of germination (Bracker, 1967). In Ustilago hordei the i n -crease i n ER seems to be confined to the pre-germinal stage of hydration. Stein (1970) reported that the outer mitochondrial mem-brane was wider than the inner i n hyphae of Ustilago hordei. This has not been substantiated i n t h i s study. But i n most other respects, including the p a r a l l e l grouping and l o n g i -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 promyoelial mito-chondria are s i m i l a r to the hyphal ones. The tendency f o r the c r i s t a e to extend across the e n t i r e organelle, e f f e c -t i v e l y d i v i d i n g i t into compartments, has also been described fo r Microspor um canis (Werner et a l . , 1966) and f o r the t i p region of young sporangia of Phycomyces (Peat and Banbury} 1967). There i s no evidence that the mitochondria of the promycelium ever undergo the complex changes which have been reported i n d i f f e r e n t i a t e d regions of the hyphae. An i n t e r e s t i n g aspect of the migration of organelles from spore to germ tube i s the spontaneous a l t e r a t i o n of shape which some of them undergo (Figs* 5 and 7)* Por the nu-cleus such a change i s necessitated by the narrow dimen-sions of the neck and germ tube, but most i f not a l l of the mitochondria could apparently proceed i n either form. Thus, an elongate shape must possess other physiological advantages. Once germination has begun i n Ustilago hordei the amount of endoplasmic reticulum does not increase s i g n i -f i c a n t l y , nor does the number and size of l i p i d bodies and mitochondria. With the exception of the extreme a p i c a l region which contains only ribosomes, the above organelles are d i s t r i b u t e d randomly throughout the promycelium thus ensuring that, following septation and bud formation, the s p o r i d i a share equally. The only organelles which s i g n i -f i c a n t l y a l t e r i n number, siz e and appearance are the spherosome-like and vacuolar organelles. In the promycelia the number of "permanganate-type" spherosomal bodies decreases, and t h e i r size range remains the same, while the number and s i z e range of the "gluta-raldehyde -osmium- type" bodies both increase. With the l i g h t microscope vacuoles can be observed forming at the base of 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 perman-ganate f i x e d material are present a f t e r promycelia have been prepared by method A ( F i g . 18a) but no " d i s t i n c t i v e " organ-e l l e s s a t i s f y i n g the necessary c r i t e r i a are observably present a f t e r methods B and C. What i s observed at the base of the promycelium are large spherosome-like bodies apparently i n the process of fusion (Pig. 20c). This suggests that the large spherosome-like organelles and fusion products observed i n glutaraldehyde-osmium fixed tissue are i d e n t i c a l to the "va-cuoles" of permanganate fixed t i s s u e . I f t h i s i s true one might expect to f i n d t r a n s i t i o n a l bodies i n KMnO^ fixe d ma-t e r i a l which might i l l u s t r a t e the manner i n which the sphero-some-like bodies give r i s e to vacuole-like bodies. At the top right of the promycelium depicted i n Figure 18a the three or-ganelles marked 1. i i , and i i i suggest such a sequence. The arrow i n Figure l8a also indicates a point at which a vacuole i s p ossibly engulfing a smaller spherosome-like body. In part I the "primary hydration vacuoles" seem to o r i g i -nate from d i l a t i o n s of the ER. Subsequent extension of the primary vacuoles during promyoelial extension undoubtedly occurs through fusion of these with the spherosome-like organ-e l l e s . The l a t t e r also seem to be derived 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 several electron dense cores (Figs. 12 and 13). The widths of the ER membranes, the tonoplast, and.the spheroso-mal membranes are almost i d e n t i c a l (Average widths = 91+ A 0, 96 A 0, and 90 A 0 r e s p e c t i v e l y ) . , The vacuoles which develop i n the promycelium are formed by expansion and fusion of the spherosomal bodies (Figs. 20a and 20c). Formation of vacuoles by the expansion and fusion 75 of smaller elements, has been suggested f o r a large number of fungi (Buckley et a l . , 1966; Hyde and Walkinshaw, 1 9 6 6 ; Hawker and Abbott. 1963a; Hawker and Abbott, 1963c; Smith and Marchant, 1968). Such vaouoles are a u t o l y t i c and often contain fragments of other c e l l organelles. In Ustilago hordei t h e i r l y t i c na-ture i s also demonstrated through the observation that where t h e i r membranes have been damaged during f i x a t i o n the sur-rounding cytoplasm i s digested ( F i g . 20a). During fungal spore germination large vacuoles commonly develop, f i r s t i n the emptying spore case and l a t e r at the base o f the extending germ tube or promycelium (Hyde and Walkinshaw, 1 9 6 6 ; Hawker and Abbott, 1963a). A s i m i l a r e f f e c t has also been noted during the extension of pollen tubes (Jensen et a l . , 1968). That the expansion of vacuoles at the bases o f such tube-like 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 extension has been suggested by Bu l l e r (1933) and Corner ( 1 9 4 8 ) . The obser-vations i n Ustilago hordei support th i s hypothesis. F i r s t , the mass of the protoplast does not appear to increase s i g n i f i -cantly during germination and budding. Second although the promycelium ceases to extend a p l c a l l y i n a manner which i n d i -cates that the "pressure" f o r forward movement has tempora-r i l y ceased, the subsequent production o f buds occurs almost explosively i n a matter of minutes. This suggests that some form of pressure i s generated within the tube after l t has ceased to extend. The promycelium i s undergoing extensive va-cuolation during t h i s period. However, although this hypo-V6 thesis i s tempting, vacuoles may, i n some or a l l such systems, be the consequence, rather than the cause, of protoplasmic movement (Hyde and .Walkinshaw, 1 9 6 6 ; Robertson, 1 9 6 8 ) . THE SPHEROSOMAL VACUOLAR SYSTEM During t h i s study the term "spherosome-like" hac been applied somewhat a r b i t r a r i l y to two groups of organelles: one group occurring i n KMnO^ fixed t i s s u e , and the other group, with a quite d i f f e r e n t appearance and size range ( i . e . i n the promycelium), occurring i n glutaraldehyde-osmium fi x e d t i s s u e . This term has been applied f o r h i s t o r i c a l reasons rather than from adherence to any p a r t i c u l a r school of thought concerning the structure and function of these organelles. Por many years l i g h t microscopists have referred to the motile, spherical, r e -f r a c t i l e droplets which are common i n higher plants and fungi as spherosomes (Armentrout et a l . , 1 9 6 8 ; Sorokin and Sorokin, 1 9 6 6 ) . Frey-Wyssling (1963) f i r s t described bodies s i m i l a r to those found i n KMnOj^  f i x e d spores and promycelia of Ustilago  hordei and named them "spherosomes." However, according to the various s t r u c t u r a l and functional features which these organ-e l l e s exhibit at various stages i n development they might equally well have been ca l l e d microbodies (Frederick et a l . , 1968; Mollenhauer et a l . , 1966), or cytosomes (Frederick and Newcomb, 1 9 6 9 ) . or lysosomes (Matile, 1 9 6 8 ; Matile* and Spichiger 1 9 6 8 ; Wilson et a l . , 1 9 7 0 ) . Wilson et a l . (1970) have recent-l y reviewed the usage and signigicance of these terms as ap-p l i e d to fungi and have j u s t l y pointed out that what i s now required are exhaustive studies on the structure, d i s t r i b u t i o n , 77 and biochemistry of one or more of these bodies i n the same higher plant or fungus* The l i t e r a t u r e pertaining to the function o f these sphero-some-like bodies i s at best confusing. The confusion arises l a r g e l y from the f a c t that they are common to two systems which seem diametrically opposed. On the one hand, they are very nu-merous i n degenerating systems such as higher plant coleop-t i l e s and free c e l l cultures (Cronshaw, I96I4.), and aging and dying fungal tissues (Wilson et a l . , 1 9 7 0 ) . On the other hand they are also numerous In plant meristematic and d i f f e r e n t i a t -ing tissues (Frederick et a l . , 1963) and fungal t i p c e l l s (Wilson et a l . , 1 9 7 0 ) * From th i s observation the conspicuous presence of such organelles i n the spores and promycelia o f Ustilago hordei i s not surprising* Such a system i s not only a young d i f f e r e n t i a t i n g system but, i n another sense, an aging systemj Biochemically oriented studies have indicated that sphero-some-like bodies i n plants contain protein 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 of these studies have been ca r r i e d out i n higher plant systems (Matile, 1969)* Typ i c a l lysosomal hydrolases have been detected i n spherosomes of to-bacco (Balz, 1 9 6 6 ; Matile and Spichiger, 1 9 6 3 ) and of corn (Semadeni, 1 9 6 7 ; Matile, 1 9 6 8 ) . Matile (1968) also found a transaminase i n lar g e r spherosomes of corn and two oxido-reductases i n the smaller ones. Although there have been fewer studies of this type i n fungi several histocheraical studies have Indicated that fungal spherosomes contain a c i d phosphatases (Armentrout et a l . , 1 9 6 8 ; Buckley et a l . , 1 9 6 8 ) . Wilson et a l . (1970) surveyed the Ispherosbmes" of seven d i -fferent fungi and concluded, on a purely c y t o l o g i e a l basis, 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 organelles are compatible with t h e i r equivalence to animal lysosomes. The yeast c e l l does not contain spherosomes and what has been c l a s s i c a l l y referred to as the"vacuole" has the appearance of being the lysosomal equivalent. A number of studies have indicated that i n other fungi, bodies s i m i l a r to the sphero-some-like bodies of Ustilago hordei also contain l i p i d (Allen et a l . , 1971; Matile and Spichiger,» 1 9 6 8 ; McKeen, 1970) , and more p a r t i c u l a r l y phospholipid (Buckley et a l . , 1966; Sorokin and Sorokin, 1 9 6 6 ) . Prey-wyssling et a l . (1963) and Semadeni (1967) have suggested that spherosomes give r i s e to l i p i d bodies i n higher plants. Gay and Greenwood (196I4.) and Buckley et a l . (1968) have associated fungal spherosomes with the mobilization of l i p i d to form membranes. In addition to the above functions v e s i c l e s which resemble the smaller "sphero-somes" of Ustilago hordei seem to be involved i n hyphal t i p extension i n Pythium ultimum ( G r o v e 5 e t s a l l , 1970); i t has been suggested that they may also transport the enzymes and other materials required f o r w a l l synthesis and wall p l a s t i -c i z a t i o n . The m u l t i p l i c i t y of functions i n which fungal spherosomes are t e n t a t i v e l y implicated i s at f i r s t disturb-ing; however, obviously i n organisms as c y t o l o g i c a l l y "simple" as most fungi, some of the organelles must perform more than one function. In Ustilago hordei the structure and function of the orga-n e l l e s which we have termed spherosome-like bodies and vacuoles suggest that these structures a c t u a l l y represent stages i n the d i f f e r e n t i a t i o n and development of a single spherosomal-vacu-olar system. The differences i n the structure of these organ-e l l e s a f t e r various 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 during germination can be accommodated by the following hypothesis. 1. The spherosome-like bodies i n Ustilago hordei con-t a i n proteins and l i p i d s (perhaps i n the form of a l i p o -protein complex). The proteins are, at least i n part, enzymes. 2. The permanganate-type spherosome-like bodies (Dia-meter = 0.11 - o.56 u) are i d e n t i c a l to the small sized glutaraldehyde-osmium f i x e d ones (Diameter = 0.21+ - 0.86u). The vacuoles observed i n promycelia fi x e d by method A are the permanganate equivalent of the larger glutaraidehye-osmium fixed spherosome-like bodies (Diameter more than 3. E i t h e r the contents of the spherosome-like bodies change throughout development or else d i f f e r e n t contents are activated at d i f f e r e n t points i n time. Consequently these organelles are involved i n a variety of a c t i v i t i e s , some of which may be a u t o l y t i c , synthetic or both. S i -multaneously controlled a l t e r a t i o n i n the contents and l o c a l i z a t i o n of these bodies have profound e f f e c t on the d i f f e r e n t i a t i o n of the organism. 80 i+. A l t e r a t i o n i n the structure of the c y t o l o g i c a l l y ob-servable contents can be correlated with changes i n enzyme a c t i v i t y . These four points are elaborated i n the, following discussion. As has been previously indicated, ample evidence exists 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 matrix i n spherosome-like bodies (Sorokin and Sorokin, 1966). That the "spherosomes" of Ustilago hordel contain l i p i d , at l e a s t i n some stages of t h e i r development, i s evident from the f a c t they often contain pseudomyelin-like figures (Pig. 11). Buckley et a l . (1966) and Gay and Greenwood (1966) described such figures i n the "spherosomes" of other fungi and i n t e r -preted them as a stage i n the mobilization of l i p i d f o r mem-brane synthesis. Figures 18b and 19 i l l u s t r a t e more c l e a r l y that spherosome-like organelles i n Ustilago hordei can e v i -dently give r i s e to membranes which are spontaneously blebbed o f f i n the form of v e s i c l e s i n the d i r e c t i o n of the plasma-lemma. These membranes appear to have a regular t r i p a r t i t e structure and t h e i r width i s s i m i l a r to the width of the plas-ma membrane. During a recent study i n which a freez-etch technique was applied to the dormant teliospores of the smut T i l l e t i a caries A l l e n et a l . (1971) described a body ( i . e . u n i d e n t i f i e d organelle) which resembles the spherosome-like bodies of Ustllago hordei. L i p i d was demonstrated to be pre-sent i n these organelles. As no cytochemistry has been at-tempted, the presence of protein has been i n f e r r e d f i r s t from the apparently h y d r o l y t i c (and therefore enzymatic) a c t i v i t i e s S i of these bodies and secondly from known reactions of the d i f -ferent preparatory techniques on various biochemical 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 spherosome-l i k e bodies w i l l depend upon the use of appropriate biochemical or histochemical techniques. 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 usually designated as spherosomes i n permanganate fixed material are quite d i f f e r e n t i n appear-ance from those so designated i n glutaraldehyde-osmium fixed material. Unfortunately few worker have simutaneously em-ployed both techniques, causing doubt that the conclusions drawn from d i f f e r e n t studies a c t u a l l y apply to the same organ-e l l e s . The following factors suggest that i n Ustilago hordei 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 pre-paratory methods are the same: 1. The appearance of these bodies aft e r methods A, B, and C i s compatible with the expected effects of each technique on a l i p i d - p r o t e i n containing body. 2. Their numbers, d i s t r i b u t i o n , and size range i n hy-drating 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 to one side of the developing beak i n germinating spores. I}.. The a l t e r a t i o n i n number, si z e , and appearance i n promycelia i s compatible with the hypothesis that the permanganate-type spherosomal body i s i d e n t i c a l with the small-sized glutaraldehyde-osmium ones and that the permanganate-type vacuoles which develop i n aging pro-raycelia are i d e n t i c a l with the large-sized glutaraldehyde-osmium spherosome-like bodies which also develop i n aging promycelia. Assuming that these various manifestations a c t u a l l y repre-sent stages In the d i f f e r e n t i a t i o n of a single system the ac-t i v i t i e s i n which the system has so f a r been implicated can be summarized, i n the order of occurrence, as follows: 1. degradation of l i p i d bodies during pre-germinal de-velopment (Pt. I, Pig. 8.) 2. spore wall degradation and possible synthesis of the new promycelial Wall (Pigs. 1 and 2 ) . 3 . cytoplasmic degradation occurring outside the ER-sac i n a germinating spore (Pig. 8 ) . 1L. promycelial wall p l a s t i c i z a t i o n and possibly synthesis, 5. formation of "vacuoles" by organelle expansion and/or fusion. A l l of these a c t i v i t i e s seem to involve a hydrolytic com-ponent .and on t h i s basis i t Is tempting to suggest that U s t i - lago hordei the spherosomal-vacuolar system i s also the l y s o -somal system. The actual i d e n t i f i c a t i o n of these organelles as lysosomal equivalents awaits future biochemical and h i s -tochemical evidence. Functions 1, 2, 3 , and if, mainly i n -volve the smaller class of thse bodies ( i . e . those which appear as spherosome-like aft e r KMnO^ f i x a t i o n ) ; function 5 i s confined to the larger class ( i . e . those which appear as vacuole-like a f t e r KMnO^). An in t e r e s t i n g observation i s that during studies on the lysosomes of root t i p c e l l s of corn 83 seedlings Matile (1968) has i d e n t i f i e d two classes of lysosomes which correspond i n size - range and a c t i v i t i e s to the two classes of spherosome-like bodies discussed i n Ustilago hordei. The heavy lysosomes which contain hydrolases, transaminases, and oxidoreductases are small spheres 0.1 to 0.3 u i n diameter, with membranes resemling the ER membranes. The " l i g h t l y s o -somes" or "small vacuoles" whioh contain hydrolases and one transaminase range i n s i z e from 0,3 to 1.5 u i n diameter. The contents of the spherosome-like bodies condense into dense cores, t h i c k f i b r i l s ( i . e . i n pre-germinal spores), or fi n e f i b r i l s . The contents of the large "spherosome" poster-i o r to the apex of the newly formed promycelium i n Figure 6 are o f the f i b r i l l a r type. The larger spherosomes have dense cores, but they also contain a higher proportion of f i b r i l l a r material than the smaller ones. In fusing spherosomal bodies (Fig. 20) and large vacuoles (Figs. 12, 13 and 11+) the con-tents are usually also present. The c y t o l o g i c a l l y f i b r i l l a r state may r e f l e c t the biochemically state of the enzymatic contents• CONCLUSION I n i t i a t i o n of promycelial development i n Ustilago hordei involves the controlled integration of three separate proces-ses : l o c a l i z e d degradation of the spore w a l l , swelling of the protoplast, and synthesis of a new layer of wall material. Further extension o f the promycelium requires a balanced system of wall p l a s t i c i z a t i o n , wall accretion, and plasma membrane synthesis i n the region of the apex. No c y t o l o g i c a l evidence has been obtained i n this organism to indicate what the st r u c -t u r a l basis f o r t h i s system might be. During the promycelium extension most of the cytoplasm flows into the tube; the remaining spore cytoplasm becomes highly vacuolate. The endoplasmic reticulum, mitochondria and l i p i d bodies are d i s t r i b u t e d randomnly and do not increase notice-ably i n t o t a l quantity. One of the major functions of the ER i n hydrating and germinating spores seems to be the f o r -mation, d i r e c t l y or i n d i r e c t l y , of bodies s a t i s f y i n g the c l a s s i c a l d e f i n i t i o n of the fungus vacuole. In part I, a sug-gestion was made that the "primary hydration vacuole" i s formed d i r e c t l y v i a d i l a t i o n of the ER intermembrane space. Subse-quent "vacuole" formation occurs by the expansion and fusion of smaller "spherosome-like" organelles, which also may be derived i n some manner from the endoplasmic reticulum. Dur-ing the t r a n s i t i o n from the spherosome-like state to the vacuole-like state these bodies evidently perform a number of functions a l l of which involve a hydrolytic component, and a l l of 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 with the hypothesis that the spherosomal-vacuolar system l n Ustilago  hordei i s the f u n c t i o n a l equivalent of the lysosomal system i n animal c e l l s (DeDuve and Wattiaux, 1966). However, as some evidence indicates that fungal spherosome-like bodies may peform anabolic functions ( i . e . w a l l synthesis) as well as catabolic the adoption of a more " r e s t r i c t i v e " nomenclature does not seem to be j u s t i f i e d at present. I I . PLATE 1 Figure 1. A general view of a teliospore at germ tube i n i t i a -t i o n . Note the large perforations (p) i n the inner spore wall opposite the protoplasmic beak. The new promycelial wall (pW) has begun to form over the beak. ER and spherosome-like bodies (S) cluster i n the beak region. The protoplast contains many large matochondrial and primary vacuoles. Note that the protoplast has pulled away from the inner spore wall over most of the surface except i n the beak region. Method A. ca. X 25,2000. Figure 2. The beak at the e a r l i e s t stages of germ tube i n i t i a -t i o n . The f i r s t promycelial wall (pW) material i s present at the extreme t i p of the beak. Large per-forations (p) with f i b r i l l a r electron dense contents are already present on the inner spore wall opposite the beak. Note the c h a r a c t e r i s t i c presence of the spherosome-like bodies (S). Method C. ca. X 41}.,600. I I . PLATE 2 Figure 3* A general view of a teliospore at a s l i g h t l y more advanced stage of promycelium formation. Note the presence of the continuous ER-sac with i t s associ-ated spherosome-like bodies (S) and knots of endo-plasmic reticulum (arrows). Most of l i p i d bodies (L) vacuoles (V) and mitochondria l i e inside of the sac. Method A, ca. X 19,000. Figure lj.a. The beak region just p r i o r to spore wall rupture. Note that the th i n layer of new wall material (pW) completely surrounds the beak. Also note the ex-tensive perforations (p) i n the innersspore wall opposite the beak and the indications of stress in the outer spore wall layers opposite the beak. Peripheral ER elements are present. The d i p l o i d nucleus (dN) has moved into p o s i t i o n behind the beak. Method B. ca. X 22,£00. Figure l+b. A thiok section prepared by method A and stained in Toluidine blue. 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 nucleus, ca. X b , ,400 . Figure 5» Migration of the d i p l o i d nucleus (dN) and mitochon-d r i a (M) into the young promycelium. Note the e l -ongate forms. Also note the c h a r a c t e r i s t i c p o s i -tions of the nuclealus (Nu) and the c e n t r i o l a r -kinetochore-equivalent (CKE). The promycelial wall (pW) extends through the neck of the ruptured spore wall but does not completely encase the spore. Method B. ca. X 17,800. I I . PLATE 3 Figure 6. Spore wall rupture. A well-formed wall layer (pW) i s present about the new promycellial t i p . Note the "exploded" spore wall debris. Several large sphero-some-like bodies (S) containing electron dense cores and fine f i b r i l l a r material are present Just behind the promycelial apex. L i p i d (L) and the inner spore wall perforations (p) are indicated. Method C. ca. X 2 9 , 7 5 0 . Figure 7 . Migration into the promycelium. Note the large spore vacuole (V), the elongated mitochondria (M) and the presence of microtubules (mt). The pro-mycelial wall (pW) and a young spherosome-like body are also indicated. Method C. ca. X 3 5 - 5 5 0 . I I . PLATE k Figure 8. Part of a germinating spore showing the fusion of ER elements to form the ER-sac and showing the r e l a t i o n s h i p of the sac membranes to the spherosome-l i k e bodies (S) and vacuoles (V). Method A. ca. X 35,700. Figure 9 . A section 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 bodies (arrow) and the smooth ER and the mature spherosome-like bodies (S). Method C. ca. X 35,7000. Figure 10. A lon g i t u d i n a l section through a part of a promy-celium showing a d i p l o i d nucleus (dNf), spherosome-l i k e bodies (S), l i p i d bodies (L), and mitochon-d r i a (M), and a large number of unid e n t i f i e d v e s i c l e s . Method C. ca. X 23,700. I I . PLATE 5 Figure 1 1 . Figure 1 2 . Figure 1 3 . Figure ll^ar. Figure llfb. Large spherosome-like bodies with dense cores and pseudomyelinate figures (arrow) i n an aging germ-inated spore. Method C. ca. X 29 , 4 7 5 . Large vacuole (V) containing multiple electron dense cores and large quantities of electron dense f i b r i l l a r material in an aging germinated spore. Method C. ca. X 1 5 , 7 5 0 . Large spore vacuole (V) containing electron dense f i b r i l l a r material and large electron transparent patches. Free spherosome-like bodies (S) are numerous in the v i c i n i t y of the vacuole. Method A. ca. X 1 9 , 9 5 0 . Basal c e l l of a f o u r - c e l l e d promycelium showing the presence of a large vacuole (V) with electron dense f i b r i l l a r contents. Method C. sea. X 1 4 , 0 0 0 . A l i v i n g two-celled promycelium viewed withyphase optics. Note the presence of the basal vacuoles and the r e f r a c t i l e appearance of the spherosome-l i k e bodies, ca. X 2 , 5 0 0 . I I . PLATE 6 Figure 1 5 . A longitudinal section through a promycelium showing stacked ER l y i n g near to and ln p a r a l l e l with the promycelial w a l l . V e s i c l e s (ve) are often located near such ER stacks. Method G. ca. X 4 8 , 0 0 0 . A l o n g i t u d i a l section through a one-celled promy-celium in which many pores can be seen opening from the protoplast through the c e l l wall to the outside. Method C. ca. X 3 2 , 7 5 0 . Figure 16b. An enlarged view of one of the pores depicted i n Figure 16a. Method C. ca. X 5 2 , 4 0 0 . Figure 16a. Figure 16c. Figure 17. Two other pores from the same promycelium as in 16a. Method C. ca. X 5 2 , 4 0 0 . A haploid nucleus (hN) within the spore after germ-ina t i o n . The arrow indicated the chromatin-nucleolar connection. Note that on the side of the connection opposite the nucleolus 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 close to the nuclear envelope. Meth-od C. ca. X 5 3 , 5 5 0 . I I . PLATE 7 Figure 18a. Figure 18b. Figure 19. A general view of one of the c e l l s i n a four-c e l l e d promycelium. Note the haploid nucleus (hN) with simple nuclear pores (NP) and the mitochondria.L^JER and l i p i d (L) i s scant. On the r i g h t a spherosome-like body (S) i s being engulfed by a large vacuolar-structure (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 possible sequence of conver-sion of spherosome-like bodies ( i ) to vacuole-l i k e bodies ( i i i ) . V esicles (ve) are often conspicuous between the vacuoles (V) or the spherosome-like bodies (S) and the c e l l w a l l . Method A. ca. X 53,£00. An enlarged view of one of the spherosome-l i k e bodies (S) i n Figure Ida which seems to be giving r i s e to v e s i c l e s which pass towards the septum. Method A, ca. X 95,200. Part of a promycelium showing membrane formation within a spherosome-like body and the blebbing of v e s i c l e s (ve) from the spherosome-like body towards the c e l l w a ll. Short segments of rough ER (arrow) are present near the haploid nucleus (hN). ER i s often also associated with the spherosome-like bodies ( i . e . lower r i g h t ) . Method B. ca. X 1+1.000. I I . PLATE 8 Figure 20a. A longitudinal section 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 bodies (L) and spherosome-like bodies (S). Method C. ca. X 13,800. Figure 20b. The apex of the promycelium depicted in Figure 20a. Note the lack of c y t o l o g i c a l l y d i s t i n c t structures i n the extreme t i p . Method C. ca. x 32,200. Figure 20c. Fusion of two spherosome-like bodies to form a large vacuolar structure. Note the two electron dense cores and the large amount of f i b i l l a r material in these organelles ( s ) . Part of the bounding membranes between the two fusing organ-e l l e s (s) i s s t i l l present. Method C. ca X 32,200. S 6 ; BIBLIOGRAPHY Al l e n , J.V., Hess, W.M., and Weber, D.J. 1 9 7 1 . 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Enzymatlsche charakterisierung des lysosOmena quivalent (Spharosomen) von Maiskeimlingen. Planta 7 2 : 91-118. Smith, P.G. and Marchant, R. 1 9 6 8 . L i p i d inclusions i n the vacuoles of Saccharomyces cerevisi ae. Arch. Mikrobiol. 6 0 : 3 4 0 - 3 4 7 . ; Sorokin, H.P. and Sorokin, S. 1 9 6 6 . The spherosomes of Cam- panula p e r s i c l f o l i a L. A l i g h t and electron microscope'-stuay. protoplasms 6 2 : 2 1 6 - 2 3 6 . Stein, C.W. 1 9 7 0 . An electron microscope study of a mycelial mutant of Ustilago hordei (Pers.) Lagerh. M.Sc. Thesis. U.B.C., Vancouver, UAJN. Stocks, D.L . jand Hess, W.M. 1970. Ultrastructure of dormant and germinated basidiospores of a species of Psilocybe. Mycologia 62: 176-191. Walkinshaw, G.H., Hyde, J.M., and Zaridt, J. van 1967. Fine® structure of quiescent and germinating aecioapores of Cronartium fuslforme. 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 observations of the fin e structure of Micro-sporum canls• 3 Invest. Dermatol. I+Jb: 130-13U. Werner, H.J. and Llndberg, G.D. 1966 Electron microscope observations of Helminthosporium v i c t o r i a e . J. gen. Micro b i o l , hgx 123=I2TT * Wilson, C&L•, S t i e r s , D.L., and Smith, G.G. 1970. Fungal lysosomes or spherosomes. Phytopathology 60: 216-227. PART I I I Promycelial Septation and S p o r i d i a l Formation TABLE OF CONTENTS Page ABSTRACT 92 INTRODUCTION 92 MATERIALS AND METHODS 9 ^ OBSERVATIONS 9*1 Septation 9k-Sporidium Formation ••••••••• 98 DISCUSSION 100 Septation « 100 Sporidium Formation e 108 CONCLUSION 110 BIBLIOGRAPHY 112 PART I I I I n i t i a t i o n of the Promycelium and Promycelial Extension ABSTRACT In the promycelia of Ustllago hordel cross walls are complete* The pattern of septation resembles that found i n actinomycetes, i n Coocidloides lmmitia, and i n E x i d i a  nucleata. Elaborate membrane complexes have been a s s o c i -ated with the i n i t i a t i o n of the cross wall* Two variati o n s of the normal septation pattern are described and the r e -su l t s of these v a r i a t i o n s are discussed. The i n i t i a t i o n of spo 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 promycelial wall followed by degradation of the old wall and subsequent synthesis of new wall mate-r i a l . At present no information i s available t o indicate the manner i n which the mature sporidium i s separated from the parent c e l l . INTRODUCTION When the promycelium of Ust l l a g o hordei has almost attained i t s masimum length the d i p l o i d nucleus undergoes meiosis. The f i r s t nuclear d i v i s i o n &e. reduction d i v i -sion) i s followed immediately by the formation of a single septum giv i n g r i s e t o a two-celled promycelium. Each of the haploid n u c l e i again divides (&e. equational 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 with the almost simultaneous establishment of two more septa. Once the f o u r - c e l l e d state has been attained each c e l l gives r i s e t o an ad d i t i o n a l daughter c e l l formed by a process of y e a s t - l i k e budding. During production of the bud ( i . e . sporidium or basidiospore) the parent c e l l nucleus divides again, m i t o t i c a l l y , one of the daughter n u c l e i becoming situated i n the sporidium and the other i n the parent c e l l . This study i s concerned with the u l t r a s t r u c t u r a l cytoplasmic events which occur during septation and budding, A sporidium i s of one of two mating types designated 4- and -• Generally a promycelium gives r i s e to equal num-bers of each type. The i n f e c t i v e dlkaryon i n Ustilago  hordei i s usually formed by the fusion of two sporidia of opposite mating type i n the presence of a suitable host. However, among smuts an alternative mechanism e x i s t s whereby a dikaryon may be established (Dickinson. 1927; Duran and Safeeulla, I960), A cytoplasmic bridge forms d i r e c t l y be-tween a plus and a minus promycelial c e l l . In t h i s p a r t i c -u l a r smut species such bridges only form between immediately adjacent c e l l s , and are often referr e d to as "knee-joints". The apex of the bridge then assumes the properties of a hyphal apex estending to produce a branch. The undivided n u c l e i of the confluent c e l l s migrate i n t o the branch region to e s t a b l i s h a dikaryon. During t h i s study the u l t r a s t r u c -t u r a l basis of bridge formation has been established. MATERIALS AND METHODS The materials and techniques are i d e n t i o a l to those previously described (Fts. I and I I ) , the tissue being f i x e d a f t e r 5> to 7% hours of incubation. The promycelium i n Figure 10 was f i x e d f o r 12 hours i n 2.0$ glutaraldehyde buffered at pH 7.2 with 0.01 M« cacodylate buffer and ob-served d i r e c t l y using phase contrast optics as described i n part I I . OBSERVATIONS SEPTATION: In U s t i l a g o hordei a simple Invagination of the plasma membrane i n i t i a t e s septum formation ( F i g . l a , arrows). F i g -ure l b c l e a r l y demonstrates the u n i t membrane del 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 to be somewhat narrower (Average width = 100 A 0) than the plasma membrane l i n i n g the l a t e r a l promycelial walls or the mature septum (Average width = 121+ A 0 ) . At the f r o n t a l edge of the e a r l y invagination the membranes are c l o s e l y ap-pressed f o r a distance of $0 t o 100 mu. and then are separated by an electron transparent lamella Ij. t o 10 mu. i n diameter which merges with the l a t e r a l w a l l . An electron 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 invagination", somewhat a f t e r the fashion of a c l o s i n g i r i s diaphragm. Amorphous electron dense material continues t o encase the advancing edge (Fig.3 ) and once the membranes have met and fused c e n t r a l l y remnants of t h i s substance can be seen l y i n g beside the fused plate (Pig. l+). The septa within the promycelia of Ustilago hordei are complete ( i . e . septal pores are absent). Just p r i o r to, or during, plate completion synthesis of the septal wall begins at the l a t e r a l edges and i s formed i n such a way as to be continuous with the inner layers of the promycelial w a l l . In appearance they are i d e n t i c a l . The new w a l l material i s l a i d down simultaneously a l l around the inside edge of the invaginated plasma membrane, thus forming a tube of w a l l material inside the invagination. The electron transparent lamella within the tube i s not occluded. During septal development i t retains the constant diameter (Ij. - 10 mu) f i r s t defined by the septum i n i t i a l . Once the plate i s complete t h i s central region i s continuous through-out the cross w a l l . Subsequent thickening of the w a l l at any point r e s u l t s from the accretion of new material along the plasma membrane on either side of the plate. Soon a f t e r plate completion, synthesis of the septal wall material spreads r a p i d l y along the inner surface (Pig. ij.). Prom t h i s point on new material must be deposited synchronously throughout the septum u n t i l i t has attained i t s maximum thickness (130 - 150 mu). In cross-section the t o t a l width of the cross-wall i s r e l a t i v e l y constant across the promy-celium, (with the exception o f the triangular thickening (Bracker and Butler, 19631 at the extreme l a t e r a l edges.,, The thickness of the material on either side of the central 96 lamella i s equal. Figures 1+, 5 and 6 demonstrate sequen-t i a l l y the thickening of the septal w a l l . In Ustilago hordei the plasma membrane of the invaginat-ing septum i n i t i a l i s frequently extended into an elaborate complex (Figs. 2, 8, and 9). Although s e r i a l sections across an entire promycelium have not been obtained such complexes always seem to l i e against one of the l a t e r a l walls (Figs. 2 and 7)» and there i s apparently at l e a s t one complex per septum. They are v i s i b l e a f t e r both glutaraldehyde-osmium and permanganate f i x a t i o n s (Figs. 2, 8, 3, and 9, and F i g . 7, r e s p e c t i v e l y ) . With the l i g h t microscope prominent bodies of s i m i l a r s i z e and shape occur i n association with septa i n glutaraldehyde-fixed material ( F i g . 10). In l i v i n g t i s -sue conspicuous stationary r e f r a c t i l e bodies c l e a r l y indicate the point at which a septum w i l l l a t e r be v i s i b l e (Introduc-t i o n , Figs. l i - j ) . The ultimate fate of the septum-associ-ated membrane complexes i s unknown but there i s some evidence that they normally degenerate once the septal wall material begins to form. Not a l l the membrane complexes i n the promycelium are associated with the septum (Fig. 7) and more w i l l be said about the r e l a t i o n s h i p of these unusual struc-tures to other c e l l organelles i n part V. Figure 9 c l e a r l y demonstrates the unit structure o f the membranes composing the complex. These membranes have an aver-age width of 96A° (Range: 60 - H+.0 A ° ) . After glutaraldehyde-osmium f i x a t i o n they are arranged i n concentric whorls (Fig. 2) or fol d s (Figs. 8 and 9) but a f t e r KMnOlf-fixation they f o l -low a more random, but s t i l l compact, or i e n t a t i o n (Fig. 7) « Aside from the complexes no other organelles are con-spicuously associated with i n i t i a t i o n or thickening of the septum* Spherosome-like bodies and l i p i d bodies often occur i n the v i c i n i t y of the cross-walls but t h e i r occurence does not seem t o be any more frequent than i n other parts of the cytoplasm* Short segments of endoplasmic reticulum sometimes l i e just beneath and p a r a l l e l with the plasma membrane of a well-developed septum i n much the same fashion that the ER borders on the l a t e r a l walls (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 quite crenulate even around mature septa which have presumably at-tained t h e i r maximum thickness (Figs* l£ and 16). Two v a r i a t i o n s to the normal septation pattern have been observed* The f i r s t , depicted i n Figure l a , i s a def-i n i t e l y abnormal event i n which twin septa are l a i d down side by side. The second v a r i a t i o n i s involved i n the pro-duction of "knee-joints". This 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 wall over a l o c a l i z e d area on both sides of and adjacent t o a septum* The protoplasts of the two c e l l s involved are then "blown out" as two bulbous extensions in, a manner sim i l a r to the normal budding process. As the protoplasts bulge the l a t e r a l promycelial wall i s extended around the outer surface. On the inner surface where the two expanding regions are growing together the septal wall resumes growth u n i d i r e o t i o n a l l y and so continues to separate the two protoplasts ( F i g . 11). Spherosome-like bodies and long elements of endoplasmic 98 reticulum are very numerous and prominent on both sides of the bulbous b r i d g e - i n i t i a l during t h i s early period o f formation. What then seems to occur might best be described as a metabolic race between those elements engaged i n syn-thesizing the walls separating the two c e l l s and another set of elements engaged i n breaking down the p a r t i t i o n ( F i g . 12). In Figure 12 a portion of the wall can s t i l l be seen between the two bulbous c e l l extensions while the regions on either side of i t have become electron transparent and contain c e l l debris and v e s i c l e s which seem to be derived from the ER. F i n a l l y the degratory elements pre-dominate and the p a r t i -tioning wall i n the bulbous region i s eliminated allowing the protoplasts to fuse completely (Fig. 13). The plasma mem-brane reforms i n some manner about the f r e e end of the remain-ing portion of the septum. The net r e s u l t i s an Incomplete sep-tum p a r t i a l l y separating two c e l l s which are now joined by a cytoplasmic bridge. In Figure 13 the nucleus of one c e l l has moved into a p o s i t i o n preparatory to entering the bridge 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 of a bright r e f r a c t i l e spot on the surface of the promycelium. After f i v e to ten minutes the w a l l begins to bulge at t h i s point, and to extend r a p i d l y . During the f i r s t phase of elongation the bud i s narrow, but a f t e r several milli-microns of growth ( i . e . as measured under the elec-tron microscope i t undergoes a spontaneous ballooning of the d i a -meter to give r i s e to the elongate ovoid form of the mature sporidium (Introduction, Pigs. 9 - 15). The total development of the bud from the appearance of the ref r a c t l i e spot to the mature sporidium requires 20 to 1*0 minutes. Under the electron microscope the f i r s t sign that a bud is about to form is the disappearance of wall material from a small region of the lateral promycelial surface. The actual disappearance of the wall substance is preceded by a change in i t s structure. The semi-electron dense, f i b r i l l a r wall thickens and becomes more electron-translucent and ap-parently homogeneous, with the exception of the thin electron dense line which has been shown to separate the wall into two layers. In Figure 11* a short walless zone is shown to be & bounded by altered wall material which i s , no doubt, in the process of being degraded, and the protoplast has already begun to bulge in the direction of the walless region. Long elements of endoplasmic reticulum lying just beneath and parallel to the l a t e r a l wall seem to end in the v i c i n i t y of the electron translucent area. Figures 15$ 16, and 17 Illustrate sequentially the development of a sporidium. In Figure 15 a thin amorphous layer of material covers the bud i n i t i a l , and the fractured edges of the old promycelial wall are clearly v i s i b l e . Be-neath the new layer of wall material a large spherosome-like body is conspicuously present. Since the lateral wall of the adjacent c e l l on the other side of the septum also seems to be thinner than the usual promycelial wall' the ceils depicted in Figure 15 may actually represent a stage in bridge-formation rather than budding. Early in this devel-opmental stage It i s impossible to predict what course of 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 increases i n size part of the protoplast of the parent c e l l moves into i t . The parental nucleus divides, m i t o t i c a l l y , i n the promy-celium, i n the bud, or i n the neck between the two and the daughter n u c l e i separate i n such a way that the parent c e l l and the basidiospore obtain one nucleus each. As was noted during the passage of the d i p l o i d nucleus up the promycelium (Ft. II) the nucleolus always l i e s at the posterior end of a migrating nucleus. The mature sporidium (Fig. Id), then, contains a single haploid nucleus, a number of mltochrondla, l i p i d bodies, and spherosome-like bodies, plus a small amount of endoplasmic reticulum 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 be-come an autonomous i n d i v i d u a l . No evidence has been obtained to indicate the manner i n which this separation occurs or i n which the proximal end wall of the sporidium i s completed. DISCUSSION SEPTATION: The hyphal c e l l s of most higher fungi maintain some sort o f cytoplasmic continuity with t h e i r neighbours by virtue of one or more pores i n the cross-walls separating the c e l l s . In general, the pores of the ascomycetes are represented by "simple holes" while the pores of the basidiomycetes are characterized by elaborate structures known as the "dolipore-parenthosome" (Moore and McAlear, 1962; Bracker, 1 9 6 7 ) . A major exception to this rule occurs among the heterobasidio-mycetes (Bracker, 1 9 6 7 ; Ehrlich; et a l . , 1 9 6 8 ) . In this 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 is commonly accepted that dolipore-septa do not occur in the Ustilaglnales (Moore, 1 9 6 5 ; Bracker, 1 9 6 7 ; Ehrlich. et a l . , 1 9 6 8 ) although to the best of the author's knowledge there is only one report on smut septa in existence (Stein, 1 9 7 0 ) . Complete septa, such as those observed in the promycelium of tTstllago hordei, have occasionally been noted in phyco-myoetes, hemiascomycetes, and deuteromycetes (Bracker, 1 9 6 7 ) as well as in a mycelial mutant of Ustilago hordei (Stein, 1 9 7 0 ) and in a basidiomycete, Cryptococcus neoformis (Cutler and Erke, 1 9 7 1)• Septa without pores, are often associated with specialized situations such as sealing off injured or evacuated cells (Wells, I96I4.) and delimiting reproductive structures (Hawker and Oooday, 1 9 6 7 ) . The four-celled metabasidium of Ustilago hordei constitutes a highly special-ized system in which each of the cells contains a different set of genetic information (lie. as a result of meiosis). Of course the cytoplasm of these cells is derived from a single protoplast, and i t is unknown how long i t takes for the new nucleus to exert i t s effect on the individual c e l l , but cer-tainly an influence is manifest by the time of bridge-forma-tion and sporidlal production. However, since complete septa also occur in a mycelial mutant of this fungus (Stein, 1 9 7 0 ) the p o s s i b i l i t y e xists that 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) described three patterns of somatic c e l l ' d i v i s i o n i n mycota. The mature septum of Ustilago hordel i s of c l a s s i f i c a t i o n s type B, represented by Streptomyces  vlolaceoruber and Coccldioides immltis. It consists of two layers of septal w a l l ffce. two plates) each of which i s con-tinuous with the inner l a t e r a l wall and an electron trans-parent lamella l y i n g between the two plates (Pigs. 5. 6, 15 and 16). Cross walls of si m i l a r structure occur among the phycomycetes (Akai et a l . , 1968; Hawker and Gooday, 1967), the ascomycetes (Brenner and C a r r o l l , 1968; Kreger and Veehuis, 1969; Moore, 1962), the basidiomycetes (Bracker and Butler, 1963; J e r s i l d et a l . , 1967; O'Hern and Henry, 1956; Wells, 1961*), and a number of imperfect human pathogens (Carbonelle and Rodrigez, 1968). They also occur among the actinomycetes (Glouert and Hopwood, 1961; Moore, 1965). The dimensions of the mature septa of Ustilago hordei are almost i d e n t i c a l to those given f o r the cross-walls of Bhlzoctonia solan! (Bracker and Butler, 1963). In the former the newly-formed cross wall width i s 6 to 10 mu ( F i g . 1*), the mature cross wall width i s 130 to 150 mu. (Figures 6, 13, 15, and 16), and the diameter of the electron transparent lamella i s I* to 10 mp (Figs. 1*, 5, and 6). In the l a t t e r the respective measurements are 7 to 8 mu, 120 mu, and 10 mu. Wells: (1961+b) has noted that the septa which oleave the hypobasidium of E x l d l a nucleate (a heterobasidiomycete) i n t o four hypobasidial segments have a sim i l a r structure. During septum formation i n t h i s smut fungus the plasma-lemma invaginates f i r s t , and septal wall material i s not l a i d down u n t i l the membranes have fused, or have almost fused across the centre of the promycelium. This mode of formation most c l o s e l y resembles that of Coccidioides immitis (O'Hern and Henry, 1 9 5 6 ) , c e r t a i n species of Streptomyces (Moore, 1 9 6 5 ) , and some ba c t e r i a (Chapman, 1 9 5 9 ) , a l l of which form type B septa according to Moore's c l a s s i f i c a t i o n . A l l of these species also form complete septa. Moore (1963) and Wells (196U.br) observed a similar mechanism of complete septation during t h a l l u s formation i n the aecia of Puecinia  podophylli and during hypobasidium segmentation i n E x i d i a  nucleate, r e s p e c t i v e l y . The f a c t that a s i m i l a r structure and developmental pattern have been observed f o r complete cross-walls i n a rust :(Moore, 1 9 6 3 ) , a tremellaceous fungus (Wells, 1963b) and a smut suggests that t h i s form of septa-t i o n may be common among heterobasldiomycetes. In most other cases where septal development has been observed ( i e . exception-Hawker and Gooday, 196?) the synthesis of wall material keeps pace with the extension of the plasma mem-brane so that the septum i n i t i a l , at a l l stages i s a wedge of wall material c l o s e l y surrounded by membrane rather than a f l a t membranous plate (Brenner and C a r r o l l , 1968; Campbell, 1969; Conti and Naylor, 1959; Manocha and Shaw, 1967; Mar-chant and Smith, 1968; Moore, 1962; Wells, 1961+). The advancing l i p of the septum i n i t i a l of Ustilago  hordei i s c l o s e l y associated with a rim of electron dense material (Pigs, 3 and l a ) . A simi l a r substance surrounds the advancing edge or the pore rim of the cross walls of Ascodemis sphaerospora (Brenner and C a r r o l l , 1968), and Sordaria f i m i c o l a (Purtado, 1971). In both the l a t t e r studies endoplasmic reticulum i s associated with septum formation and/or the electron dense substance. Hawker and Gooday (1967) have suggested that the ER surrounding the septum i n i t i a l of Rhizopus:aexualis (Smith) Callen gives r i s e to v e s i c l e s which contain the new wall material, and which release t h i s material by fusing with the septal mem-brane. Lomasomes may also be involved i n the formation of cross walls (Brenner and C a r r o l l , 1968; Hawker and Gooday, 1967). The source of septal wall material i n Ust i l a g o  horde! Is as obscure as the mechanism of a p i c a l extension (Pt. I I ) . As noted by Wells f o r E x i d i a nucleata (1961*b), ves c i o l e s , endoplasmic reticulum, and lomasomes are not conspicuously associated with the invaginating membrane or thickening p l a t e . In Ustilago hordei large membrane complexes are often associated with the invaginating septum i n i t i a l . What are now required are s e r i a l sections through promycelia which are under-goint c e l l d i v i s i o n . Hopefully such studies w i l l prove or disprove the hypothesis that there i s always one or more of these structures present i n the region of c e l l d i v i s i o n . According to the current data a septal membrane complex i s an "extension" of the invaginating plasma mem-brane which l i e s against one of the l a t e r a l germ tube walls (Pigs. 2 and 8). 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 part of the ER-sac i n a germinating c e l l (Pt. II) i s unknown at present, but the farmer seems to be associated with the plasma membrane, and the l a t t e r with the endoplamic reticulum. A number of researchers have described similar membrane com-plexes among fu n g i . In the basldiomycetes A r m i l l a r l a melea (Berliner and Duff), Coprlnus micaceous (Edwards, 1969), Coprinus lagopus (Lu, 1965; Lu, 1966), and Lycoperdon per- latum (Mardhant, 1969), the ascomycetes Asp e r g i l l u s fumiculus (Edwards, 1969), Neurospora tetrasperma (Lowry and Sussman, 1968), and Neurospora crassa (Kozar and Weijer, 1969), and the imperfect fungi Paracoccidioides l o b o i (Purtado et a l . 1967) and Vertlcvjllium dahliae ( G r i f f i t h s , 1970) the respec-t i v e workers have not noted any r e l a t i o n s h i p of the complexes to the septum. The s p e c i f i c association of sim i l a r membrane systems with septa occurs i n the imperfect fungi Paracocci- dioides b r a s i l i e n s i s and Blastomyces dermatitldis (Carbonelle, 1967, and Carbonelle and Rodrigez, 1968) and i n the b a s i d -iomycete Lenzltes saepiaria (Hyde and Walkinshaw, 1966). Membrane systems derived from the plasma membrane have long been associated with septum formation among the actinomycetes (Edwards, 1970; E l l a r et a l . , 1967; Glauert and Hopwood, I960; Imaida and Ogura, 1963) where such structures have usually been r e f e r r e d to as "mesosomes" because of the s t r i k i n g s t r u c -t u r a l s i m i l a r i t y to the b a c t e r i a l organelles of that name. One of the postulated functions of the b a c t e r i a l mesosome i s to a s s i s t i n septum formation (Rogers, 1970; Ryter, 1968), 106 The membrane complex-cross wall association has suggested a number of possible functions f o r these elaborate membrane systems: synthesis of septal wall material between the c e l l and i t s environment during periods of increased metabolic a c t i v i t y (Carbonelle, 1967), and i n i t i a t i o n of c e l l d i v i s i o n (Edwards, 1970). In Ustilago hordel i t i s u n l i k e l y that the complex acts d i r e c t l y i n deposition of the wall material since such systems are most prominent in the v i c i n i t y of the invaginating septal i n i t i a l before wall thickening has begun. However, one of the ways i n which a membrane complex might i n i t i a t e septum formation would be to provide a l o c a l -ized source of pre-synthesized membrane which would then be incorporated into the developing membranous p l a t e . This hypothesis i s supported by the f a c t that the membrane of the septal i n i t i a l i s continuous with that of the complex and by the f a c t that the width of the invagination membrane (approxi-mately 100A°) i s closer to the mean width of the unit mem-branes of the complex (96A°) than to the mean width of the plasmalemma on the sides of the promycelium (121+A0). Berliner and Duff (1965), and Lu (1965 and 1966) have also postulated that similar membrane complexes may act as membrane generators in connection with other a c t i v i t i e s i n fungi. Further d i s -cussion of the smut membrane complex, i t s o r i g i n and possible functions w i l l be reserved u n t i l part V. The l i t e r a t u r e pertaining to membrane complexes i n fungi has frequently been confused by the f a i l u r e to d i s -tinguish these structures from artefacts produced i n tissue by glutaraldehyde f i x a t i o n (Fawcett and Susuma, 1 9 5 8 ; Ohal and Rohlich, 1966; Palade and Claude, 1 9 U 9 a ; Palade and Claude, 19U9b; Revel et a l . , 1 9 5 8 ) . The f a c t that stationary bodies of similar size to the membrane complexes can be observed i n l i v i n g germ tubes at points where septa w i l l subsequently form indicates that such structures 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 that these membranous whorls are formed by hyperactive membrane synthesis which r e s u l t s from growth in a r i c h medium. A comparison should be made with meta-basidi a produced i n minimal medium or d i s t i l l e d water. Fungal membrane complexes have also been confused with pseudo-myelin figures which are frequently observed in vacuoles or lysosomes (Smith and Marchant, 1 9 6 8 ; Thomas and Isaac, 1 9 6 7 ) . Intravacuolar pseudomyelinate figures also occur i n Ustilago hordei (Pt. I I ) ; however, the mem¥ brane complexes per se are c l e a r l y not associated i n any way with lysosome-like structures. Two variat i o n s on the normal septation pattern have been observed. The formation of twin septa r e s u l t s i n a promycelium with f i v e compartments one of which contains no nucleus and r a p i d l y becomes vacuolated. 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 formation of a cytoplasmic bridge between two d e l l s of opposite mating type and - ) . These appear to be similar i n structure and probably also i n o r i g i n to the pseudo-septa, or incomplete transverse septa de-scribed i n other basidiomycetes (Ehrlich et a l . , 1963; J e r s i l d et a l . , 1967; K o l t i n and Flexer, 1969). In part II long elements of endoplasmic reticulum and spherosome-l i k e bodies were associated with wall synthesis and degrada-t i o n i n the region surrounding the beak during germination. Interestingly, a s i m i l a r association exists i n the region of the bridge formation (Pigs. 11 and 12). Vesicles which seem to be derived from the ER accumulate i n the region of degradation (Fig. 12) suggesting that the ER i s more l i k e l y involved i n catabolic a c t i v i t i e s ( i . e . wall degradation) than i n anabolic ones ( i . e . wall synthesis). SPORIDIUM FORMATION: Bartnicki-Garcia and Lippman (1969) hypothesized that bud formation i n dimorphic fungi might be based on l o c a l -i z e d resumption of growth of the parent c e l l , followed by a uniformly dispersed pattern of wall synthesis i n the bud. This would r e s u l t i n spherical, 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 extension. The observations i n Ustilago hordei at le a s t support the f i r s t part o f t h i s hypothesis. The i n i t i a l thickening of the parent wall material which occurs 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 of the dimorphic fungi, Paracoccidioides b r a s i l i e n s i s and Blas- tomyces dermatltidls (Carbonelle, 1967; Carbonelle and Rodriguez, 1968), as well as several species of Histo- plasma (Edwards et a l . , 1959). However, i n Ustilago hordei the thickened w a l l region i s more electron translucent than the mature promycelial w a l l , while i n the human pathogens the thickened region i s more electron dense. The Increased homogeneity and decreased electron density in the former i s presumed to indicate an increase i n p l a s t i c i t y . Un-doubtedly the r e f r a c t i l e spot which appeas i n the l i g h t microscope before a sporidium becomes v i s i b l e represents t h i s period of wall p l a s t i c i z a t i o n and the subsequent period of wall degradation. In the promycelium of Ustilago hordei, a l o c a l i z e d portion of the l a t e r a l wall i s evidently completely r e -moved before the new bud wall i s formed (Pig. 11+). As the new material thickens about the neck i t again becomes s t r u c t u r a l l y continuous with the promycelial w a l l . This mechanism of bud formation varies from the mechanisms previously described. The bud wall i n Saccharomyces  cerevisiae i s a d i r e c t continuation of the parental c e l l wall (Marchant and Smith, 1968; McClary and Bowers, 1965; Moor, 1967). 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 several species of Histoplasma (Edwards et a l . , 1959). Sporobolomyoes-type budding involves the rupture of an outer wall l a y e r — t h e bud wall being an extension of an inner wall layer of the parent (Calonge, 1969; Prusso and Wells, 1967). In Rhodotorula g l u t l n i s a new wall layer i s synthesized beneath the old and dur-ing budding the parental wall layer ruptures, the bud being encased by completely new material (Marchant and Smith, 1967)• Rhodotorula-type budding seems to be the most sim i l a r to s p o r i d i a l formation i n Ustilago hordei. Notice should he made that the yeast-like Rhodotorulas have recently been r e c l a s s i f i e d as a new genus of the heterobasidiomycetes, the Rhodosporidluro (Banno, 1 9 6 7 ) . Long elements of endoplasmic reticulum are prominent beneath the bud i n i t i a l during the e a r l i e s t observation that during knee-joint formation v e s i c l e s which are ap-parently derived from the ER are prominent i n the region of breakdown. Moor (1967) has suggested that i n Saccharo- myces cerevisiae v e s i c l e s derived from the ER carry 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 wall of the mother c e l l , and hence i n i t i a t e bud formation. On the other hand, Marchant and Smith (1967) have associated large amounts of ER and ER-derived v e s i c l e s with active wall synthesis during budding. In Ust i l a g o hordel spherosome-like bodies are quite prominent once synthesis of the new wall has be-gun ( F i g . 1 5 ) . This observation helps to support the hypo-thes i s previously discussed i n part I I , that i n t h i s species of smut fungus the spherosome-like organelles function i n wal l synthesis. CONOLUSION Septation i n the promycelium of Ustilago hordel i s i n i t i a t e d by the " c e n t r i p e t a l invagination" of membranes which are continuous with the plasma membrane. The source of the membranes forming the i n i t i a l membranous plate seems to be a large membrane complex. Further studies are required to te s t the hypothesis that at lea s t one such complex i s associated with every developing septum i n i t i a l . Septal wall material i s not deposited i n any quantity u n t i l the i n i t i a l plate has been completed. The source of the septal wall material i s unknown. No pores occur in the mature cross walls of the metabasidium. Two va r i a t i o n s of the regular septation pattern occur, one of which gives r i s e to "knee-joints". Budding i s i n i t i a t e d by a sequential p l a s t i c i z a t i o n and degradation of the wall of the parent c e l l . This stage i s followed by an explosive protrusion of the s p o r i -dium accompanied by synthesis of new wall material. Fur-ther studies are required to determine the manner i n which the mature sporidium i s separated from the promycelium. Evidence i s presented which supports the hypothesis that i n Ustilago hordei the endoplasmic reticulum functions i n the break down of mature wall material. The spherosome-l i k e organelles, which may also be involved i n wall de-gradation and p l a s t i c i z a t i o n , probably have an addit i o n a l r o l e i n the biosynthesis of new wall material. I I I . PLATE 1 Figure l a . Figure l b . Figure 2 . Septum i n i a t i o n . An abnormal side-by-side septa-t i o n . Each septum i s formed normally. Three points at which the septum i n i t i a l s can be observed are indicated ( i . e . arrows and i n s e r t ) . Each i n i t i a l consists of an invagination of the plasma membrane bounding an electron transparent c e n t r a l lamella and ending i n an amorphous electron dense material. Method C. ca. X 2 6 , 2 0 0 . An enlarged view of one side of one of the septum i n i t i a l s in figure l a . Note the well defined plasma membrane (FM), the central lamella, and the electron dense material surrounding the f r o n t a l edge. Method C. ca. X 4 5 , 8 5 0 . Aiiraore advanced septum i n i t i a l showing a clear continuity between the plasma membrane delimiting the septal i n i t i a l and the membranes composing a membrane complex,(mc). The two sides of the sep-tum i n i t i a l are indicated 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 indicates the electron dense f r o n t a l edge material. Note septal wall material i s beginning to be deposited at the l a t e r a l edges and i s con-tinuous with the inner promycelial w a l l . L i p i d bodies (L), mitochondria (M) and spherosome-like organelles (S) are indicated. None of these or-ganelles are s p e c i f i c a l l y associated with the form-ing cross-wall. Method C. ca. X 3 2 , 7 5 0 . Figure 1*. A newly completed cross-wall. Note the very t h i n layer of septal wall material, the central lamella, 9 and the remains of the electron dense f r o n t a l edge material (arrow). Method C. ca. X 1+5,850. Figure 5 » A more advanced stage i n septal wall thickening. The two wall plates (pi) and the central lamella ( c l ) are indicated. Method C. ca. X k$,&50v Figure 6 . A mature septum. The two wall plates (pi) and the central lamella ( c l ) are indicated. Method C. ca. X 1*5,850. I I I . PLATE 3 Figure 7. A membrane complex (mc) which i a not c l e a r l y ^associated with septum formation. This complex seems to be continuous with the ER as well 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 in the upper l e f t . Method A. ca. X 35,700. Figure 8. A membrane complex (mc) associated with the form-ing septum. Method C. sea. X 35,700. Figure 9. An enlarged view of the membrane complex seen in Figure 8 showing the connection with the plasma membrane delimiting the septum and showing the structure of the complex membranes. Method C. ca. X 116,600. Figure 10. A l i g h t microscope view of a r e f r a c t i l e membrane complex (mc) associated with the formation of the f i r s t septum. The material was fi x e d in 2% glutaraldehyde and photographed unstained with phase optics, ca. X 3,000. I I I . PLATE Figure 11. Knee-joint formation. Note the protruberance on either side of the septum and the poorly defined walls regions in the zone between the two protruberances. Note the c l u s t e r i n g of ER and spherosome-like bodies (S) beneath the wall of the larger protuberance. A l i p i d body (L) i s also indicated. Method A. ca. X 22,500. Figure 12. A more advanced stage i n knee-joint formation. Note the c l u s t e r i n g of ER and shperosome-like bodies (S) in the bridge region, the apparent degradation of material i n the zone between the two bulges which are s t i l l separate. Ve s i c l e s (ve) are present i n the region of degradation and can also be seen i n the adjacent protoplast. Method A. ca. X 15,900. Figure 13• A completed knee-joint. Note that the plasma membrane has been reformed around the end og the now incomplete septum. The wall surrounding the bridge i s s t i l l thinner than the septal w a l l . Note the p o s i t i o n of the haploid nucleus (hN). Method C. ca. X 26,200. I I I . PLATE 5 Figure ll+a. The e a r l i e s t v i s i b l e Indication of bud formation [(bracketed region). A c e n t r a l , walless zone i s bounded by a modified zone of promycelial w a l l . Long ER elements end beneath the modified wall zone. Method C. ca. X 5 9 , 5 0 0 . Figure llj,b. An enlarged view of the bracketed wall region i n Figure llj-a. Method C. ca. X 81a,,600. Figure 1 5 . A more advanced stage i n bud formation. Note the appearance of new amorphous wall material covering the now protruding bud. A spherosome-like body (S) l i e s just behind the bud apex. Method C. ca. X 3 1 , 8 7 5 . Figure 16. A developing bud showing the p o s i t i o n of the hap-l o i d nucleus (hN). The bud now has a well devel-oped wall which thins towards the apex. The plasma membrane (PM) i s very d i s t i n c t . Method C. ca. X 3 1 , 8 7 5 . I I I . PLATE 6 Figure 17. A sporidium which has almost attained maximum length. Note the narrow neck region. Numerous mitachondria (M) and spherosome-like organelles (S) are present in young spo r i d i a . Method C. ca. X 19 , 5 0 0 . Figure 18. 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B a c t e r i o l . 72: 632-61+5. Ohal, E . and Rohlich, !>. 1966. Peculiar membrane config-urations af t e r f i x a t i o n i n glutaraldehyde. Acta. B i o l . Hung. 17: 65-73. Palade, G.E. and Claude, A. 191+9*. The nature of the Golgi apparatus I P a r a l l e l i s m between i n t e r c e l l u l a r myelin figures and Golgi apparatus i n somatic c e l l s . J . Morphology 85: 35-69. Palade, G.E. and Claude, A. 1 9 4 9 b . The nature of the Golgi apparatus II I d e n t i f i c a t i o n of the Golgi ap-paratus with a complex of myelin f i g u r e s . J . Morphology 8 5 : 7 1 - 1 0 3 . Prusso, D.C. and Wells, K. 1 9 6 7 . Sporobolomyoes roseus I U l t r a s t r u c t u r e . Mycologia 5 9 : 337 -34P. ' Revel, J . P 1 , Ito, S, and Pawcett, D.W. 1 9 5 8 . Electron micrographs of myelin figures of phospholipid simu-l a t i n g i n t r a c e l l u l a r membranes. J . Biophys. Biochem. C y t o l . 4: 4 9 5 - 4 9 6 . Rogers, H.£. 1 9 7 0 . 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,*34: 1 9 4 - 2 1 4 . Ryter, A. 1 9 6 8 . Association of the mucleus and the mem-brane of b a c t e r i a : a mophological study. B a c t e r i o l . Rev. 3 2 : 3 9 - 5 4 . Smith, D.G. and Marchant, R. 1 9 6 8 . L i p i d Inclusions in the vacuoles of Saocharomyces ce r e v i s i a e . Arch. Mikrobiol. 6 0 : 3 4 0 - 3 4 7 . Stein, C.W. 1 9 7 0 . An electron microscope study of a mycelial mutant of Us t i l a g o hordei (Pers.) Lagerh. M.Sc. Thesis. Univ 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 Isaac, P.K. 1 9 6 7 . An electron microscope study of intravacuolar bodies i n the uredia of wheat stem rust and i n hyphae of other fungi. Can. J . Bot. 4 5 : 1 4 7 3 - 1 4 7 8 . Wells, K. 1 9 6 4 a . The b a s i d i a of E x i d i a nucleata I U l t r a s t r u c t u r e . Mycologia 5 6 : 3 2 7 - 3 4 1 . Wells, K. 1 9 6 4 b . The b a s i d i a of E x i d i a nucleata II Development. Am. J . Bot. 5 1 : 3 6 0 - 3 7 0 . PART IV Nuclear D i v i s i o n with Special Emphasis on Meiosis TABLE OF CONTENTS Page ABSTRACT 118 INTRODUCTION 113 MATERIALS AND METHODS 120 Cultures and Culturing • 120 Light Microscopy 120 Electron Microscopy 123 OBSERVATIONS 123 Light Microscopy ••••••••• 123 Electron Microscopy • • ••••• 127 DISCUSSION 136 Nuclear Fores * 136 r Centriolar-kinetochore-equivalent 137 Cytoplasmic Microtubules .......••*. li+1 Chromosome Number •••••••••••••••••••••••• 11+2 The Model 11+7 BIBLIOGRAPHY 152 118 PART IV Nuclear D i v i s i o n With Special Emphasis on Meiosis ABSTRACT A study has been made of the ultrastructure of the nucleus and associated structures of the smut fungus, Ustilago hordel. with s p e c i a l emphasis on the p o s i t i o n and structure of the nuclear pores, the c e n t r i o l a r -kinelochore-equivalent, and cytoplasmic microtubules. E l e c -tron microscope observations of the meiotic and mitotic promy-c e l i a l d i v i s i o n s have been compared with l i g h t microscope observations of nuclear d i v i s i o n i n both Ustilago hordel and Ustilago k i l l e r i . Evidently c e r t a i n aspects of the meiotic 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 interpreted according to Brown and Stack's (1971) model f o r somatic nuclear d i v i s i o n i n some f u n g i . The problem of chrom-osome number i s discussed. INTRODUCTION The teliospore of Ustilago hordei i s the sexual spore, and contains the only d i p l o i d nucleus i n the l i f e c y cle. When the spore germinates the nucleus usually migrates into promycelium (metabasidium) i n the d i p l o i d state (Pt. I I ) , Three cycles of nuclear d i v i s i o n occur i n quick succession: the f i r s t two d i v i s i o n are the meiotic reduction d i v i s i o n and the meiotic equational d i v i s i o n , r espectively, and the l a s t i s the mitotic d i v i s i o n which gives r i s e to the f i r s t 119 haploid s p o r i d i a l n u c l e i . Chromosomes and spindles i n the nu c l e i of a smut species were f i r s t described by Harper i n 1893, and h i s r e s u l t s have since been substantiated by other workers. Most researchers have reported that the haploid chromosome number i n smuts i s two. Because of the d i f f i c u l t i e s i n preparation and i n t e r p r e t a t i o n the state of knowledge concerning the nuclear cytology of the smut fungi rests pre-c i s e l y where i t d i d i n 191+5. No photographic record e x i s t s , whether at the l i g h t or electron-microscope l e v e l and none of the d e t a i l s of nuclear 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 progress, genetic information has been accumulating r a p i d l y . Recently various Ustilago species have been used i n studies o f recombination (Holliday, 1961; Holliday I96I4.; Kozar 1969), o f ha p l o i d i z a t i o n (Day and Jones, 1969), of genetic complementation (Dinoor and Person, 1969) and of parasitism (Thomas and Person 1965; Halisky, 1965)* Furthermore several of these studies indicate that the haploid chromosome number of at l e a s t some smut species may be greater than two (Holliday, I96I4.; Day and Jones, 1969), Such studies lead to a renewed demand f o r more information, concerning the n u c l e i of the smut fu n g i . In her p r e s i d e n t i a l address to the B r i t i s h My c o l o g i c a l Society, i n 1939, Kathleen Sampson stated the case most suc c i n c t l y when she sa i d , ,,, i t would be unwise to become dazzled by the fascinations of smut genetics and to forget how few are the established facts concerning chromosome behaviour i n t h i s group of fungi. To obtain a convincing c y t o l o g i c a l picture of meiosis i n t h i s group i s no easy task ... 120 MATERIALS AND METHODS CULTURES AND CULTURING The wild-type s t r a i n s of Ustilago hordei are the same as those previously described i n part I, Included i n the light-microscope photographs, by way of comparison, are observations on the n u c l e i of a mycelial mutant of Ustilago  hordei (Stein, 1970) (Pigs, Pl-ij.), and on the meiotic and post-meiotic d i v i s i o n s i n Ustllago k o l l e r i ( W i l l i e ) (Pigs, Rows G-L), The mycelial mutant was prepared and photo-graphed by Dr. Jean Mayo, and the U, k o l l e r i samples by D. C. Wighton. A l l the material was cultured as described i n part I with the exception of the mycelial mutant which was grown on the surface of agar plates (Appendix B). LIGHT MICROSCOPY (see also Appendix C) Squash Preparations. - To prepare stained squash pre-parations a drop of medium containing the material was a i r -dried b r i e f l y on cover-slips, and f i x e d i n one of the follow-ing ways: a) f o r one hour i n ac e t i c - a l c o h o l (1:3) to which was added a few drops of chloroform. b) f o r 21*. hours i n BAC-fixative (Lu 1962). After f i x a t i o n , the material was stained by one of the following procedures: a) Peulgen (Darlington and La Cour 1962); U. hordei was hydrolyzed i n 1 N HC1 at 60° f o r 10-12 minutes: U. k o l l e r i was hydrolyzed i n 5 N HC1 at room temperature f o r 1 hour. The material was then stained f o r 30-I4.O minutes TABLE I: Summary of Light Microscope Techniques Species Stage Fixative Stain £• K o l l e i > i teliospores and acetic - alcohol Feulgen sporidia teliospores acetic - alcohol Feulgen Haematoxylin TJ. horde i sporidia spheroplasts and acetic - alcohol Haematoxylin mycelial mutant 122 and 8quashed i n 1+5 % acetic acid, b) Propiono-haematoxylin (Henderson and Lu, Method A, 1968): the material was hydrolyzed i n 1 N HC1 at 60° C for 10-12 minutes and stained f o r 1-2 minutes. A l l photographs were made from f r e s h l y prepared s l i d e s . Slides were subsequently made permanent with Euparol where desired. The procedures used f o r each c e l l type are outlined i n Table I. The c e l l depicted i n Figure 33a was f i x e d i n glutaralde-hyde and observed unstained with phase optics as described i n part I II Sections. - The material (U. hordei) was prepared ac-cording to electron microscope preparation B (Part I ) . Thick sections (0.25-0.50 u) were cut on a Sorval Porter-Blum MT-2 ultramicrotome and were stained with 1 % Toluidine blue i n 1 % borax. Spheroplaats. - Spheroplasts were prepared by Mr. M. Holmwood as follows: a monosporidial l i n e of U. hordei was evil, cultured i n modified complete broth containing 15 % dextrose and glusulase (Endolaboratories Inc.). At 22° C 18 - 21\. hours were required f o r spheroplasts to form. The material was then f i x e d i n acetic-aloohol and stained with Henderson and Lu's haematoxylin. Microscopy. - A l l the U. k o l l e r i material was observed with a L e i t z 3 V by i+V 1 bellows camera at f u l l extension. 1 2 3 A Zeiss photomicroscope was used i n a l l studies of U. hordel. Both microscopes were equipped with a $\\h mu interference f i l t e r . ELECTRON MICROSCOPY Germinating teliospores of Ustilago hordel were prepared for electron microscopy according to methods A, B and C as described i n part I. OBSERVATIONS LIGHT MICROSCOPY Dif f e r e n t stages of the l i f e cycle apparently react quite d i f f e r e n t l y to the various methods of s t a i n i n g . For studies of the promycelial d i v i s i o n s , f i x a t i o n i n a c e t i c -alcohol followed by Feulgen staining (Figs. Rows A-Lj_ gave greater chromosome contrast than did haematoxylin (Figs. Ml, NI and 0 1 ) with the techniques used. However, several haematoxylin-; preparations of meiosis i n Ustilago hordei are included to show that s i m i l a r configurations have been observed (Figs. Ml, NI and 0 1 ) . On the other hand, f i x a t i o n i n a cetic-alcohol followed by haematoxylin produced much superior r e s u l t s i n vegetative tissue, p a r t i c u l a r l y with the mycelial mutant. Haematoxylin, unlike Feulgen, also stains the nucleolus densely (Row P), allowing v i s u a l i z a t i o n of the chromatin-nucleolar r e l a t i o n s h i p . L i t t l e i s known about the early stages of meiotic pro-phase I whioh presumably occur within the forming, thick-walled t e l i o s p o r e . When the nucleus f i r s t passes into the promycelium the chromatin i s already highly contracted (Pigs. Rows A, B, and G). Normally t h i s l a t e prophase chromatin takes the form of two elongate densely-stained bodies l y i n g side by side and p a r a l l e l with the long axis of the promycelium. Occasionally, when the d i p l o i d nucleus i s Just entering the promycelium, r i n g - l i k e configurations occur (Pigs. A l , and 0), Such configurations probably represent the e a r l i e s t meiotic stages which can be seen i n the promy-celium. One c h a r a c t e r i s t i c of l a t e prophase I n u c l e i i s that the two chromatin bodies frequently appear to be closely associated at one or both ends. (Pigs. A2, A3, Alj., B3, Bij., Cif, E l , G2, Glj.). A second common feature i s that one or both bodies may be joined by a fin e Peulgen-positive thread to a small knob-like structure l y i n g against one o f the l a t e r a l promycelial walls (Pigs. A2, B l , Gif, M). In Ustilago hordei and Ustilago k o l l e r i no stage exists which v i s u a l l y compares with the metaphase plate seen at meiosis I i n higher plants and many fu n g i . The chromatin bodies contract as nuclear d i v i s i o n approaches ( i . e . compare A2, B2, and P2; also G2 and H2). As they a t t a i n t h e i r minimum d i p l o i d length (approximately 1.6 u) they begin to rotate and come to l i e at an angle to the l o n g i t u d i n a l axis of the metabasidium (Pig. E2). 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 discern-i b l e (Pig. E3). These l a s t two stages are infrequently seen and are probably short. When the chromatin bodies again become d i s t i n c t they l i e i n a well-defined anaphase 1 2 5 configuration (Pigs. Elf., H 3 - 4 . , I I , and 0 1 ) . Viewed with planar optics the Peulgen staining reaction i s considerably l e s s intense, supporting the common assumption that reduc-t i o n d i v i s i o n has occured ( i . e . compare H 2 and H 3 ) , The daughter haploid n u c l e i p u l l apart r a p i d l y and i n so doing the two chromatin bodies within each mucleus continue the contraction which was i n i t i a t e d i n the d i p l o i d state. The minimum length attained by the chromatin bodies of haploid n u c l e i i s 1 . 0 u (Pigs. P 2 , and J l - 2 ) . Occasionally the pair of anaphase chromatin bodies i n each of the separating n u c l e i appear to be joined at the "poleward" end (Pigs. E L L , and 1 2 ) . In Figures PI and 1 3 a chromatin t a i l i s lagging behind each of the chromatin pairs and projecting i n the d i r e c t i o n of i t s homologue. The t a i l s each terminate i n a conspicuous knob. During the anaphase migration the two chromatin bodies generally l i e paralle with the l o n g i t u d i n a l promycelial a x i s . Good l i g h t microscope pictures were r a r e l y obtained of the second and t h i r d promycelial 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 enough, with the exception of the reduced length of the two chromatin bodies, both these d i v i s i o n s progress i n a manner e s s e n t i a l l y ident-i c a l with the reduction d i v i s i o n . The meiotic equational d i v i s i o n i s represented here by Figures F 4 . and L (note the arrow marking the f i r s t septum) and the set of s e r i a l sections ( M 3 - 4 . , N3-I4., and 0 3 - 4 - ) . The f i r s t post-meiotic d i v i s i o n i s represented by Figure J 3 - 4 where the nucleus 126 l i e s i n the neck of the forming bud. Decondensed prophase II and III configurations (Fig. J3 -U-) a r e infrequent, suggesting either that chromatin decondensation between d i v i s i o n s i s "optional" or that i t occurs r a p i d l y . During prophase I I and II I the condensed chromatin bodies r a r e l y l i e p a r a l l e l to the long promycelial a x i s ; usually they are angled steeply across i t (Figs. Fq. and N3-lj.). L i t t l e d e t a i l i s di s c e r n i b l e i n the ti n y anaphase I I and II I n u c l e i but, i n general, they resemble anaphase I configuration ( F i g . L ) . By way o f comparison a few photographs of somatic mitotic d i v i s i o n s have been included (Rows P, Z and R). Figures Ql-lj. and Rl-2 represent 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 haploid 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 figures do occur they are located either i n the parental c e l l or, more often, i n the neck between the parent c e l l and the bud (Rl-2). Two prophase-like configurations (Ql-2 and Q3-I4.) and an anaphase (Rl-2) are shown. In a l l three c e l l s ,the two chromatin bodies are c l e a r l y v i s i b l e . Several attempts have been made to produce s p o r l d i a l sphero-plasts o f Ustilago hordei since removal of the wall affords considerable advantages f o r f i x i n g , s t a i n i n g and adequately squashing the preparations. Unfortunately most of the nuclei seen thus f a r i n spheroplast cultures seem to be l n a r e s t i n g state. R3 and Rl+ are photographs of two spheroplasts con-t a i n i n g prophase-like f i g u r e s . Again there are two d i s t i n c t chromatin bodies (Length = 1.0 u). A l l " preparations of the mycelial mutant were made with 127 haematoxylin* The nuclei and chromatin bodies of the mycelial mutant are exceptionally large, being comparable to those of the meiotic d i p l o i d state* At present the ploidy of the mycelial mutant i s unknown* The p o s s i b i l i t y exists that i t may be a true d i p l o i d ( J . Mayo, personal communication)* In any case the large size of the n u c l e i does allow clear observation of the two chromatin bodies. Haematoxylin has the advantage of staining the nucleolus as well as the chromatin; i n row P the conspicuous nucleolus, which l i e s at the end of the nucleus, i s indicated by arrows. One or both of the chromatin masses are attached to the nuc-l e o l u s . Figures PI, P2, and P3 are considered to be i n l a t e m i t o t i c prophase. A number of anomalous figures l i k e those shown i n Figure Plj. occur, and these have been i n t e r -preted as early somatic anaphase; the daughter chromatin bodies have begun to separate but the nucleolus which i s persistent, i s yet undivided. ELECTRON MICROSCOPY Ultrastructure of the Nucleus and Associated Structures. -The observations concerning the ul t r a s t r u c t u r e and a c t i v i t i e s of the nucleus and i t s associated structures are based on glutaraldehyde-osmium f i x e d material (Methods B and C) unless otherwise stated. Potassium permanganate leaches out and destroys both nucleic acids and microtubular structures which mainly compose the nuclear apparatus. Figure 5 i l l u s t r a t e s a haploid, early prophase II nucleus 128 i n a promycelium prepared by method C, The nuclear 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 possible the average double membrane width i s 300 A 0 (Range 210-1+00 Ao) with each of the unit membranes averaging 83 A 0 (Range £ 0 - 1 2 0 A 0 ) . After KMnOj^ - f i x a t i o n the respective measurements are 27£ A 0 (Range 220-31+0 A°) and 80 A 0. In spite of the tenuous nature of the nuclear membranes a f t e r g l u t a r -aldehyde-osmium f i x a t i o n the 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 outer surface of the envelope i s often accentuated by a conspicuous attachment o f r i b -osomes (Fig. £ ) , Second, the nucleoplasm i s l e s s dense than the cytoplasm (Figs. 7 , ll+, and 28). The nuclear envelope of Ustllago hordel i s frequently interrupted by nuclear pores. In material prepared by meth-ods A and B, these pores appear as simple "holes", allowing d i r e c t continuity 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 clear that the pores are occluded by electron-dense material. In cross-section, t h i s material i s rather amorphous (Fig. 2 3 ) , but i n face-view the c i r c u l a r pore region, which has an average diameter of 1 , 0 6 0 A° (Range: 950 1100 A°), i s highly structured (Fig. 6 a-c). A r i n g of granular or microtubular elements l i e s just inside the pore periphery and i s apparently embedded i n the electron-dense substance. One or two large granules (or microtubules) l i e i n the centre (Average diameter 190 -2U+ AO), Fine 129 f i b r i l l a r material (Average diameter = 1+2 A 0) radiates from the c e n t r a l element i n a l l directions towards the pore periphery. Pores of t h i s d escription are most prominent i n n u c l e i which are close to d i v i s i o n (Pig. 23 and Pt. V, P i g . 3&) and cytoplasmic microtubules are in e v i t a b l y present i n the peripheral cytoplasm near such pores (Pig. 6a-to, and 2 3 ) . The nucleolus i s usually the most conspicuous structure i n the nucleoplasm (Pigs. f>, 2 0 , 2 2 , 2 3 , 2l+, 2 7 - 9 , and 3 1 ) . In shape i t i s spherical to ovoid; i t s average diameter i s 1 . 0 6 u i n both d i p l o i d and haploid n u c l e i . In structure i t consists of loosely organized, electron-dense, granular material interspersed by patches of less dense nucleoplasm (Pigs. 22 and 2 3 ) . One often has the impression that It i s divided into two separate sections by a t h i n , l e s s -granular zone (Pig. 5, 2 3 , and 2 7 , arrows). The nucleolus i s always c l o s e l y appressed on one side to the inner surface of the nuclear envelope. As has been noted previously (Pts. I I and 111$, the nucleolus always l i e s at the poster-i o r end of the migrating nucleus ( i . e . during migration up • jfehe promycelium or into the bud, or during anaphase r e p u l -sion) . At the anterior end of the migrating nucleus i s 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 centriolar-4cenetochore-equivalent, (CKE). (Pigs. 7 and 2 2 ) . The CKE Is, s t r i c t l y speaking, extra-nuclear ( i . e . the main body of the CKE l i e s outside 130 the nuclear envelope); however, i t s structure and a c t i v i t i e s are intimately associated with nuclear events. Migrating n u c l e i are s l i g h t l y elongate (Pig. 7 ) . When the migration has ceased, the n u c l e i round-up insofar as i s possible (Pig. 2 3 ) , and the CKE assume a po s i t i o n c l o s e l y appressed to the outer surface of the nuclear envelope and d i r e c t l y opposite to the nucleolus. Usually i n non-dividing n u c l e i , the CKE l i e s i n a cytoplasmic well protruding into the nucleoplasm. During prophase I, this w ell i s shallow (Pig. 3 , 2 2 , and 2 3 ) , but i t increases l n depth during prophase II and III (Fig. 7* and 2 0 ) corresponding with an increase i n the size of the CKE. The w e l l seems to be a r e l a t i v e l y stable part of prophase nuclear structure whether or not the CKE l i e s within i t . Figures 17a-b and l 8 a-b, i l l u s t r a t e respectively a nuclear well without a CKE and a nuclear well with a CKE. Usually the CKE i s surrounded by a halo of low density cytoplasm from which other cytoplasmic organelles i n c l u -ding ribosomes are excluded. The adjacent nuclear membrane remains Intact (Fig. 3 and 1 1 ) . On i t s inside surface i s a cap of electron dense nucleoplasmic material ( i . e . chromatin) which i s associated with the presence of the c e n t r i o l a r -kinetochore-equivalent (Figs. 7 , 9 , 1 8 b , and 2 0 ) . In Figure 7 , the chromatin cap i s c l e a r l y b i p a r t i t e . Notably, t h i s i s the only point on the nuclear envelope to which the chromatin can be shown to attach. Structures resembling the CKE have never been observed i n ungerminated spores but whether t h i s i s due to the actual 131' absence of She body or to the f a c t that i t i s masked by the density of the cytoplasm i s unknown. A single c e n t r i o l a r -Mnetbchore-equivalent i s located at the anterior of the d i p l o i d nucleus when i t f i r s t begins to migrate up the promy-— celium. #Ft. I I , P i g . 5 ) In the cross-section, the CKE i s oval, ( 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 , or 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 . Its maximum siae i n prophase I i s approximately 1 6 5 by 9 5 mu (Pig. 8 ) but during prophase I I and I I I , i t increases to as much as 37© by 370 mu (Figg 9 ) . This increase i s r e a d i l y observed by comparing Figures 8 and 9 which are at approximately the same magnification. S t r u c t u a l l y , the centrlolar-^kenetochore- — equivalent consists of a tangle of f i n e f i b r i l s with an average diameter of 28.1* A 0 (Range: 3 : 8 - ^ 9 A 0) embedded i n an amorphous electron-dense matrix (Figs § - 1 3 ) * In prophase II and I I I , i t often develops what appears to be a dense elongated core that follows the contours of the outer s u r -face of the body on the side opposite to the nucleus (Figs. 9 and 1 2 ) . Occasionally, as i n Figures 10 and 1 1 , there are two d i s t i n c t zones i n the CKE, a dense f i b r i l l a r zone on one side and a region i n which the f i b r i l s are "spun-out" on the other. This e f f e c t may be produced when the section passes through, and i s i n the same plane as the dense core/ In promycelia of Ustilago hordei, each prophase I, I I , and III nucleus i s associated with a single small c e n t r i o l a r -kinetochore-equivalent .tsuObviously f o r t h i s to be so the centriolaj?-kinet ©chore-equivalent must r e p l i c a t e once per nuclear d i v i s i o n . Occasionally elongate f i b r i l l a r structures 132 have been observed l y i n g p a r a l l e l with the nuclear envelope (Figs. I4.-I6) and these have been interpreted as d i v i d i n g centriolar-klnetochore-equivalents. In Figure 15?, f i n e f i b r i l s , s i m i l a r i n structure to those constituting the CEE, seem to connect the d i v i d i n g CKE to the nuolear en-velope. During l a t e prophase, cytoplasmic microtubules develop i n association with the nucleus and the CKE. The tubules have an average diameter of 244 A 0 (Range: 2 0 0 - 2 5 0 A°) with an electron transparent central core (Average diameter = 102 A 0 ) . For the most part these microtubules l i e i n the lon g i t u d i n a l axis of the promycelium or at a s l i g h t angle to i t (Figs. 1 9 , 20 and 23 ) 5 and radiate from the v i c i n i t y o f the CKE (Figs. 19 and 2 0 ) . The tubules do not orig 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 globular electron-dense structures (mtoc) i n the v i c i n i t y of the CKE (Figs. 2 1 ) . Other micro-tubules end i n s i m i l a r electron dense structures (Fig. 2 0 ) . Nuclear D i v i s i o n . - Figures 22 to 33 are arranged sequentially to demonstrate the main stages of the promy-c e l i a l d i v i s i o n s according to the author's i n t e r p r e t a t i o n . Figures 22 to 26 i l l u s t r a t e meiosis I ( i . e. reduction d i v i -sion) and Figures 27 to 33* meiosis I I ( i . e. equational d i -v i s i o n ) . The sequence of 27 to 33 i s a oomposite of observa-tions made from d i v i s i o n I I and II 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 with the exception that when d i v i s i o n I I I I occurs i n the parent c e l l the nulear axis ( i . e . an Imaginary l i n e drawn through the two poles) 133, l i e s at an angle of about 45° to the longitudinal promycelial axis while the nuclear axis of division II lies roughly par-a l l e l to the longitudinal c e l l axis. Unless the nuclear axis can be determined, septa counted, or a bud is visible i t is impossible to distinguish division II and III nuclei. The diploid fusion nucleus in Figure 22 is about 4 . 2 5 u by 1.70 p. The arrow indicates the direction 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 directly opposite to the nucleolus and about one-third of the nuclear length behind the anterior nuclear tip is the centriolar-kinetochore-equivalent. Between the nucleolus and the CKE is a band of nucleoplasm of increased density ( i . e. chromatin) about 0.7 p wide and 1 .3 p long. This nucliolar-ehromatin-JC'KE configuration is characteristic of the migrating prophase I nucleus. It is represented schematically in Figure 35a* When the nucleus has ceased Its forward motion i t shortens and rounds-up insofar as is possible. In Figure 23 the CKE has shifted i t s position posteriorly with respect to the nucleus so that i t now l i e s midway along the nuclear length and at the same time the nucleolus<shas begun to move from a posterior to a lateral position. The impression is that the entire nucleus is rDe-feating. The nucleolar-CKE distance is shortened to 0.6 p. Such late prophase I nuclei are associated with microtubules lying parallel with the length of the nucleus ( i . e. with the promycelial axis), on the side occupied by the CKE. Figure 23 is represented acheraatically in Figure 3 5 b . No data are available on the d i v i s i o n of the c e n t r i o l a r -kinetochore-equivalent at reduction d i v i s i o n . In the next c h a r a c t e r i s t i c stage that i s observed the nucleus elongates and constricts i n the center, becoming dumbbell shaped ( F i g . 21+). In Figure 23 the two bulbous ends of the nucleus have v i r t u a l l y separated. The nucleolus has divided and one of the CKE's i s v i s i b l e on the r i g h t side of the lower daughter nucleus. Figure 2l\ i s represented diagrammatically i n Figure 35>d. As the two daughter n u c l e i p u l l apart the nuclear mem-brane undergoes a very short period of p a r t i a l breakdown (Figures 2$ and 26). In Figure 26 the centriolar-kinetochore-equivalent has 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 of the chromatin-nucleolar material i n Figures 2$ and 26. After KMnO^ f i x a t i o n the nucleic a c i d containing material ( I . e. nucleolus and chromatin) i s represented by electron-transparent patches (Figs. 2$ and 3 3)• Figure 7 I l l u s t r a t e s a haploid daughter nucleus i n repulsion. The nuclear envelope has been reconstituted and the centriplar - T-kinetochore-equivalent i s i n the lead p o s i t i o n . IhYiFigure 27 the haploid daughter nucleus ( Average diameter - 1.8 u) has come to rest and has presumably entered prophase I I . The CKE l i e s i n a c h a r a c t e r i s t i c postion d i r e c t l y opposite the nucleolus. VA line.drawn through the . CKE and b i s e c t i n g the nucleolus would cross the l o n g i t u d i n a l promycelial axis almost perpendicularly. Figure £8 i l l u s t r a t e s a l a t e prophase II nucleus just p r i o r to CKE d i v i s i o n . The section has passed d i r e c t l y through the centre of the nucleus 1 3 5 i n the same plasne as the chromatin which i s condensed and c l e a r l y v i s i b l e . One chromatin body d i r e c t l y joins the CKE to the nucleolus, and the second c o l l s v e r t i c a l l y upwards i n Figure 28 (arrows). Both o f the chromatin bodies are joined d i r e c t l y to the CKE by a p a i r of f i b r i l s which have an average diameter of 8 0 A 0 (Range: 7 8 - 8 3 A 0 ) . Figure 28 i s represented schematically l n Figure 35®. Shortly a f t e r t h i s stage the centriolar-kinetochore-equivalent moves out o f the nuclear well, elongates and divides as described previously (Figs, 29 and 3 5 f ) . In Figure 30 the daughter CKE'a are migrating around the nuclear envelope which i s outlined by the presence of nuclear pores, and the nucleus has already begun to elongate i n the d i r e c t i o n of the promy-celiaJl a xis. Figure 30 i s represented schematically i n Figure 3 5 g . Figure 31 shows the two CKE's at the poles of a d i v i s i o n I I I nucleus ( i . e. note the p o s i t i o n of the nuclear axis with respec* to the promycelial a x i s ) . The nucleolus 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 side of the nuclear exis and midway between the poles (Fig. 3 5 M . As i n Meiosis I the nuclear envelope p a r t i a l l y breaks down and the daughter n u c l e i move apart (Figs. 32 and 3 5 k ) . In Figure 32 microtubules can be seen passing into the open daughter nucleus. Presumably the microtubules represent part of the d i v i s i o n spindle which i n inadequately preserved by the techniques employed. Under the l i g h t microscope using phase contrast o p t i c s , t h i s spindle appears i n glutaraldehyde f i x e d material 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 Callan and Torolin (1950) f i r s t discovered "pores" or "holes" i n the nuclear envelope of amphibian oocyte n u c l e i i t has become increasingly clear that these regions are not just simple "spaces" allowing the free d i f -f usion of substances between the nucleus and cytoplasm (Feldherr, 1 9 6 5 ) . When properly prepared the nuclear pores are f i l l e d with a v a r i e t y of s t r u c t u r a l components embedded i n an electron opaque substance. These pores seem to be ubiquitous among eukaryotic organisms and a large volume of l i t e r a t u r e pertaining to t h e i r chemical and st r u c t u r a l properties now exists (Abelson and Smith 1970; prank and Scheer, 1970; Koshiba et a l . , 1970; Yoo and Hayley, 1967). There are many reports of nuclear pores i n fungi (Bracker, I967) but i n most cases they appear as simple "holes" i n the nuclear envelope. In Ustilago hordei preparatory methods A and B (Pt I) evidently destroy the complex pore apparatus which i s so prominent i n tissue 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-ations on the nuclear pores o f this fungus agree with the findings i n other plants and animals i n respect of the pre-sence, appearance, and size of the pore regions, peripheral granules, c e n t r a l granules, and r a d i a t i n g filaments. A complex pore structure of t h i s type has also been described i n Cop- rinus lagopus (Lu, 1965) and Ascobolus stercorarius (Wells, 1970). 137 In Ustilago hordel there i a an increase i n the number and prominence of nuclear pores as the nu c l e i approach d i v i s i o n . GENTRIOLAR-KENETOCHORE -EQUIVALENT Although true centrioles have been found i n phycomycetes with a motile;stage i n the l i f e cycle (Bracker 1967) they do not occur among da oomycetes and basidiomycetes. Yet. when appropriately stained t i s s u e ' i s observed i n the l i g h t microscope t i n y s p h e r i c a l , elongate, or rectangular bodies frequently l i e at the poles of d i v i d i n g fungal n u c l e i and i f these bodies are observed at various stages of the d i v i s i o n cycle they behave i n a " c e n t r i o l a r " manner. Under the electron microscope these c e n t r i o l e - l i k e structures con-s i s t of semi-electron dense, usually amorphous, material. During d i v i s i o n they l i e at the poles of the spindle micro-tubules and are the hub of the a s t r a l rays. Wells (1970) has recently reviewed the l i t e r a t u r e pertaining to these bodies and nuclear d i v i s i o n i n the Ascomycetes: notable , among these studies are those of Beckett and Wilson (1968) * n Podospora anserina, Robinow and Marak (1966) i n Sacchar--omyces cerevisiae, Schrantz (1967) i n Pustularia cupularls , Wells (1970) i n Ascobolus stercorarlus and Z i c k l e r (1970) i n two species of Ascobolus and two species of Podospora. Probably because of the d i f f i c u l t y of f i x i n g basidiomycetous fungi with appropriate E. M. techniques ( i . e. glutaraldehyde smium) l i t t l e information i s available concerning the "centriolar-equivalent" i n these f u n g i . Girbardt (1968), 138 Lu (1965 and 1967b), and Motta (1967 and 1969) have described structures which appear to be the " c e n t r i o l a r -equivalent" i n Polystiotus v e r s i c o l o r , Coprinus 1agopus, and A r m i l l a r i a mellea r e s p e c t i v e l y . In Ustilago hordei the body which l i e s at the poles of the d i v i s i o n spindle has been termed the centriolar-kinetochore-equivalent (CKE) for reasons which w i l l be subsequently d i s -cussed. The CKE of Ustilago hordei c l o s e l y resembles i n shape and size the "centriolar-equivalent" i n Coprinus lagopus and A r m i l l a r i a melleaj that o f Polystictus 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 both Ustilago hordei and Coprinus lagopus (Lu, 1967b) t h i s body has a f i b r i l l a r sub-structure. The diameter of the f i b r i l s (Average diameter = 28.4. A°) and the probable Peulgen-positive nature of the CKE i n t h i s smut fungus suggest that the centriolar-kinetochore-equivalent may be composed pa r t l y of DNA. Why the CKE should increase i n size during prophase of the second meidtic d i v i s i o n and f i r s t post-meiotic mitotic d i v i s i o n i s , at present, unknown; but the phenomenon i s widespread among fungi (Berkson, 1970; Lu, 1967a; Olive, 1965; Singleton, 1953; Z i c k l e r , 1970). As i n most other basidiomycetes and ascomy-cetes studied u l t r a s t r u e t u r a l l y , the "centriolar-kinetochore-equivalent" of Ustilago hordei i s c l o s e l y associated with the outer surface of the nuclear envelope at a l l times, and except during the very b r i e f period of d i v i s i o n i t s e l f , i t l i e s within a cytoplasmic well protruding into the nucleus (Girbardt, 1968; Lu, 1965; Motta, 1969; Robinow and Marak, 1966; Wells, 1970; Z i c k l e r , 1970). 139 In Ustilago an organelle-free zone surrounds the CKE. This i s also 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 (Girbardt, 1968) and Ascobolus steroorarius (Wells, 1970) and, as pointed out by Wells i s reminiscent of the robosome-free zone which f r e -quently surrounds true c e n t r i o l e s . The large variety of names which have been applied to the bodies that act as c e n t r i o l a r equivalents during fungal nuclear d i v i s i o n i s at best confusing. Wells (1970) has recently reviewed the terminology as i t has been applied to ascomycetes. The most commonly used terms are " c e n t r i o l e " (Lu, 1967a), "centrosome" (Lu, 1965; Beckett sand Wilson, 1968; Z i c k l e r , 1970) and " c e n t r i o l a r plaque" (Robinow and Marak, 1966; Wells, 1970). However, as pointed out by Pickett-Heaps (1969c) i n an a r t i c l e on the evolution o f the m i t o t i c apparatus, the use of terms that suggest true centrioles when applied to these fungal structures may be misleading. Recently Girbardt (1968) has coined the phrase "kinetochore-equivalent" as a more appropriate term i n P o l y s t i c t u s v e r s i c o l o r . In t h i s fungus the chromatin appears to be d i r e c t l y attaohed to t h i s body which Is apparently involved i n d i r e c t i n g independent motions of both the nucleus as a whole, and the chromatin within. The "chromosomes" of Polystictus do not act as independent e n t i t i e s but pass through the d i v i s i o n cycle as i f they are c o l l e c t i v e l y attached to t h i s structure which acts as a common kine to chore. Hence the term "kinetochore-equivalent;!' This i s not a new concept. I t has long been observed with 11+0 the l i g h t microscope that l n many fungi the chromatin Is attached by a fine filament to a small knob at the edge of 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 nuclear d i v i s i o n , either meiotic (Berkson and B r i t t o n , 1969; Berkson, 1970; Olive, 19U9) or mitotic (Girbardt, 1968; Lu, 1967a, Marks, 1965; M i t c h e l l and McKeen, 1970). During d i v i s i o n , as i n P o l y s t i c t u s , the "chromosomes" of many of these fungi do not form a c l a s s i c a l metaphase plate but behave as i f controlled s o l e l y by the d i v i d i n g knob to which they are joined; the knob acting as both a c o l l e c t i v e kinetochore and a centriolar-equivalent. The meiotic "chromosomes" of Ustilago hordei are also attached to a small Peulgen-positive knob l y i n g against the l a t e r a l promycelial wall (Figs. A2, B l , Gl+, and Ml) and t h i s association persists up to and perhaps during d i v i s i o n (Figs. M3-1+, N3-1+, and 03-1+). Presumably the knob i s either the c e n t r i o l a r - k i n e -tochore-equivalent i t s e l f or the condensed chromatin cap that l i e s just beneath the CKE (Fig. 7), This association has been observed u l t r a s t r u c t u r a l l y at telophase (Fig. 7), prophase (Fig. 22), and l a t e prophase (F i g . 28), but not yet at metaphase. The p o s s i b i l i t y that a single structure may at once f u l f i l the functions of both centriole and kinetochore i s not as unusual as i t may at f i r s t appear. F i r s t both structures are known to give r i s e d i r e c t l y or i n d i r e c t l y to microtubules. Second, some evidence exists that 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 into c e n t r i o l e s ( P o l l i s t e r and P o l l i s t e r , 1943). Third, the "kinetochore-equivalent" i n Ustilago  hordei resembles very c l o s e l y i n structure the fkinetochore" of some animal c e l l s (Brinkley, 1966) since i t i s composed of an electron dense a x i a l filament (Figs. 9, 12, and 1^) surrounded hy f i n e f i b r i l l a r material. CYTOPLASMIC MICROTUBULES In size and appearance the microtubules of Ustilago  hordei are s i m i l a r to those of higher plants (Newcombe, 1968). The p o s i t i o n and late prophase development of these aggregations are reminiscent of the nuclear-associated "prophase bands" of the cytoplasmic microtubules which herald the onset of nuclear d i v i s i o n i n many plants (Burgess and Northcote, 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 mitotic spindle (Burgess, 1970a) or they may be a rese r v o i r of presynthesized microtubules which move intact into the spindle (PIckett-Heaps, 1969a). Most of the cytoplasmic microtubules of Ustilago do not radiate d i r e c t l y from the CKE (Pigs. 19 and 20) but seem to terminate i n globular, electron-dense regions of cytoplasm (Pig. 21). Similar densities (fatbe) often occur at the, d i s t a l inds of other tubules (Pig. 20). Recent evidence suggests that these dense regions of cytoplasm which occur i n association with microtubules i n many plants (Burgess, 1970b) and animals (Tilney and Goddard, 1970) are the actual s i t e s of microtubules synthesis ( i . e. microtubule-organizing centres). U|2 CHROMOSOME NUMBER: When Harper (1898) f i r s t described chromosomes and spindles i n n u c l e i of Ustilago aoabiosae. he stated that the chromosome number was eight to ten for t h i s species. With the exception of Dickinson ( 1 9 3 1 ) , however, subsequent c y t o l o g i c a l studies among the U s t i l a g i n a l e s have not sup-ported t h i s high chromosome count. Kharbush ( 1 9 2 7 ) , Wang, (193U) and Hirschhorn (191+5) whose investigations c o l l e c -t i v e l y dealt with f i v e d i f f e r e n t genera including eleven Ustllago species concluded that the haploid number i s two. These chromosome counts were made mainly at meiosis, but i n f i v e species at mitosis as w e l l . This count has also been confirmed by Rawitscher ( 1 9 2 2 ) , Wang (1914-3), Das (191+9), and Person and Wighton (I96I4.). Our observations i n Ustilago  hordei and Ustilago k o l l e r i indicate that two elongate chromatin bodies are present i n meiotic and mitotic prophase n u c l e i up to, and including d i v i s i o n , and that during ana-phase two chromatin bodies constitute each daughter comple-ment. However, considering current investigations of fungal cytology, the presence of two chromatin bodies does not necessarily indicate that n = 2J Undoubtedly i n the n a j o r i t y of fung i , meiosis occurs c l a s s i c a l l y (Olive, 1 9 6 5 ; Lu, 1 9 6 5 ; Lu, 19.67a and b; Singleton, 1 9 5 3 ; Westergaard and von Wettstein, 1 9 6 5 ) . The chromosomes pass through the leptotene, zygotene, pachytene, diplotene, and diakinesis prophase stages, l i n e up on a t y p i c a l metaphase plate, p u l l apart on a normal spindle and regroup into daughter haploid n u c l e i at telophase. However, i n Ustilago hordei, and several other fungi^ meiosis apparently does not conform to t h i s t r a d i t i o n -a l pattern. In these species meiosis and mitosis look ident-i c a l except that the quantity of prophase chromatin i s great-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 figures i n these species strongly resemble the d i v i s i o n figures i n the many fungi (ascomycetes and b a s i d -iomycetes) which demonstrate a normal meiotic pattern but an abnormal mitotic one. Robinow and Caten (1969) have recently reviewed the l i t e r a t u r e pertaining to t h i s l a t t e r class of fungi which includes such well-studied species as Aspergillus nldulans (Robinow and Caten, 1969), Schizophyllum  commune (Bakerspiegel, 1959), Neurospora crassa (Namboodiri and Lowry, 1967), and Fusarium oxysporum (Robinow, personal communication). These apparently unusual d i v i s i o n figures a l l have the following 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 chrom-osome-like bodies l y i n g p a r a l l e l with or at a s l i g h t angle to the longitudinal c e l l axis i n prophase n u c l e i , anaphase and telophase n u c l e i . 2. At a l l observable stages, each of the two chromosome—Like bodies i s joined by a f i n e filament to a knob-like structure which seems to precede the nucleus. This knob-like structure divides, gives m r i s e to the d i v i s i o n spindle, and generally behaves i n a manner suggesting that i t i s the c e n t r i o l a r -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-osis i s not, the meiotic complement consists of more than two chromosome p a i r s . Por example, i n Aspergillus nidulans eight bivalents 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 this number agrees with the number of linkage groups established g e n e t i c a l l y (Kafer> 1958)* To emphasize the point even more dramatically, somatic n u c l e i i n a d i p l o i d variety of A. nidulans which must have sixteen chromosomes, only two chromatin bodies are observable and these look just l i k e those of the haploid (Robinow and Caten, 1968). Clearly the presence of two 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 reason to conclude that n = 21 That unusual nuclear configurations occur i n many fungi i s a f a c t . The problem that aofifronts cytologists i s to determine the significance of t h i s f a c t . Two pos-s i b i l i t i e s e x i s t : either these configurations r e f l e c t mere-the inadequacy of available c y t o l o g i c a l techniques as applied to fungal n u c l e i or else they r e s u l t from genuine behavor-i a l v a r i a t i o n . Uhfortunately, fungal n u c l e i are f o r the most part very small; they are usually constrained i n very narrow tubes with thick wa11s .that;defy squashing, and the chromatin frequently f a i l s to s t a i n with the usual chromosome stains (11 e, Feulgen, haematoxylin, methyl-green pyronine). These factors have, very j u s t i f i a b l y , l e d fungal cytologists to be highly s k e p t i c a l of these unusual configurations. However, recent evidence suggests that the 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 fungi i n which unusual chromosomal c o n f i g -urations are observed these configurations occur consistently and they have t h e i r own equally consistent c h a r a c t e r i s t i c s . 2. These configurations arethe same i n both sectioned material (Pigs. M3-U, N3-J4, and 03-Aj) and squashed material and therefore probably do not r e s u l t from squashing the material i n a confined space. 3. The yeast, Saccharomyoes c e r v i s i a e , has at least eighteen linkage groups and presumably, therefore, eighteen chromosomes. Protoplasts of yeast are r e a d i l y produced, eliminating the problem of the wall and yet no amount of squashing w i l l further resolve the two chromatin bodies commonly seen at anaphase. A s i m i l a r s i t u a t i o n exists i n Ustilago  hordei (Figs. R3, Rlj). Therefore the problem does not seem,to be e n t i r e l y on of constraint. 1|. As has been discussed previously, considerable, l i g h t microscope evidence indicates that i n these fungi the two elongate chromosomal bodies are attached perm-anently to the " c e n t r i o l a r equivalent". Although s t i l l inconclusive, electron microscopic observations i n Schizophyllum commune (Girbardt, 1968), Saccharo- myoes pombe (Robinow, personal communication) and Ustilago hordei (Pigs. 7, 22, and 28) support t h i s hypothesis. Clearly, i f i t i s true that the "chromo-somes" are permanently attached to the c e n t r i o l a r -kinetochore-equivalent d i v i s i o n could not conceivably occur i n the c l a s s i c a l fashion. If the usual chromosome configuration i n species l i k e Aspergillus nidulans, Neurospora crassa, Schizophyllum commune, and Fusarium oxysporum are not due to technical inadequacies then one i s forced to conclude that the chromosomes v i s i b l e at meiosis become linked together i n some manner i n two groups i n somatic n u c l e i . The class of fungi which demonstrate the double chromatin-body configuration at meiosis as well as mitosis i s quite small to date. Included i n the class are certain heterobasidiomycetes such as the smuts Ustilago hordei and Ustilago k o l l e r i , the rusts Puecinia lobata (Berkson, 1970), and Coleosporlum vernonia (Olive, 19U9) and the ceratobasldiaceous heterobasidiomycete Ceratobasidium practlcolum (Saksena, 1961). Also included are several species of the horaobasidio-mycete Marasmius (Duncan and MacDonald, 1965) and the asco-mycete Saccharomyces cerevisiae (Robinow, personal commun-i c a t i o n ) . At present there does not seem to be any conclusive way i n which to determine c y t o l o g i c a l l y the true chromosome number of these fungi, p a r t i c u l a r l y i n species l i k e Ustilago  hordei and Ustilago k o l l e r i where the early meiotic prophase stages have not been seen. 247 Recent gentle studies indicate that some Ustllago species may have more than two linkage groups. Holliday ( I 9 6 I 4 ) has presented evidence f o r the existence of f i v e chromosomal arms i n Ustilago maydis and Day and J o n e s ( 1 9 6 9 ) have concluded from haploidization studies that Ustllago  violacea has at least ten chromosome per genome. Unfortun-ately the c y t o l o g i c a l data i n these species i s not a v a i l -able f o r comparison with Ustilago hordei. In spite of the analysis of a r e l a t i v e l y large volume of recombination data a r i s i n g out of many di f f e r e n t crosses i n Ustilago hordei only one linkage group has been found (Person, unpublished), supporting the suggestion that Ustilago hordei may have a low chromosome numbers. Interestingly only one centromere has been mapped i n both Ustilago hordei and Ustilago violacae. This i s i n agreement with the p o s s i b i l i t y that the "chromo-somes" share a common kinetochore. THE MODEL Brown and Stack ( 1 9 7 1 ) have recently formulated an alternative theory of somatic d i v i s i o n on fungi, ( s p e c i f i c a l l y ] Aspergillus mldulans), s t a r t i n g from the assumption that the double chromatin body configuration r e f l e c t s the actual chromosomal behaviour pattern. Their model, depicted i n Figure 3U with the kind permission of the authors, i s designed to s a t i f y two c r i t e r i a : 1 . It must incorporate the c h a r a c t e r i s t i c features of the unusual nuclear configurations as they have been observed i n the l i g h t and electron 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 Ustilago hordei SEPTUM i ) the chromosomes are joined end-to-end i n two groups, i i ) the two chromatin bodies are permanently joined to a common kinetochore-equivalent, i i i ) the kinetochore also acts as the c e n t r i o l a r -equivalent and gives r i s e to the spindle, i v ) at d i v i s i o n the chromosomes do not form a meta-phase plate but remain stretched out i n two l i n e s . v) the chromosomes reach t h e i r maximum degree of contraction at telophase. 2. The model must account f o r the f a c t that during somatic d i v i s i o n i n the hyphae of Aspergillus nidulans s i s t e r chromatid segregation and nuclear migration are non-random (Rosenberger and Kessel, 1968), In t h e i r most i n t r i g u i n g experiment Rosenberger and Kessel found t h e i r r e s u l t s were compatible with the view that chromatids containing DNA strands of the same age segregate as a uni t during mitosis. Figure 35 depicts schematically meiosis i n Ustilago hordei as i t would be interpreted according to Brown and Stack's model. The o r i g i n a l model has been modified only insofar as necessary to make i t compatible with a meiotic s i t u a t i o n and with the d e t a i l s of structure and function i n the par-t i c u l a r fungus. For example Figure 35 takes into account both nuclear r o t a t i o n and the f a c t that at l e a s t one of the chromatin bodies of Ustilago hordei d i r e c t l y l i n k s the centriolar-kinetochore-equivalent to the nucleolus. How-ever neither of these situations i s l i k e l y to be unique to Ustilago. Nuclear r o t a t i o n i s a well-known concomitant of nuclear d i v i s i o n of many fungi (Aist and Wilson, 1968) and evidence i n both Schizophyllum commune (Girbardt, 1968) and Saccharomyce3 pombe (Robinow, personal communication) suggests that the centriolar-kinetochore-equivalent and nucleolus are joined via at least one of the chromatin bodies The diagrammatic representation ( F i g . 35>) i s supported by Figures 22 and 33a which depict the major stages i n the d i v i s i o n process. At present the author considers the data i n Ustilago hordei to be more compatible with Brown and Stack's model f o r fungal nuclear d i v i s i o n that with the c l a s s i c a l pattern. 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 material i s ce 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 nuclear d i v i s i o n i n Aspergillus nidulans was conceived by Dr. R. M. Brown (University of Texas at Austin, U. S. A.) and was based on the previous work of many others. The author i s indebted to Dr. Brown f o r h i s kind permission to make use of t h i s model i n the preparation of t h i s t h e s i s . Thanks i s also due to Dr. C. Robinow (University of Western Ontario, Canada) whose kindly c r i t i c i s m s , information, and advice have contributed greatly to the ideas expressed i n part TV. IV. PLATE 1 Meiotic d i v i s i o n s following teliospore germination i n Ustilago hordei. Feulgen stained. Phase optics. Figures A l - lj., B l - I;, C l . Mid-prophase I. Figures C 2 I ; , DI - l|, E l . Late prophase I. Figure E2, Late prophase I. Figures E3 — lj., FI — 3« Anaphase I. Figure Fl*. Metaphase I I . Note: i n plates 1 - 3 (a) the lon g i t u d i n a l axes of the promyce-l i a are h o r i z o n t a l . (b) one scale d i v i s i o n represents 1 mics* ron. (Total = 10 u.) ca. X 5,000. 1 2 3 4 *> 4 • J I «•» ^ — • -»^ or* •i) IV. PLATE 2 Meiotic and mitotic d i v i s i o n s succeeding teliospore germination i n Ustilago k o l l e r i . Feulgen stained. Planar optics. Figures Gl - 4 . Mid-prophase I. Figures HI - 2 . Late prophase I. Figures H 3 - 4 , II - 4 , J l - 2 , K. Anaphase I. Figure L. Anaphase IIX (arrow indicates f i r s t septum). Figure J3 - 4 . Mitosis I I I . Note: ca. X 5,000. I M ' i i i i i i I IV. PLATE 3 Rows M, N, and 0 . Meiotic d i v i s i o n s succeeding teliospore germination i n Ustilago hordei. Planar optics. Group a: Haematoxylin. Figure Ml. Mid-prophase Figure N l . Late prophase I Figure 0 1 . Anaphase I Group b: s e r i a l sections stained in T. blue. Figures M3 - Lt, N3 - l\ and 03 - 4 . Metaphase I I . Row P. Meiotic mycelial d i v i s i o n s (arrows indicate nucleolus). Planar optics. Figures PI - 3 - Mid-prophase. Figure Pii. Metaphase. Rows Q, and R. Mitotic s p o r i d i a l and spheroplast d i v i s i o n . Planar opt ic s. Figure Q l -2. Prophase. Figure Q3 - k» Metaphase. Figure R l - 2. Anaphase. Figure R3 - k» Metaphase. Note: ca. X 5 , 0 0 0 . IV. PLATE 1+ Figure $, A general view of a haploid nucleus (hN) showing the nucleolus (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 to one side of the nucleus and against the nuclear envelope (NE). The arrow indicates: a t h i n e l e c -tron transparent zone apparently d i v i d i n g the nu-cleolus into two. Note the attachment of ribosomes to the outer surface of the nuclear envelope. Method G. ca. X 1+0,600. Figure 6 a . Nuclear pores in cross-section. Note the micro-tubule i n the upper l e f t . Method C. ca. X 3 7 , 6 5 0 . f Figure 6 b . A lo n g i t u d i n a l section through a promycelium show-ing the p o s i t i o n of the nuclear pore region depict-ed i n Figure 6 a . Method C. ca. X 1+0,600. Figure 6 c . An enlarged view of two of the nuclear pores from figure 6 a . Note the central granules and the f i n e filaments r a d i a t i n g from the c e n t r a l granule to a r i n g of peripheral granules. Method C. ca. X 9 5 , 2 0 0 . Note: the black scale represents only 0 . 1 u. IV. PLATE 5 Figure 7. Figure 8 . Figure 9. Figure 1 0 . Figure 1 1 . Figure 1 2 . Figure 1 3 • A migrating haploid 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 sition of the centriolar-kinetochore-equivalent (CKE) at the narrow end. The chromatin cap l y i n g on the i n -ner side of the nuclear 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. ca. X 1*7,600. The centriolar-kinetochore-equivalent at prophase I. Note the small size of the CKE and i t s position within a shallow cytoplasmic w e l l . The nuclear envelope (NE) appears to be i n t a c t . This i s an enlarged view of the CKE seen i n Figure 22. Method B. ca. X 60,1+00. The centriolar-kinetochore-equivalent at prophase I I . Note the size of the CKE i n comparison with the CKE i n Figure 8 . Also note the narrow electron dense zone within the CKE and the chromatin cap. Method C. ca. X 60 , 7 0 0 . The CKE at prophase I I . Note the f i b r i l l a r appear-ance. Method C. ca. X 129,900. The CKE at prophase I I . Note the f i b r i l l a r appear-ance and the two zones of varying electron density. Method C. ca. X 90,000. The CKE at prophase I I . Note the f i b r i l l a r appear-ance of the CKE and the t h i n electron dense zone, within the CKE. Method C. ca. X 1 8 8 , 2 5 0 . The CKE at prophase I I . Note the surrounding less electron dense halo. Method C. ca. X 126,900. Note: The black scale on Figures 8 to 13 represents 0 . 1 u only. IV. PLATE 6 Figure 11+. Figure 1 5 . Figure 16. Figure 17a. Figure 17b. Figure l 8 a . Figure l8b. The d i v i s i o n of the centriolar-kinetochore-equivalent (CKE) during the t h i r d promyoelial nuclear d i v i s i o n which gives r i s e to the f i r s t s p o r l d i a l nucleus. Note that the CKE has l e f t the nuclear well and i s now elongate and p a r a l l e l with the nuclear envelope. Method C. ca. X 1 3 , 0 0 0 . An enlarged view of the elongate CKE i n Figure 11+. Note the f i b r i l l a r nature of the CKE, the nuclear envelope (NE) and the f i n e f i b r i l s pas-sing between the CKE and the nuclear envelope. Method C. ca. X 1 2 7 , 5 0 0 . An elongate CKE which has been interpreted as a stage i n CKE-division. Note the f i b r i l l a r nature of the CKE. Method C. ca. 1 2 7 , 5 0 0 . A section through the nuclear region showing an enlarged nuclear pore which may be a permanent CKE-well. Method C. ca. X 5 3 , 5 5 0 . An enlarged view of the well-region seen i n F i g -ure 1 7 a . Method C. ca. X 158,600. A section through the haploid nucleus showing the CKE within the w e l l . Method C. ca. X 29 , 7 5 0 . An enlarged view of the CKE within the nuclear well seen i n Figure l 8 a . Note the appearance of the chromatin cap i n cross-section. Method C. ca. X 1 5 9 , 0 0 0 . Note: the black scale on Figures 17b and l 8 b represents 0 . 1 u only. IV. PLATE 7 Figure 19. Microtubules (mt) which often originate from the v i c i n i t y of the centriolar-kinetochore-equivalent (CKE) appear to end i n dense amorphous structures resembling the microtubule-organizing centres (mtoc) of higher animals and plants. Note that the 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 side of the haploid nucleus (hN) d i r e c t l y opposite the nu-cleolus (Nu). Method C. ca. X 36,000. Figure 20. Microtubules (mt) rad i a t i n g from the v i c i n i t y of the centriolar-kinetochore-equlvalent (CKE). Method C. ca. X 51,250. Figure 21. Microtubules (mt) ending i n the v i c i n i t y of the centriolar-kinetochore-equivalent (CKE) do not terminate at the CKE i t s e l f but rather end i n electron dense bodies (mtoc) surrounding the CKE. Method C, ca. X 96,200. Figure 22. D i p l o i d prophase I nucleus (dN) l y i n g i n the meta-basidium. The arrow indicates the d i r e c t i o n of the promycelial apex. Note the posterior p o s i t i o n of the nucleolus and the po s i t i o n of the c e n t r i o l a r -kinetochore -equivalent (CKE) on the side of the nucleus opposite the nucleolus. The CKE l i e s i n a shallow cytoplasmic well approximately one-third of the nuclear length behind the nuclear apex. A band of electron dense chromatin connects the nu-cleolus to the nuclear envelope just beneath the CKE. Method B. ca. X 31+,000. 2? IV. PLATE 8 Figure 2 3 , Figure 2I4, Figure 2 5 . Figure 26. Dip l o i d nucleus (dN) in late prophase I. The CKE has moved to a po s i t i o n midway along the nuclear length and the nucleolus (Nu) has shifted l a t e r -a l l y to the side opposite the CKE. The nuclear envelope has become very i n d i s t i n c t and i s mainly outlined by the position of the nuclear pores (NP). Microtubules (mt) l i e p a r a l l e l with the l o n g i t u -dinal c e l l axis on the CKE-side of the nucleus. Method 0 . ca. X 5 1 , 1 0 0 . E a r l y anaphase I. Note the dumb-bell shaped nu-clear p r o f i l e , and the apparently intact nuclear envelope. A nucleolus (Nu) i s d i s t i n c t in each haploid nucleus (hN). One of the CKE's i s v i s i -ble on the ri g h t side of the lower nucleus. Method C. ca. X 19 , 1 2 5 . E a r l y anaphase I suggesting a period of p a r t i a l breakdown of the nuclear envelope (NE). Note that the electron-transparent regions represent nucleic acid containing material. Method A. ca. X 2 0 , 7 0 0 0 . Telophase I nucleus. The CKE has moved to the leading end of the migrating nucleus. Note the density of the chromatin and p a r t i a l breakdown of the nuclear envelope on the r i g h t side of the f i g u r e . Method C. ca. X 3 5 , 7 0 0 . IV. PLATE 9 Figure 27. Figure 28. Figure 29. Figure 3 0 . E a r l y prophase II nucleus. (hN). Note the charac-t e r i s t i c positionsisof the CKE and the nucleolus (nu). Arrow Indicates a t h i n electron transparent zone apparently d i v i d i n g the nucleolus i n two. Method C. ca. X 1 0 , 9 5 0 . Late prphase II nucleus (hN). Microtubule-like strauctures seem to be r a d i a t i n g from the enlarged CKE. One very condensed chromatin body hoins the CKE d i r e c t l y to the nucleolus (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 the 1 f i g u r e . The two arrows indicate the positions of the chromatin bodies. Each chromatin body i s joined d i r e c t l y to the CKE by at least one p a i r of chromatin strands. Method C. ca. X 1+7,600. CKE-replication during nuclear d i v i s i o n I I . The nucleolus (Nu) also seems to be b i p a r t i t e . Method C. X ca. 1+7,600. Migration of the daughter CKE's during d i v i s i o n I I . The nuclear region i s demarcated by the p o s i t i o n of the nuclear pores (NP). Note that the nucleus i s becoming elongate. Method c. ca. X 1+0,650. IV. PLATE 10 Figure 31. Figure 32. Figure 33a, Figure 33b, The metaphse or early anaphase equivalent at d i v i -sion I I I . The two enlarged GKE's die at the poles of the nucleus. Note that the nuclear axis l i e s at an angle of about 1+5° to the promycelial axis. The nucleolus (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 to one side of the nuclear axis and midway between the poles. Method c. ca. X. 1+2,000. Anaphase I I . Microtubules (mt) penetrate the daugh-ter nucleus on the side opposite the lead end dur-ing nuclear migration. The nuclear envelope i s broken on the side through which the microtubules pass into the nucleus. Method c. ca. X 32,7£0. Anaphase I I . Note the incomplete nuclear 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 ca. 17,700. A l i g h t microscope view of anaphase II i n material fixed i n GA and viewed with phase optics. The ' second round of nuclear d i v i s i o n i s not always synchronous. The single arrow indicates a nucleus i n prophase II; the double arrow indicates a nu-cleus i n anaphase I I . The t h i n dense l i n e i n the lon g i t u d i n a l axis of the d i v i d i n g nucleus repre-sents the spindle, ca. 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Preprophase microtubular bands i n some abnormal mitotic c e l l s of wheat. J . C e l l S c i . It: 3 9 7 - 4 2 0 . Pickett-Heaps, J.D. 1 9 6 9 c . The evolution of the mitotic apparatus, an attempt at comparative u l t r a s t r u c t u r a l cytology i n d i v i d i n g plant c e l l s . Cytobios 2 5 7 - 2 8 0 . P o l l i s t e r , A.W. and P o l l i s t e r , P.F. 1943* The r e l a t i o n between ce n t r i o l e and centromere i n a t y p i c a l spermato-genesis of v i v i p a r i d s n a i l . Ann. N.Y. Acad. S c i . jt£: 1 - 4 8 . Rawitscher, F. 1 9 2 2 . Beitrage zur Kentnis der Ustilagineen. II Z e i t . Bot. 14. : 2 7 3 - 2 9 6 . Robinow, C.F. and Caten, C.E. 1 9 6 9 . Mitosis i n Aspergillus  nidulans. J . C e l l S c i . £: 4 0 3 - 4 3 1 . Robinow, C.F. and Marak, J. 1 9 6 6 . A f i b e r apparatus i n the nucleus of the yeast c e l l . J . C e l l B i o l , 29_: 129 - 1 5 1 . Rosenberger, R.F. and Kessel, M. 1 9 6 8 . 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Cytol. 7 : 5 8 3 - 5 8 8 . Tilney, L.G. and GOddard, J. 1 9 7 0 . Nucleating s i t e s f o r the assembly of cytoplasmic microtubules i n the ectodermal c e l l s of Arbacla punctulata. J . C e l l B i o l . Ij^: 561+-575* Wang, C.S. 1 9 4 3 . Studies on the cytology of Ustilago cramerl! Phytopathology 3 3 : 1 1 2 2 - 1 1 3 3 . Wang, D.T. 193U- Contribution a 1»etude des Ustilaginees (CytolOgie du parasite et pathologie de l a c e l l u l e hote). Le Botanists 2h: 5 3 9 - 6 7 0 . Wells, K. 1 9 7 0 . Light and electron microscope studies of Ascobolus stercoarius I Nuclear d l v i i i o n s i n the ascus. Mycologia 62: 7 6 1 - 7 V U . Westergaard, M. and von Wettstein, D. 1 9 6 5 . Studies on the mechanism of crossing-over I I I On the ultra s t r u c t u r e Of the chromosomes In N e o t i e l l a r u t i l a n s (Fr.) Dennis. C. R. Lab. Carlsberg 5 3 : 201-2ot>. Yoo, B.Y. and Bayley, S.T. 1 9 6 7 . The structure of pores i n is o l a t e d pea n u c l e i . J. U l t r a s t r u c t . 1 8 : 651-660. Zickler,~D. 1 9 7 0 . D i v i s i o n spindle and centrosomal plaques during mitosis and meiosis i n some ascomycetes. Chromosome 3 0 : 287 - 3 0 4 . 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 TABLE OF CONTENTS Page ABSTRACT 158 INTRODUCTION 158 MATERIALS AND METHODS 159 OBSERVATIONS 160 DISCUSSION 1 6 1 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 previously been associated with septum i n i t i a t i o n in Ustllago hordei. Evidently similar complexes are formed in the v i c i n i t y of the nucleus during meiotic prophases I and I I . The hypothesis i s presented that the complexes are part of a system governing 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 in the promycelium. A review i s presented of the l i t e r a t u r e pertaining to fungal membrane complexes, and t h e i r r e l a t i o n s h i p to mesosomes of bacteria and the Golgi apparatus of higher plants and animals. INTRODUCTION In part III membrane complexes were described in association with the septum i n i t i a l of Ustilago hordei and the suggestion was made that these membranous struc-tures are involved in the i n i t a t i o n of cross wall forma-t i o n . The observations presented.in t h i s paper indicate that the membrane complexes form from the plasma membrane in association with prophase n u c l e i . Their subsequent a c t i v i t i e s are compatible with the hypothesis that they are an important part of the mechanism which regulates the positioning of c e l l and nuclear d i v i s i o n with respect to each other. In higher plants the c e l l wallIdelimiting two daughter c e l l s forms as a di r e c t telophasic continuation of the nu-clear d i v i s i o n which gave r i s e to the two new nuclei,-the microtubular spindle apparatus of the parent nucleus being reorganized to pa r t i c i p a t e in the formation of the c e l l plate *(Neweombe, 1969). The microtubular apparatus asso-ciated with many fungal n u c l e i i s of very limited duration and disappears completely as soon as the daughter nuclei have separated. In septate fungi cross walls subsequently form, usually v i a c e n t r i p e t a l invagination of the l a t e r a l wall and plasma membrane. Microtubules do not seem to be involved and there are no obvious connections between t h i s process and nuclear d i v i s i o n . Yet some method of ensuring that the septum separates the daughter nuclei 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 that of Ustilago hordei where f a i l u r e to main-t a i n a one-to-one r e l a t i o n s h i p between the nucleus and the c e l l would greatly decrease the meiotic e f f i c i e n c y . MATERIALS AND METHODS The materials and techniques used are the same as those previously described (Pt. I ) . Methods A, B, and C, for preparing tissue for electron microscopy are out-l i n e d i n part I, Table I. 160 OBSERVATIONS Membrane complexes, similar in appearance to those associated with septum i n i t i a t i o n (Pt. I l l ) , also occur contiguous to the nu c l e i of Ustllago hordei. Figure 1 shows a cross-section through a very prominent complex which seems to be in d i r e c t connection with the nuclear envelope. The nucleus to which t h i s complex i s attached i s a d i p l o i d nucleus which f i x e d while migrating into 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 0.2 u removed from the f i r s t . In the promycelium, whomever similar membranous structures are observed i n association with the undivided fusion nucleus, they are found 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 . The complex i s always pressed close to the promycelial wall at the posterior end of the nucleus ( i . e . the end closest to the spore: F i g s . 3a and 1*), and consequently i s adjacent to the nucleolus ( F i g . k U ) . It also l i e s on the side of the nucleus opposite to the single centriolar-kinetochore-equiva-lent ( F i g . U ) . A group of ve s i c l e s of various dimensions i s inevi t a b l y located i n the v i c i n i t y of the complex and also at the posterior end of the nucleus (Figs. 3a, 3b, and 1*). These v e s i c l e s may be associated with microtubules. Figure 5> i s interpreted as a section through a dumb-b e l l shaped nucleus which i s i n the process of reduction d i v i s i o n and which has probably proceeded to a phy s i o l o g i c a l state equivalent to early anaphase I. 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 cross-section i n the middle of the c o n s t r i c t i n g region between the daughter n u c l e i . At a s l i g h t l y l a t e r stage when the daughter nuolei of the f i r s t meiotic d i v i s i o n have v i r t u a l l y separated the membrane complex with 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 internuclear zone (Fig. 6) Although membranous systems have not yet been observed i n close proximity to metaphase I I or anaphase II nuolei they do occur i n the perinuclear region during prophase II (Figs. 7a and 7b), and prophase I I I (Fig. 8). Figure 7a i l l u s t r a t e s the rare case i n which two separate complexes seem to be associated with a single nucleus. The o r i g i n of the membrane complexes i s s t i l l uncertain. However, i n several oases where nuolei have been observed close to the plasmalemma, the plasma membrane invaginates i n the d i r e c t i o n of the nucleus. The invaginating membrane does not t r a v e l i n a straight l i n e but c o i l s on i t s e l f i n double oonoentric layers (Figs. 9a and 9b). It seems reasonable that continuation of t h i s kind of a c t i v i t y would give r i s e to structures resembling membrane complexes. DISCUSSION Membranous structures, s i m i l a r i n appearance to the membrane complexes of Ustilago hordei, have been observed i n at least a dozen d i f f e r e n t fungi (Ft. I l l ) including phycomycetes, basidiomycetes, ascomycetes, and imperfect forms (Table I ) . The complexes seem to be associated with 162 septa i n two human pathogens, Paracoccidioides b r a s i l i e n s i s and Blastomyces dermatitidis (Carbonelle, 1967; Carbonelle and Rodrigez, 1 9 6 8 ) and i n two basidiomycetes Lenzltes  aaepiaria (Hyde and Walkinshaw, 1966) and Ustilago hordei (Pt. I I I ) . In both of the preceding basidiomycetes, mem-brane complexes also occur i n proximity to the n u c l e i ; a simi l a r r e l a t i o n s h i p has been noted i n Paracoccidiodes l o b o i (Purtado et a l , , 1967) and Coprinus lagopus (Lu, 1965; Lu, 1966), To date, the only studies which have considered th i s association i n any d e t a i l are those i n Coprinus. Ac-cording to Lu (1965 and 1966) a single membrane complex forms i n the basidium of Coprinus logopus during prophase I of meiosis. At t h i s time the complex which i s 0.9 to 1.0 u i n diameter l i e s close to the d i p l o i d fusion nucleus and gives r i s e to large numbers of v e s i c l e s . The Ustilago com-plex described i n this paper resembles the Coprinus complex both i n time and place of o r i g i n and i n i t s association with v e s i c l e s (Pigs. 3a, 3D, and i+). In Ustilago hordel i t has not been proven that the complex ac t u a l l y produces the ves-i c l e s . However, at metaphase I the Coprinus complex p r o i f e r -ates r a p i d l y to almost f i v e times i t s prophase size while that of Ustilago remains unchanged. Whether t h i s i s due to a difference i n function or perhaps only i n the degree o f function i s unknown. Considering the r e l a t i v e l y wide d i s t r i b u t i o n of this type of membranous body among fungi, one wonders why so l i t t l e attention has been paid to i t . There are probably three basic reasons for t h i s neglect. F i r s t , as discussed i n part I I I , there has been a general tendency to avoid these bodies because of the p o s s i b i l i t y that they may simply be f i x a t i o n artefacts or else that they may r e s u l t from the aut o l y t i c degradation of other "normal" organelles. E v i -dence has previously been presented which indicated that neither of these p o s s i b i l i t i e s i s l i k e l y to be the case i n Ustilago hordei. Second, s t r e s s f u l conditions are known to cause the r o l l i n g up of ER-elements in higher plants (Whaley et a l . , 1961+) and animals (Fawcett and Susuma, 1958). This p o s s i b i l i t y should be examined c r i t i c a l l y in fungal studies because most of the tissue prepared for electron microscopy has been a r t i f i c i a l l y cultured. However whorls of endo-plasmic reticulum also occur 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 true in embryonic tissues (Fawcett and Susuma, 1958; Robertson, 1961). In Ustilago hordei a further point to consider i s that the complex seems to arise from a r o l l i n g up of the plasma mem-brane and not of the endoplasmic reticulum. A t h i r d reason that l i t t l e information i s available pertaining to fungal membrane complexes i s that no one, with the exception of Lu (1965)» appears to have obtained s u f f i c i e n t information i n d i c a t i n g that these unusual structures might perform some phy s i o l o g i c a l function. I submit that membrane complexes in Ustilago hordei do serve a d e f i n i t e and important func-tion,—namely 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 s . The observations i n the promycelia of t h i s smut fungus suggest the following hypothesis: 1. During late meiotic 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 basal portion of the d i p l o i d nucleus. 2 . The p r o l i f e r a t i n g membrane invaginates and c o i l s on i t s e l f (Pigs-*; 9a and 9b) to f i v e r i s e to a reasonably large structure consisting of concentric unit membrane layers (Pigs. 3 a , 3b and 4 ) . 3 . The membrane complex, in some way, establishes f i r m connection with the nucleus. The nature of t h i s contact i s unknown. Possibly the nuclear envelope i s involved in formation of the complex but there i s no d i r e c t evidence f o r t h i s . In Figure 1 the unit membrane of the complex, and the nuclear envelope seem to be in di r e c t connection. 4 . The nuclear r o t a t i o n described i n part IV which brings the centriolar-kinetochore-equivalent and the nucleolus into a medial p o s i t i o n with respect to the length of the deploid nucleus also centres the mem-brane complex. 5>. During reduction d i v i s i o n the daughter haploid n u c l e i separate and the membrane complex, having now l o s t contact with the nuclear envelope, remains i n the middle of the lengthening internuclear zone (Figs. £ and 6) . 6. The free membrane complex then begins to " u n r o l l " providing the membrane to form the i n i t i a l septal plate (Pt. I l l , Pigs. 2 and 5). ?• Having completed i t s function the membrane complex i s destroyed. 8. Hew complexes form i n association with the prophase II n u c l e i and the cycle begins again. This tentative hypothesis w i l l demand considerable t e s t i n g . One of the immediate requirements i s to obtain s e r i a l sec-tions since there i s no other way to accurately judge the closeness of the complex-nucleus r e l a t i o n s h i p or the one-to-one-to-one r e l a t i o n s h i p of the nucleus, membrance com-plex, and septum. Should t h i s hypothesis prove correct there i s no reason to assume that the same mechanism i s necessarily active during somatic mitotic d i v i s i o n s although l i m i t e d e v i -dence indicates that i t may be. Possibly t h i s mechanism has evolved as a spe c i a l i z e d adaptation to the peculiar problems of undergoing meiosis i n a metabasidium. Other authors have proposed d i f f e r e n t functions which such membranous structures might f u l f i l l i n fungi and the complexes i n Ustilago hordei may carry out one or more of these i n addition to that of posit i o n i n g . In Coprinus  1 ago pus. Lu (1965) has postulated that since the meiotic d i v i s i o n s occur i n rap i d succession the membrane complex, which p r o l i f e r a t e s enormously at metaphase I, might be the generator of additional nuclear envelope. This does not seem to be the case i n Ustilago since the complex-associated 166 v e s i c l e s have not been observed to fuse with the nuclear membrane, and most of the vesi c l e s remain i n the internuclear zone following nuclear d i v i s i o n (Pig. 6). A second p o s s i -b i l i t y i s that the complex acts as an anchor which brings the nucleus to a stationary p o s i t i o n p r i o r to d i v i s i o n . In part IV i t was suggested that Brown and Stack's theory (1971) of fungal nuclear d i v i s i o n i s at present the most l i k e l y model to account for the phenomena which occur in Ustilago  hordei during meiosis. This theory i s p a r t l y based on the e a r l i e r work by Rosenberger and Kessel (1968) which showed non-random s i s t e r chromatid segregation and nuclear migration in the hyphae of Aspergillus nidulans. Interestingly the l a t t e r authors postulated that in order to obtain the r e s u l t s which they did the oldest of the segregating units should be anchored to some stationary part of the c e l l u l a r membrane system.-The Ustilago complex resembles s t r i k i n g l y the mesosomal system of b a c t e r i a (Rogers, 1970; Ryter, 1968) and actino-mycetes (Edwards, 1970; and Pitz-James, I960). S t r u c t u r a l l y both the complex and the mesosome consist of c o i l e d membranes which are derived from the plasma membrane. They both occur in association with the nuclear material and with the forming septa; consequently they have both been implicated in main-ta i n i n g c e r t a i n s t r u c t u r a l and functional r e l a t i o n s h i p s be4-tween c e l l and nuclear d i v i s i o n . The obvious s i m i l a r i t y between the two structures has prompted some workers to r e f e r to these structures as "fungal mesosomes" (Edwards, 1969; Kozar and Weijer, 1969). This i s hazardous because i t tends to imply some sort of evolutionary significance which may not exist at a l l . According to the current data there i s reason to assume that the undeniable resemblance of the two systems i s not by way of i d e n t i t y but rather by way of ana-logy. F i r s t the current model of the b a c t e r i a l mesosome indicates that i t i s composed of tubular or v e s i c u l a r mem-branes (Ryter, 1968) although some proponents of the lamellar membrane theory s t i l l exist (Highton, 1970). Of course the problem i n b a c t e r i a l cytology has long been that d i f f e r e n t f i x a t i o n procedures r e s u l t i n a d i f f e r e n t appearance of the mesosomes (Burdett and Rogers, 1970; Ryter, 1968), and one must admit that the issue i s not yet closed. Obviously the same s i t u a t i o n exists i n Ustilago hordel (Figs. 1, 3*> andllO). A further complication i s that most of the u l t r a s t r u c t u r a l studies have described gram-positive b a c t e r i a , and there i s good evidence that the lamellar conformation probably occurs among gram-negative bacteria (Kakefuda et a l . , 1967; Ryter, 1968) and actinomycetes. Second, while both systems are de-r i v e d from the plasma membrane they are probably not formed in the same manner (Imaeda and Ogura, 1963; Rogers, 1970). Third, the b a c t e r i a l mesosome ca r r i e s out a v a r i e t y of other important c e l l u l a r functions such as c e l l u l a r r e s p i r a t i o n (Ryter, 1968), wall secretion (Rogers, 1970) and exoenzyrae secretion (Beaton, 1968). Whether the fungal complex c a r r i e s out any of these additional functions w i l l remain unknown u n t i l the appropriate biochemical and/or histochemical i n f o r -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 to be u n l i k e l y . Lu (1965 and 1966) has noted that fungal membrane complexes also resemble the Golgi apparatus i n some aspects of structure and function, and has hence referred to these structures as G o l g i . Undoubtedly the membrane complex formed in Coprinus lagopus during meiotic prophase I bears a greater resemblance to a Golgi than does the complex i n Ustilago hor- dei i n i t s structure, i t s production of v e s i c l e s , and i t s probable functional significance as a nuclear membrane gen-erator.' The structure of the fungal membrane complex i s p a r t i c u l a r l y reminiscent of the Golgi apparatus described in cer t a i n types of algae (Bouck, 1965; Brown, 1969; Millington and Gawlik, 1970). Another feature which the two systems share i n common i s a tendency to be situated i n the p e r i -nuclear region. Beams and Kessel (1968) and Mollenhauer and Morre (1966) have reviewed the various functions of the Golgi apparatus—the l a t t e r with p a r t i c u l a r reference to plants. Notably, functions which Golgi serve i n animals, higher plants, and some fungi are also functions which have been suggested for fungal membrane complexes, for example enzyme secretion ( G r i f f i t h s , 1970) and membrane generation and transformation (Berliner and Duff, 1965; Lu, 1965 and 1966). In further support of the hypothesis that fungal membrane complexes are the Golgi-equivalent i s the observa-t i o n that membrane complexes are usually not observed i n fungi which possess the usual plant dictyosomes (Table I I ) . TABLE I Membrane Complexes and Golgi in Fungi Membrane Complexes Phycomycetes: Peronaspora Chou (1970) parasitica Golgi Phycomycetes: Albugo Candida Nowakowskiella prorusa Peronospora P a r a 3 l t T c a  Peronospora m a n a h u r T c a  Phytophthora  erythroseptica  i^nytopntnora mrestans"" Phytopnthora parasitica Pythium debTry-anum  Pytnium ult imum Saprolegna ferax Berlin & Bowen (196!+) Chambers et a l . (1967) Davison (1968) Peyton & Bowen (1963) Chapman & Vujicic (1965) Ehrlich & Ehrlich (1966) t! II II Hawker (1963) Grove et a l . (1967) Heath & Green-wood (1971) Ascoraycetes: Neurospora crassa Neurospora tetrasperma Kozar & Weijer (1969) Lowry & Sussman (1968) Neobulgar la pur a Moore & McAlear (1963) Basidiomycetes: Armillaria mellea  Coprinus lagopus Lenzites saepiaria Lycoperdon perlatum  Blastomyces derma tl^HTd is Parac occ idiocies hrasiliensis Paracoccidiodes loboi  Verticillium dahliae Berliner & Duff (1965) Lu (1965 & 1966) Hyde & Walk-inshaw (1966) Marchant (1969) Puccinia podophylli Moore (1963) Carbonelle Rodrigez ibid & (1968) a l . Furtado et (1967) Griffiths (1970) 17© At present too l i t t l e information i s available concern-ing fungal membrane complexes to attempt to Identify them with either the mesosomal system or the Golgi apparatus. Consequently we should perhaps r e t a i n the simple term "fungal membrane complex" f o r these structures and so avoid a nomen-clature which might r e s u l t i n misleading implications. Cer-t a i n l y the complexes resemble mesosomes and some types of Golgi i n certa i n aspects of structure and function. However, the ontogeny of the three systems i s d i f f e r e n t , i n d i c a t i n g a separate evolutionary o r i g i n . What seems most l i k e l y to be the case i s that c o i l e d membrane systems of t h i s type have optimal properties for ce r t a i n kinds of phy s i o l o g i c a l functions r e l a t i n g to secretion, and membrane generation and storage, and that these properties have been ^ u t i l i z e d " i n -dependently i n d i f f e r e n t classes of organisms. V. PLATE 1 Figure 1 A large membrane complex (mc) i n d i r e c t connection with the nuclear envelope of a prophase I nucleus (dN). Method A. ca. X 21}.,750. Figure 2 A consecutive section about 1000 A 0 removed from figure 1 showing the po s i t i o n of the whole nucleus with respect to the membrane complex. Method A. ca. X 13,500v V. PLATE 2 Figure 3a. A prophase I nucleus (dN) i n the promycelium show-ing the posterior p o s i t i o n of 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 ves i c l e s (ve). The CKE l i e s i n a position opposite the complex. The nuclear bound-ary i s demarcated by nuclear pores (NP). Method A. ca. X 37,200. Figure 3b An enlarged view of the membrane complex and vesicle region seen i n figure 3a. Method A. ca. X 7h»k-Q0, Figure 4, A prophase I nucleus (dN) showing the c h a r a c t e r i s t i c positions of the membrane complex (mc) ves i c l e s (ve) and nucleolus (Nu). The arrow indicates one of the chromatin-nucleolar connections. Method C. ca. X 47,500. Figure 5. D i v i s i o n I nucleus i n the dumb-bell stage. The nu-cleus i s the same as that shown i n part IV figure 24. Note the po s i t i o n of the membrane complex (mc). Method c. ca. X 18,000. V. PLATE 3 Figure 6. Figure 7a. Figure 7b. Figure 8. 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 divides the membrane comples (mc) comes to l i e i n a central p o s i t i o n between the two separating daughter n u c l e i . Also note the vesicles i n the internuclear zone. Method B. ca. X 18,700. A haploid nucleus associated with two membrane com-plexes (mc's). The membrane complex on the lower l e f t i s associated with v e s i c l e s . Method C. ca. X 30,000. An enlarged view of the membrane complex (mc) and vesicles seen i n figure 7a. Method C. ca. X 61;,400. A haploid nucleus showing the intimate r e l a t i o n s h i p between the membranes of the complex and the nuclear envelope. Method B. ca. X 60,600. V. PLATE k Figure 9a. Formation of a membrane complex i n vagination of the plasma membrane. Method B. ca. X 31,000. Figure 9b. An enlarged view of the forming complex seen i n Figure 9a. Method B. ca. X 81,200. Figure 10. A l o n g i t u d i a l section 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 thickening septum. Method B. ca. X 37,000. 1 7 1 BIBLIOGRAPHY Beams. H.W. and Kessel, R.G. 1968. The Golgie apparatus: structure and function. Int. Rev. Cytol. 2 ^ : 209 -176. Beaton, C.D. 1968. An electron microscope study o f the mesosomes of a penicilllnase-producing Staphyloco ecus. J . gen. Microbiol. £ 0 : 37-1+2* B e r l i n , J.D, and Bowen, C.C. 1961+. The most-parasite i n -terface of Albugo Candida on Raphanus sativus. 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 . Ultrastructure of A r m i l l a r i a mellea hyphae. Can. J . Bot. b^: 171-172. Bouck, G.B. 1 9 6 5 . Fine structure and organelle associa-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 the re l a t i o n s h i p of the Golgi apparatus to wall formation i n a marine Chryso-phycean alga, Pleurochrysis s c h e r f f e l i i Pringsheim. J. C e l l B i o l . Ifcl; 109 -123. Brown, R.M. and Stack, S.M. 1971. Personal communication (unpublished)• Burdett, I.D.J, and Rogers, H.J. 1 9 7 0 . Modification o f the appearance of mesosomes i n sections of B a c i l l u s l i c h e n i -forrais according 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. Carbonelle, L.M. 1 9 6 7 . C e l l wall changes during the bud-ding process of Paracoccidioides b r a s i l i e n s l s and Blastomyces dermatitidis. J . B a c t e r i o l . 91+: 213-223. Carbonelle, L.M. and Rodrigez, J . 1 9 6 8 . Mycelial phase of Paracoccidiodes 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 electron microscope study. J . Bacteriol.~96: 533-51+3. Chambers, T.C., Markus, K., and Willoughby, L.G. 1 9 6 7 . The f i n e structure of the mature zoosporangium of Nowakowskiel- l a profusa. J . gen. Microbiol, 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 structure of sporangia of Phytophthora erythroseptica Pehtyb. J . gen. Microbiol. 1+1.: 2 7 5 - 2 9 o T Chou, O.K. 1 9 7 0 . An electron microscope study of host penetration and early stages of haustorium formation of Peronospora p a r a s i t i c a (Fr.) T u l . on cabbage c o t y l e -dons. Ann. Bot. 3k: 189-201+. 172 Davison, E.M. 1968. Cytochemistry and ultrastructure of hyphae and haustoria of Pieronospora p a r a s i t i c a . Ann. Bot. 3 2 : 613-621. Edwards, M. 1969. Mesosome-like structures i n blue-green algae and lower eukaryotic forms. Biophys. J. 9} PA 176 (Abstract). Edwards, R.P. 1970. Electron microscope i l l u s t r a t i o n s of d i v i s i o n i n Mycobacterium leprae. J. Med. Microbio. 3 : 4 9 3 - 4 9 8 . ~ ; E n r l i e b , M.A. and E h r l i c h , H.G. 1966. Ultrastructure of the hyphae and haustoria of Phytophthora infestans and hyphae: of 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 cytoplasmic membranes of t e s t i c u l a r c e l l s , examined by phase contrast and electron microscopy. J . Biophys. C y t o l . ly: 1 3 5 - l U l * Fitz-James, P.C. I960. P a r t i c i p a t i o n of the cytoplasmic membrane «iin the growth and spore formation of b a c i l l i . J. Biophys. Biochem. Cy t o l . B : 5 0 7 - 5 2 8 . Furtado, J.S., B r i t o , T. de, and Freymuller, E. 1967* Struc-ture and reproduction of Paracoccidioides l o b o i . Mycologia 5 9 : 2 8 6 - 2 9 4 . G r i f f i t h s , D.A. 1970. The f i n e structure of V e r t i c i l l i u m dahliae Kleb colonizing cellophane. Can. J. Microbiol. 17: 7 9 - 8 1 . Grove, S.N., Morre, D.J., and Bracker, C E . 1967. The Golgi apparatus as a s i t e of endomembrane d i f f e r e n t i a t i o n i n Pythium ultimum. Am. J . Bot. 5 4 : 638 (Abstract). Hawker, L.E. 1963. Fine structure of 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 observations On the kinetosome, and Golgi bodies during the asexual l i f e cycle of Saprolegnia. Z e i t . Z e l l f o r s c h . 112: 371-389. ~ Hlghton, P.J, 1970. An electron microscope study of the structure of mesosomal membranes in 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 basldiospores and mycelium of Lenzites saepiaria. J. B a c t e r i o l . 92: 1218-1227. Imaeda, T. and Ogura, M. 1963. Formation of intracytoplasmic membrane systems of Mycobacteria re l a t e d to c e l l division;' J. B a c t e r i o l . 8 5 : 15'0-163. Kakefuda, T., Holden, J.T., and Utech, N.M. 1 9 6 7 . U l t r a s t r u c -ture of tbe membrane system i n Lactobacillus plantarum. J. B a c t e r i o l . 9 3 : 1*72-1*82. Kozar, F. and Weijer, J. 1 9 6 9 . Electron dense structures i n Neurospora crassa. 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 during germination of ascospores. J. gen. M i c r o b i o l . 5 1 : 1*03-4*09. Lu, B.C. 1 9 6 5 . Fine structure i n f r u i t i n g bodies of Coprinus, with s p e c i a l emphasis on chromosome structures. Ph.D. Thesis, The University of Alberta, Edmonton, Alberta, CAN. Lu, B.C. 1 9 6 6 . Golgi apparatus of the basidiomycete Coprinus  lagopus. J. B a c t e r i o l . 92: I831 -183I*. Marchant, R. 1 9 6 9 . The fine structure and development of the f r u c t i f i c a t i o n of Lycoperdon perlaturn. Trans. B r i t . Mycol. 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 . Ultrastructure and i n i t i a t i o n of wall pattern i n Pediaetrum boryanum. Am. J. Bot. 5 7 : 5 5 2 - 5 6 1 . " Mollenhauer, H.H. and Morre, D.J. 1 9 6 6 . Golgi apparatus and plant secretion. Ann. Rev. PI. Physiol. I J : 27-1*6. Moore, R.T. 1 9 6 3 . Fine structure of mycota. XI Occurrence of the Golgi dictyosome i n the heterobasidlomycete Pucci- nia podophylli. J. B a c t e r i o l . 8 6 : 8 6 6 - 8 7 1 . — Moore, R.T. and McAlear, J.H. 1 9 6 3 . Fine structure of mycota !*• The occurrence of the Golgi 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 . Plant Microtubules. Ann. Rev. PI. Physiol. 2 0 : 2 5 3 - 2 8 7 . Peyton, G.A. and Bowen, C G . 1 9 6 3 . The host-parasite i n t e r -face of Peronospora manshurica on Glycine max. Am. J. Bot. 5 0 : 7 8 V - 7 9 9 . Robertson, J.D. 1 9 6 1 . New unit membrane organelle of Schwann ce11. In Biophysiologlcal and Pharmacological Actions, ed. Shane, A.M., Am. ASSOC. AOV. or s c i . m Rogers, H.J. 1970. Bacteria-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 Kessel, M. 1968. Non-random s i s t e r chromatid segregation and nuclear migration i n hyphae o f Aspergillus nidulans. J. B a c t e r i o l . 96: 1208-1213. Ryter, A. 1968. Association of the nucleus and the membrane of b a c t e r i a : a morphological 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 of cytoplasmic membranes during development. In C e l l u l a r Membranes i n Development, ed. Locke, M., H#H2?lol!aj..j. Acaaemic .rress, inc. ppT 135-157. 175 GENERAL CONCLUSION "The things that are not yet done." (Isaiah, 1+6.10) A generalized c y t o l o g i c a l study of an entire stage i n the l i f e cyole of an organism has c e r t a i n advantages and ce r t a i n disadvantages; however, i n approaohing an organism as l i t t l e known as Ustilago hordei, t h i s step i s both ne-cessary and valuable. Casual observation of organelles at miscellaneous stages i n the l i f e cycle of an organism re-veals the remarkable v a r i e t y of fas c i n a t i n g structures to be found. Unfortunately, t h i s approach does l i t t l e to es-t a b l i s h the equally i n t e r e s t i n g and perhaps more relevant i n t e r r e l a t i o n s between organelles and organelle systems. In most cases, as i n Ustllago hordei, the e s s e n t i a l orga-n e l l e s are always present. It i s the changes i n the s i z e , contents, d i s t r i b u t i o n and in 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 of d i f f e r e n t i a t i o n i n the organism. C l e a r l y a ge* nera l i z e d study enoourages the observation of development i n i t s e n t i r e t y , thus allowing one to peroeive exactly which organelles and systems may be important at c e r t a i n develop-mental stages. Perhaps the most important results of such a study are, f i r s t , the formulation of hypotheses concerning exaotly what i s important and when and where i t i s important; and, second, the formulation of methods by which these hypo-theses might be tested. 176 This study indicates the f e a s i b i l i t y o f studying pre-germinal mlcroanatomieal changes i n hydrating spores. Con-sidering that many of the fundamental events which lead up to metabasidium formation begin long before germination i t -s e l f , the paucity of information i n t h i s developmental stage i s astounding] What i s now required are extensive studies on a variety of spores, coupled with the appropriate biochemical and histochemical techniques. In Ustilago hordei. two im-portant aspects to study are the composition of the " f l o c -culent cytoplasm" and the development of the respiratory pattern 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 of the mitochon-d r i a l population. Another general area deserving of further study i s that of promycelial extension. In part I, the evidence suggests that the nuclear enve-lope gives r i s e to the endoplasmic reticulum, and the endo-plasmic reticulum i t s e l f gives r i s e to the "primary hydra-t i o n " vacuoles by d i l a t i o n of the intermembranous space. More study i s required to determine the exact manner i n which thi s happens; information from glutaraldehyde-osmium f i x e d material i s necessary. Certainly one of the next steps i n pursuing t h i s question should be the a p p l i c a t i o n of the Gomori reaction (or some equivalent technique) to determine whether the primary vacuoles have acid-phosphate a c t i v i t y , ( i . e. are l y t i c i n nature). Vacuoles i n Ustilago hordei are formed by several me-chanisms. In the metabasidium, spherosome-like bodies seem to give r i s e It© vacuolar structures by d i l a t i o n and/or f u -sion. This would imply that they areequivalent i n function to the animal lysosome. However, this cannot be decided for c e r t a i n l y without appropriate histochemlcal tests for l y s o -somal a c t i v i t y ( i . e . the Gomori re a c t i o n ) . The evidence i n Ustilago hordei suggests that these organelles may be i n -volved i n other functions as well as l y t i c ones; thus, even i f the spherosomal bodies should prove to have lysosomal a c t i v i t i e s , the author f e e l s that one would be well advised not to conclude that that i s t h e i r only function. Much yet remains to be learned about the n u c l e i of smut fungi. To date, the evidence suggests that the meiotic d i v i s ion figures are most compatible with Brown and Stack's model for somatic nuclear d i v i s i o n i n some fungi. However, more" information i s required on a l l stages of nuclear d i v i s i o n p a r t i c u l a r l y at the electron microscope l e v e l , and s e r i a l sectioning through entire nuclei i s r e q u i s i t e . Studies of the somatic s p o r i d i a l nuclear d i v i s i o n s should also be made. One also suspects that the techniques could be improved suf-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 nucleus. Some e f f o r t should be made to determine the extent to which th i s mechanism of d i v i s i o n occurs among fungi and any genetic im-p l i c a t i o n s should be investigated (especially at meiosis). There now seems to be l i t t l e doubt that membrane com-plexes are common among fungi, and i t seems unreasonable that these complexes should not be studied more seriously. In Ustilago hordei. these complexes appear to play some part i n establishing 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 proceeding nuclear d i v i s i o n . S e r i a l sectioning i s required to 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, nucleus, and septum. Studies on c e l l d i v i s i o n mutants may prove useful i n determining the s i g n i -ficance of the complex. The disadvantage i n a generalized study of t h i s type i s that although a multitude of hypotheses suggest them-selves, few d e f i n i t e conclusions can be drawn. Such a study merely points the way. The author hopes that the observations and hypotheses presented are s u f f i c i e n t l y well-documented and of s u f f i c i e n t i n t e r e s t to encourage others, as well to pursue answers to these problems. APPENDIX A Culture Medium (A) COMPLETE BROTH Vogel's solution (dilute) 20 ml. D i s t i l l e d water 1 1 . Tryptophane £0 mg. Casein hydrolysate 5 gm. Yeast extract (Difco) 55 gnu Sucrose (20 gm. or ( Dextrose (10 gm. Vitamin solution 10 ml. Note: 1. Vitamin solution to be added afte r auto-claving. 2. To make complete plates add 20 gm. Bacto-agar before autoclaving. (B) VOGEL'S SOLUTION (concentrate) Nao c i t r a t e . 2H2O 123 gm. KH^PO. anhydrous 2$0 gm. NHi NO-J anhydrous 100 gm. MgSOh^. 7H20 10 gm. CaClg . 2 H 2 0 5 gm. Trace element solution 5 ml. D i s t i l l e d water 750 ml. Chloroform 2 ml. Note: 1. Add chemicals successively with s t i r r i n g . 2. Store at room temperature. 3. D i l u t e 50-fold with d i s t i l l e d water before use. (C) VITAMIN SOLUTION Thiamin 100 mg. Rib o f l a v i n £0 rag. Pyridoxin 50 mg;. Calcium pantothenate 200 mg.. Benzoic acid £0 mg. N i c o t i n i c acid 200 mg. Choline chloride 200 mg. I n o s i t o l 1*00 mg. F o l i c acid £0 mg. D i s t i l l e d water to a t o t a l of 1 1. Note: 1. Store at 1*°C. 2. Use 10 ml. of vitamin solution per l i t r e of s t e r i l e medium. (D) TRACE ELEMENT SOLUTION C i t r i c acid . 1H 20 5 gm* ZnSOj. . 7H2O $ gm. FpfNliJpSO). . 6HpO 1 gmi C U S O K . 5R20 0.25 gm. MnSOJT , lHpO 0.05 gm. H3POR anhydrous O.OS^gBU N S 2 M 0 O 1 , • 2H 20 0.05 gnu Chloroform 1 ml. H20 d i s t i l l e d 95 ml. Note: 1. Store at room temperature. APPENDIX B Preparation of Material f o r Electron Microscopy i NOTE: Tbe enti r e procedure i s carried out at room temperature. 1. C o l l e c t i o n . - Resting spores are obtained by s p l i t t i n g open the kernels of smutted heads and shaking the spores into a test tube containing a few m i l l i - l i t r e s of d i s t i l l e d water. These are shaken v i o l e n t l y for several seconds to wet the spores, and are centrifuged immediately at low speeds for 1-2 minutes on a P h i l l i p s Drucker Combination centrifuge L-708, The water i s decanted o f f and the spores resuspended i n the desired f i x a t i v e . The time i n which the r e s t i n g spores are i n water must be kept to a minimum i n order to avoid 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 hydrated half-an-hour or more i n broth, a suitable aliquot of the spore suspension (this de-pends on' the concentration of spores,- usually 10-20 ml. i s s u f f i c i e n t ) i s centrifuged down. The broth 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 appropriate b u f f e r , for 1-2 minutes. After r e p e l -l e t i n g , the l i q u i d i s poured o f f and the material resuspended i n the desired f i x a t i v e . 2. F i x a t i o n . - The two f i x a t i o n procedures used are: 1) 1.5$ KMnO^ (aqueous) - Potassium permanganate i s dissolved i n d i s t i l l e d water over-night and f i l t e r e d before use. F i x a t i o n time i s 10 - 20 minutes. 2) 2.0$ Glutaraldehyde - 1 part with 34 parts 0.01 M cacodylate buffer (Sabatini et a l . , 1961j Bracker and Grove, personal communication). The pH of the buffer i s adjusted to 7.0-7.2 with HC1 before use. Fix a t i o n time i s 12-16 hours (the longer time i s required for the resting spores). When using glutaraldehyde as a f i x a t i v e the osmolarity of the f i x a t i v e i s important i n obtaining optimal r e s u l t s for Ustilago hordei. The osmolarity of the growth medium, the growth medium plus the material, and the f i n a l 2% glutaraldehyde solution were determined on an Advance Osmometer (Model 3W). The r e s u l t s (Table I) indicated that the osmolaritjres of a l l three are within £0 milli-osmoles of each other. I t i s advisable to carry out the f i r s t hour of f i x a t i o n under vacuum. This i s p a r t i c u l a r l y true of r e s t i n g spores which tend to f l o a t . The material i s agitated continuously throughout f i x a t i o n . 3. Washing. - The fi x e d material i s washed for a minimum of lh hour i n water or buffer (whichever i s appropriate) changing the washing f l u i d 6-10 times. The material i s always recovered by centrifugation. I*. P o s t - f i x a t i o n . - Only the glutaraldehyde-fixed material i s post-fixed i n OsO^. OsO^ i s prepared by mixing equal parts of either 2% or" \\% OsO^ i n d i s t i l l e d water with the appropriate b u f f e r . P o s t - f i x a tion time i s 3 to 3^ g hours. 5>. Washing. - Post-fixed material i s washed as i n step 3« 6. Uranyl acetate s t a i n i n g . - Glutaraldehyde-osmium f i x e d material maybe pre^atained with 0.3$ Ur(Ae) 2 i n d i s t i l l e d water f o r 2-J* hours. Note t h i s step i s OPTIONAL and i s 183 usually not desirable f o r germinated material (pt. I I ) . I f t h i s step i s used the material should be rewahed as i n steps 3 and 5 . 7 . Agar embedding. - At thi s stage, the spores are co 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 material i s coated on the free side by dropping 2$ water agar (I4.70 C) onto the surface. The f i l t e r i s stripped o f f onoe the agar has s o l i d i f i e d and the under surface i s s i m i l a r i l y coated. The p e l l e t i s cut into small pieces f o r embedding. 8 . Alcohol dehydration. - A l l material (KMnO^fixed and GA-Os fixed) i s dehydrated through a standard ethanol s e r i e s : 3 0 $ , 5 0 $ , 7 0 $ , 8 5 $ , 9 5 $ , 1 0 0 $ . The agar-embedded tissue i s passed through the f i r s t 5 solutions i n approximately 1 hour, and given 3 changes i n absolute alcohol o f 30 minutes each (Total = I3g h r . ) . NOTE: In the text, 3 basic preparatory methods are distinguished, noted A, B and C. Method A involves KMnOi, f i x a t i o n . Methods B and ;C involve GA-Os f i x a t i o n and remain the same u n t i l the end of the alcohol dehydration. B and C are distinguished on the basis of the embedding p l a s t i c . Methods A and B are com-pleted by following steps 9 and 1 0 ; method C by step 1 1 only. 9 . Propylene oxide dehydration.- Propylene oxide i s added drop-by-drop to the f i n a l absolute ethanol change u n t i l the sol u t i o n i s half-and-half (Time = 3g hour). Three changes are made i n Propylene oxide (Time = 1 hour). 1 0 . Embedding i n Epon 8 1 2 . - The material i s i n f i l t r a t e d by Epon 812 (Ladd or Shell O i l ) ; (Luft, 1 9 6 1 ; 7A:3B). P l a s t i c minus accelerator i s added drop-wise to the blast propylene ox-ide change over a period of 1 ^ - 2 hours to a f i n a l half-and-half s o l u t i o n . The material i s l e f t overnight, uncovered and fresh p l a s t i o i s made (including the accelerator, 1.5#-DMP 30). The fresh p l a s t i o i s changed 3 times (3g hour each) before embedding. The Epon 812 i s hardened by polymerizing the blocks 12 hours at 37° C and 28-32 hours at 60° C. 11. Embedding i n Spurr's media. - The material i s i n f i l t r a t e d by Spurr's standard embedding medium (Folysciences Incorporated) (Spurr, 1969; Standard medium A f i r m ) . I t i s brought to the p l a s t i c through immersion i s f i v e standard solutions o f increas-ing p l a s t i c concentration: Spurr's : ETOH = 1 : 3, 1 : 2, 1 : 1, 2 : 1, 3 : 1. I t i s l e f t i n the l a s t s olution overnight; then i s put through 3 changes of p l a s t i c (^ hr. each) and em-bedded i n blocks. Harden Spurr's by polymerizing the blocks 6-7 hr. at 70° C. 12. Post-embedding technique. - S i l v e r to grey sections are cut on a S o r v a l l Porter-Blum MT-2 ultramicrotome using glass of diamond knives (Dupont) knives, and are picked up on carbon-coated g r i d s . The material i s post-stained i n a saturated solu-t i o n o f uranyl acetate i n $0% ethanol followed by lead c i t r a t e (Reynolds, 1963). After methods A and B the staining times are 30-lj.O minutes and 20-30 minutes respectively; while a f t e r method G the times are 3-k- hours (37° C) and 30-ij.O minutes respe c t i v e l y . 185 TABLE I Osmolarity of Broth, Broth plus Material, and 2$ Glutaraldehyde Material Measurement (milli-osmoles) Complete broth 290 .5 2 8 9 . 8 2 9 1 . 0 5 5 3 H i ' . 5 Broth plus 7 hr. spores 313 315 320 2% G.A. (cacodylate buffer) 336 Broth plus 0 hr. spores 312 312 3 3 5 0 3 3 5 . 5 3 3 9 . 5 NOTE: Measurements were made on an Advance Instrument Osmometer (Model 3W). BIBLIOGRAPHY Luft, J.H. 1961. Improvements i n epoxy r e s i n embedding methods. J . Biophys. Bioohem. Cytol. £: I+.09-I|.llij.-Reynolds, E.S. 1963* The use of lead c i t r a t e at high pH as an electron opaque s t a i n i n electron microscopy. J. C e l l B i o l . 1 2 : 208-212. Sabatini, D.D., Bensoh, K.G. and Barrnett, R.J. 1963. C y t o -chemistry and electron mioroscopy - preservation of 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 -t i o n / J . C e l l B i o l . 12.: 19-58. Spurr, 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 electron microscopy. J . U l t r a s t r u c t . Res. 26: 31-43. 186 APPENDIX C Light Microscope F i x i n g and Staining Procedures SQUASH PREPARATIONS In a l l cases a drop of medium containing the material i s a i r - d r i e d b r i e f l y on coverslips before being treated by one of the following methods, a) (Acetic-alcohol)-Feulgen Acetic-alcohol f i x a t i v e : Mix: 1 pt. g | a c i a l acetic acid with Feulgen Stain (Darlington and 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 Basic Fuchsin and shake. Cool to 50°C; f i l t e r into brown or darkened stock bot t l e with ground glass stopper and add 2 0 cc. I N H C 1 (Hydrochloric a c i d ) . Cool to 25°C and add either 1 gm. anhydrous sodium b i s u l f i t e (NaHSOj^) or 1-3 gm. potassium metabisulfite ( K 2 S 2 O 4 ) . Keep i n the dark. After 21+ hr. i t w i l l decolourize. If i t does not decolorize properly add 0 . 5 - 1 . 0 gm of Activated charcoal, shake, and f i l t e r . S O 2 water: Mix: 2 0 0 ml. of d i s t i l l e d water with 1 gm. po-tassium metabisulphite and 1 0 cc. 1 N H C 1 . Method: 1 . Wash two times i n d i s t i l l e d H 2 O - 5 min. each. 2 . Fix i n acetic-alcohol - 1 hr. 3 pt. absolute alcohol. Add: a few drops of chloroform to every 10 ml. of above solution. 1*. 3. Wash six times i n d i s t i l l e d H 20 - 5 min. each. Hydrolyse i n 1 N HC1 @ 60° C. - 8 min. or i n 18? $ N HC1 @ 22° C. - 1 hr. 5 . Wash 2 times i n tap water - 5 min. each. 6. Wash four times i n d i s t i l l e d water - £ min. each 7. Feulgen - 1-2 hr. 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 water - 5 min. each 10. Store cold u n t i l use. To squash the coverslip i s inverted ( i . e . material down) on a glass s l i d e on which a drop of h&% accetic acid has been placed, and pressure applied. A l l material was photographed from f r e s h l y prepared s l i d e s . After observation the s l i d e i s floated o f f i n absolute alcohol and remounted i n Euparol. b) BAC-Propi onic-haemat oxylin BAC-fixative (Lu, 1962); Mix: 9 pt. n-butyl alcohol, 6 pt. g l a c i a l acetic acid, and 2 - 3 pt. 10$ aqueous chromic acid. Use: fresh. Propionic-haematoxylin (Henderson and Lu, 1968): Stock solutions: A) 2$ haematoxylin i n 5 0 $ propiacid. B) 0 . 5 $ iron alum i n 5 0 $ propiacid. Mix: equal volumes of A and B. If stock solutions are fresh mix together one day before use. Aged stock solutions ( i . e . 3 mon. or more) can be mixed and used immediately. Method: 1. Wash two timed i n d i s t i l l e d water - £ min. each. 2. F i x i n BAC-fixative @ room temperature - 2l± hr. 3. Wash six times i n d i s t i l l e d water ( l a s t change at 60° C.) - 5> min. each. i+. Hydrolyze i n 1 N HC1 @ 60° C. - 12 min. 5. Wash two times i n tap water - 5 min. each. 6. Wash four times i n d i s t i l l e d water - 5 min. each. 7. Drain c o v e r s l i p and apply a few drops of staim macerating the tissue with an iron needle. 8. Invert onto a glass s l i d e and apply pressure. Material i s photographed from f r e s h l y prepared s l i d e s and i s made permanent as described i n procedure a. c) (Acetic-alcohol)-(Propiono-haematoxylin) Preparation of the f i x a t i v e ( i . e . acetic-alcohol) i s des-cribed i n part a and preparation of the, stain i n part b. Method: The method i s the same as that described i n part b substituting acetic-alcohol for BAC-fixative i n step 2, and shortening the f i x a t i o n time to 1 hr. SECTIONS F i x a t i v e : Method A, B, or C as described f o r electron micros-copy (Appendix B). Toluidine blue: Mix 1 gm. Toluidine blue i n 100 ml. 1$ borax. Method: 1. Thick sections (0 .25 - 0.50 u) are picked up with a small copper loop (1-2 mm diameter), and trans-ferred to a drop of water on a clean glass s l i d e . 2. Evaporate water by heating gently over a flame. 3. Place a drop of st a i n d i r e c t l y on sections. 1+. Heat u n t i l the edge of the drop turn yellow. 5. Wash the stain o f f i n tap water. 6. Dehydrate 1 min. ihir.i70# ethanol followed by 1-2 min. i n absolute alcohol. 7. Pass s l i d e through xylene f o r 1 min. 8. Mount i n immersion o i l and seal with n a i l p o l i s h . BIBLIOGRAPHY Darlington, CD. and La Cour, L.P. 1962. The Handling of  Chromosomes. George A l l e n and Unwin Ltd., London. Henderson, S.A. and Lu, B.C 1968. The use of haematoxylin f o r squash preparations of chromosomes. Stain Technology U3 : 233-236. Lu, B.C. 1962. A new f i x a t i v e and improved propi on o-c amine squash technique f o r staining fungus n u c l e i . Can. J. Bat. kO : 8I4.3-847. 

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