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The Nucleolus of wheat stem rust uredospores. Mitchell, Stephen Richard 1969

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THE NUCLEOLUS OF WHEAT STEM RUST UREDOSPORES by STEPHEN RICHARD MITCHELL B.Sc., University of V i c t o r i a , 1964. A THESIS SUBMITTTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department ' of Plant Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Plant Science The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date June 1 0 , 1 9 6 9 i ABSTRACT A c y t o l o g i c a l study of sporogenesis, mature uredo-spores and hydrated uredospores of Puccinia graminis t r i t i c i was made to determine i f a d e f i n i t i v e nucleolus was present. Electron microscopy has shown that the nuclei i n immature uredospores and associated fungal tissue of the uredosorus possess prominent n u c l e o l i . Nucleoli whose average diameter was 1 .7 microns were observed i n 5$ percent of the nuclear sections from immature uredospores. Presence of a nucleolus i n u l t r a t h i n sections of mature uredospores i s established. Nucleoli whose average diameter was 0 . 5 microns were observed i n 6 percent of the nuclear sections from mature uredospores. Nucleoli were not observed i n hydrated uredospores which had resumed active metabolism. The reduction i n the size of the n u c l e o l i i n mature uredospores and absence of n u c l e o l i i n hydrated uredospores may indicate that ribosomal RNA synthesis i s repressed as uredospores mature. i i ACKNOWLEDGEMENTS I am grateful to Dr. Michael Shaw fo r i n i t i a t i n g and d i r e c t i n g my work i n the c e l l biology of plant diseases and f o r his assistance i n preparation of the manuscript. I wish to acknowledge the members of the Electron Microscope Committee, Department of Biology, Saskatoon, f o r t h e i r i n s t r u c t i o n . I am grate f u l to Dr. M. Weintraub, CDA Research Station, Vancouver, f o r the use of electron microscope f a c i l i t i e s and to Miss Esther Lo f o r her assistance. I also thank Dr. Eaton and Frank Eady f o r help with the s t a t i s t i c a l analysis and the many other people who have enriched my work. This study was supported by an NRC Research assistantship and extramural research' grants from the Canada Department of Agriculture made to Dr. Shaw, i i i TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i i TABLE OF CONTENTS i i i LIST OF TABLES i v LI ST OF FIGURES v INTRODUCTION 1 LITERATURE REVIEW 3 MATERIALS AND METHODS 19 A. Uredospore c u l t u r e 19 1. Plant m a t e r i a l s 19 2 . Spore i n o c u l a t i o n and c o l l e c t i o n 19 B. Cytology 20 1. Tissues 20 2 . F i x a t i o n 20 3 . Dehydration and embedding 21 4 . S e c t i o n i n g , s t a i n i n g and microscopy 21 RESULTS 23 A. P r e l i m i n a r y i n v e s t i g a t i o n 23 B. Cytology of spore development, maturation and hy d r a t i o n 24 1 . Immature uredospores 24 2. Mature uredospores 33 3. Hydrated uredospores 37 4 . Observation of a nucleolus 40 DISCUSSION 41 SUMMARY 48 LITERATURE CITED 49 i v LIST OF TABLES TABLE Page I Diameters of Nuclei and N u c l e o l i . 39 I I Dimensions of Uredospore Structures. 39 V LIST OF FIGURES Figure Page 1 Pustule development on wheat l e a f . 25 2 Sporulating rust fungus i n pustule. 25 3 Zones of increasing d i f f e r e n t i a t i o n . 25 4 Apical hyphal c e l l i n pustule. 26 5 Incipient spore i n pustule. 27 6 Immature uredospore i n pustule. 2$ 7 Nuclei of a p i c a l c e l l with prominent n u c l e o l i . ' 30 3 Nuclei of i n c i p i e n t spore 31 9 Nuclei of immature uredospore 3 2 10 Nuclei of immature uredospore with nucleolus.in each. 32 11 Cytoplasm of mature uredospore. 34 12 Nucleus of mature uredospore with reduced nucleolus. 3 5 13 Nucleus of mature uredospore with doughnut-like nucleolus. 36 14, Cytoplasm of hydrated uredospore showing germ process. 3 8 1 INTRODUCTION Rust fungi are dependent on host tissue f o r success-f u l growth and reproduction. No biochemical lesions which may cause rusts to be obligate parasites have been defined and success i n axenic culture of rusts has been li m i t e d . Uredospores are the repeating spore form of the graminicolous rusts. These spores are produced on the grass host from a binucleate mycelium con s t i t u t i n g a uredosorus beneath the epidermis of the host. The formation of uredospores ruptures the epidermis of. the host and dissemination i s accomp-l i s h e d by wind or r a i n . The a b i l i t y of the spores to germinate at maturity makes the uredial stage the most destructive aspect i n the rust l i f e cycle and consequently has stimulated much in v e s t i g a t i o n . Uredospores w i l l germinate on water at the expense of endogenous reserves. U n t i l recently, d i f f e r e n t i a t i o n of the germ tube of the sporeling could be achieved only on the host,plant. D i f f e r e n t i a t i o n of germ tubes into i n f e c t i o n structures has been induced with l e a f c u t i c l e hydrocarbons, and heat treatment. However, uredospore inoculation of defined media w i l l not y i e l d the luxuriant growth characteris-t i c of saprophytic fungi. The concept has emerged from biochemical studies of the u t i l i z a t i o n of exogenous carbon sources that germinating uredospores have a defective mechanism of protein synthesis. This was reinforced by the apparent absence of n u c l e o l i i n 2 the nuclei of mature uredospores as revealed by electron microscopy. The nucleolus-protein synthesis relationship of rust spores i s further complicated by reports of extrusion of n u c l e o l i from the nuclei of germinating uredospores. The present study was undertaken to determine whether the nuclei In immature, mature and hydrated uredospores of wheat stem r u s t , Puccinia graminis t r i t i c i . contain d e f i n i t i v e n u c l e o l i and to re-examine other e a r l i e r c y t o l o g i c a l findings. Electron microscopy was the primary t o o l f or a c y t o l o g i c a l study of the above tissues. 3 LITERATURE REVIEW Cytology Cyto l o g i c a l studies of rusts extend back to 1$80 and i n the i n t e r v a l from that date Savile (1939) recognized three phases of i n t e r e s t . The f i r s t was the recognition of a d i p l o i d stage, the te l i o s p o r e , and subsequent reduction to haploidy. The second was i n i t i a t e d by the discovery of the o r i g i n of the dikaryotic state i n the aecium. The t h i r d phase was ushered i n by the discovery that the locus of nuclear mixing was the pycnium (Craigie 1927). Current research publications reveal that fungal cytologists and plant pathologists have embraced u l t r a -s t r u c t u r a l and biochemical studies and such studies may be considered to represent the fourth phase of rust cytology. Current investigations are being conducted at magnifications and with a degree of refinement unimaginable to the early c y t o l o g i s t s . Uredospores of Puccinia graminis t r i t i c i E r i k s s . and Henn. (wheat stem rust) are ovoid, s i n g l e - c e l l structures 27|-i i n length and l 6 u . wide. The thick c e l l w a l l , 1.5u - 2u, i s covered with spines which develop beneath the wall and move to the exterior as the spore develops (Thomas and Isaac 1967). Williams and Ledingham (1964), Strobel (1965) and Thomas and Isaac (1967) described the c e l l wall as consisting of three layers. The inner and outer layers were electron dense and the intermediate layer was electron transparent. The c e l l wall of f l a x rust uredospores has been described by Manocha and Shaw (1967) as a multi-layered structure. Thin areas i n the spore 4 wall were termed germ pores (Williams and Ledingham 1964, Manocha and Shaw 1967). A reduction i n c e l l wall thickness when spores were hydrated was reported by Manocha and Shaw (1967) but a s i m i l a r change i n spores of Puccinia s t r i i f o r m i s ( s t r i p e rust) was not observed by Strobel (196-5). The plasma membrane of spore cytoplasm of f l a x rust uredospores has been described by Manocha and Shaw (1967) a s a single membrane. The same zone i n wheat stem rust uredospores was v i s u a l i z e d by Williams and Ledingham (1964) as a layer of v e s i c l e s . Hyde and Walkinshaw (1966) described the plasma membrane i n basidiospores of Lenzites as a single membrane with numerous invaginations. Most cytoplasmic organelles and inclusions of fungi are common to other plants and animal tissues (Bracker 1967). Nuclei, mitochondria, endoplasmic reticulum, ribosomes and l i p i d bodies have been found i n rust uredospores. Lipids i n the form of large central droplets or small, rounded membrane-bound bodies were observed by Strobel (1965) to occupy a considerable volume of spore cytoplasm. Small l i p i d bodies i n uredospores have also been described by Williams and Ledingham (1964) and Manocha and Shaw (1967). L i p i d bodies have been described i n basidio-spores (Hyde and Walkinshaw 1966), aeciospores (Walkinshaw et a l . 1967) and i n spores of other fungal groups (Remsen et a l . 1967, McKeen et a l . 1967, Marchant 1966). I t has been stated (Shaw 1964, Staples and^Wynn 1965, Allen 1965) that l i p i d s are an important source of metabolites f o r spore maintenance and germination. At the onset of germination of rust spores Strobel 5 (1965) observed that the mobilization of l i p i d reserves re-sulted i n the reduction i n siz e of large l i p i d bodies. The very dense cytoplasm of spores with low water content (uredospores, aeciospores) has proved d i f f i c u l t to f i x and embed (Williams and Ledingham 1964, Walkinshaw et a l . 1967) and many features have remained obscure. Williams and Ledingham (1964) and Manocha and Shaw (1967) reported that the endoplasmic reticulum took the form of v e s i c l e s and ribbons i n mature spores. The former authors observed increased amounts of the same form of endoplasmic reticulum i n germ tubes where-as the l a t t e r observed elongated threads of endoplasmic reticulum i n germ tubes. Walkinshaw et a l . (196?) described the endoplas-mic reticulum of aeciospores as i r r e g u l a r v e s i c l e s while germ tubes a r i s i n g from these spores possessed elongate endoplasmic reticulum covered with ribosomes. Strobel (1965) did not des-cribe endoplasmic reticulum i n dormant uredospores but i n hydrated spores he did observe a vesiculate endoplasmic reticulum the appearance of which was correlated with a decrease i n soluble protein. Hyde and Walkinshaw (1966) did not f i n d endoplasmic reticulum i n basidiospores of Lenzites fi x e d i n either permanganate or aldehyde although ribosome groupings were observed. The above condition was observed i n germ tubes. Associations of endoplasmic reticulum with cytoplasmic elements other than ribosomes have been reported by Williams and Ledingham (1964) who observed endoplasmic reticulum clo s e l y associated with small l i p i d i n c l u s i o n s . McKeen et a l . (1967) described an 6 apparent association of endoplasmic reticulum and the tono-p l a s t or vacuolar membrane, i n conidia of Erysiphe. The mitochondria of mature uredospores have been des-cribed as rounded and about one micron i n diameter (Williams and Ledingham 1964, Manocha and Shaw 1967). Williams and Ledingham noted that c r i s t a e within these mitochondria are few i n number and l i e i n random d i r e c t i o n s . During germina-t i o n c r i s t a e have been v i s u a l i z e d as d i s t i n c t p a r a l l e l mem-branes (Williams and Ledingham 1964, Manocha and Shaw 1967). Walkinshaw et a l . (1967) observed the aforementioned changes i n c r i s t a e and the presence of tubules or rdbosomes on the surface of mitochondria i n dormant spores whereas the mito-chondria i n germ tubes exhibited smooth outer membranes. Increased r e s p i r a t i o n associated with spore germina-t i o n (Shaw 1964, Staples and Wynn 1965, Allen 1965) i s consis-tent with reports of increased numbers of mitochondria (Marchant 1966, Remsen et a l . 1967, Lowry and Sussman 1968, Buckley et a l . 1968). Marchant (1966) described a reduction i n size of mitochondria, suggesting d i v i s i o n , whereas Lowry and Sussman (1968) suggested that a change i n biochemical function might cause a reduction i n s i z e . Enlarged mitochondria have been reported i n the germination of Rhizopus sporangiospores and i t was suggested that t h i s i s a response to the requirement f o r membrane subunits (Buckley et a l . 1968). An alignment of mitochondria along the jplasma membrane when spores were 7 hydrated was noted by Strobel (1965) who suggested that t h i s might f a c i l i t a t e gas exchange. Metabolic a c t i v i t i e s within wheat stem rust uredo-spores are mediated by two haploid n u c l e i . The haploid chromosome number was determined to be s i x (McGinnis 1953). Savile (1939) presented an extensive study of nuclear be-havior throughout the l i f e cycle i n the Uredinales. Savile (1939) described the uredospore nuclei as f u l l y expanded with chromatin i n an 'ectosphere' and a clear homogeneous inner area termed the Tendosphere T. The 'endosphere' was stated to be non-homologous to the nucleolus of higher plants. It was concluded that during uredospore germination, the 'endosphere' •moved to the membrane of the 'ectosphere'. Subsequently the membrane broke down at the point of association,and reformed behind the extruded 'endosphere' -which disintegrated i n the cytoplasm. Distorted n u c l e i , henceforth known as 'unexpanded' n u c l e i , were observed to round up and migrate into the germ tube. Similar observations were made at aeciospore germination. The presence of an unexpanded nucleus at various points within the rust l i f e cycle prompted Savile to conclude that the trans-formation of an 'expanded' nucleus to an 'unexpanded' nucleus occurred at any point where a nucleus had to pass through a r e s t r i c t i n g pore. The existence of two nuclear forms was concluded to be f u n c t i o n a l . Im a more recent study, Graigie (1959) observed n u c l e o l i i n the nuclei of immature uredospores but not i n germ tube n u c l e i . It was concluded that extrusion of n u c l e o l i from 8 the nuclei of germinating spores occurred i n the manner des-cribed by Savile (1939). Williams and Ledingham (1964) did not observe n u c l e o l i i n Puccinia graminis t r i t i c i uredospores i n one of the f i r s t f i n e structure studies of rust spores. There was no i n d i c a t i o n that a nucleolus was present i n basidiospores of Lenzites but promi-nent n u c l e o l i were shown i n germ tube nuclei (Hyde and Walkinshaw 1966). Manocha and Shaw (196?) did not report n u c l e o l i i n nuclei of mature hydrated uredospores and i n t e r c e l l u l a r mycelium but structures interpreted as n u c l e o l i occurred i n immature uredo-spores. A -study of the development of echinulations on spore walls (Thomas and Isaac 1967) revealed that n u c l e o l i were present i n immature uredospores of Puccinia graminis t r i t i c i . At the onset of sporogenesis i n Plasmodiophora brassicae 4 n u c l e o l i have been reported to disappear (Williams and McNabola 1967). Shaw (1967) has speculated that i f the nucleolus i s absent from mature uredospores or cannot be reformed after nuclear d i v i s i o n during germination then subsequent p r o l i f e r a t i o n of the fungus may depend on stimulation from the host directed at synthesis of ribosomes and protein synthesis' i n the fungus. The conidia of Erysiphe cichoracearum exhibit a nucleolus adjacent to the nuclear membrane (McKeen et al.1967). Neither n u c l e o l i nor chromosome figures were found i n dormant conidia or germinating conidia of Fusarium (Marchant 1966). In contrast, Heale e t ' a l . (1968) observed that a nucleolus was absent i n the r e s t i n g nucleus of V e r t i c i l l i u m conidia. During 9 germination and. nuclear d i v i s i o n , a nucleolus appeared, increased i n size and persisted u n t i l l a te d i v i s i o n . Nucleolus Since the primary objective of the investigation was to determine i f d e f i n i t i v e n u c l e o l i were present i n uredospores i t i s desirable to discuss the nucleolus b r i e f l y . The nucleolus i s a chromatin-associated organelle concerned with synthesis of ribosomal RNA (Waddington 1 9 6 6 ) . The major macromolecules of the nucleolus are nucleolar DNA, nucleolar RNA and proteins (Waddington 1 9 6 6 , B i r n s t i e l 1 9 6 7 ) . Nucleolar DNA i s that part of the genome containing the cistrons for precursors of rib o -somal RNA. Nucleolar RNA exists as two species which are precursors of ribosome subunits. Nucleolar protein consti-tutes 8 0 - 9 0 $ of the dry matter .in the nucleolus and exists i n two classes, l y s i n e - r i c h protein and residual proteins, the l a t t e r resembling ribosomal protein ( B i r n s t i e l 1 9 6 7 ) . There i s general agreement that the nucleolus has four s t r u c t u r a l components: granules, f i b r i l s , protein matrix and chromatin f i b r i l s ( B i r n s t i e l 1 9 6 7 ) . In plants, the f i b r i l l a r portion, termed the parsamorpha, i s c e n t r a l l y located i n the nucleolus while the granular portion, or nucleonema, i s peripheral. U t i l i z i n g enzymatic digestions i t has been shown that the granules, which resemble ribosomes, and the f i b r i l s are composed of RNA and protein ( B i r n s t i e l 1 9 6 7 ) . At mitosis the nucleolus begins to lose i n t e g r i t y i n midprophase with the dispersion of the granular f r a c t i o n 1 0 into the nucleoplasm (Lafontaine 1 9 6 8 ) . RNA synthesis stops at l a t e prophase ( B i r n s t i e l 1 9 6 7 ) . Dissolution of the f i b r i l l a r material coincides with the breakdown of the nuclear membrane. RNA and protein synthesis are resumed during telophase and the s t r u c t u r a l elements of the nucleolus reappear i n the reverse order of t h e i r d i s s o l u t i o n (Ghouinard 1 9 6 6 ) . The behavior of the nucleolus i n mature and germinating uredospores may be better understood when viewed within the context of the spore physiology and biochemistry. Uredospore Physiology The physiology of dormant and germinating rust uredo-spores has been reviewed by Shaw ( 1 9 6 4 ) and Staples and Wynn ( 1 9 6 5 ) . A l l e n ( 1 9 6 5 ) has reviewed fungal metabolism pertain-ing to spore germination. The general composition of wheat stem rust uredospores i s as follows: carbohydrate 2 2 $ , protein 2 6 $ , l i p i d 2 0 $ , c h i t i n 1 . 4 $ and water 1 0 - 1 5 $ (Shaw 1 9 6 4 , Staples and Wynn 1 9 6 5 ) . A r a b i t o l , mannitol and trehalose were found to be the major soluble carbohydrates (Wynn et_ a l . 1 9 6 6 , Daly et a l . 1 9 6 7 ) and lesser amounts of arabinose, fructose, galactose, glucose, maltose, mannose, ribose, xylose and glycer o l have been reported (Shaw 1 9 6 4 , Staples and Wynn 1 9 6 5 ) . A polysaccharide extracted from,uredospores was shown (Kloker et a l . 1 9 6 5 ) to consist of D-mannose, D-galactose, and D-glucose, with pentoses and glucosamine as probable side-chain additions. The nature of uredospore proteins remains open to 11 i n v e s t i g a t i o n . Many enzymes have been demonstrated i n c e l l homogenates and c e l l - f r e e b r e i s (Shaw 1964, S t a p l e s and Wynn 1965). A c i d h y d r o l y s i s of uredospore p r o t e i n s y i e l d e d a l l t h e common amino a c i d s ( K l o k e r et a l . 1965). T o t a l p r o -t e i n c o n t e n t of u r e d o s p o r e s may change under v a r i o u s e n v i r o n -m e n t a l c o n d i t i o n s and t h i s may r e f l e c t t h e n u t r i e n t l e v e l i n h o s t t i s s u e ( E y a l et a l . 1967). Wynn et a l . (1966) found t h a t t h e predominant amino a c i d s i n u r e d o s p o r e s were g l u t a m a t e , a l a n i n e , c y s t i n e , a s p a r t a t e and s e r i n e . W i t h a d i f f e r e n t e x t r a c t i o n p r o c e d u r e S t e f a n y e and B r o m f i e l d (1965) i s o l a t e d g l u t a m i n e , g l u t a t h i o n e , a l a n i n e , ammonia and g l u t a m i c a c i d as t h e main n i n h y d r i n - p o s i t i v e sub-s t a n c e s . L i p i d s comprise a p p r o x i m a t e l y 20% o f uredospore f r e s h w e i g h t i n wheat stem r u s t (Shaw 1964, D a l y et a l . 1967) and 14% i n f l a x r u s t ( J a c k s o n and F r e a r 1967). The l i p i d s have been f o u n d t o be composed l a r g e l y of g l y c e r i d e s , and a c o n s i d e r a b l e f r a c t i o n o f u n s a p o n i f i a b l e l i p i d s ( S t a p l e s and Wynn 1965) whic h a r e m a i n l y s t e r o l s ( J a c k s o n and F r e a r 1968). Imp o r t a n t f a t t y a c i d s f o u n d were p a l m i t i c , s t e a r i c , o l e i c , l i n o l e i c , l i n o l e n i c and c i s - 9 , 1 0 - e p o x y o c t a d e c a n o i c a c i d ( J a c k s o n and F r e a r 1967, D a l y et a l . 1967.) . One- s t e r o l of wheat stem r u s t , and two s t e r o l s of f l a x r u s t have been r e p o r t e d ( J a c k s o n and F r e a r 196$). The u n s a p o n i f i a b l e f r a c t i o n of wheat stem r u s t u r e d o s p o r e s has been found t o c o n t a i n ^ - c a r o t e n e , - c a r o t e n e , l y c o p e n e and phytoene w h i c h a re p a r t l y r e s p o n s i b l e f o r t h e br o w n i s h c o l o r o f s p o r e s (Shaw 1964, S t a p l e s and Wynn 1965). Bush (1967) 12 supported the above findings and showed that carotene content of spores changes with season and age of spores. He suggested that carotenes serve to protect the spore protoplast from u l t r a -v i o l e t radiation damage. The unsaponifiable l i p i d f r a c t i o n from f l a x rust uredospores has been separated into /9 -carotene, */ -carotene and an uni d e n t i f i e d pigment (Kelley I965). The many other substances which .have been is o l a t e d from uredospores include organic acids, phenolics, vitamins, nucleotides and auxins (Shaw 1964, Staples and Wynn 1965). Existence of the Embden-Meyerhof-Parnas pathway, hexose monophosphate pathway, t r i c a r b o x y l i c acid cycle and the glyoxylate pathway has been demonstrated. Detection of the appropriate enzymes forms part of the evidence for the presence of these pathways. Shaw (1964) and Staples and Wynn (1965) have comprehensively reviewed the respiratory metabolism of uredospores. In order to determine the nature of respiratory path-ways and substrates being u t i l i z e d , uredospores were allowed to re s p i r e i n the presence of C-^-labelled sugars, sugar alcohols, organic, amino and f a t t y acids. I t was found that uredospores assimilate exogenous substrates very slowly and that the incorporation of l a b e l l e d carbon into c e l l carbon was very low. Short chain f a t t y acids (C2 to C Q) were u t i l i z e d more rapidl y than other substrates and increased chain length promoted higher oxygen consumption. Some features of uredospore r e s p i r a t i o n included (a) low oxygen consumption, (b) a low 13 C^ /C-|_ r a t i o suggesting that much CO^ i s released through the pentose phosphate pathway and (c) no i n h i b i t i o n of oxygen up-take by malonate or sodium f l u o r i d e but (d) i n h i b i t i o n by cyanide and 2 , 4-dinitrophenol (Shaw 1964, Staples and Wynn 1965). I n a b i l i t y of o b l i g a t e l y p a r a s i t i c fungi to grow on simple media prompted comparative studies of exogenous substrate u t i l i z a t i o n by saprophytic and obligately p a r a s i t i c fungi (Staples et a l . 1961, Staples et a l . 1962). Non-germinating spores of the l a t t e r group of fungi did not synthesize net protein to a greater extent than the obligate fungi. It was concluded that the differences were due i n part to the a b i l i t y of the spores to remove the l a b e l l e d precursors from the exogenous medium. When the studies were extended to germinating spores, Staples et_ a l . (1962) found no net synthesis of protein or nucleic acids by the germinating uredospores. In contrast to germinating spores of Aspergillus. Neurospora and Glomerella .which assimilated more substrate and synthesized more i n t e r -mediates, the uredospores incorporated into protein i n a manner suggesting that proteins were formed by a turnover •process. The nature of the block i n protein synthesis i n uredospores was not known. More recently Staples et a l . (1966) demonstrated that ribosomes extracted from dormant and germinating uredospores were equally e f f e c t i v e i n incorporating phenylalanine i n a p o l y u r i d y l i c acid directed system which included a c t i v a t i n g 14 enzymes prepared from r i c e embryos. Subsequently Staples and Bedigian (1967) described an amino acid incorporating system prepared from bean rust uredospores i n which both the ac t i v a t i n g enzymes and ribosomes were derived from uredospores. From the evidence f o r -polyribosomes i n dormant uredospores Staples et a l . (1968) suggested that an intact mechanism for -protein synthesis i n uredospores i s under r e s t r a i n t . In a re-view of the above work and current research, Staples (1968) re-ported that cessation of germ tube growth correlated with de-creasing template a c t i v i t y of spore messenger RNA and decreasing amino acid incorporation by ribosomes and suggested that i n t e r -action with a host may allow maintenance of these nucleic acid a c t i v i t i e s . In addition to the evidence presented f o r the exis-tence of an intact protein synthesis mechanism, uredospores are also capable of incorporating ammonia nitrogen into amino acids which are i n turn u t i l i z e d i n protein synthesis (McConnell and Underhill 1966). Uredospores are able to f i x carbon dioxide into organic acids (Staples and Weinstein 1959, Rick and Mirocha 1963). The former workers found that l a b e l l e d carbon was dist r i b u t e d r a p i d l y throughout intermediates of the Krebs cycle and postulated that phosphoenolpyruvate carboxylase was the mechanism of incorporation. Rick and Mirocha (1968) demon-strated that the malic enzyme present i n uredospores catalyzed the formation of malate from pyruvate and CO2 i n c e l l - f r e e preparations. The reverse reaction was also demonstrated and no evidence f o r the mechanism of CCu f i x a t i o n postulated by 15 Staples and Weinstein (1959) was found. The sign i f i c a n c e of COg f i x a t i o n by rust spores i s not clear, although i t may en-hance the Krebs cycle or engage i n hydrogen transport by u t i l i z i n g NADPH from the pentose cycle f o r the reductive carboxylation of pyruvate to malate. Oxidation of malate i n the Krebs cycle would produce NADH. which could par t i c i p a t e i n the electron transport system (Rick and Mirocha 1968). Germination of rust spores and many other spores i s preceded by.hydration which may be effected by free water or high humidity (Staples and Wynn 1965). Hydration coincides with an abrupt increase i n oxygen uptake, the QOg r i s i n g from 0.5 to 12 and de c l i n i n g to 5 a f t e r the f i r s t hour (Williams and Allen 196?). Bush (I968) observed an i n i t i a l high rate of oxygen consumption by uredospores but detected the decline i n oxygen uptake after ten minutes incubation. Germination and oxygen uptake have been stimulated by many exogenous substrates but short chain (C^ - C^) f a t t y acids have proven most ef f e c t i v e (Shaw 1964) and probably r e f l e c t the u t i l i z a t i o n of endogenous l i p i d reserves. Similar findings have been obtained by Bush (1968) and Loesl ( 1 9 6 7). In contrast, Walkinshaw (1968) ob-served that oxygen uptake by aeciospores was i n h i b i t e d by short chain f a t t y acids but stimulated by C-^ g unsaturated f a t t y acids. Considerable evidence has been obtained (Shaw 1964, Staples and Wynn 1965, Allen 1965) which suggests that uredo-spores germinate at the expense of l i p i d reserves. This evidence rests mainly on the low respiratory quotient of germinating spores and the disappearance of l i p i d s . Jackson and Frear (1967) found that t o t a l l i p i d s decreased but unsaponifiable l i p i d s and l i p i d phosphorus ( i n d i c a t i v e of phopholipid 16 synthesis) increased during the germination of f l a x rust uredospores. There was also a rapid transformation of c i s -9,10-dihydroxyoactadecanoic acid to threo -9.10-dihydroxyocta-decanoic acid. In an e f f o r t to obtain a better understanding of the r o l e of l i p i d s and carbohydrates i n spore germination, Daly et a l . (1967) u t i l i z e d uredospores l a b e l l e d with C"^ fed to host plants during spore formation. An average of 7% of the spore carbon, which was mostly carbohydrate i n nature, was found i n the water on which the spores germinated. Soluble carbohydrates were reduced by 30% within the f i r s t hour and at seven hours approximately one-half the carbohydrate pool had been metabolized to other compounds. Free f a t t y acids changed markedly within one hour with a sharp reduction i n the amount of cis-9.IQ-epoxyoctadecanoic acid. Total l i p i d s did not change greatly during seven hours. I t i s noted that the carbohydrate der i v a t i v e s , c h i t i n and glucosamine, were found to increase during germination (Gottlieb 1966). Protein synthesis i n germinating uredospores i s apparently very low compared to that i n saprophytes according to the work from Staple's laboratory which has.been mentioned e a r l i e r i n t h i s review. Dunkle ejt a l . (1969) have shown by the use of i n h i b i t o r s that completion of i n f e c t i o n structure forma-t i o n i s dependent upon protein synthesis. According to Gottlieb (1966) increases i n c e l l protein are equivalent to increases i n enzymes. Increased enzyme a c t i v i t y , p a r t i c u l a r l y r e s p i r a t o r y enzymes, has been reported by Shaw (1964) and Allen (1965). P_e novo synthesis of enzymes during germination has 17 been detected i n many fungi, but Ustilago i s a notable example since enzymes of the EMP, PP, and TCA pathways were synthesized (Gottlieb 1966). Hemicellulase, c e l l u l a s e and polygalacturonase were found to increase i n concentration during uredospore germination (Shaw 1964). In contrast to Staples et a l . (1966), Van Etten (1968) found that an i n v i t r o amino acid incorporating system prepared from resting spores of a f a c u l t a t i v e parasite was inactive whereas the same system prepared from germinating spores was very active. It was con-cluded that the enzyme f r a c t i o n was defective i n ungerminated spores and subsequently, Van Etten and Brambl (1963) demonstrated that the s p e c i f i c a c t i v i t y of the aminoacyl-s RNA synthetases i s low i n ungerminated spores but increases during germination. Net synthesis of nucleic acids i n germinating spores has been documented i n species of Aspergillus and Penicillium (Gottlieb 1966, Allen 1965). Staples et a l . (1962) did not observe any net synthesis of nucleic acids i n germinating uredospores supplied with acetate. However, the germ tube nu c l e i observed by Maheshwari et a l . (1967) indicated a net synthesis of DNA and the arrest of i n f e c t i o n structure d i f f e r -e n t i a t i o n by i n h i b i t o r s of RNA synthesis has suggested that synthesis of a new RNA i s a prerequisite to t h i s d i f f e r e n t i a t i o n (Dunkle et a l . 1969). Gottlieb et a l . (1968) have described a complexed RNA i n teliospores of Ustilago which i s enzymatically changed to normal RNA. The appearance of the normal RNA coincided with synthesis of missing enzymes. 18 Germination of uredospores i s affected by s e l f -i nhibitors, and many external f a c t o r s . Allen (1955) discovered that a water soluble, v o l a t i l e substance, synthesized de novo, i n h i b i t e d germination of dense spore masses f l o a t i n g on buffer. Dinitrophenol, methyl naphthoquinone and coumarin were effec-t i v e i n counteracting the i n h i b i t i o n . In attempting to puri f y aqueous extracts of the i n h i b i t o r , Allen (1957) found a d i s -t i l l a t e f r a c t i o n which stimulated germination and d i f f e r e n t i a -t i o n of germ tubes to i n f e c t i o n structures. The stimulant was e f f e c t i v e either as a solution i n direct contact with spores or as a gas. There are suggestions that a relat i o n s h i p between l i g h t and i n h i b i t o r s may exist (Givan and Bromfield 1964, Wiese and Daly 1967). The depression i n germination of spores exposed to 400 foot candles of l i g h t i s temperature se n s i t i v e , suggesting photochemical and enzymatic controls (Givan and Bromfield 1964). Temperature optima for uredospore germination of Puccinia graminis t r i t i c i was 22°C (Wiese and Daly 1967), 7°C for Puccinia s t r i i f o r m i s (Sharp 1965) and 20° - 30°C for Puccinia cynodontis (Vargas et a l . 1967). Surface secretions by host tissues and factors derived from microflora have been implicated as other influences on spore germination (Sharp 1965). 19 MATERIALS AND METHODS A. Uredospore culture 1. Plant materials Uredospores of Puccinia graminis Pers". f . sp. t r i t i c i E r i k s s . and Henn., race 15B (wheat stem rust) were obtained from the Department of Biology', University of Saskatchewan, Saska-toon. These spores were increased on L i t t l e Club wheat, Triticum aestivum s. sp. compactum (Host) Mackey, grown i n a sandy loam i n ten-inch p l a s t i c pots. Healthy and rusted plants were grown i n a Sherer growth cabinet at 2 2 - 2 4°C during the day and 17-19°C during the night. An illumination of approximately 700 f t - c 18 inches from the source (top of pot) and 1100 f t - c 6 inches from the source was available for 16 hours d a i l y . 2. Spore inoculation and c o l l e c t i o n Plants one week to one month old were inoculated by rubbing uredospores onto the leaves using a moistened thumb and for e f i n g e r . Leaves were then sprayed with d i s t i l l e d water from an atomizer. A p l a s t i c bag was placed over the plants i n order to maintain high humidity and pr i o r to sealing the bag around the pot, the plants were watered. Inoculated plants were l e f t i n a dark growth chamber overnight, after which the p l a s t i c covering was removed. Flecking i n leaf tissues was evident at 5 days afte r inoculation and sporulation began at 7 to 10 days a f t e r . i n o c u l a t i o n . Mature spores were collected by tapping the leaves over a manila folder or by suction into a glass c o l l e c t o r . 20 B. Cytology 1. Tissues Immature uredospores i n s i t u i n leaf t i s s u e , mature uredospores and hydrated uredospores were studied i n thi n sec-tions i n the electron microscope. Immature uredospores, sampled on two occasions, were obtained by excising unbroken pustules immersed i n f i x a t i v e . Hydrated uredospores were prepared by heat-shocking (5 min at 40°C) spores stored i n l i q u i d nitrogen f o r 7 days. Spores were then (a) floated on 20 ml d i s t i l l e d water i n a 9 cm p e t r i dish i n darkness at room temperature f o r •1.25 hr or, (b) placed i n aluminum f o i l boats f l o a t i n g on water i n a saturated atmosphere for 21+ hr at room temperature i n dark-ness. In a t h i r d method, f r e s h l y collected spores were sprayed onto 2% agar i n 9 cm p e t r i dishes and incubated i n darkness at room temperature f o r 25 min. Mature uredospores no more than 48 hr old were obtained d i r e c t l y from a c t i v e l y sporulating pustules on two occasions. A v a i l a b i l i t y of fresh spores of approximately the same age was assured by c o l l e c t i n g spores or shaking plants every two days. 2. Fixation A l l tissues were fix e d i n a mixture of 5% acrolein and 5% glutaraldehyde ( f i n a l concentrations) i n 0.1M phosphate buffer pH 7.2. A trace of 1% Rexol (non-ionic wetting agent) was added to the f i x a t i v e to ensure wetting of the spores. Excised pustules were f i x e d i n 2 dram s h e l l v i a l s . Mature uredospores were s t i r r e d into the f i x a t i v e i n 10 x 75 mm culture tubes and sank within f i v e minutes. Hydrated uredospores were fix e d by 21 flooding the p e t r i dishes with f i x a t i v e and after one hour the spores were pipetted into a culture tube. Fixation was done at room temperature overnight but never exceeded 20 hours. After primary f i x a t i o n , tissues were rinsed at least three times with equal parts of buffer and water. Postfixation was accomplished with 1 part 2% KMnO^, 1 part 0 .1M phosphate buffer •pH 7.2 (Buckley et a l . 1968) and 1 or 2 drops of 1% Rexol. Tissues were fix e d for 1 hour at room temperature then rinsed with d i s t i l l e d water at least three times over 15 to 30 minutes. Mature spores and hydrated spores were embedded i n 1% •agar to f a c i l i t a t e handling during dehydration and embedding. 3.•Dehydration and embedding An ethanol series s t a r t i n g with 30% ethanol was employed fo r dehydration of tiss u e s . Transition to the embedding medium was made with propylene oxide. Maraglas 605 used according' to B i s l p u t r a and Weier (1963) and Epon-Araldite (Skvarla 1966) were the most s a t i s f a c t o r y epoxy r e s i n embedding media. Tissues were soaked overnight i n a mixture consisting of 1 part propylene oxide and 1 part r e s i n . The i n f i l t r a t i o n mix-ture was then adjusted to 3 parts r e s i n and 1 part propylene oxide and l e f t for 3 - 4 hours. Leaf tissues were flat-embedded i n pure res i n medium i n a miniature ice cube tray. Agar blocks containing spores were placed at the top of polyethylene (BEEM) capsules f i l l e d with r e s i n . When the tissues had sunk i n the r e s i n , the containers were placed i n a 60-65°C oven f o r 24 hours. 22 4. Sectioning, s t a i n i n g and microscopy Polymerized blocks were trimmed to a pyramidal face le s s than 0.5 mm on a side. Sections were cut with glass knives on a So r v a l l MT.-l ultramicrotome and were flattened and expanded with xylene vapor. Parlodion-carbon coated grids (3 mm, 200 mesh) were used to c o l l e c t sections. Staining was done with 5$ aqueous uranyl acetate (40 min) and Reynold's lead c i t r a t e (20 min). Sections were viewed and photographed at 60KV i n a P h i l i p s EM 100B electron microscope. Micrographs were made on a 35 mm f i l m , Recordak M i c r o f i l e . Measurements of organelles were made on negatives using an 8X L e i t z lens. Sections f o r l i g h t microscopy were cut 0.5 u thick and stained for 5 min at 55°C with 1% t o l u i d i n e blue i n 1% sodium borate. 23 RESULTS A. Preliminary Investigation Investigation into the structure of uredospores commenced July 1966 at the Department of Biology, University of Saskatchewan, Saskatoon. The investigation began with a l i g h t microscope study of sections of mature uredospores em-bedded i n epoxy r e s i n . Adequate f i x a t i o n , embedding, and stain d i f f e r e n t i a t i o n were not achieved. Fixatives used were: KMnO^, acrolein-glutaraldehyde plus OsO^ (Hess 1966), and acrolein or glutaraldehyde alone followed by OsO^. Epon 812 used according to Luft (I96l) did not i n f i l t r a t e spores adequately and extensive tearing during sectioning occurred. Richardson's s t a i n (Azure 2 and methylene blue i n borax s o l u t i o n ) , Azure B (Dodge 1961+), c r y s t a l v i o l e t , and haematoxylin were t r i e d i n numerous attempts to achieve suitable s t a i n i n g and d i f f e r e n t i a t i o n of spore cyto-plasm. The use of l i g h t microscopy was eventually abandoned i n favour of electron microscopy. Preliminary u l t r a s t r u c t u r a l investigations at Saskatoon were terminated i n la t e summer 1967 when the author moved to the Di v i s i o n of Plant Science, Faculty of A g r i c u l t u r a l Sciences, University of B r i t i s h Columbia. The use of Maraglas epoxy res i n was brought to the author's attention by T. Bisalputra, Department of Botany, U.B.C., and i t has proven to be quite s a t i s f a c t o r y . Sections of spores postfixed i n OsO^ were found to have a grey overcast which did 24 not allow stai n d i f f e r e n t i a t i o n of cytoplasm. The use of OsO^ was discontinued i n favour of KMnO^ (Buckley et a l . 1968) and a l l results presented i n th i s thesis have been obtained with a double f i x a t i o n using aldehydes and KMhO . 4 B. Cytology of spore development, maturation and hydration 1. Immature uredospore Uredospores of Puccinia graminis t r i t i c i arise from a binucleate layer of mycelium beneath the epidermis of host t i s s u e s . The extent of the i n f e c t i o n court and early pustule development on the abaxial leaf surface are shown i n Figure 1. The nature of the sporulating tissue and i t s association with the host tissue can be seen i n Figure 2. Nucleoli are evident i n nuclei of the basal c e l l s and sporogenous hyphae. Figure 3 delimits areas of increasing d i f f e r e n t i a t i o n which were found i n a study of th i n sections made through a young pustule which had not ruptured the host epidermis. Stages i n the ontogeny of uredospores are represented by Figures 4-6. An enlargement ( F i g . 7) of the ap i c a l c e l l i n Figure 4 shows that prominent n u c l e o l i occur i n both n u c l e i . Increased c e l l volume i s a feature of spore formation as shown by Figures 5 and 6. Thickness of the c e l l wall increased from 0.2LL i n the ap i c a l c e l l to approximately 111 i n the immature spore. Protrusions of the c e l l wall into the cytoplasm are seen i n Figure 5. The complexity of the spore wall increased, as shown i n Figures 4-6, with the appearance of spines (Figure 5) and a layering as seen i n the upper l e f t part of Figure 6. 25 Figure 1. A b a x i a l surface of wheat l e a f showing i n f e c -t i o n courts (IC) and pustules ( P ) . Figu r e 2. S p o r u l a t i n g r u s t fungus i n p u s t u l e . Note a s s o c i a t i o n of fungus (F) and host (H). Mara-glas s e c t i o n , t o l u i d i n e b l u e , phase c o n t r a s t . X940. Figure 3. Stages i n d i f -f e r e n t i a t i o n shown i n Figures 4-6. 26 F i g u r e l+. A p i c a l c e l l s i n p u s t u l e . Note n u c l e o l i (Nu) i n n u c l e i (N) and d i v i d i n g m i t o -c h o n d r i o n (M). X 7 6 O O . * S c a l e l i n e 111 i n l e n g t h . ' S c a l e l i n e s on a l l m i c r o g r a p h s r e p r e s e n t d i s t a n c e of 111 . 27 Figure 5. I n c i p i e n t uredospore showing two n u c l e i (N) each w i t h a nucleolus (Nu). Note p r o t r u s i o n s (Pt) of c e l l w a l l (W), i n c r e a s i n g number of l i p i d d r o p l e t s (L) and d i v i d i n g mitochondrion (M). X7800. 2 8 F i g u r e 6 . Immature uredospore w i t h prominent nucleo l u s (Nu) i n one nucleus (N). Note u n d u l a t i n g plasmalemma (Pm). Compare c e l l volume and c e l l w a l l w i t h previous f i g u r e s . X8400. 29 L i p i d droplets, v i s u a l i z e d as clear white areas, increased markedly i n number as spores developed. Some l i p i d droplets appeared to be membrane bound whereas others were bounded only -by cytoplasm (Figs. 7-9). Mitochondria i n the d i f f e r -e n t i a t i n g c e l l s appeared elongate with d i s t i n c t , . p a r a l l e l c r i s t a e . Dividing mitochondria appeared elongate but con-s t r i c t e d near the middle (Figs. 4 and 5). Extensive v a r i a -t i o n i n the length of mitochondria was found, the shortest being 0.4M- i n length and 0.2LL wide and the longest being 1.5M-by 0.2\x. Dimensions of c e l l walls, l i p i d droplets and mito-chondria of the three spore stages investigated are presented i n Table I I . Endoplasmic reticulum was present i n the form of elongate ribbons. A close association was observed between the ER (Fig. 9) and one nucleus and the double membrane nature of the ER was evident. The plasma membrane appeared as a thi n electron dense layer adjacent to the spore wall (Fig. 6) but did not appear smooth at some points. Prominent n u c l e o l i (Figs. 9 and 10) were present i n immature uredospores and each nucleus had a single nucleolus (Figs. 8-10). The n u c l e o l i were granular and more dense at the center than the periphery or non-homogeneous. The mean diameter of a nucleus i n an immature spore was 3.1u and the mean diameter of the nucleolus was 1.7n (Table I ) . Discon-o t i n u i t i e s i n the nuclear, envelope were approximately 500A i n length. 30 Figu r e 7. Enlargement of n u c l e i (N) i n a p i c a l c e l l ..shown i n Figure 4 . Note prominent n u c l e o l i (Nu). X17400. 31 Figure 8. Enlargement of nuclei (N) i n i n c i p i e n t uredospore shown i n Figure 5. X17400. 32 Figure 9 . Enlargement of n u c l e i (N) i n immature uredospore shown i n Figure 6 . Note prominent, nonhomo-geneous nucleo l u s (Nu) i n one nucleus, c l o s e a s s o c i a t i o n of endoplasmic r e t i c u l u m (ER) and nucleus, and d i s c o n t i n u i t i e s (D) i n nuclear envelope (NE). X 1 6 6 0 0 . F i g u r e 1 0 . Section of immature uredospore which may be s e r i a l to s e c t i o n i n Figure 9 . Note nucleolus (Nu) i n each nucleus (N). X I 6 4 O O . 33 2. Mature uredospores Mature uredospores were very d i f f i c u l t to f i x , em-bed, and section due to the impermeability of the thick spore w a l l . L i p i d droplets occupied a considerable portion of the cytoplasm (Fig. 11). Endoplasmic reticulum was pre-sent i n the form of short, t h i n or vesiculate ribbons. The double membrane nature of the ER was evident (Figs. 11, 12). In contrast to immature uredospores, the mitochondria of mature spores were nearly sph e r i c a l . The cr i s t a e exhibited a p a r a l l e l p r o f i l e . The plasma membrane appeared as a t h i n electron dense layer adjacent to the spore wall but was separated from the wall at many points ( F i g . 11). Nucleoli occurred i n the nuclei of mature uredospores but were much reduced i n s i z e . The mean diameter of the nucl was 2.7n and that of the n u c l e o l i was 0.5LL (Table I ) . The n u c l e o l i of mature uredospore nuclei were non-homogeneous (Fig. 12), certain areas being more electron dense than others, but'the o v e r a l l density was not as great as that of n u c l e o l i i n immature spores. One nucleolus had a doughnut-l i k e configuration (Fig. 13). The double membrane nature of the nuclear envelope i s evident ( F i g . 12). D i s c o n t i n u i t i e s i n the nuclear envelopes of the nuclei of mature uredospores had the same dimensions as those given f o r immature uredospores. 34 Figure 1 1 . Cytoplasm of mature uredospore. Note abundance of l i p i d d r o p l e t s ( L ) , s p h e r i c a l mitochondria ( M ) w i t h w e l l developed c r i s t a e (Cr) and endo-plasmic r e t i c u l u m (ER). Plasmalemma ( R M ) has separated from c e l l w a l l at some p o i n t s . XlgOOO. 35 Figure 12. Nucleus (N) of mature uredospore showing nucleolus (Nu) cl o s e to nu c l e a r envelope (NE). Note double membrane of nuclear envelope, d i s -c o n t i n u i t i e s (D) i n nuclear envelope and endoplasmic r e t i c u l u m (ER). X20800. 36 F i g u r e 13. Nucleus (N) of mature uredospore showing doughnut-like nucleolus (Nu). X21,300. 3 7 ' 3. Hydrated uredospores Uredospores hydrated on 2% agar exhibited germ processes aft e r 25 minutes incubation i n darkness whereas spores floa t e d on d i s t i l l e d water or i n aluminum f o i l boats were apparently i n h i b i t e d . In the spores which appeared to be i n h i b i t e d mitochondrial elongation was the only i n d i c a -t i o n of enhanced metabolic a c t i v i t y . In the spores which had been sprayed onto the agar plates, one or two germ processes developed i n many spores and occasionally three i n others. Figure 14 i s representative of spores hydrated on agar and further 'description w i l l be r e s t r i c t e d to these spores. The spore wall, which had two zones, did not appear to swell with hydration but loosening and d i s s o l u t i o n of outer w a l l material prevented adequate measurements. Mitochondria assumed an elongate shape s i m i l a r to that found i n the immature spore. Extensive fusion of the l i p i d droplets occurred although a t h i n p a r t i t i o n could be seen between many droplets. The plasma membrane was a t h i n , i r r e g u l a r , electron dense layer adjacent to the spore wall but separated from i t . Continuity of the plasma membrane of the spore and of the germ'process was very d i s t i n c t . Vesicles occupied a considerable portion of the germ process cytoplasm. In a t o t a l of 3 8 nuclei observed, no nu c l e o l i were found. The mean diameter of the nuclei i n hydrated spores was 3 . I L L , the same as that of nuclei i n immature spores (Table I ) . 3 8 Figure 14. Cytoplasm of hydrated uredospore. Plasma-lemma (Pm) of germ process (&P) i s c o n t i n -uous wi t h plasmalemma of spore. Mitochondria (M) appear elongate. Note f u s i o n of l i p i d d r o p l e t s (L) and v e s i c l e s (V) i n germ process. Xl6,600. 39 Table I. Diameters of Nuclei and Nucleoli Nuclei Nucleoli No. Mean diameter (u) No Mean diameter (L I ) Immature uredospores 20 2.1 ± 0 . 6 * 15 1.7 ± 0.4 Mature uredospores 30 2.7 ± 0.8 3 0.5 Hydrated uredospores 24 3.1 ± 0.5 0 0 Standard deviation. Table I I . Dimensions of Uredospore Structures Spore Stage Immature Mature Hydrated C e l l wall thickness ( L I ) 0.9 ( 3 0 ) * 2.0 (30) 1.8 (8) L i p i d droplet diameter ( L I ) 0.5 (30) 0.7 (30) 0.5 (30) Mitochondria length x width 0.7x0.3(30) 0.7x0.6(30) 0.7x0.3(30) Mitochondria length/width 2 . 6 4 a * * 1.15b 2.73a 'Number of observations. Means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (p = 0.1). 40 4. Observations of a nucleolus Nucleoli were observed i n immature uredospores i n 33 out of 57 or 58$ of nuclear sections and i n 3 out of '55 or 6$ of the nuclear sections from mature uredospores. An estimate of the p r o b a b i l i t y of observing a nucleolus i n any p a r t i c u l a r nucleus i s given by the expression mean diameter nucleolus/mean diameter nucleus using the data i n Table I. Thus, the expected r e l a t i v e frequency with which n u c l e o l i were observed i n nuclear sections was 55$ f o r immature and 18$ f o r mature uredospores. The apparent discrepancy between the estimate of 18$ and the observed frequency of 6$ for mature uredospores may be due p a r t l y to lack of sections which pass through the diameter of the nucleus. 4 1 DISCUSSION D i f f e r e n t i a t i o n of an organism i s r e f l e c t e d by a change i n composition or structure, the l a t t e r being the most obvious (Baldwin and Busch 1 9 6 5 ) . The c y t o l o g i c a l and biochemical changes which occur i n fungi during the d i f f e r e n -t i a t i o n of the vegetative state to a spore state have been widely studied i n simple systems such as slime molds and yeasts. In these systems, manipulation of growth media i s the t o o l for inves t i g a t i o n of metabolic changes. Biochemical inv e s t i g a t i o n of the formation of rust uredospores i s hampered by the intimate association of the fungus and host. Uredospore germination, which i s a form of d i f f e r e n t i a t i o n , i s not subject to the above l i m i t a t i o n and numerous studies of dormant and germinating spores have been c i t e d . The i n a b i l i t y of germinating uredospores to synthesize net protein and nucleic acids has been implicated as a possible metabolic l e s i o n (Shaw 1 9 6 7 , Brian 1 9 6 7 , Staples 1 9 6 8 ) . An u l t r a s t r u c t u r a l study of sporogenesis, mature uredospores and hydrated uredospores was therefore under-taken to determine i f d e f i n i t i v e n u c l e o l i were present. The large n u c l e o l i seen i n stages of sporogenesis (Figures 4 - 9 ) are associated with the ribosomal RNA requirements of a d i f f e r e n t i a t i n g and growing c e l l . The n u c l e o l i i n the immature uredospore, (Figs. 6 , 9 and 1 0 ) support the f i n d i n g of. Thomas and Isaac ( 1 9 6 7 ) . The nucleolus i n these developmen-t a l stages may be analogous to the nucleolus i n maturing am-phibian oocytes,which exhibit intense synthesis of ribosomal RNA (Brown 1 9 6 6 ) . In primary root tissues of PIantago (Hyde 42 1967) n u c l e o l i are small i n the quiescent meristematic zone but increase ten to twelve times i n diameter i n the a c t i v e l y d i v i d i n g c e l l s of the cortex, epidermis and root cap. Demands f o r p r o t e i n synthesis during spore formation are vi s u a l i z e d i n c e l l enlargement (Figs. 4-6), c e l l wall synthesis (Fig. 5), and the ,accumulation of l i p i d droplets (Figs. 4-6), The l a t t e r probably involves the conversion of carbohydrates to l i p i d s . The protrusions of c e l l wall into the cytoplasm (Fig. 5) are possible s i t e s of c e l l wall synthesis. During maturation of the spore on the stalk, the spore wall increased i n thickness. The average thickness of 2LX, given i n Table I I , may be high due to many spore walls being sectioned obliquely. The number of l i p i d droplets i n mature spores appears to increase beyond that i n immature spores. A layer of v e s i c l e s which Williams and Ledingham (1964) observed as the plasmalemma may have been a re s u l t of f i x a t i o n damage. In the current study the plasmalemma of the mature spore (Fig, 11) was seen as a t h i n electron-dense layer which had pulled away from the spore wall at some points. This separation of the membrane and spore wall may have been f i x a t i o n damage i n part but Hawker (1965) states that the plasma-lemma i n the young fungal c e l l s while normally i n close contact with the c e l l wall may undulate or show invaginations i n older c e l l s . L i p i d droplets observed i n mature spores have the same size as those described i n e a r l i e r reports (Williams and 43 Ledingham 1964, Manocha and Shaw 1967). Unlike Williams and Ledingham (1964) the dense layer surrounding some l i p i d droplets (Figs. 9, 14) i s not interpreted as a membrane. Fawcett (1966) states that the dense l i n e (product of f i x a -t i o n at cytoplasm-lipid interface) around the periphery of the droplet i s thicker and denser than ordinary membranes and does not have the trilaminar structure of li p o p r o t e i n membranes. Endoplasmic reticulum'(Fig. 12), v i s u a l i z e d as t h i n ribbons, corresponds to that reported by other workers. No evidence of ribosome groupings on the ER was observed but the f i l m magnification was low (,<2500X) and the use of KMnO^ may have disrupted ribosomes. Mitochondria i n mature uredospores tended to be spherical and i n contrast to the observations of Williams and Ledingham (1964) and Manocha and Shaw (1967) the cri s t a e appeared well developed and were arranged i n p a r a l l e l arrays. The occurrence of reduced n u c l e o l i i n mature uredo-spores i s the most s i g n i f i c a n t f i n d i n g of the study. Williams and Ledingham (1964) did not observe a nucleolus i n any nucleus although they admitted that the number of sections viewed was too small to exclude the existence of a nucleolus. I t i s very doubtful however, that t h e i r preparations would have revealed n u c l e o l i since KMnO^ preserves l i t t l e more than l i p o p r o t e i n membranes. Manocha and Shaw (1967) were unable to detect n u c l e o l i i n mature f l a x rust uredospores but t h i s may have resulted from the use of KMnO^ as a primary f i x a t i v e or an i n s u f f i c i e n t number of sections viewed. In the present 44 study a nucleolus was found i n 3 out of 55 nuclear sections. The d i f f i c u l t y of sectioning mature spores with glass knives reduces the number of adequate sections. A reduced nucleolus, as shown i n Figure 12, may be present i n every nucleus. A l -t e r n a t i v e l y , the observed n u c l e o l i may s i g n i f y degeneration of a l l n u c l e o l i with increasing spore age. Decreased size of the nucleolus or complete absence of the nucleolus could r e s u l t from repression of that part of the genome concerned with synthesis of ribosomal precursors. The nucleolus shown i n Figure 13 had a doughnut-like configuration for which no explanation i s av a i l a b l e . There i s no evidence to suggest that i t i s an intermediate stage i n the reduction of the nucleolus from the size presented i n Figure 9, to the size i n Figure 12. Doughnut-like configurations of n u c l e o l i have been reported i n interphase and prophase nuclei i n V i c i a faba (Lafontaine and Chouinard 1963), cortex c e l l s of PIantago (Hyde 1967) and i n young and old leaves of S t e l l a r i a media (Weintraub et a l . 1968). Nucleoli with two zones which were d i f f e r e n t i a t e d by cytochemical tests or -staining methods have been described by Snoad (1956). Hyde (1967) suggests that the doughnut-like and angular configurations i n Plantago may r e s u l t from (a) synthetic a c t i v i t y of nucleolus, (b) mechanical rel a t i o n s h i p of nucleolus and chromosomes, and (c) rearrangement of nucleolus during duplication of chromo-somes . Hydration of uredospores i n non-inhibiting conditions resulted i n rapid i n i t i a t i o n of germination as seen by the 45 presence of germ processes i n approximately one-half hour (F i g . 14). Mitochondrial elongation and fusion of l i p i d droplets were obvious c y t o l o g i c a l changes. The shape of mitochondria at sporogenesis and hydra-t i o n i s s i g n i f i c a n t l y d i f f e r e n t , at the 1% l e v e l of s i g n i f i -cance, from the mitochondria of mature spores (Table I I ) . It i s concluded that the elongate mitochondria were associated with a c t i v e l y r e s p i r i n g t i s s u e . Nucleoli were not found i n any of the nuclei of hy-drated spores i n the present study and the germ tube nuclei described by Manocha and Shaw (1967) did not possess any n u c l e o l i . This evidence suggests that nucleolar function i s completely repressed through the early stages of germination. I t i s s i g n i f i c a n t that dormant conidia of V e r t i c i l l i u m albo-atrum. a f a c u l t a t i v e parasite, do not exhibit a nucleolus but that during germination a nucleolus appears and increases i n size (Heale et a l . 1968). This i s consistent with the demand f o r protein synthesis i n the germling. Immediate enzymatic functions within the germinating uredospore would be the mobilization of l i p i d s and carbohydrates f o r synthesis of membranes and c e l l wall of the germ tube, and d i s s o l u t i o n of the germ pore. Vesicles i n the germ processes may be transporting substrates f o r synthesis of c e l l components. Wynn et a l . (1966) have suggested that the germinability of uredospores depends on the a b i l i t y of the spores to mobilize food reserves. Hemicellulase, c e l l u l a s e and polygalacturonase have been reported to increase during spore germination (Shaw 46 1964) and i t i s conceivable that they could be synthesized from ex i s t i n g protein. The p o t e n t i a l f o r protein synthesis i n germinating uredospores has been likened (Shaw 1967) to the anucleolate mutant of Xenopus (South African clawed toad), described by Brown (1964). The anucleolate mutant of Xenopus develops to the swimming stage, u t i l i z i n g preformed ribosomes, then dies f o r lack of new ribosomes. Amino acid incorporating systems derived wholly or i n part from uredospores have been described by Staples and h i s co-workers and i t has been concluded that uredospores possess a complete protein synthesizing apparatus which operates at very low rates. More recently (Dunkle et a l . 1969), develop-ment of i n f e c t i o n structures was shown, by use of appropriate i n h i b i t o r s , to be dependent on RNA and protein synthesis, although the species of RNA that i s affected i s not known. The germ tube nuclei observed by Maheshwari et a l . (1967) are evidence that a functional system of nucleic acid and pro-t e i n synthesis e x i s t s . The presence of a diminished nucleolus i n mature uredospores as described e a r l i e r suggests that r i b o -somal RNA may normally l i m i t growth of t h i s obligate parasite i n the absence of a susceptible host t i s s u e . I t has been suggested (Shaw 1967) that i f indeed the nucleolar apparatus of uredospores i s repressed, i n t e r a c t i o n of the fungus with the host may provide the stimulus to reactivate synthesis of ribosomal RNA and protein. Presumably some component of the media used i n the successful axenic culture of wheat and 47 f l a x rusts (Williams et a l . 1966, Williams et a l . 1967, Bushnell 1968, Scott 1968, Turel 1969, and Coffey and Bose 1969) f u l f i l l s the role normally played by a susceptible host. The formation of uredospores and t h e i r subsequent germination i s an i n t r i g u i n g problem i n c e l l biology since normal nucleolar function i n the mature spore appears to be repressed. Further studies of rust spore formation and spore germination could include a high resolution study of the nucleolus although improved f i x a t i o n and embedding must be attained. Light microscope studies of nuclear behavior u t i l i z -i ng whole amounts of germ tubes and i n f e c t i o n structures or half-micron thick g l y c o l methacrylate sections would be f r u i t -f u l . I t i s desirable to study spores germinating i n a manner si m i l a r to that occuring i n nature. Attention i s drawn to the heat-shock induction of i n f e c t i o n structures by Maheshwari et a l . (1967). Studies of nucleic acid and protein synthesis i n the germling, using i n h i b i t o r s , should also provide insight into the nature of nuclear control. The axenic culture of rusts can provide tissues for c y t o l o g i c a l study and i t i s expected that improvements i n culturing w i l l y i e l d s u f f i c i e n t quantities of tissue for biochemical study. 48 SUMMARY 1. An electron microscope study of immature uredospores, mature uredospores and hydrated uredospores of Puccinia  graminis t r i t i c i (Race 15B) was undertaken to determine i f d e f i n i t i v e n u c l e o l i were present. 2. Prominent n u c l e o l i (l . 7 u diam.) were present i n the d i -karyotic fungal tissue giving r i s e to and including immature uredospores. 3. Reduced n u c l e o l i (0.5u diam.), which were less dense than n u c l e o l i found i n immature uredospores, were found i n 3 out of 55 nuclear 'sections from mature uredospores. Two n u c l e o l i had a normal shape while the t h i r d had a dough-nut - l i k e configuration. 4 . Nucleoli were not observed i n nuclei of hydrated uredo-spores . 5. Increasing c e l l volume, increasing c e l l wall thickness and accumulation of l i p i d droplets were evident parameters of growth and development during sporogenesis. 6.. Elongate mitochondria i n immature and hydrated uredospores were d i s t i n c t from the round mitochondria of mature uredo-spores . 49 LITERATURE CITED Alexopoulos, C.J. 1962. Introductory Mycology. John Wiley & Sons, Inc., New York. j i A l l e n , P.J. 1955. The role of a s e l f - i n h i b i t o r i n the germina-t i o n of rust uredospores. Phytopathology 45: 259-266. A l l e n , P.J. 1957. Properties of a v o l a t i l e f r a c t i o n from uredo-spores of P. graminis var. t r i t i c i a f f e c t i n g t h e i r germination and development. 1. B i o l o g i c a l a c t i v i t y . Plant Physiol. 32: 385-389. A l l e n , P.J. 1965. Metabolic aspects of spore germination. Ann. Rev. Phytopath. 3: 313-342. Baldwin, H.H. and H.P. Busch. 1965. The chemistry of d i f f e r e n -t i a t i o n i n lower organisms. Ann. Rev. Biochem. 34: 565-594. B i r n s t i e l , M. 1967. The nucleolus i n c e l l metabolism. Ann. Rev. Plant Physiol. 18: 25-58. Bisalputra, T. and T.E. Weier. 1963. 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