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UBC Theses and Dissertations

Ultrastructure of aplanospore production and germination and the role of calcium in germination of aplanospores… Fitch, Robert Scott 1986

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ULTRASTRUCTURE OF APLANOSPORE PRODUCTION AND GERMINATION AND THE ROLE OF CALCIUM IN GERMINATION OF APLANOSPORES OF VAUCHERIA LONGICAULIS VARIETY MACOUNII BLUM (CHRYSOPHYTA, TRIBOPHYCEAE) by ROBERT SCOTT FITCH B.A., UNIVERSITY OF CALIFORNIA, SANTA BARBARA, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE DEPARTMENT OF BOTANY We accept t h i s t h e s i s as con f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August 1986 @ ROBERT SCOTT FITCH, 1986 I n 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 t h e 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 o l u m b i a , I agree t h a t t h e 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 s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r 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 purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r 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 o r 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 n o t 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 BOTANY The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 SEPTEMBER 30, 1986. Date ABSTRACT i i V a u c h e r i a l o n g i c a u l i s v a r . m a c o u n i i (Blum) i s the most common of the f o u r b r a c k i s h water s p e c i e s of Vaucher i a known t o occur i n B r i t i s h Columbia and n o r t h e r n Washington. T h i s v a r i e t y i s easy t o m a i n t a i n i n c u l t u r e w i t h minimum n u t r i t i o n a l r e q u i r e m e n t s . I t s u r v i v e s c u l t u r e c o n d i t i o n s f o r extended p e r i o d s of time (up t o 2 y e a r s or more) w i t h o u t l o s i n g i t s r e p r o d u c t i v e c a p a b i l i t i e s . A p l a n o s p o r o g e n e s i s i s e a s i l y i n d u ced by t r a n s f e r r i n g p l a n t s t o f r e s h medium w i t h almost every v e g e t a t i v e f i l a m e n t p r o d u c i n g one a p i c a l aplanosporangium c o n t a i n i n g a s i n g l e a p l a n o s p o r e . A p l a n o s p o r o g e n e s i s has been examined i n s e c t i o n s p r e p a r e d f o r l i g h t and e l e c t r o n m i c r o s c o p i e s . As the v e g e t a t i v e f i l a m e n t t i p expands, s i g n a l l i n g the b e g i n n i n g of a p l a n o s p o r o g e n e s i s , the l a r g e c e n t r a l v a c u o l e i s d i s p l a c e d from the t i p . An i n n e r w a l l i s s e c r e t e d w i t h i n the e x i s t i n g c e l l w a l l v i a e x o c y t o s i s of numerous f i b r i l l a r - c o n t a i n i n g v e s i c l e s . S e p t a t i o n of the aplanosporangium from the v e g e t a t i v e f i l a m e n t i s a c c o m p l i s h e d by the c e n t r i p e t a l i n f u r r o w i n g of t h i s newly s e c r e t e d i n n e r w a l l at the base of the e n l a r g e d f i l a m e n t t i p . Each aplanosporangium produces a s i n g l e , m u l t i n u c l e a t e d w a l l e d a p l a n o s p o r e , w i t h unique o r g a n e l l e m o r p h o l o g i e s and a s s o c i a t i o n s . The importance of these o r g a n e l l e s i n the mechanism of a p l a n o s p o r e i i i r e l e a s e and g e r m i n a t i o n are d i s c u s s e d . G e r m i n a t i o n of a p l a n o s p o r e s i s i n i t i a t e d a t s i t e s c h a r a c t e r i z e d by low o p t i c a l d e n s i t y and l e a d s t o the f o r m a t i o n of f i l a m e n t s . The most prominent u l t r a s t r u c t u r a l f e a t u r e s c h a r a c t e r i z i n g g e r m i n a t i o n a r e the r a p i d e x p a n s i o n of the c e n t r a l v a c u o l e , the a c c u m u l a t i o n of d i c t y o s o m e - d e r i v e d v e s i c l e s a t the t i p of the g e r m i n a t i n g f i l a m e n t i n a s s o c i a t i o n w i t h c e l l w a l l e x p a n s i o n , and the i n c r e a s e i n the number and r e d i s t r i b u t i o n of m i c r o t u b u l e s . The p o s s i b l e f u n c t i o n of unique o r g a n e l l e a s s o c i a t i o n s , such as those o c c u r r i n g among the m i t o c h o n d r i a - e n d o p l a s m i c r e t i c u l u m - d i c t y o s o m e a s s o c i a t i o n , are d i s c u s s e d as w e l l . In a d d i t i o n , q u a n t i t a t i v e changes i n the volume d e n s i t y of some s u b c e l l u l a r compartments and o r g a n e l l e s are e v a l u a t e d u s i n g morphometric a n a l y s i s . U s i n g the f l u o r e s c e n t probe c h l o r o t e t r a c y c l i n e as an i n d i c a t o r of i n t r a c e l l u l a r C a 2 + , the r o l e ( s ) of c a l c i u m on a p l a n o s p o r e g e r m i n a t i o n and f i l a m e n t growth are s t u d i e d . These s t u d i e s are supplemented w i t h the use of drugs t o d i s r u p t the a v a i l a b i l i t y and d i s t r i b u t i o n of e x t r a - and i n t r a c e l l u l a r C a 2 + . The r e s u l t s a r e then a n a l y z e d i n terms of the c o m p l e x i t i e s of the mechanisms i n v o l v e d i n the g e r m i n a t i o n and growth of V a u c h e r i a a p l a n o s p o r e s . TABLE OF CONTENTS i v ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES V LIST OF FIGURES v i ACKNOWLEDGEMENTS x INTRODUCTION 1 MATERIAL AND METHODS 6 RESULTS 11 COLLECTION AND CULTURING 11 APLANOSPOROGENESIS 12 APLANOSPORE RELEASE AND GERMINATION 16 CALCIUM LOCALIZATION WITH CHLOROTETRACYCLINE . 22 CALCIUM PERTURBATIONS 25 DISCUSSION 29 APLANOSPOROGENESIS 29 APLANOSPORE RELEASE AND GERMINATION 35 CALCIUM LOCALIZATION WITH CHLOROTETRACYCLINE . 39 CALCIUM PERTURBATIONS 46 CONCLUSION 55 KEY FOR FIGURES 57 FIGURES 58 LITERATURE CITED 77 LIST OF TABLES V TABLE I . A p l a n o s p o r o g e n e s i s v s . z o o s p o r o g e n e s i s ... 34 LIST OF FIGURES v i FIGURE 1 V a u c h e r i a f i l a m e n t s p r o d u c i n g a p l a n o s p o r e s . .. 58 2 E a r l y a p l a n o s p o r o g e n e s i s 58 3 I n i t i a t i o n of s e p t a t i o n 58 4 Co m p l e t i o n of s e p t a t i o n 58 5 Mature a p l a n o s p o r e 58 6 S i n g l e c e l l w a l l and c h l o r o p l a s t s 59 7 M i t o c h o n d r i o n - E R - d i c t y o s o m e complex 58 8 L o n g i t u d i n a l s e c t i o n of e a r l y a p l a n o s p o r o g e n e s i s 59 9 A u t o p h a g i c v a c u o l e 59 10 C r y s t a l l i n e i n c l u s i o n 59 11 F i b r i l l a r m a t e r i a l i n paramural space 59 12 L o n g i t u d i n a l s e c t i o n of m i d - s e p t a t i o n 60 13 D e t a i l of e a r l y s e p t a t i o n 60 14 L o n g i t u d i n a l s e c t i o n of l a t e s e p t a t i o n 60 15 F i n a l s t a g e of s e p t a t i o n 60 16 A p l a n o s p o r e and aplanosporangium w a l l s 61 17 Th i c k e n e d a p l a n o s p o r e w a l l 61 18 C h l o r o p l a s t morphology 61 19 L o n g i t u d i n a l s e c t i o n of mature a p l a n o s p o r e . .. 61 20 Mature a p l a n o s p o r e i n a p l a n o s p o r angium 61 21 Schematic drawing of a p l a n o s p o r o g e n e s i s 62 v i i 22 G e r m i n a t i o n of a p l a n o s p o r e p r o d u c i n g f o u r f i l a m e n t s 63 23 Empty a p l a n o s p o r e body 63 24 Emergence of a p l a n o s p o r e 63 25 L o n g i t u d i n a l s e c t i o n through newly r e l e a s e d a p l a n o s p o r e 63 26 In s i t u g e r m i n a t i o n 63 27 L o n g i t u d i n a l s e c t i o n through a p l a n o s p o r e at the onset of g e r m i n a t i o n 63 28 L o n g i t u d i n a l s e c t i o n of two f i l a m e n t s a r i s i n g from one a p l a n o s p o r e 63 29 Morphology of newly r e l e a s e d a p l a n o s p o r e . ... 64 30 M i t o c h o n d r i o n - E R - d i c t y o s o m e complex 64 31 D e t a i l of u l t r a s t r u c t u r e of g e r m i n a t i o n p r o t r u s i o n 64 32 D e t a i l of paramural space at the t i p of a g e r m i n a t i o n p r o t r u s i o n 64 33 E x o c y t o s i s at g e r m i n a t i o n p r o t r u s i o n . 64 34 S m a l l m i c r o t u b u l e bundle 65 35 L a r g e r m i c r o t u b u l e bundle 65 36 E n d o p h y t i c b a c t e r i a near a n u c l e u s 65 37 P a r t i a l l y d i g e s t e d b a c t e r i a 65 38 B a c t e r i u m embedded i n a p l a n o s p o r e c e l l w a l l . . 65 39 Graph of the volume d e n s i t y changes of major a p l a n o s p o r e compartments d u r i n g g e r m i n a t i o n . . 66 v i i i 40 Graph of the volume d e n s i t y changes of c y t o p l a s m i c compartments d u r i n g g e r m i n a t i o n . . 67 41 Growth c h a r t of the e f f e c t of CTC on o v e r a l l f i l a m e n t l e n g t h 68 42 Growth c h a r t of the e f f e c t of CTC on f i l a m e n t growth r a t e 69 43 Graph of the area where CTC f l u o r e s c e n c e i s seen 70 44 Graph of the d i s t r i b u t i o n and i n t e n s i t y of CTC f l u o r e s c e n c e 71 45 G e r m i n a t i o n i n c o n t r o l growth medium 72 46 G e r m i n a t i o n i n 10"^5 CTC 72 47 G e r m i n a t i n g a p l a n o s p o r e ( u n t r e a t e d ) 72 48 CTC f l u o r e s c e n c e of a p l a n o s p o r e from F i g u r e 47 72 49 OTC-treated a p l a n o s p o r e 72 50 F l u o r e s c e n c e observed i n 10~4M CTC two hours a f t e r g e r m i n a t i o n 73 51 F l u o r e s c e n c e observed i n 10 _5M CTC two hours a f t e r g e r m i n a t i o n 73 52 CTC f l u o r e s c e n c e of g e r m i n a t i n g a p l a n o s p o r e 73 53 CTC f l u o r e s c e n c e f o u r hours a f t e r g e r m i n a t i o n 73 i x 54 CTC f l u o r e s c e n c e observed between two and f o u r hours a f t e r g e r m i n a t i o n 73 55 CTC f l u o r e s c e n c e of f i e l d - c o l l e c t e d f i l a m e n t . 73 56 Growth c h a r t of the e f f e c t of EGTA on growth r a t e . 74 57 Growth c h a r t of the e f f e c t of A23187 on growth r a t e 74 58 Growth c h a r t of the e f f e c t of TFP on growth r a t e 74 59 Combined growth c h a r t of the e f f e c t s of EGTA, A23187 and TFP on growth r a t e 74 60 Morphology of c o n t r o l f i l a m e n t 75 61 CTC f l u o r e s c e n c e of f i l a m e n t from F i g . 60. .. 75 62 Morphology of EGTA-treated f i l a m e n t 75 63 CTC f l u o r e s c e n c e of f i l a m e n t from F i g . 62. .. 75 64 Morphology of A 2 3 1 8 7 - t r e a t e d f i l a m e n t s 75 65 CTC f l u o r e s c e n c e of A 2 3 1 8 7 - t r e a t e d f i l a m e n t s 75 66 Morphology of 10~5M A 2 3 1 8 7 - t r e a t e d f i l a m e n t s 76 67 Morphology of 10~6M A 2 3 1 8 7 - t r e a t e d f i l a m e n t s 76 68 Morphology of 1% DMSO-treated f i l a m e n t s 76 69 CTC f l u o r e s c e n c e of T F P - t r e a t e d a p l a n o s p o r e 76 ACKNOWLEDGEMENTS x The author g r a t e f u l l y acknowledges the f i n a n c i a l s u p p ort and the i n s i g h t f u l guidance of Dr. L. O l i v e i r a , h i s t h e s i s s u p e r v i s o r . H i s p a t i e n c e , encouragement and wisdom were i n v a l u a b l e i n the c o m p l e t i o n of t h i s work. He wishes t o thank Dr. T. B i s a l p u t r a f o r f u l l and u n r e s t r i c t e d use of a l l h i s r e s e a r c h f a c i l i t i e s d u r i n g the course of t h i s s t u d y . S i n c e r e g r a t i t u d e i s a l s o extended t o Dr. M. Hawkes and Dr. D. Garba r y ; t h e i r p h y c o l o g i c a l i n s i g h t s and academic enthusiasm were c o n t a g i o u s . He a l s o e x p r e s s e s h e a r t f e l t g r a t i t u d e t o o f f i c e p a r t n e r s and f e l l o w s t u d e n t s whose f r i e n d s h i p s , l a u g h t e r and support h e l p e d r e a l i z e t h i s t h e s i s . Most i m p o r t a n t l y , he thanks h i s w i f e , Kathy, whose un c e a s i n g l o v e , p a t i e n c e and commitment kept him g o i n g . INTRODUCTION 1 Vaucher i a i s a w i d e l y d i s t r i b u t e d a l g a l genus o c c u r r i n g i n both f r e s h w a t e r and marine h a b i t a t s . In temperate r e g i o n s i t i s a common f l o r a l element i n s a l t marshes and e s t u a r i e s . V a u c h e r i a has been a g e n e r a l l y o v e r l o o k e d member of the marine a l g a l f l o r a of B r i t i s h Columbia and n o r t h e r n Washington. In a comprehensive l i s t of marine a l g a e from the a r e a , S c a g e l (1957) i n c l u d e d ^. l i t o r e a (Agardh) based on the e a r l i e r r e p o r t of Jao (1937) and an a d d i t i o n a l p e r s o n a l c o l l e c t i o n . Blum (1971) l a t e r d e s c r i b e d V.. l o n g i c a u l i s v a r . m a c o u n i i and v.. i n t e r m e d i a ( N o r d s t a d t ) from n o r t h e r n Washington, and l a t e r r e f e r r e d t o V.. t h u r e t i i (Woronin) from the P a c i f i c c o ast of the U n i t e d S t a t e s (Blum, 1972). In a d d i t i o n , Pomeroy (1977) and Pomeroy and St o c k n e r (1976) r e p o r t V.. dichotoroa (Agardh) from B r i t i s h Columbia. For a g e n e r a l r e v i e w of the ecolo g y and d i s t r i b u t i o n of m a r i n e / b r a c k i s h Vaucher i a spp., see Simons (1975). The genus V a u c h e r i a has been w i d e l y s t u d i e d s i n c e the p l a n t s were f i r s t d e s c r i b e d by Vaucher (1801). A s e x u a l r e p r o d u c t i o n i s a c c o m p l i s h e d by means of a k i n e t e s , m u l t i f l a g e l l a t e d zoospores or n o n - m o t i l e a p l a n o s p o r e s depending upon the s p e c i e s (Venkataraman 1961, Blum 1972, P i e t h 1980). P r e v i o u s u l t r a s t r u c t u r a l s t u d i e s have d e a l t w i t h the v e g e t a t i v e f i l a m e n t (Ott and Brown 1974a, Ott 1979), m i t o s i s (Ott and Brown, 1972), 2 s p e r m a t o g e n e s i s (Moestrup 1970, Ott and Brown 1978) and the c h l o r o p l a s t (Dangeard 1939, Descomps 1963a, 1963b, Marchant 1972). U n i q u e l y m u l t i f l a g e l l a t e d and m u l t i n u c l e a t e d zoospores and z o o s p o r o g e n e s i s have been e x t e n s i v e l y s t u d i e d by l i g h t ( T r e n t e p h o l 1807, Unger 1843, Thuret 1843, P r i n g s h e i m 1855, Schmitz 1878, S t r a s b u r g e r 1880, 1890, Koch 1951) and e l e c t r o n m i c r o s c o p i e s (Greenwood £t_ a_l 1957, Greenwood 1959, Ott and Brown 1974b, 1975) . D e s p i t e the f a c t t h a t a p l a n o s p o r e s are r e p o r t e d i n a p p r o x i m a t e l y t w i c e as many s p e c i e s of V a u c h e r i a as are z o o s p o r e s , i n f o r m a t i o n on a p l a n o s p o r o g e n e s i s , r e l e a s e and subsequent g e r m i n a t i o n of a p l a n o s p o r e s i s o n l y very b r i e f l y d e a l t w i t h by F r i t s c h (1935), Smith (1950), T a y l o r (1952), Chopra (1971), Knutzen (1973), Simons (1974) and Garbary and F i t c h (1984). However, none of these s t u d i e s i n v o l v e d u l t r a s t r u c t u r a l work. In a p o r t i o n of t h i s t h e s i s , t h e r e f o r e , the events l e a d i n g t o the d i f f e r e n t i a t i o n of the v e g e t a t i v e f i l a m e n t apex i n t o the a p l a n o s p o r e w i l l be r e p o r t e d . The mechanism of a p l a n o s p o r e r e l e a s e and the u l t r a s t r u c t u r a l events c h a r a c t e r i z i n g a p l a n o s p o r e g e r m i n a t i o n i n ^. l o n g i c a u l i s v a r . m a c o u n i i w i l l a l s o be s t u d i e d . In a d d i t i o n , morphometric a n a l y s i s i s used t o q u a n t i f y the u l t r a s t r u c t u r a l events o c c u r r i n g i n a p l a n o s p o r e s d u r i n g 3 the e a r l y s t a g e s of g e r m i n a t i o n . Recent s t u d i e s emphasize t h a t Ca2+ i o n s p l a y many im p o r t a n t r o l e s i n c e l l u l a r growth p r o c e s s e s i n p l a n t s (see Quatrano 1978, W e i s e n s e e l and K i c h e r e r 1981, S i e v e r s and Schnepf 1981, P o l i t o 1985 and Marme 1985 f o r r e v i e w s ) . P l a n t c e l l p r o c e s s e s r e g u l a t e d by Ca2+ i o n s i n c l u d e bud f o r m a t i o n (Saunders and H e p l e r 1981, Saunders 1986) , c y t o p l a s m i c v i s c o s i t y ( P i c t o n and S t e e r 1982 , Goodwin and T r a i n o r 1985 ), m i t o s i s (Wolniak £i .a_l 1980, Saunders and He p l e r 1981, Hepler and Wayne 1985), wound h e a l i n g i n g i a n t a l g a l c e l l s (La C l a i r e , 1983, 1984), c e l l volume (Kauss and Fausch, 1984) and r e c o v e r y from f r e e z i n g i n j u r y (Woods si. a i r 1984). Vaucher i a i s a c o e n o c y t i c a l g a i n which g e r m i n a t i o n and subsequent f i l a m e n t growth i s i n i t i a t e d by o r i e n t e d e x o c y t o s i s of d i c t y o s o m e - d e r i v e d v e s i c l e s a t s p e c i f i c r e g i o n s a l o n g the a p l a n o s p o r e ( F i t c h and O l i v e i r a , 1986b) and a t the t i p of the growing f i l a m e n t (Ott and Brown 1974, 1975a, 1975b, Kataoka 1982, F i t c h and O l i v e i r a 1986a, 1986b). C o n t r o l of p o l a r i z e d growth of p o l l e n tubes i s known t o i n v o l v e t i p - l o c a l i z e d Ca2+ c o n c e n t r a t i o n g r a d i e n t s a r i s i n g from the i n f l u x of Ca2+ i o n s a c r o s s membranes (Quatrano 1978, Chen and J a f f e 1979, We i s e n s e e l and K i c h e r e r 1981, P o l i t o 1985). These 4 g r a d i e n t s have been r e p o r t e d i n many o t h e r t i p - g r o w i n g p l a n t and f u n g a l c e l l s and a r e known t o s u s t a i n o r i e n t e d e x o c y t o s i s of p o l y s a c c h a r i d e - s t o r i n g v e s i c l e s n e c e s s a r y f o r a p i c a l w a l l e x p a n s i o n and subsequent growth ( J a f f e £± &1 1975, Herth 1978, Quatrano 1978, R e i s s and Herth 1978, 1979a, 1979b, 1982, Saunders and H e p l e r 1981, We i s e n s e e l and K i c h e r e r 1981, M e i n d l 1982, P i c t o n and S t e e r 1982 , 1985 , Hausser and Herth 1983 , R e i s s £± a_l 1983, 1985, Goodwin and T r a i n o r 1985, P o l i t o 1985, Wayne and H e p l e r 1985, McKerracher and Heath 1986). The d i s t r i b u t i o n of c e l l u l a r o r g a n e l l e s i n p o l l e n tubes a l s o seems t o be r e g u l a t e d by a t i p - l o c a l i z e d d i s t r i b u t i o n of Ca2+ i o n s (Weisenseel and K i c h e r e r 1981, M e i n d l 1982, P i c t o n and S t e e r 1982, R e i s s and Herth 1982, Grotha 1983). C y t o p l a s m i c Ca2+ i s a l s o known to p a r t i c i p a t e i n the r e g u l a t i o n of c y t o p l a s m i c s t r e a m i n g i n a l g a e (Hepler and Wayne, 1985) and f u n g a l c e l l s (McKerracher and Heath, 1986) . The f l u o r e s c e n t probe c h l o r o t e t r a c y c l i n e (CTC) has o f t e n been used as an i n d i c a t o r of membrane-bound Ca2+ i n l i v i n g c e l l s ( f o r a re v i e w see C a s w e l l , 1979). By employing CTC t o l o c a l i z e i n t r a c e l l u l a r Ca2+ and combining t h i s data w i t h u l t r a s t r u c t u r a l o b s e r v a t i o n s of g e r m i n a t i o n i n V a u c h e r i a . an attempt i s made t o p r e s e n t an i n t e g r a t e d p i c t u r e of the r o l e of Ca2+ i n the complex 5 mechanism of p o l a r i z e d g e r m i n a t i o n and f i l a m e n t e x t e n s i o n i n ^. l o n g i c a u l i s v a r . m a c o u n i i . C a l c i u m i s a l s o known t o be i n v o l v e d i n the r e g u l a t i o n of the v i s c o e l a s t i c i t y of the g e l - l i k e c y t o p l a s m r i c h i n c y t o s k e l e t a l elements found a d j a c e n t t o the plasma membrane (Goodwin and T r a i n o r , 1985). O v e r a l l , the p o l a r i z i n g c a p a c i t y of the p e r i p h e r a l c y t o p l a s m i s thought t o be based on the f o r m a t i o n of i o n g r a d i e n t s and e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s w i t h Ca24 i o n s p l a y i n g a major r o l e i n t h i s system (Schnepf, 1 986). L o c a l v a r i a t i o n s i n c a l c i u m d i s t r i b u t i o n a r e , t h e r e f o r e , p a r t of the mechanism a f f e c t i n g l o c a l i z e d growth i n p l a n t c e l l s ( P i c t o n and S t e e r 1982, Goodwin and T r a i n o r 1985) . By u s i n g s p e c i f i c drugs i t i s p o s s i b l e t o i n t e r f e r e w i t h both e x t r a - and i n t r a c e l l u l a r Ca2+ a v a i l a b i l i t y and d i s t r i b u t i o n ; hence t o a s c e r t a i n some a s p e c t s of the i n v o l v e m e n t of these i o n s i n t i p - o r i e n t e d growth. T h e r e f o r e , p e r t u r b a t i o n s i n the i n t r a c e l l u l a r b a l a n c e of Ca2+ f caused by the drugs e t h y l e n e g l y c o l t e t r a a c e t i c a c i d (EGTA), c a l c i u m ionophore A23187 and t r i f l u o p e r a z i n e (TFP), a r e d i s c u s s e d i n terms of those mechanisms known t o p a r t i c i p a t e i n t i p - o r i e n t e d growth i n V a u c h e r i a . MATERIAL AND METHODS 6 Vaucher i a spp. were c o l l e c t e d p r i m a r i l y i n Mud Bay ( B l a c k i e ' s S p i t ) , the F r a s e r R i v e r e s t u a r y , Vancouver Harbor ( B u r r a r d I n l e t ) and the S t r a i t of G e o r g i a i n so u t h e r n B r i t i s h Columbia. Most c o l l e c t i o n s were made i n s a l t marshes where V a u c h e r i a spp. grow at the bases o f , or i n bare patches among S a l i c o r n i a v i r g i n i c a and v a r i o u s o t h e r e s t u a r i n e phanerogams. V a u c h e r i a spp. produced dense mats from s e v e r a l c e n t i m e t e r s t o many meters i n l a t e r a l e x t e n t . P l a n t s were r e t u r n e d t o the l a b o r a t o r y f o r i d e n t i f i c a t i o n . Venkataraman (1961), Blum (1972) and R i e t h (1980) were the p r i m a r y taxonomic a u t h o r i t i e s . I f p l a n t s were s t e r i l e , p o r t i o n s of the a l g a l mats were p l a c e d i n g l a s s p e t r i d i s h e s and moistened w i t h c u l t u r e media. C u l t u r e s u s u a l l y became r e p r o d u c t i v e w i t h i n one month. P o r t i o n s of r e p r o d u c t i v e mats were mounted f o r d e p o s i t i n the U n i v e r s i t y of B r i t i s h Columbia Herbarium (UBC) or f i x e d w i t h 5% f o r m a l i n and m a i n t a i n e d i n l i q u i d p r e s e r v a t i v e (70% e t h a n o l ) . Vaucher i a spp. have been m a i n t a i n e d i n c u l t u r e f o r as l o n g as one and one h a l f y e a r s . Media u t i l i z e d was h a l f s t r e n g t h I n s t a n t Ocean (Aquarium Systems I n c . , E a s t l a k e , Ohio) supplemented w i t h minor elements (Lewin, 1966) and s o i l e x t r a c t . C u l t u r e s were kept at 10oc, w i t h c o o l w h i t e l i g h t of photon f l u x d e n s i t y of jc_a. 25 7 umol m-2 s ~ l under a 16-8 h l i g h t - d a r k p h o t o p e r i o d . C u l t u r e s of V.. l o p g j c a u l A s v a r . roacounii were t r a n s f e r r e d i n t o f r e s h media to induce a p l a n o s p o r o g e n e s i s . T h i s u s u a l l y o c c u r r e d w i t h i n 24 h o u r s . O b s e r v a t i o n s of a p l a n o s p o r o g e n e s i s and a p l a n o s p o r e g e r m i n a t i o n u s i n g l i v i n g m a t e r i a l were made w i t h a L e i t z D i a l u x 20 EB compound l i g h t m i c r o s c o p e . Growth of f i l a m e n t s g e r m i n a t i n g from a p l a n o s p o r e s was r e c o r d e d u s i n g a W i l d MPS 11 d i s s e c t i n g m i c r o s c o p e equipped w i t h an e y e p i e c e micrometer. For u l t r a s t r u c t u r a l s t u d i e s , f r e s h l y c o l l e c t e d a p l a n o s p o r e s and g e r m i n a t i n g f i l a m e n t s were p l a c e d i n 1% (v/v) g l u t a r a l d e h y d e made from an 8% s t o c k s o l u t i o n ( P o l y s c i e n c e s I n c . , W a r r i n g t o n , PA), b u f f e r e d i n 0.1 M phosphate b u f f e r s a l i n e (PBS), pH 6.9, f o r 45 minutes at room temperature ( 2 0 - 2 3 O C ) . A f t e r t r a n s f e r t o a f i n a l s o l u t i o n of 4% (v/v) g l u t a r a l d e h y d e i n the same b u f f e r f o r 2 hours a t 2 o c , the m a t e r i a l was washed and l e f t o v e r n i g h t a t 2 o c i n c o r r e s p o n d i n g b u f f e r . P o s t f i x a t i o n was done i n 2% (v/v) Os0 4 (Stevens M e t a l l u r g i c a l Corp., N.Y.) b u f f e r e d by 0.1 M PBS, pH 6.9, f o r 6 hours at 2°C, a f t e r which the specimens were washed and l e f t o v e r n i g h t i n b u f f e r at 2°C. The m a t e r i a l was then d e h y d r a t e d through a methanol s e r i e s . For s c a n n i n g e l e c t r o n m i c r o s c o p y , samples were then c r i t i c a l p o i n t d r i e d u s i n g 8 l i q u i d CC>2, s p u t t e r c o a t e d w i t h g o l d and examined w i t h a Cambridge S t e r e o s c a n 250T s c a n n i n g e l e c t r o n m i c r o s c o p e . For t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y , samples were f u r t h e r d e hydrated i n t o p r o p y l e n e o x i d e and i n f i l t r a t e d w i t h Epon 812 ( P o l y s c i e n c e s I n c . , W a r r i n g t o n , P A ). Throughout f i x a t i o n , d e h y d r a t i o n and i n f i l t r a t i o n , the specimens were g e n t l y a g i t a t e d v i a a m e c h a n i c a l r o t a t o r (Taab L a b o r a t o r y Equipment L t d . , B e r k s h i r e , England) at 4 R.P.M. S i l v e r s e c t i o n s were cut on a F e i c h e r t OMU3 u l t r a m i c r o t o m e and s t a i n e d i n s a t u r a t e d m e t h a n o l i c u r a n y l a c e t a t e and l e a d c i t r a t e ( R e y n olds, 1963). S e c t i o n s were examined w i t h a Z e i s s EM9S, a Z e i s s EM10 or a P h i l i p s 400 e l e c t r o n m i c r o s c o p e s . T h i c k s e c t i o n s (0.75-1.0 pm) were a l s o cut and d r i e d onto g l a s s s l i d e s . These s e c t i o n s were s t a i n e d w i t h 1% t o l u i d i n g b l u e f o r 10 seconds over an a l c o h o l lamp and examined w i t h a L e i t z D i a l u x 20 EB l i g h t m i c r o s c o p e . Morphometric a n a l y s e s of l i g h t and e l e c t r o n m i c r o g r a p h s of t h i s m a t e r i a l were c a r r i e d out as d e s c r i b e d by B r i a r t y (1980) w i t h the f o l l o w i n g m o d i f i c a t i o n : o n l y v o l u m e t r i c a n a l y s e s were performed. A W e i b e l - t y p e t e s t system ( W e i b e l , 1973) w i t h a 21 l i n e (7 x 3) a r r a y was employed t o measure the c e l l u l a r compartments at 1435X ( l i g h t m i c r o s c o p y ) . Test p o i n t s 9 l y i n g over c y t o p l a s m , c e l l w a l l , n u c l e i , c e n t r a l v a c u o l e and c h l o r o p l a s t s were counted. C y t o p l a s m i c compartments were measured at 6000X ( e l e c t r o n m i c r o s c o p y ) u s i n g a W e i b e l - t y p e t e s t system w i t h an 84 l i n e (14 x 6) a r r a y . Test p o i n t s l y i n g over c h l o r o p l a s t s , v e s i c l e s , n u c l e i , d ictyosomes and m i t o c h o n d r i a were counted. Volume d e n s i t y c a l c u l a t i o n s were performed a c c o r d i n g t o Weibel (1973). A p l a n o s p o r e s were a l s o c o l l e c t e d and t r a n s f e r r e d t o s m a l l p e t r i d i s h e s (12 per sample) c o n t a i n i n g c h l o r o t e t r a c y c l i n e (Sigma, S t . L o u i s , MO) d i s s o l v e d i n c u l t u r e medium t o c o n c e n t r a t i o n s r a n g i n g from 10~3M t o 10"7M. C o n t r o l t e s t s w i t h a p l a n o s p o r e s g e r m i n a t i n g i n CTC-free c u l t u r e medium were run p a r a l l e l t o the CTC experiments t o determine the e f f e c t of d i f f e r e n t c o n c e n t r a t i o n s of CTC on g e r m i n a t i o n . For CTC-dependent C a 2 + l o c a l i z a t i o n , a f r e s h l y p r e p a r e d s o l u t i o n of 10~4M CTC was a p p l i e d t o m a t e r i a l t r a n s f e r r e d from CTC-free c u l t u r e s . C T C - t r e a t e d m a t e r i a l was then observed under a L e i t z D i a l u x 20 EB compound microscope equipped w i t h e p i f l u o r e s c e n c e o p t i c s . A l l CTC f l u o r e s c e n c e m i c r o g r a p h s were taken 1 to 10 minutes a f t e r exposure t o CTC. The p o s s i b i l i t y t h a t CTC might f l u o r e s c e b r i g h t l y even when not complexed w i t h Ca2+ was checked by u s i n g the 10 C a 2 + - i n s e n s i t i v e probe o x y t e t r a c y c l i n e [OTC] (Sigma, S t . L o u i s , MO). Morphometric a n a l y s i s of the r e l a t i v e a rea o c c u p i e d by CTC f l u o r e s c e n c e was c a r r i e d out by t r a c i n g m i c r o g r a p h s e n l a r g e d t o the same f i n a l m a g n i f i c a t i o n u s i n g a H i Pad D i g i t i z e r (Houston I n s t r u m e n t s , A u s t i n , TX) and an IBM p e r s o n a l computer. The d i s t r i b u t i o n and i n t e n s i t y of CTC f l u o r e s c e n c e a l o n g the t e r m i n a l 200 pm a x i s of the CT C - t r e a t e d f i l a m e n t s was a r b i t r a r i l y a s s e s s e d from the same m i c r o g r a p h s used t o q u a n t i f y the r e l a t i v e area o c c u p i e d by CTC f l u o r e s c e n c e . A p l a n o s p o r e s were a l s o s u b j e c t e d t o the e f f e c t s of t h r e e d i f f e r e n t a n t a g o n i s t s of c a l c i u m i n v a r i o u s c o n c e n t r a t i o n s : the c h e l a t o r EGTA (10~3M, 10" 4M, 10~ 5M, 10-6M), the c a l c i u m ionophore A23187 (10~4M, 10"5M, 1 0 - 6 M ) , and the c a l m o d u l i n a n t a g o n i s t t r i f l u o p e r a z i n e [TFP] (10-4M, 5 x 10" 5M, 2 x l O ^ M , 1 0 " 5 M ) . A l l c h e m i c a l s were o b t a i n e d from Sigma ( S t . L o u i s , MO). EGTA and TFP were added d i r e c t l y t o the growth medium at the d e s i r e d c o n c e n t r a t i o n s . The ionophore A23187 was f i r s t d i s s o l v e d i n DMSO b e f o r e a d d i t i o n t o the growth medium. A c o n t r o l w i t h 1% DMSO added t o the growth medium was run p a r a l l e l t o the experiments u s i n g the ion o p h o r e A23187. The growth of g e r m i n a t i n g f i l a m e n t s and CTC-dependent Ca2+ l o c a l i z a t i o n were then r e c o r d e d over a 24 h r . p e r i o d . RESULTS 11 COLLECTION AND CULTURING M o r p h o l o g i c a l l y the f i e l d c o l l e c t e d p l a n t s of t h i s v a r i e t y of V a u c h e r i a l o n g i c a u l i s a r e s i m i l a r t o those d e s c r i b e d by Blum (1971, 1972) f o r n o r t h e r n Washington. None of the B r i t i s h Columbian p l a n t s p o s s e s s the ext r e m e l y l o n g a n t h e r i d i a c h a r a c t e r i s t i c of the s p e c i e s d e s c r i b e d from C a l i f o r n i a (Hoppaugh, 1930). V a u c h e r i a l o n g i c a u l i s v a r . ma c o u n i i i s the most common b r a c k i s h water s p e c i e s of V a u c h e r i a i n B r i t i s h Columbia and c o l l e c t i o n s were made a t a number of l o c a l i t i e s i n the S t r a i t of G e o r g i a (Garbary and F i t c h , 1984) . At many s i t e s V. l o n g i c a u l i s v a r . m a c o u n i i was the o n l y apparent s p e c i e s of V a u c h e r i a i n the f i e l d and t h i s was c o n f i r m e d through l a b o r a t o r y c u l t u r e of s e v e r a l n o n - r e p r o d u c t i v e p o p u l a t i o n s . However, i t was o f t e n p r e s e n t i n t e r m i n g l e d w i t h o c c a s i o n a l f i l a m e n t s of 3Z. t h u r e t i i . P l a n t s form e x t e n s i v e mats i n the h i g h i n t e r t i d a l zone p r i m a r i l y on muddy s u b s t r a t e where they c o -occur w i t h S a l i c o r n i a and oth e r marsh phanerogams. E x t e n s i v e c o l l e c t i o n s were made from a p o p u l a t i o n i n North Vancouver, a p p r o x i m a t e l y 100 meters east of the n o r t h span of the L i o n ' s Gate B r i d g e . In t h i s p a r t i c u l a r l o c a t i o n , h e a l t h y and e x t e n s i v e mats of l o n g i c a u l i s v a r . ma c o u n i i have been found year round 22 throughout the f o u r year d u r a t i o n of t h i s s t u d y . At B l a c k i e ' s S p i t , an a d d i t i o n a l l a r g e p o p u l a t i o n was found i n the h i g h i n t e r t i d a l zone on a sand beach. When p l a c e d i n t o c u l t u r e , V.. l o n g i c a u l i s v a r . m a c o u n i i produced abundant a p l a n o s p o r e s t h a t have not been p r e v i o u s l y r e p o r t e d f o r t h i s v a r i e t y , a l t h o u g h T a y l o r (1952) and Pecora (1979) r e p o r t a p l a n o s p o r e s i n l o n g i c a u l i s from C a l i f o r n i a and L o u i s i a n a , r e s p e c t i v e l y . T a y l o r r e p o r t s t h a t a p l a n o s p o r e s measure 225 pm i n l e n g t h and 90-120 pm i n diameter comparing w i t h 114-252 pm i n l e n g t h and 80-170 pm i n diameter i n the p r e s e n t m a t e r i a l . Over 90% of a p l a n o s p o r e s g e r m i n a t e d w i t h i n 48 hours of r e l e a s e and showed i n i t i a l growth r a t e s of 200-250 pm/h. The g e r m l i n g s o f t e n produced a d d i t i o n a l a p l a n o s p o r e s and/or oogonia and a n t h e r i d i a w i t h i n one week of g e r m i n a t i o n . APLANOS POP, OG ENESIS A d d i t i o n of f r e s h medium t o f i e l d - c o l l e c t e d c u l t u r e s r e s u l t e d i n abundant a p l a n o s p o r e p r o d u c t i o n ( F i g . 1 ) . A p l a n o s p o r o g e n e s i s i s u s u a l l y so p r o l i f i c t h a t n e a r l y every f i l a m e n t produces an a p l a n o s p o r e . 13 S i n g l e a p l a n o s p o r e s are d i f f e r e n t i a t e d t e r m i n a l l y on v e g e t a t i v e f i l a m e n t s ( F i g s 1-5) . However i n d i v i d u a l f i l a m e n t s o f t e n produce many a p l a n o s p o r e s over a p e r i o d of days. The o r g a n i z a t i o n of the n o n - s e p t a t e , c o e n o c y t i c v e g e t a t i v e f i l a m e n t of V.. j o n g i c a u l i s v a r . macpunij, i s e s s e n t i a l l y the same as t h a t r e p o r t e d by Ott and Brown (1974a) f o r e i g h t o t h e r s p e c i e s of V a u c h e r i a . The f i l a m e n t a p i c e s can be s u b d i v i d e d i n t o t h r e e zones: the a p i c a l zone, s u b - a p i c a l zone and zone of v a c u o l a t i o n . The same o r g a n i z a t i o n can be r e c o g n i z e d a t the b e g i n n i n g of a p l a n o s p o r o g e n e s i s ( F i g s 8 and 2 1 ) . U l t r a s t r u c t u r a l l y , both the a p i c a l zone and s u b a p i c a l zone c o n t a i n many n u c l e i , m i t o c h o n d r i a , c h l o r o p l a s t s and l a r g e numbers of v e s i c l e s c o n t a i n i n g f i b r i l l a r m a t e r i a l ( F i g s 6 and 2 1 ) . The f i b r i l l a r m a t e r i a l - c o n t a i n i n g v e s i c l e s a re p a r t i c u l a r l y abundant at the a p i c e s of each f i l a m e n t . Another c o n s p i c u o u s f e a t u r e i s the presence of l a r g e numbers of dictyosomes always seen c l o s e l y a s s o c i a t e d w i t h endoplasmic r e t i c u l u m elements and m i t o c h o n d r i a . M i c r o b o d y - l i k e o r g a n e l l e s a re a l s o seen i n c l o s e a s s o c i a t i o n w i t h these o r g a n e l l e s ( F i g . 7 ) . A l a r g e c e n t r a l v a c u o l e extends from the s u b a p i c a l zone. A s i n g l e - l a y e r e d c e l l w a l l s u r r o unds the e n t i r e f i l a m e n t a t the b e g i n n i n g of 14 a p l a n o s p o r o g e n e s i s ( F i g s 8 and 1 1 ) . A g r a d u a l d a r k e n i n g and s w e l l i n g of the v e g e t a t i v e f i l a m e n t t i p i n d i c a t e s the onset of a p l a n o s p o r o g e n e s i s ( F i g s 1 and 2 ) . As the a p i c a l area expands, the c e n t r a l v a c u o l e i s d i s p l a c e d t o a more s u b a p i c a l p o s i t i o n ( F i g s 8 and 2 1 ) . A u t o p h a g i c d i g e s t i o n of m a t e r i a l s of c y t o p l a s m i c o r i g i n ( F i g . 9) and c r y s t a l l i n e i n c l u s i o n s ( F i g s 8 and 10) a r e observed w i t h i n the c e n t r a l v a c u o l e . T h i s s i n g l e l a r g e c e n t r a l v a c u o l e i s e v e n t u a l l y r e p l a c e d by a network of s m a l l e r v a c u o l e s which l e n d t o the c y t o p l a s m a h i g h l y r e t i c u l a t e d morphology ( c f . F i g . 12 w i t h F i g . 8 ) . The m i t o c h o n d r i a - E F - d i c t y o s o m e complexes as w e l l as the f i b r i l l a r m a t e r i a l - c o n t a i n i n g v e s i c l e s remain abundant even though e l o n g a t i o n of the v e g e t a t i v e f i l a m e n t has ceased a t t h i s t i m e . Some of the f i b r i l l a r m a t e r i a l - c o n t a i n i n g v e s i c l e s r e l e a s e t h e i r c o n t e n t s by e x o c y t o s i s adding new m a t e r i a l t o the e x i s t i n g c e l l w a l l . T h i s p r o c e s s extends some d i s t a n c e down the v e g e t a t i v e f i l a m e n t and produces a second i n n e r w a l l ( F i g s 16 and 1 7 ) . S e p t a t i o n of the a p l a n o s p o r e from the v e g e t a t i v e f i l a m e n t i s i n i t i a t e d by the c e n t r i p e t a l i n f u r r o w i n g of the newly formed i n n e r w a l l at the base of the e n l a r g e d t i p of the v e g e t a t i v e f i l a m e n t ( F i g s 3, 12 and 1 3 ) . C y t o p l a s m i c c o n t i n u i t y remains i n t a c t u n t i l the 15 i n f u r r o w i n g i n n e r w a l l s e v e r s the c y t o p l a s m of the d e v e l o p i n g a p l a n o s p o r e from the c y t o p l a s m of the r e m a i n i n g v e g e t a t i v e f i l a m e n t ( F i g s 14 and 1 5 ) . At t h i s s t a g e , the o r i g i n a l c e l l w a l l at the t i p of the v e g e t a t i v e f i l a m e n t becomes the aplanosporangium w a l l (ASW) w h i l e the e n c i r c l i n g i n n e r w a l l becomes the a p l a n o s p o r e w a l l (AW) ( F i g s 16 and 1 9 ) . Both c e l l w a l l s are composed of m i c r o f i b r i l s a r r a n g e d p a r a l l e l t o one another ( F i g s 16 and 1 7 ) . The w a l l s of the a p l a n o s p o r e and the aplanosporangium are u s u a l l y of s i m i l a r t h i c k n e s s ( F i g . 1 6 ) , but a t h i c k e r a p l a n o s p o r e w a l l i s not uncommon ( F i g . 17) . Between the a p l a n o s p o r e and aplanosporangium w a l l s t h e r e i s a r e g i o n of v a r i a b l e w i d t h ( F i g . 17, * ) c o n t a i n i n g f i b r i l l a r m a t e r i a l comparable i n morphology t o t h a t observed i n the paramural space of the v e g e t a t i v e f i l a m e n t at the onset of a p l a n o s p o r o g e n e s i s ( F i g s 6 and 11, * ) . T h i s m a t e r i a l i s absent from the r e g i o n s e p a r a t i n g both w a l l s i n more advanced s t a g e s of a p l a n o s p o r e development ( F i g . 16) and hence i t s d i s i n t e g r a t i o n may be i n v o l v e d i n the f i n a l i s o l a t i o n of the a p l a n o s p o r e w i t h i n the aplanosporangium which o c c u r s p r i o r t o i t s r e l e a s e ( F i g . 2 0 ) . As the p r o c e s s of s e p t a t i o n p r o c e e d s , l e a d i n g t o the f o r m a t i o n of the a p l a n o s p o r e , a new c e n t r a l v a c u o l e 16 d e v e l o p s c o n c o m i t a n t l y w i t h the d i s a p p e a r a n c e of the h i g h l y r e t i c u l a t e d morphology of the c y t o p l a s m ( F i g s 14 and 19; compare w i t h F i g . 1 2 ) . N u c l e i are seen s c a t t e r e d throughout the a p l a n o s p o r e i n no apparent p a t t e r n . C h l o r o p l a s t s , numbered by the hundreds, are seen throughout the c y t o p l a s m , a l t h o u g h they are p r e f e r e n t i a l l y a r r a n g e d a g a i n s t the plasma membrane of the a p l a n o s p o r e ( F i g s 6 and 1 5 ) . T h i s arrangement becomes more pronounced as the new c e n t r a l v a c u o l e c o n t i n u e s t o expand w i t h i n the m a t u r i n g a p l a n o s p o r e ( F i g s 14 and 1 9 ) . Large prominent p y r e n o i d s a r e l o c a t e d c e n t r a l l y or t e r m i n a l l y w i t h i n the c h l o r o p l a s t s ( F i g s 6 and 1 8 ) . Numerous dictyosomes c o n s i s t i n g of 3-7 s t a c k e d c i s t e r n a e are observed always i n a s s o c i a t i o n w i t h endoplasmic r e t i c u l u m elements and m i t o c h o n d r i a ( F i g . 1 6 ) ; a c o n d i t i o n t h a t p e r s i s t s throughout a p l a n o s p o r o g e n e s i s . A summary of the most i m p o r t a n t events l e a d i n g t o the i n d i v i d u a l i z a t i o n of the a p l a n o s p o r e w i t h i n the a p l a n o s p o r angium i n V.. l o n g i c a u l i s v a r . m a c o u n i i i s p r e s e n t e d i n F i g u r e 21. APLANOSPORE RELEASE AND GERMINATION Emergence of mature a p l a n o s p o r e s from a p l a n o s p o r a n g i a was m o n i t o r e d w i t h the d i s s e c t i n g 17 m i c r o s c o p e . An opening formed at the a p i c a l p o r t i o n of the aplanosporangium w a l l through which the a p l a n o s p o r e emerged. Movement of the a p l a n o s p o r e out of the aplanosporangium was slow but c o n t i n u o u s . Once f r e e , i t l e f t b e h i n d an empty aplanosporangium w a l l c a s i n g ( F i g . 24, s m a l l arrowhead) and s l o w l y sunk t o the bottom of the c u l t u r e d i s h . Mature a p l a n o s p o r e s measured 114-282 urn i n l e n g t h and 80-170 pm i n diameter (Garbary and F i t c h , 1984), w h i l e r e t a i n i n g the e l l i p s o i d , obovate or oblong shape imp a r t e d d u r i n g a p l a n o s p o r o g e n e s i s ( F i g s 24 and 25, compare w i t h F i g s 5 and 2 0 ) . The study of the e a r l y phases of a p l a n o s p o r e g e r m i n a t i o n can be d i v i d e d i n t o 3 main s t a g e s : Stage I = newly r e l e a s e d a p l a n o s p o r e ( F i g . 2 5 ) ; Stage I I = onset of g e r m i n a t i o n ( F i g . 2 7 ) ; Stage I I I = a p p r o x i m a t e l y 1 hour a f t e r the i n i t i a t i o n of g e r m i n a t i o n ( F i g . 2 8 ) . S e c t i o n s through the c e n t e r of a newly r e l e a s e d a p l a n o s p o r e (stage I) r e v e a l randomly s c a t t e r e d n u c l e i and hundreds of c h l o r o p l a s t s , many of which are p e r i p h e r a l l y a r r a n g e d ( F i g s 25 and 2 9 ) . M i t o c h o n d r i a - E R - d i c t y o s o m e a s s o c i a t i o n s , w i t h v e s i c l e s o f t e n seen b l e b b i n g o f f from the i n t e r s p e r s e d s t r a n d of the ER i n t r a n s i t to the c i s - r e g i o n of the d i c t y o s o m e s , a r e a l s o observed ( F i g . 3 0 ) . F i b r i l l a r - m a t e r i a l c o n t a i n i n g v e s i c l e s , produced by the t r a n s - r e g i o n of the 18 di c t y o s o m e s ( F i g . 30, ar r o w h e a d s ) , accumulate i n the p e r i p h e r a l c y t o p l a s m of the a p l a n o s p o r e i n l a r g e q u a n t i t i e s ( F i g . 31, V ) . No d i s c h a r g e of v e s i c u l a r m a t e r i a l t o the paramural space i s seen at t h i s s t a g e . A c e l l w a l l of u n i f o r m t h i c k n e s s c o n t a i n i n g two m o r p h o l o g i c a l l y d i s t i n c t l a y e r s surrounds the newly r e l e a s e d a p l a n o s p o r e . The t h i n n e r , amorphous o u t e r w a l l l a y e r c o n t r a s t s w i t h the t h i c k e r , m i c r o f i b r i l l a r one due t o i t s h i g h e r e l e c t r o n d e n s i t y ( F i g . 29) . G e r m i n a t i o n (stage I I ) i s i n i t i a t e d by a p r o t r u s i o n of the c e l l w a l l at one or more l o c a t i o n s a l o n g the s u r f a c e of the a p l a n o s p o r e ( F i g s 26 and 27, arrowheads, F i g . 2 8 ) . Up t o f o u r f i l a m e n t s have been seen a r i s i n g from a s i n g l e a p l a n o s p o r e ( F i g . 2 2 ) . N e a r l y a l l a p l a n o s p o r e s germinate s u c c e s s f u l l y w i t h i n 48 hours of r e l e a s e . In the meantime, the u n d e r l y i n g q u i e s c e n t v e g e t a t i v e f i l a m e n t resumes i t s growth through the empty a p l a n o s p o r a n g i a l case and may produce s e v e r a l more a p l a n o s p o r e s . T h i s l e a v e s a node i n the f i l a m e n t i n d i c a t i n g where r e p e a t e d a p l a n o s p o r o g e n e s i s has o c c u r r e d ( F i g . 24, l a r g e arrowhead). In s i t u g e r m i n a t i o n of a p l a n o s p o r e s w i t h i n a p l a n o s p o r a n g i a has a l s o been obser v e d when a p l a n o s p o r e r e l e a s e i s d e l a y e d or f a i l s t o occur ( F i g . 2 6 ) . The t i p of the g e r m i n a t i n g p r o t r u s i o n ( s t a g e s I I 19 and I I I ) i s c h a r a c t e r i z e d by the presence of many mi t o c h o n d r i a - E R - d i c t y o s o m e complexes, d e n s e l y - p a c k e d v e s i c l e s and a c e l l w a l l of i r r e g u l a r t h i c k n e s s ( F i g . 3 1 ) . Two d i s t i n c t w a l l l a y e r s a r e s t i l l r e c o g n i z a b l e , however the outer w a l l l a y e r shows s i g n s of d i s r u p t i o n ( F i g . 31, c f . w i t h F i g . 2 9 ) . S i g n s of e x o c y t o s i s of v e s i c u l a r c o n t e n t s t o the paramural space and subsequent i n c o r p o r a t i o n of t h i s n e w l y - r e l e a s e d m a t e r i a l i n t o the i n n e r l a y e r of the a p l a n o s p o r e w a l l are a l s o observed and account f o r the i r r e g u l a r t h i c k n e s s c h a r a c t e r i s t i c of these s t a g e s ( F i g s 32 and 3 3 ) . F i g u r e 39 compares the volume d e n s i t i e s of major c e l l u l a r compartments d u r i n g the t h r e e s t a g e s of a p l a n o s p o r e g e r m i n a t i o n observed w i t h l i g h t m i c r o s c o p y . The g r e a t e s t o v e r a l l changes observed i n these compartments occur i n the v a c u o l e and c h l o r o p l a s t s . The volume of the v a c u o l e i n c r e a s e s d u r i n g e a r l y g e r m i n a t i o n from 23% (±4%) t o 35% (±5%), and reaches 47% (±3%) of the t o t a l volume of the a p l a n o s p o r e by s t a g e I I I . The volume d e n s i t y of t h i s compartment keeps on i n c r e a s i n g as g e r m i n a t i o n proceeds u n t i l the a p l a n o s p o r e body i s f i n a l l y emptied of i t s c o n t e n t s ( F i g . 2 3 ) . C r y s t a l l i n e i n c l u s i o n s and l a r g e l i p i d b o d i e s , absent s i n c e a p l a n o s p o r o g e n e s i s , reappear c o n c o m i t a n t l y w i t h the c e n t r a l v a c u o l e ' s development and e x p a n s i o n ( F i g s 25 and 20 2 7 ) . Meanwhile, the volume d e n s i t y of the c h l o r o p l a s t compartment d e c r e a s e s from 47% (±4%) i n stage I t o 40% (±5%) i n stage I I , and reaches a low v a l u e of 26% (±2%) i n s tage I I I . The volume d e n s i t y of the c e l l w a l l d e c r e a s e s by a p p r o x i m a t e l y one h a l f through s t a g e s I I and I I I of g e r m i n a t i o n , w h i l e t h a t of the n u c l e a r f r a c t i o n remains f a i r l y c o n s t a n t at a p p r o x i m a t e l y 3% of the t o t a l a p l a n o s p o r e volume. The c y t o p l a s m i c f r a c t i o n a l s o remains r e l a t i v e l y unchanged a t 20-23% of the volume of the a p l a n o s p o r e d u r i n g the e a r l y s t a g e s of germ ina t i o n . Volume d e n s i t y changes i n r e l a t i o n t o t o t a l c y t o p l a s m i c volume d e n s i t y were a l s o measured i n s e v e r a l c e l l u l a r compartments ( F i g . 4 0 ) . The volume d e n s i t y of the c h l o r o p l a s t compartment d e c r e a s e d d u r i n g e a r l y g e r m i n a t i o n from 49% (±6%) t o 41% (±4%) and f i n a l l y t o 34% (±4%) of the c y t o p l a s m i c volume d e n s i t y at stage I I I ( c f . w i t h F i g . 3 9 ) . The volume d e n s i t i e s of the m i t o c h o n d r i a l compartment i n c r e a s e d from 5% (±2%) t o 9% (±2%) and t h a t of v e s i c l e s from 39% (±4%) t o 52% (±5%) c f the t o t a l c y t o p l a s m i c volume d e n s i t y . The l a r g e i n c r e a s e i n the volume d e n s i t y of the v e s i c u l a r compartment c o i n c i d e s w i t h the a c c u m u l a t i o n i n the p e r i p h e r a l c y t o p l a s m of l a r g e numbers of f i b r i l l a r - m a t e r i a l c o n t a i n i n g v e s i c l e s d u r i n g stage I 21 and a t an e a r l y phase of stage I I ( F i g . 3 1 ) . N u c l e a r and d i c t y o s o m a l compartments remain f a i r l y c o n s t a n t a t 2-4% of the t o t a l c y t o p l a s m i c volume d e n s i t y . N o n - p r e f e r e n t i a l l y o r i e n t e d bundles of a few m i c r o t u b u l e s were observed i n the c y t o p l a s m of newly r e l e a s e d (stage I) a p l a n o s p o r e s ( F i g . 34, a r r o w h e a d s ) . Upon g e r m i n a t i o n ( s t a g e s I I and I I I ) , the number of m i c r o t u b u l e s per bundle i n c r e a s e s and the bundles become p r e f e r e n t i a l l y a r r a n g e d p a r a l l e l t o the plasma membrane of the p r o t r u d i n g f i l a m e n t s ( F i g . 35, a r r o w h e a d s ) . In a d d i t i o n , these l a r g e r m i c r o t u b u l e bundles were o f t e n seen i n c l o s e p r o x i m i t y to n u c l e i ( F i g . 35) and c h l o r o p l a s t s . In some a p l a n o s p o r e s , l a r g e numbers of b a c t e r i a were seen w i t h i n the c y t o p l a s m , o f t e n i n c l o s e p r o x i m i t y t o the n u c l e u s ( F i g . 36) . No apparent c y t o p l a s m i c damage was observed a l t h o u g h s m a l l v a c u o l e s c o n t a i n i n g b a c t e r i a i n v a r i o u s s t a g e s of d e g r a d a t i o n were noted ( F i g . 3 7 ) . B a c t e r i a were a l s o o c c a s i o n a l l y seen p e n e t r a t i n g the c e l l w a l l ( F i g . 3 8 ) . I n t e r e s t i n g enough i s the f a c t t h a t these e x t e n s i v e s i g n s of the o c c u r r e n c e of b a c t e r i a l i n f e c t i o n i n a p l a n o s p o r e s of V a u c h e r i a have no apparent e f f e c t on the f r e q u e n c y or r a t e of g e r m i n a t i o n of these r e p r o d u c t i v e c e l l s . CALCIUM LOCALIZATION WITH CHLOROTETRACYCLINE 22 Growth of g e r m i n a t i n g f i l a m e n t s of V.. l o n g i c a u l i s v a r . m a c o u n i i i n v a r i o u s c o n c e n t r a t i o n s of CTC i s shown i n F i g . 41. O v e r a l l f i l a m e n t growth was g r e a t e s t i n the CTC-free c o n t r o l t e s t and g r a d u a l l y d e c r e a s e d i n C T C - t r e a t e d m a t e r i a l t o 71% ±7% ( 1 0 - 7M ) , 58% ±5% ( 1 0 " 6M), 40% ±5% ( 1 0 - 5 M ) and 31% ±5% (10~4M) of the c o n t r o l over a 24 hour p e r i o d , w i t h no growth observed at 10 "3M CTC. Over p e r i o d s of time of 6 hours or l e s s , f i l a m e n t growth was v e r y s i m i l a r t o the c o n t r o l i n a l l but 1 0 - 3M CTC. When 1 0 - 3M CTC i s added t o g e r m i n a t i n g f i l a m e n t s , growth i s imme d i a t e l y a r r e s t e d and the f i l a m e n t t i p s tend t o b u r s t . F i g u r e 42 shows the growth r a t e s of f i l a m e n t s t r e a t e d i n v a r i o u s c o n c e n t r a t i o n s of CTC d u r i n g the f i r s t 6 hours of g e r m i n a t i o n . D u r i n g the f i r s t 2 ho u r s , the growth r a t e of the m a t e r i a l i n c u b a t e d i n 10~4M CTC v a r i e s from 42% t o 52% of t h a t observed i n f i l a m e n t s t r e a t e d w i t h lower c o n c e n t r a t i o n s of CTC and r e p r e s e n t s o n l y 39% (±6%) of t h a t of the c o n t r o l . From 2 to 4 hours a f t e r g e r m i n a t i o n , the growth r a t e of the m a t e r i a l i n c u b a t e d i n 10~4M CTC remains v i r t u a l l y unchanged w h i l e the growth r a t e s of a l l o t h e r C T C - t e s t e d m a t e r i a l and the c o n t r o l s t e a d i l y d e c l i n e s . From f o u r t o s i x hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n , the d e c l i n e i n growth r a t e c o n t i n u e s . In c o n t r a s t , a r e c o v e r y o c c u r s 23 i n 10"4M C T C - t r e a t e d m a t e r i a l l e a d i n g t o growth r a t e v a l u e s s i m i l a r t o t h a t observed i n the c o n t r o l by the end of t h i s p e r i o d . S t a b l i z e d growth r a t e s c h a r a c t e r i z e subsequent p o s t - g e r m i n a t i v e growth i n a l l C T C - t r e a t e d m a t e r i a l and the c o n t r o l . The absence of l o n g term i l l e f f e c t s on the V a u c h e r i a f i l a m e n t s from exposure t o 10-4M CTC can a l s o be g a t h e r e d from o b s e r v a t i o n s showing t h a t t h i s c o n c e n t r a t i o n of CTC does not a f f e c t the o r i e n t a t i o n or b r a n c h i n g p a t t e r n s of the g e r m i n a t i n g f i l a m e n t s when compared t o t h a t of the c o n t r o l m a t e r i a l ( c f . F i g . 45 w i t h F i g . 4 6 ) . M a t e r i a l i n c u b a t e d i n 1 0 - 4 M CTC p o s s e s s e s the added advantage of showing h i g h e r f l u o r e s c e n c e i n t e n s i t y than t h a t observed w i t h o t h e r c o n c e n t r a t i o n s ( F i g . 51, compare w i t h F i g . 5 0 ) . B r i g h t f i e l d o b s e r v a t i o n s of g e r m i n a t i n g a p l a n o s p o r e s r e v e a l r e g i o n s of lower o p t i c a l d e n s i t y c h a r a c t e r i s t i c of the g e r m i n a t i o n s i t e ( s ) ( F i g . 4 7 ) . These c o i n c i d e w i t h the o c c u r r e n c e of l o c a l i z e d CTC f l u o r e s c e n c e ( F i g . 4 8 ) . In c o n t r a s t , m a t e r i a l t r e a t e d w i t h o x y t e t r a c y c l i n e (OTC), a C a 2 + - i n s e n s i t i v e probe which i s an a n a l o g of CTC (Wolniak £i M ! 1980 , Wise and Wolniak 1984), e x h i b i t s no f l u o r e s c e n c e ( F i g . 4 9 ) . Pi s h a r p l y - d e l i m i t e d r e g i o n of CTC f l u o r e s c e n c e i s o b s e r v e d at the f i l a m e n t t i p d u r i n g the f i r s t 2 hours of 24 g e r m i n a t i o n ( F i g s 50 and 52) and a g a i n from a p p r o x i m a t e l y 4 hours a f t e r g e r m i n a t i o n and beyond ( F i g . 5 3 ) . A more d i f f u s e p a t t e r n of CTC f l u o r e s c e n c e , e x t e n d i n g b a s i p e t a l l y up t o 200 urn from the apex of the g e r m i n a t i n g f i l a m e n t s i s observed between 2 and 4 hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n ( F i g . 5 4 ) . A s i m i l a r p a t t e r n of CTC f l u o r e s c e n c e can a l s o be d e t e c t e d i n specimens of V a u c h e r i a f r e s h l y c o l l e c t e d from the f i e l d ( F i g . 5 5 ) . T r a n s i t i o n a l s t a g e s showing the t r a n s f o r m a t i o n of w e l l l o c a l i z e d t o more d i f f u s e f l u o r e s c e n c e a re observed ( F i g . 5 1 ) . The morphometric a n a l y s i s of the r e l a t i v e area o c c u p i e d by CTC f l u o r e s c e n c e d u r i n g g e r m i n a t i o n and f i l a m e n t e x t e n s i o n i s shown i n F i g u r e 43. In F i g u r e 44, the d i s t r i b u t i o n and i n t e n s i t y of CTC f l u o r e s c e n c e a l o n g the t e r m i n a l 200 um of g e r m i n a t i n g f i l a m e n t axes a r e graphed. D u r i n g the f i r s t 2 hours of g e r m i n a t i o n , when the growth r a t e of c o n t r o l f i l a m e n t s i s i n c r e a s i n g ( F i g . 4 2 ) , CTC f l u o r e s c e n c e o c c u p i e s an area e q u i v a l e n t t o 8% (±3%) of the t e r m i n a l 200 um of the f i l a m e n t l e n g t h ( F i g . 4 3 ) ; almost a l l of which i s l o c a l i z e d at the t i p ( F i g . 4 4 ) . From between 2 to 4 hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n , when f i l a m e n t growth r a t e s a r e r a p i d l y s l o w i n g down and CTC f l u o r e s c e n c e extends f u r t h e r away from the t i p i n a more d i f f u s e manner ( F i g s 25 44 and 5 4 ) , t h i s a rea i n c r e a s e s to 33% (±6%) of the t o t a l a rea of the growing f i l a m e n t ( F i g . 4 3 ) . In f i l a m e n t s g e r m i n a t i n g f o r 4 or more h o u r s , CTC f l u o r e s c e n c e i s a g a i n reduced t o 8.5% (±4%) of the t o t a l area of the growing f i l a m e n t ( F i g . 4 3 ) , w i t h almost a l l of the f l u o r e s c e n c e once more c o n f i n e d t o the t i p ( F i g . 4 4 ) . T h i s p a t t e r n of t i p - l o c a l i z e d CTC f l u o r e s c e n c e does not change s i g n i f i c a n t l y d u r i n g subsequent p o s t - g e r m i n a t i v e growth. CALCIUM PERTURBATIONS U n t r e a t e d a p l a n o s p o r e s and f i l a m e n t s used as c o n t r o l m a t e r i a l f o r these experiments grew at r a t e s r a n g i n g from 180 t o 250 um/hr. These v a l u e s are comparable t o those p r e v i o u s l y r e p o r t e d (see a l s o Garbary and F i t c h , 1984). The m o r p h o l o g i c a l f e a t u r e s and the s h a r p l y - d e l i m i t e d p a t t e r n of t i p - l o c a l i z e d Ca2+, as v i s u a l i z e d d u r i n g the f i r s t 2 hours of g e r m i n a t i o n w i t h CTC f l u o r e s c e n c e , a l s o resembles t h a t p r e v i o u s l y r e p o r t e d f o r V a u c h e r i a ( F i g s 60 and 61, compare w i t h F i g s 47 and 5 2 ) . F i g u r e 56 shows the e f f e c t of EGTA on both g e r m i n a t i o n and f i l a m e n t growth i n V a u c h e r i a . The i n h i b i t o r y e f f e c t of 1 0 _ 3 M EGTA on f i l a m e n t s i s 26 i r r e v e r s i b l e as r e c o v e r y from growth i n h i b i t i o n by r e p e a t e d washings and i n c u b a t i o n i n EGTA-free growth medium was u n s u c c e s s f u l . Over the 6 hour p e r i o d of t h i s e x p e r i m e n t , the growth r a t e s of newly g e r m i n a t e d a p l a n o s p o r e s g r a d u a l l y d e c r e a s e d i n the EGTA-treated m a t e r i a l t o 92% ±8% ( 10-6M), 78% ±6% (10"5M) and 45% ±5% (10-4M) of those o b s e r v e d i n the c o n t r o l . No abnormal growth p a t t e r n s or m o r p h o l o g i c a l a l t e r a t i o n s were observed as a r e s u l t of the t r e a t m e n t s w i t h EGTA ( F i g . 62 c f . w i t h F i g . 6 0 ) . F i l a m e n t s which had been i n c u b a t e d i n 10~^M EGTA and exposed t o CTC two hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n , r e s u l t e d i n a more d i f f u s e o v e r a l l p a t t e r n of f l u o r e s c e n c e d i s t r i b u t i o n . N o n e t h e l e s s , most of the f l u o r e s c e n c e i s s t i l l c o n c e n t r a t e d near the t i p ; a l t h o u g h , the f l u o r e s c e n c e i n t e n s i t y i s weaker than t h a t of the c o n t r o l ( F i g . 63 compare w i t h F i g . 6 1 ) . Treatment w i t h the ionophore A23187 r e s u l t e d i n v a r y i n g degrees of growth i n h i b i t i o n . Growth r a t e s d e c r e a s e d i n the i o n o p h o r e - t r e a t e d m a t e r i a l t o 78% ±5% (10~6M), 28% ±5% (10"5M) and 8% ±4% ( I O - ^ M ) of the c o n t r o l ( F i g . 5 7 ) . M a t e r i a l i n c u b a t e d i n growth medium w i t h 1% D M S O grew at a r a t e v i r t u a l l y i d e n t i c a l t o t h a t of the c o n t r o l ( F i g . 5 7 ) . G e r m i n a t i n g f i l a m e n t s from a p l a n o s p o r e s t r e a t e d w i t h A23187 are o f t e n broadened at 27 the base and of i r r e g u l a r d i a m e t e r . The a b n o r m a l i t i t e s a r e more pronounced i n m a t e r i a l i n c u b a t e d i n 10~4M A23187, where s w o l l e n a p i c e s and b u d - l i k e p r o t u b e r a n c e s c o r r e s p o n d i n g t o r e g i o n s of low o p t i c a l d e n s i t y are abundant ( F i g . 6 4 ) . In lower c o n c e n t r a t i o n s of A23187, the a p i c a l s w e l l i n g and b u d - l i k e p r o t r u s i o n s become l e s s pronounced ( F i g s 66 and 6 7 ) . F i l a m e n t s grown i n 1% D M S O d i s p l a y growth p a t t e r n s and m o r p h o l o g i c a l f e a t u r e s s i m i l a r t o those of the c o n t r o l ( F i g . 68, compare w i t h F i g . 60) . When m a t e r i a l i n c u b a t e d i n 10~4M A23187 i s s t a i n e d w i t h CTC, f l u o r e s c e n c e i s l o c a l i z e d p r i m a r i l y i n the abn o r m a l l y s w o l l e n a p i c e s and b u d - l i k e p r o t r u s i o n s o b s e r v e d a l o n g the g e r m i n a t i n g f i l a m e n t s ( F i g . 6 5 ) . The presence of f l u o r e s c e n c e c o i n c i d e s w i t h r e g i o n s of lower o p t i c a l d e n s i t y observed w i t h b r i g h t - f i e l d m i c r o s c o p y ( F i g . 6 4 ) . A p l a n o s p o r e s d i d not g e r m i n a t e , nor d i d f i l a m e n t s grow i n any c o n c e n t r a t i o n of TFP t e s t e d ( F i g . 58) . T r a n s f e r r i n g T F P - t r e a t e d m a t e r i a l i n t o normal medium d i d not l e a d t o r e c o v e r y from growth i n h i b i t i o n or g e r m i n a t i o n , i n d i c a t i n g the i r r e v e r s i b i l i t y of the e f f e c t s of TFP on V a u c h e r i a . C o n t r o l specimens grew at r a t e s s i m i l a r t o c o n t r o l s from the EGTA and A23187 experiments and c o n t i n u e d to grow when s u b j e c t e d t o the 28 same t r a n s f e r p r o c e d u r e as the T F P - t r e a t e d m a t e r i a l . When T F P - t r e a t e d m a t e r i a l was s t a i n e d w i t h CTC, no f l u o r e s c e n c e was observed ( F i g . 6 9 ) . F i g u r e 59 i s a comparison of the growth r a t e s graphed a g a i n s t the v a r i o u s c o n c e n t r a t i o n s of each of the t h r e e growth i n h i b i t o r s used. TFP i s c l e a r l y the most p o t e n t growth i n h i b i t o r f o l l o w e d by A23187 and EGTA, r e s p e c t i v e l y . A23187 i s more t o x i c than EGTA at each of the c o n c e n t r a t i o n s t e s t e d . The potency of A23187 i s more pronounced than t h a t f o r EGTA at 10~4M. However, the t o x i c i t y of TFP i s n e a r l y the same as t h a t of EGTA at 10-5M and a t 10"6M. DISCUSSION 29 APLANOSPOROGENESIS Venkataraman (1961) and Chopra (1971) r e p o r t zoospores as the most common method of a s e x u a l r e p r o d u c t i o n i n V a u c h e r i a c e a e . H i b b e r d (1980) s t a t e s t h a t a s e x u a l r e p r o d u c t i o n i n V a u c h e r i a i s e x c l u s i v e l y by the p r o d u c t i o n of synzoospores formed i n z o o s p o r a n g i a . N e i t h e r Lee (1980) or B o l d and Wynne (1985) acknowledge the presence of a p l a n o s p o r e s i n V a u c h e r i a when d e s c r i b i n g t h i s genus' modes of a s e x u a l r e p r o d u c t i o n i n t h e i r t e x t b o o k s ; however, Smith (1950) and C h r i s t e n s e n (1980) do. Zoospores are a c t u a l l y r e p o r t e d i n fewer s p e c i e s of V a u c h e r i a than a p l a n o s p o r e s . D e s p i t e t h i s d e s c r e p a n c y , no s t u d i e s o u t s i d e the taxonoroic and e c o l o g i c realms have been conducted on a p l a n o s p o r e s or a p l a n o s p o r o g e n e s i s u n t i l now. In f a c t , u l t r a s t r u c t u r a l s t u d i e s of a p l a n o s p o r o g e n e s i s or a p l a n o s p o r e s i n the p h y c o l o g i c a l l i t e r a t u r e as a whole a r e v i r t u a l l y n o n e x i s t e n t . The b a s i c o r g a n i z a t i o n of the v e g e t a t i v e f i l a m e n t of V.. l o n g i c a u l i s v a r . m a c o u n i i and the i n i t i a l s t a g e s of s p o r o g e n e s i s are s i m i l a r t o t h a t r e p o r t e d by Ott and Brown (1974a, 1974b) f o r o t h e r s p e c i e s of V a u c h e r i a . However, the mechanism of s e p t a t i o n , l e a d i n g t o the f o r m a t i o n of the s p o r e , i s s t r i k i n g l y d i f f e r e n t i n 30 z o o s p o r o g e n e s i s and a p l a n o s p o r o g e n e s i s . F r i t s c h (1935) and Ott and Brown (1974b) d e s c r i b e a t r a n s v e r s e b r i d g e of c o l o r l e s s c y t o p l a s m s e p a r a t i n g the zoosporangium from the v e g e t a t i v e c y t o p l a s m p r i o r to a c t u a l s e p t a t i o n . W i t h i n a few m i n u t e s , these two s e p a r a t e d c y t o p l a s m i c masses rea p p r o a c h one another w i t h each f o r m i n g a membrane r e s u l t i n g i n a septum t h a t i s o l a t e s the zoosporangiuro from the v e g e t a t i v e f i l a m e n t . The s e p a r a t i o n and reapproachment of c y t o p l a s m i c masses a r e not seen i n a p l a n o s p o r o g e n e s i s . F a t h e r , o b s e r v a t i o n of s e p t a t i o n v i a the c e n t r i p e t a l i n f u r r o w i n g of the i n n e r w a l l at the base of the e n l a r g e d t i p of r e p r o d u c t i v e f i l a m e n t s o c c u r r e d . T h i s i n f u r r o w i n g mechanism resembles c l e a v a g e f u r r o w s d e s c r i b e d i n s p e r m a t o g e n e s i s of Vaucher i a (Ott and Brown, 1978) and i n c y t o k i n e s i s , t e t r a s p o r o g e n e s i s and s p e r m a t o g e n e s i s of red a l g a e ( S c o t t 1983, Pueschul 1979, Vesk and B o r o w i t z k a 1984, C o l e and Sheath 1980). T h i s c o n s t r i c t i n g i n n e r w a l l i s o l a t e s the mature a p l a n o s p o r e from the v e g e t a t i v e f i l a m e n t , w h i l e becoming p a r t of the c e l l w a l l of the m a t u r i n g a p l a n o s p o r e . T a y l o r (1952) r e p o r t e d t h a t t h e r e was no w a l l p r e s e n t i n a p l a n o s p o r e s from l o n g i c a u l i s c o l l e c t e d i n C a l i f o r n i a ; a s i t u a t i o n s i m i l a r t o the one observed i n zoospores of f o n t i n a l i s ( C h r i s t e n s e n ) p r i o r t o g e r m i n a t i o n (Ott and Brown, 1975). However, 31 the presence of a complete w a l l around the a p l a n o s p o r e p r i o r t o r e l e a s e and s e t t l e m e n t has been r e p o r t e d i n the genus V a u c h e r i a by F r i t s c h (1935) and R i e t h (1980) and i s f u r t h e r c o n f i r m e d i n the p r e s e n t s t u d y . P r e v i o u s l y r e p o r t e d i n v e g e t a t i v e f i l a m e n t s of s e s s i l i s (Vauch.) DeCandolle (Greenwood, 1959), ^. f o n t i n a l i s (L.) C h r i s t e n s e n and d i l w y n i i (Weber et Mohr) C. A. Agardh (Ott and Brown, 1974a) , the m i t o c h o n d r i o n - d i c t y o s o m e a s s o c i a t i o n i s a l s o p r e s e n t i n .y. l o n g i c a u l i s v a r . m a c o u n i i . T h i s a s s o c i a t i o n p e r s i s t s throughout a p l a n o s p o r o g e n e s i s and c o n t r a s t s w i t h z o o s p o r o g e n e s i s where the m i t o c h o n d r i a - d i c t y o s o m a l a s s o c i a t i o n s g r a d u a l l y d i s a p p e a r (except i n the apex) u n t i l g e r m i n a t i o n of the zoospore o c c u r s (Ott and Brown, 1974b, 1975). The band of endoplasmic r e t i c u l u m and v e s i c l e s seen i n t e r s p e r s e d between the m i t o c h o n d r i o n and dictyosomes appear i n a l l V a u c h e r i a s p e c i e s s t u d i e d t o d a t e . A s i m i l a r m i t o c h o n d r i a - E R - d i c t y o s o m a l a s s o c i a t i o n has a l s o been r e p o r t e d i n the Oomycete S a p r o l e g n i a (Heath and Greenwood, 1971). The a s s o c i a t i o n of a m i t o c h o n d r i o n a d j a c e n t t o a dictyosome may be f u n c t i o n a l l y i m p o r t a n t i n a coenocyte l i k e V a u c h e r i a where v i g o r o u s c y t o p l a s m i c s t r e a m i n g might o t h e r w i s e cause these o r g a n e l l e s t o become s e p a r a t e d by c o n s i d e r a b l e d i s t a n c e s . Y e t , other c o e n o c y t e s l a c k t h i s 32 type of a s s o c i a t i o n ( P e n i c i l l u s f Turner and Friedman 1974, Dichotomosiphon, Moestrup and Hoffman 1973, Pseudodichotoroosiphon f H o r i £t £l. 1979, B r y o p s i s . Burr and West 1970) and s t i l l remain f u n c t i o n a l . Heath and Greenwood (1971) s u g g e s t e d t h a t t h i s a s s o c i a t i o n may f a c i l i t a t e e f f i c i e n t energy t r a n s f e r between the o r g a n e l l e s . The c o u p l i n g of an energy p r o d u c t i o n s i t e (the m i t o c h o n d r i o n ) w i t h energy u t i l i z a t i o n s i t e s ( dictyosomes and EP) c o u l d then be f u n c t i o n a l l y s i g n i f i c a n t f o r the i n t e n s i v e s e c r e t o r y a c t i v i t y of dictyosomes l e a d i n g t o the f o r m a t i o n of the a p l a n o s p o r e w a l l as suggested f o r t e t r a s p o r o g e n e s i s i n c e r t a i n r e d a l g a e ( P u e s c h u l , 1979) . However, the a c t u a l r o l e and f o r c e s r e t a i n i n g t h i s a s s o c i a t i o n remain e n i g m a t i c . The f o r m a t i o n of a new c e n t r a l v a c u o l e w i t h i n the d e v e l o p i n g a p l a n o s p o r e p r i o r t o r e l e a s e from the aplanosporangium was not seen i n z o o s p o r o g e n e s i s (Ott and Brown, 1974b). The c o a l e s c e n c e of many s m a l l v a c u o l e s i n t o a new c e n t r a l v a c u o l e may b u i l d t u r g o r p r e s s u r e w i t h i n the a p l a n o s p o r e so t h a t i t can germinate i m m e d i a t e l y upon r e l e a s e through l o c a l i z e d t u r g o r - d r i v e n c e l l w a l l e x p a n s i o n ( F i t c h and O l i v e i r a , 1986b). P y r e n o i d s were r e p o r t e d l a c k i n g i n V a u c h e r i a f i l a m e n t s ( F r i t s c h 1935, Ott and Brown 1974a) and zoospores (Greenwood 1959, Ott and Brown 1974b, 33 1975). T h i s i s i n c o n t r a s t w i t h our f i n d i n g s and those of Marchant (1972) i n a p l a n o s p o r e s of ^. w o r o n i n i a n a . In z o o s p o r o g e n e s i s , n u c l e i w i t h a s s o c i a t e d c e n t r i o l e s converge on the plasma membrane l e a d i n g t o the f o r m a t i o n of f l a g e l l a r p o o l s (Ott and Brown, 1974b). T h i s c o n t r a s t s w i t h a p l a n o s p o r o g e n e s i s where n u c l e i remain s c a t t e r e d throughout the c y t o p l a s m and n e i t h e r f l a g e l l a r i n i t i a t i o n or assembly are o b s e r v e d . T a b l e I c o n t r a s t s a p l a n o s p o r o g e n e s i s i n ^. l o n g i c a u l i s v a r . m a c o u n i i w i t h z o o s p o r o g e n e s i s i n iZ. f o n t i n a l i s and r e v e a l s t h a t a p l a n o s p o r e s may be more than s i m p l y " o n t o g e n e t i c a l l y p o t e n t i a l z o o s p o r e s " ( B o l d and Wynne, 1985). C h r i s t e n s e n 1 s (1980) d e s c r i p t i o n of a p l a n o s p o r e s i n V a u c h e r i a as " . . . r e p r o d u c t i v e c e l l s t h a t s t a r t t h e i r development i n the same way as zoospores but then assume an a p p r o x i m a t e l y s p h e r i c a l shape and s u r r o u n d themselves w i t h w a l l s w i t h o u t h a v i n g formed f l a g e l l a " more a c c u r a t e l y d e s c r i b e s the a p l a n o s p o r e s i n H. l o n g i c a u l i s v a r . m a c o u n i i . A l t h o u g h some u l t r a s t r u c t u r a l and d e v e l o p m e n t a l s i m i l a r i t i e s a re seen i n a p l a n o s p o r o g e n e s i s and z o o s p o r o g e n e s i s , s i g n i f i c a n t d i f f e r e n c e s e x i s t between these two p r o c e s s e s as w e l l . Such d i f f e r e n c e s warrant f u r t h e r study of the p r o c e s s of a p l a n o s p o r o g e n e s i s i n the T r i b o p h y c e a e and i n a l l a p l a n o s p o r e - p r o d u c i n g a l g a e i n g e n e r a l . 34 TABLE I APLANOSPOROGENESIS VS ZOOSPOROGENESIS ULTRUSTRUCTURAL FEATURE APLANOSPOROGENESIS* ZOOSPOROGENESIS** C e l l W a l l M i t o c h o n d r i a - E R -Dictyosome F i b r i l l a r m a t e r i a l -c o n t a i n i n g v e s i c l e s C e n t r a l V a c u o l e C h l o r o p l a s t s N u c l e i P r e s e n t P r e s e n t P r e s e n t i n l a r g e numbers; c o n t a i n w a l l m a t e r i a l V a c u o l e d i s p l a c e d , t r a n s f o r m e d i n t o r e t i c u l a t e p a t t e r n , r e a p p e a r s Many p e r i p h e r a l l y a r r a n g e d , p y r e n o i d p r e s e n t Always randomly s c a t t e r e d Absent G r a d u a l l y d i s a p p e a r s (except at apex) G r a d u a l l y d i s a p p e a r Absent Randomly s c a t t e r e d , P y r e n o i d absent P a r i e t a l l y p o s i t i o n e d i n l a t e z o o s p o r o g e n e s i s F l a g e l l a Absent P r e s e n t * V.. l o n g i c a u l i s v a r . m a c o u n i i ( F i t c h and O l i v e i r a , 1986a) ** 3Z. f o n t i n a l i s (Ott and Brown, 1974b) APLANOSPORE RELEASE AND GERMINATION 35 T a y l o r (1952) r e p o r t e d t h a t d i s s o l u t i o n of the d i s t a l aplanosporangium w a l l l e a d s t o l i b e r a t i o n of a p l a n o s p o r e s i n V.. l o n g i c a u l i s . Enzymatic d e g r a d a t i o n of the zoosporangium w a l l was r e p o r t e d d u r i n g zoospore r e l e a s e i n ^ . f o n t i n a l i s (Ott and Brown, 1975). In our m a t e r i a l , however, p o l y s a c c h a r i d e - w a l l d e g r a d i n g enzymes would tend t o break down ( i n a d d i t i o n t o the aplanosporangium w a l l ) the a l r e a d y s e c r e t e d a p l a n o s p o r e w a l l . T h i s would a f f e c t the s t r u c t u r a l i n t e g r i t y of the a p l a n o s p o r e and hence i t i s u n l i k e l y t o o c c u r . We have observed an a c c u m u l a t i o n of m u c i l a g i n o u s - l i k e m a t e r i a l between the mature a p l a n o s p o r e and the aplanosporangium w a l l of l o n g i c a u l i s v a r . m a c o u n i i ( F i t c h and O l i v e i r a , 1986a). S w e l l i n g of the m u c i l a g i n o u s m a t e r i a l s u r r o u n d i n g the a p l a n o s p o r e c o u l d then r u p t u r e the d i s t a l end of the aplanosporangium and f a c i l i t a t e a p l a n o s p o r e r e l e a s e . In a d d i t i o n , the c o a l e s c e n c e of many s m a l l v a c u o l e s i n t o the new c e n t r a l v a c u o l e , observed d u r i n g a p l a n o s p o r o g e n e s i s ( F i t c h and O l i v e i r a , 1986a), may h e l p b u i l d up s u f f i c i e n t t u r g o r p r e s s u r e w i t h i n the a p l a n o s p o r e to a i d i n b u r s t i n g open the aplanosporangium. These o b s e r v a t i o n s a re i n agreement w i t h those of F r i t s c h (1935) and Venkataraman (1961) 36 s u g g e s t i n g t h a t o s m o t i c p r e s s u r e b u i l d - u p w i t h i n the i n t a c t aplanosporangium l e a d s t o the a p i c a l r u p t u r e f o l l o w e d by c o n t r a c t i o n of the aplanosporangium w a l l and the subsequent r e l e a s e of the a p l a n o s p o r e . B e s i d e s i t s i n v o l v e m e n t i n a p l a n o s p o r e r e l e a s e , t u r g o r p r e s s u r e has a l s o been c i t e d as the moving f o r c e behind p l a n t ( P i c k e t t - H e a p s , 1977, Burns e_t a l , 1982) and f u n g a l ( B u l l e r , 1958) c e l l e l o n g a t i o n . In V.. l o n g i c a u l i s v a r . m a c o u n i i , the c o a l e s c e n c e of s m a l l v a c u o l e s w i t h the c e n t r a l v a c u o l e d u r i n g a p l a n o s p o r o g e n e s i s ( F i t c h and O l i v e i r a , 1986a) and the r a p i d volume ex p a n s i o n of the c e n t r a l v a c u o l e d u r i n g the e a r l y s t a g e s of a p l a n o s p o r e g e r m i n a t i o n support t h i s p r o p o s a l . The c e l l w a l l at the t i p of the g e r m i n a t i n g f i l a m e n t must m a i n t a i n i t s s t r u c t u r a l i n t e g r i t y w h i l e y i e l d i n g t o the t u r g o r - d r i v e n f o r c e of the r a p i d l y expanding c e n t r a l v a c u o l e . The presence of a non-uniform c e l l w a l l at the g e r m i n a t i o n s i t e ( s ) on the a p l a n o s p o r e t o g e t h e r w i t h the morphometric data showing a decrease i n the volume d e n s i t y of the c e l l w a l l compartment d u r i n g the e a r l y s t a g e s of g e r m i n a t i o n , r e f l e c t s the d u a l r o l e t h a t the c e l l w a l l must p e r f o r m . G e r m i n a t i o n and t i p growth can, t h e r e f o r e , be c o n s i d e r e d a dynamic b a l a n c e between w a l l l y s i s t o y i e l d t o t u r g o r 37 p r e s s u r e development, as e v i d e n c e d by d i s r u p t i o n of the o u t e r w a l l l a y e r , and new w a l l s y n t h e s i s t o m a i n t a i n f i l a m e n t i n t e g r i t y , as e v i d e n c e d by i n c o r p o r a t i o n of r e l e a s e d m a t e r i a l s t o the i n n e r w a l l l a y e r (Garraway and Evans, 1984). Heath and Greenwood (1971), Ott and Brown (1974a) and A g h a j a n i a n (1979) have p o s t u l a t e d t h a t a c l o s e a s s o c i a t i o n between m i t o c h o n d r i a and dictyosomes f a c i l i t a t e s e f f i c i e n t energy t r a n s f e r w i t h i n the c e l l . The p e r s i s t e n c e of t h i s a s s o c i a t i o n , f i r s t o b served i n V a u c h e r i a by Ott and Brown (1974a, 1974b, 1975) and c o n f i r m e d by our work on a p l a n o s p o r o g e n e s i s ( F i t c h and O l i v e i r a , 1986a), may be n e c e s s a r y t o support the r a p i d growth r a t e (apprcx. 250 um/hr.) of the f i l a m e n t s d u r i n g the e a r l y s t a g e s of g e r m i n a t i o n . A l t h o u g h d i c t y o s o m a l volume d e n s i t y does not i n c r e a s e d u r i n g g e r m i n a t i o n , an i n c r e a s e i n d i c t y o s o m a l e f f i c i e n c y i s suggested by both an a c c u m u l a t i o n of v e s i c l e s at the t i p of the g e r m i n a t i n g f i l a m e n t s ( F i g . 10) and an i n c r e a s e i n v e s i c l e volume d e n s i t y t h a t remains s i g n i f i c a n t l y h i g h d u r i n g the a c t i v e p e r i o d s of s t a g e s I I and I I I and i n subsequent g e r m i n a t i o n ( F i g . 1 9 ) . These o b s e r v a t i o n s t o g e t h e r w i t h the i n c r e a s e observed i n the m i t o c h o n d r i a l volume d e n s i t y d u r i n g s t a g e s I I and I I I i n d i c a t e a h i g h m e t a b o l i c r a t e f o r the s y n t h e s i s and e x p o r t of m a t e r i a l s 38 to the i n n e r l a y e r of the c e l l w a l l by the m i t o c h o n d r i a - E R - d i c t y o s o m a l complexes ( F i g . 9 ) . D u r i n g stage I , s m a l l bundles of m i c r o t u b u l e s were seen s c a t t e r e d i n the c y t o p l a s m . T h i s i s i n c o n t r a s t w i t h g e r m i n a t i o n ( s t a g e s I I and I I I ) and subsequent f i l a m e n t e x t e n s i o n i n which m i c r o t u b u l e bundles appear more numerous and show p r e f e r e n t i a l arrangement near the plasma membrane. I n c r e a s e s i n m i c r o t u b u l e numbers have been noted i n the g e r m i n a t i n g spores of the moss F u n a r i a (Schnepf £± a l . , 1 982), i n p o l l e n tubes of L i l i u m ( R e i s s and H e r t h , 1979a), i n the t i p s of a c t i v e l y growing f u n g a l hyphae (Howard and A i s t , 1980) and i n budding y e a s t c e l l s (Garraway and Evans, 1984). I n c r e a s e s i n m i c r o t u b u l e number accompanied by t h e i r a s s o c i a t i o n w i t h n u c l e i and c h l o r o p l a s t s were r e p o r t e d by Ott and Brown (1974a) i n V a u c h e r i a l i t o r e a and Schnepf £i a l (1982) i n the moss F u n a r i a h y g r o m e t r i c a . These o b s e r v a t i o n s seem to i n d i c a t e t h a t d i r e c t i o n a l movement of o r g a n e l l e s by m i c r o t u b u l e s and/or m i c r o f i l a m e n t s i s e s s e n t i a l i n e s t a b l i s h i n g the p o l a r i t y of g e r m i n a t i o n and i n o r c h e s t r a t i n g the development of r e p r o d u c t i v e s t r u c t u r e s i n vaucheyia. E n d o s y m b i o t i c b a c t e r i a have p r e v i o u s l y been r e p o r t e d i n t h r e e s p e c i e s of V a u c h e r i a ( O t t , 1979). T h i s author b e l i e v e s t h i s may be a m u t u a l i s t i c 39 r e l a t i o n s h i p . T h i s i n t e r p r e t a t i o n i s p a r t i c u l a r l y a t t r a c t i v e i n l i g h t of c u r r e n t r e s u l t s showing the o c c u r r e n c e of a s i g n i f i c a n t decrease i n the volume d e n s i t y of the c h l o r o p l a s t compartment d u r i n g g e r m i n a t i o n . T h i s c o u l d c o n c e i v a b l y be t r a n s l a t e d i n t o lower p h o t o s y n t h e t i c c a p a c i t y and the need t o supplement the h i g h e n e r g e t i c demands imposed on the g e r m i n a t i n g a p l a n o s p o r e by other means. B a c t e r i a l d i g e s t i o n w i t h i n v a c u o l e s may then be i m p o r t a n t i n s u p p l y i n g the a p l a n o s p o r e w i t h some n u t r i e n t s . The f a c t t h a t t h e r e i s no apparent e f f e c t on the r a t e of a p l a n o s p o r e g e r m i n a t i o n due t o l a r g e numbers of i n t a c t b a c t e r i a l y i n g i n the c y t o p l a s m c l o s e t o n u c l e i l e n d s support t o t h i s i n t e r p r e t a t i o n . More work i s r e q u i r e d , however, on t h i s s u b j e c t s i n c e the o b s e r v a t i o n i n our m a t e r i a l of some b a c t e r i a w i t h i n d i g e s t i v e v a c u o l e s may be p a r t of a d e f e n s i v e response to b a c t e r i a l i n f e c t i o n by the ap l a n o s p o r es. CALCIUM LOCALIZATION WITH CHLOROTETRACYCLINE V a r i o u s c o n c e n t r a t i o n s of CTC have been used t o observe the d i s t r i b u t i o n of c a l c i u m i n l i v i n g p l a n t and an i m a l c e l l s (Wolniak £i &1 1980 , M e i n d l 1982 , Kiermayer and M e i n d l 1984, Kauss and Rausch 1984, 40 Glowacka £ i a_l 1985), w i t h a c o n c e n t r a t i o n of 10~4M b e i n g the most f r e q u e n t l y u t i l i z e d (Chandler and W i l l i a m s 1978a,b, F e i s s and H e r t h 1978, 1979b, 1982, 1985 , F e i s s £± a l 1983 , F e i s s j_t a l 1985). C a s w e l l (1979) s u g g e s t s t h a t CTC s h o u l d not be used above a c o n c e n t r a t i o n of 10-4M s i n c e h i g h e r l e v e l s may d i s r u p t the response b e i n g m o n i t o r e d . However, the s u i t a b i l i t y of t h i s CTC c o n c e n t r a t i o n must be t e s t e d i n each case s i n c e c e l l s from d i f f e r e n t organisms d i s p l a y v a r y i n g degrees of s e n s i t i v i t y t o CTC as an a n t i b i o t i c (Baloun and Hudak 1979, F e i s s and H e r t h 1979b, Saunders and H e p l e r 1981). For example, 1 0 - 4 H CTC i s r e p o r t e d t o have no d e l e t e r i o u s e f f e c t s on the fungus A c h l y a and the a l g a A c e t a b u l a r i a ( R e i s s and H e r t h , 1979b), the l i v e r w o r t F i e l l a ( G r o t h a , 1983), and the moss F u n a r i a (Saunders and H e p l e r , 1981). However, Lepidiuro ( c r e s s ) r o o t h a i r s show a tendency t o b u r s t when t r e a t e d w i t h 10~4M CTC ( F e i s s and H e r t h , 1979b). P r o l o n g e d t r e a t m e n t w i t h 10~4M CTC a l s o causes d i s o r i e n t e d growth, abnormal w a l l t h i c k e n i n g s and a p i c a l s w e l l i n g i n L i l i u m p o l l e n tubes a f t e r 30 minutes of exposure and e v e n t u a l l y r e s u l t s i n complete c e s s a t i o n of growth a f t e r 2 hours of treatment ( F e i s s and H e r t h , 1982). In the desmid M i c r a s t e r i a s . growth i s stopped w i t h i n m i n u t e s , the w a l l i s a b n o r m a l l y t h i c k e n e d and death o c c u r s a f t e r 6 to 8 hours i n 10~4M 41 CTC (Hausser and H e r t h , 1983). In V a u c h e r i a , t o t a l f i l a m e n t l e n g t h i s d i m i n i s h e d but growth i s not stopped i n 1 0 - 4 M CTC, w h i l e the growth r a t e shows a s i g n i f i c a n t r e c o v e r y between 4 t o 6 hours a f t e r i n i t i a t i o n of g e r m i n a t i o n p r i o r to becoming s t a b i l i z e d . In a d d i t i o n , the morphology and o r i e n t a t i o n of V a u c h e r i a f i l a m e n t s remain u n a f f e c t e d by t h i s t r e a t m e n t . C o n c e n t r a t i o n s of CTC g r e a t e r than 10~4M show b r i g h t e r f l u o r e s c e n c e . However, n e i t h e r g e r m i n a t i o n nor growth o c c u r r e d at these l e v e l s , w h i l e the f l u o r e s c e n c e i n t e n s i t y produced by u s i n g lower c o n c e n t r a t i o n s of CTC becomes reduced. S i n c e a l l o b s e r v a t i o n s were made w i t h i n 1 to 10 minutes of CTC a p p l i c a t i o n , a time when the growth r a t e of t r e a t e d f i l a m e n t s i s s i m i l a r to t h a t of the c o n t r o l , the 10~4M CTC c o n c e n t r a t i o n i s the best compromise between b r i g h t f l u o r e s c e n c e and h e a l t h y m e t a b o l i c a c t i v i t y i n V a u c h e r i a . C a l c i u m a c c u m u l a t i o n s at the a p i c e s of L i l i u m p o l l e n tubes have been demonstrated u s i n g a u t o r a d i o g r a p h y ( J a f f e £i a_l, 1975), a p r o t o n - m i c r o p r o b e ( P e i s s e_t 1983) and CTC f l u o r e s c e n c e ( R e i s s and H e r t h , 1978, 1982). J a f f e £i &1 (1975) found t h a t the a c c u m u l a t i o n of Ca2+ was 2 to 4 times h i g h e r at the apex than i n the bulk of the p o l l e n tubes where Ca2+ a c c u m u l a t i o n s d i d not o c c u r . R e i s s je_fc a_l (1983) 42 r e p o r t e d t h a t i n L i l i u m p o l l e n t u b e s , the maximum c a l c i u m c o n t e n t l i e s w i t h i n the t e r m i n a l 7 um; a r e g i o n r i c h i n endoplasmic r e t i c u l u m , m i t o c h o n d r i a and g o l g i - d e r i v e d v e s i c l e s , o r g a n e l l e s known t o p a r t i c i p a t e i n the s t o r a g e of Ca2+ i o n s (Herth 1978, P i c t o n and S t e e r 1982 , P e i s s £i a_l 1 9 83). S i n c e CTC i s known t o accumulate and b i n d Ca2+ t o c e l l u l a r membranes ( C a s w e l l 1979, R e i s s and Herth 1982), those r e s u l t s concur w i t h our f i n d i n g s t h a t , i n V a u c h e r i a . the maximum f l u o r e s c e n c e , and hence Ca2+ c o n c e n t r a t i o n , l i e s w i t h i n the t i p of the g e r m i n a t i n g f i l a m e n t . T h i s r e g i o n i s a l s o r i c h i n o r g a n e l l e s such as v e s i c l e s and m i t o c h o n d r i a ( F i t c h and O l i v e i r a , 1986b) known to be c a p a b l e of s t o r i n g c e l l u l a r c a l c i u m . The more d i f f u s e d i s t r i b u t i o n of CTC f l u o r e s c e n c e seen i n V a u c h e r i a f i l a m e n t s between 2 and 4 hours a f t e r g e r m i n a t i o n may i n d i c a t e a r e l e a s e of Ca2+ from i n t r a c e l l u l a r s t o r a g e and/or an i n f l u x of Ca2+ from the e x t r a c e l l u l a r compartment i n t o the c y t o p l a s m . I n f l u x of Ca2+ would be r e q u i r e d f o r the maintenance of an area of h i g h i n t e n s i t y f l u o r e s c e n c e at the t i p of the f i l a m e n t s , w h i l e r e l e a s e of Ca2+ from i n t r a c e l l u l a r s t o r a g e c o u l d m a i n l y account f o r the p r o g r e s s i v e i n c r e a s e i n the area o c c u p i e d by CTC f l u o r e s c e n c e . A wides p r e a d i n c r e a s e i n Ca2+ d i s t r i b u t i o n , due to a r i s e i n f r e e c y t o p l a s m i c 43 Ca2+, l e a d s t o growth i n h i b i t i o n i n L i l i u m p o l l e n tubes (Herth 1978, F e i s s and Herth 1979a,b, P i c t o n and S t e e r 1982). The same phenomenon may then be r e s p o n s i b l e f o r the reduced growth r a t e observed between 2 and 4 hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n i n our m a t e r i a l . From a p p r o x i m a t e l y 4 hours a f t e r g e r m i n a t i o n and beyond, CTC f l u o r e s c e n c e r e t u r n s t o a t i p - l o c a l i z e d p a t t e r n s u g g e s t i n g t h a t excess f r e e c y t o p l a s m i c Ca2+ may have been r e s e q u e s t e r e d i n t o known s u b - c y t o p l a s m i c Ca2+ p o o l s ( F e i s s and Herth 1978, Wolniak £i £l 1980 , Saunders and Hep l e r 1981, Wise and Wolniak 1984, Kauss and Fausch 1984) . In F u n a r i a , bud f o r m a t i o n i s t r i g g e r e d by the a c c u m u l a t i o n at the growing t i p of Ca2+ c h a n n e l s . These promote Ca2+ i n f l u x l e a d i n g t o l o c a l i z e d microdomains of i n c r e a s e d Ca2+ c o n c e n t r a t i o n (Saunders, 1986). There i s ev i d e n c e i n A c e t a b u l a r i a f and i t has been suggested i n S p h a c e l a r i a , t h a t the e s t a b l i s h m e n t of growth r e g i o n s a l s o c o i n c i d e s w i t h e x t e r n a l a c i d i f i c a t i o n of the c e l l w a l l due t o the a c t i o n of p r o t o n pumps (Burns e_t &1 1984, Goodwin and T r a i n o r 1985). I n c r e a s e d e x t e r n a l p r o t o n c o n c e n t r a t i o n i s known t o cause d i s p l a c e m e n t of Ca2+ i o n s from the c e l l w a l l r e s u l t i n g i n l o c a l l y i n c r e a s e d Ca2+ i n f l u x a c r o s s the plasma membrane (Goodwin and T r a i n o r , 1985). Such a t r a n s i e n t i n c r e a s e 44 i n c e l l u l a r c a l c i u m has been suggested t o r e s u l t i n e x o c y t o s i s and i n s e r t i o n i n t o the s u r f a c e membrane of H+-ATPase t r a n s p o r t systems (van A d e l s b e r g and A l - A w q a t i , 1986). F u r t h e r a c i d i f i c a t i o n of the c e l l w a l l c o u l d then t i l t the b a l a n c e of Ca2+ d i s t r i b u t i o n towards a temporary a c c u m u l a t i o n of Ca2+ i n the p e r i p h e r a l c y t o p l a s m . The l o c a l i z e d CTC f l u o r e s c e n c e i d e n t i f y i n g the g e r m i n a t i n g r e g i o n s of a p l a n o s p o r e s and the f i l a m e n t t i p s of V a u c h e r i a may then r e f l e c t the e x i s t e n c e of s i m i l a r mechanisms l e a d i n g t o the d i s p l a c e m e n t of Ca2 + i o n s . Indeed, l o c a l i z e d i o n c u r r e n t s a c r o s s the plasma membrane have p r e v i o u s l y been r e p o r t e d i n V a u c h e r i a s e s s i l i s and o t h e r t i p - g r o w i n g c e l l s (Quatrano 1978, W e i s e n s e e l and K i c h e r e r 1981). These c o n t r i b u t e t o the a c c u m u l a t i o n of g o l g i - d e r i v e d v e s i c l e s a t the growth p o l e ( s ) and promote t h e i r f u s i o n w i t h the c e l l membrane (Weisenseel and K i c h e r e r , 1981), r e s u l t i n g i n o r i e n t e d e x o c y t o s i s of p o l y s a c c h a r i d e - s t o r i n g v e s i c l e s n e c e s s a r y f o r a p i c a l w a l l e x p a n s i o n (Herth 1978, F e i s s and Herth 1978, 1979a, 1979b, 1982, W e i s e n s e e l and K i c h e r e r 1981). The a c c u m u l a t i o n and subsequent e x o c y t o s i s of v e s i c l e s at the t i p of V a u c h e r i a f i l a m e n t s (Ott and Brown 1974, Kataoka 1982, F i t c h and O l i v e i r a 1986a,b), concomitant w i t h a w e l l - d e f i n e d t i p - l o c a l i z e d CTC f l u o r e s c e n c e 45 o b s e r v e d d u r i n g p e r i o d s of r a p i d i n c r e a s e i n growth r a t e , s u ggests t h a t d i f f e r e n c e s i n e l e c t r i c a l p o t e n t i a l due, at l e a s t p a r t i a l l y , to Ca2+ i o n r e d i s t r i b u t i o n , a r e a l s o r e s p o n s i b l e f o r the e s t a b l i s h m e n t of p o l a r i z e d g e r m i n a t i o n and growth i n V a u c h e r j a . C a l c i u m i s known t o be i n v o l v e d i n the r e g u l a t i o n of the v i s c o e l a s t i c i t y of the g e l - l i k e c y t o p l a s m r i c h i n c y t o s k e l e t a l elements found a d j a c e n t t o the plasma membrane (Goodwin and T r a i n o r , 1985). L o c a l i z e d h i g h l e v e l s of Ca2+ causes d e p o l y m e r i z a t i o n of m i c r o t u b u l e s and d e c r e a s e s the v i s c o s i t y of the c y t o p l a s m ( P i c t o n and S t e e r 1982, Goodwin and T r a i n o r 1985). T h i s l o o s e n i n g of c y t o p l a s m i c s t r u c t u r e i n c o n j u n c t i o n w i t h v a c u o l e - d e r i v e d t u r g o r p r e s s u r e due to the r a p i d e x p a n s i o n of the c e n t r a l v a c u o l e of g e r m i n a t i n g f i l a m e n t s ( F i t c h and O l i v e i r a , 1986b) w i l l then f a c i l i t a t e growth. C o n c o m i t a n t l y , c y t o p l a s m i c i n t e g r i t y at the t i p of growing f i l a m e n t s i s m a i n t a i n e d by a network of m i c r o f i l a m e n t s which a re s t a b i l i z e d by h i g h l e v e l s of Ca2+ i o n s ( P i c t o n and S t e e r , 1982). A l t h o u g h m i c r o f i l a m e n t s have not been documented i n our m a t e r i a l , due p o s s i b l y t o t h e i r d i s r u p t i o n by g l u t a r a l d e h y d e f i x a t i o n , t h e i r presence has p r e v i o u s l y been r e p o r t e d i n V a u c h e r i a (Ott and Brown, 1974). T h i s d u a l opposing a c t i o n s of Ca2+ on d i f f e r e n t components 46 of the c y t o s k e l e t a l a p p a r a t u s may then p l a y an i m p o r t a n t r o l e i n r e g u l a t i n g the b a l a n c e between i n t e g r i t y and f l e x i b i l i t y t h a t must occur at the t i p ( s ) of the a p l a n o s p o r e and g e r m i n a t i n g f i l a m e n t s f o r growth t o ta k e p l a c e w i t h o u t b u r s t i n g the c e l l w a l l ( F i t c h and O l i v e i r a , 1986b) . CALCIUM PERTURBATIONS B e s i d e s d i r e c t o b s e r v a t i o n of the i n t r a c e l l u l a r l o c a l i z a t i o n of Ca2 + by CTC, another way t o o b t a i n i n f o r m a t i o n on the r o l e of Ca2+ i n these phenomena i s to use compounds which a l t e r the i n t r a c e l l u l a r Ca2+ g r a d i e n t s and a v a i l a b i l i t y , and t o observe whether or not m e t a b o l i c p a r a m e t e r s , such as growth r a t e s , a re changed as a consequence of a l t e r a t i o n s i n the d i s t r i b u t i o n of Ca2+ (Kauss and Rausch, 1984). For t h i s p urpose, we used t h r e e growth i n h i b i t o r s known t o a l t e r the a v a i l a b i l i t y of Ca2+ t o the c e l l : the c h e l a t o r EGTA, the ionophore A23187 and the p h e n o t h i a z i n e - t y p e c a l m o d u l i n a n t a g o n i s t TFP. EGTA s e l e c t i v e l y c h e l a t e s Ca2+ e x t e r n a l t o the plasmalemma y e t i s unable t o p e n e t r a t e b i o l o g i c a l membranes ( B l i n k s e_£ a_l 1982 , A l - K h a z z a r e_t a_l 1984 , van A d e l s b e r g and A l - A w q a t i 1986, V o l b e r g £i a_l 1986). EGTA 47 c o n c e n t r a t i o n s of 10~5M or g r e a t e r have been shown t o i n h i b i t growth i n the desmid M i c r a s t e r i a s (Lehtonen, 1984), p r e v e n t hyphal e x t e n s i o n i n flchlya b i s e x u a l i s ( H a r o l d and H a r o l d , 1986) , i n h i b i t p r o t o p l a s t f u s i o n i n Daucus c a r o t a (Grimes and Boss, 1985), p r e v e n t Ca2+ uptake by human e r y t h r o c y t e s (Foder £i a_l, 1984) and d e l a y the onset of anaphase i n Haemanthus endosperm c e l l s (Wise and Wo l n i a k , 1984). R e s u l t s o b t a i n e d i n t h i s work show t h a t EGTA c o n c e n t r a t i o n s of l e s s than 10"^M reduces growth r a t e s , w h i l e EGTA c o n c e n t r a t i o n s g r e a t e r than 10~ 4M c o m p l e t e l y i n h i b i t both g e r m i n a t i o n and growth. The data suggest t h a t the a v a i l a b i l i t y of e x t r a c e l l u l a r Ca2+ i s e s s e n t i a l t o a p l a n o s p o r e g e r m i n a t i o n and growth i n V a u c h e r i a . EGTA has a l s o been found to reduce or quench CTC f l u o r e s c e n c e i n the r h i z o i d s of the f e r n Onoclea  s e n s i b i l i s ( M i l l e r £i a_l, 1983) , i n gemmalings of the l i v e r w o r t R i e l l a h e l i c o p h y l l a ( G r o t h a , 1983), i n Haemanthus endosperm c e l l s (Wolniak £_t a_l, 1980) and i n p o l l e n tubes of v a r i o u s p l a n t s ( P o l i t o , 1985). P r e s e n t o b s e r v a t i o n s i n which the i n t e n s i t y of the CTC f l u o r e s c e n c e i s d i m i n i s h e d i n EGTA-treated m a t e r i a l p r o v i d e s e v i d e n c e t h a t much of the CTC f l u o r e s c e n c e i n u n t r e a t e d c e l l s i s due t o the t i p - l o c a l i z e d i n f l u x of Ca2+. T h e r e f o r e , a decrease i n CTC f l u o r e s c e n c e i n 48 EGTA-treated c e l l s seems to i n d i c a t e a r e d u c t i o n i n Ca2+ i n f 1 u x . EGTA-treated m a t e r i a l a l s o showed a more d i f f u s e d i s t r i b u t i o n of CTC f l u o r e s c e n c e r e s e m b l i n g t h a t o b s e r v e d i n u n t r e a t e d specimens between two t o f o u r hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n . T h i s o c c u r s c o n c o m i t a n t l y w i t h a r e d u c t i o n i n growth r a t e s s u g g e s t i n g an a l t e r a t i o n i n the i n f l u x of Ca2 + i n t o the c y t o p l a s m and/or the r e l e a s e of Ca2+ from i n t r a c e l l u l a r s t o r a g e as w e l l . T h i s temporary a l t e r a t i o n i n the d i s t r i b u t i o n and a v a i l a b i l i t y of c e l l u l a r Ca2+ i n d i c a t e s d i s r u p t i o n s i n normal Ca2+ g r a d i e n t s , hence of a s s o c i a t e d i o n i c c u r r e n t s . These o b s e r v a t i o n s concur then w i t h those of W i e s e n s e e l and K i c h e r e r (1981) showing t h a t the growth r a t e of V a u c h e r i a i s dependent upon the i n f l u x of s p e c i f i c e x t r a c e l l u l a r i o n s , p o s s i b l y Ca2 + i o n s , which produces the transmembrane i o n i c c u r r e n t n e c e s s a r y f o r m a i n t a i n i n g t i p - o r i e n t e d e x o c y t o s i s which i s known to p l a y a major r o l e i n a p l a n o s p o r e g e r m i n a t i o n and growth ( F i t c h and O l i v e i r a , 1986b) . The ionophore A23187 i s known t o i n h i b i t growth i n L i l i u m p o l l e n tubes (Herth 1978, F e i s s and Herth 1979a) and i n the u n i c e l l u l a r a l g a M i c r a s t e r i a s (Lehtonen, 1984). Abnormal growth and b r a n c h i n g p a t t e r n s due to 49 A23187 are r e p o r t e d i n L i l i u m p o l l e n tubes (Herth 1978, R e i s s and Hert h 1979a), i n the water mold A c h l y a  b i s e x u a l i s ( H a r o l d and H a r o l d , 1986), i n M i c r a s t e r i a s (Lehtonen, 1984) and i n the caulonema of the moss F u n a r i a ( S i e v e r s and Schnepf, 1981). In V a u c h e r i a , t h i s compound l o w e r s the growth r a t e of the g e r m i n a t i n g f i l a m e n t s and s t o p s growth when a p p l i e d at c o n c e n t r a t i o n s of 10" 4M or above. The treatment a l s o c r e a t e s m o r p h o l o g i c a l a b n o r m a l i t i e s i n the growing f i l a m e n t s . The ionophore A23187 i s known t o r e l e a s e Ca2 + from i n t e r n a l s t o r a g e , such as m i t o c h o n d r i a and endoplasmic r e t i c u l u m (Chandler and W i l l i a m s 1978b, Herth 1978, P e i s s and H e r t h 1979a, A l - K h a z z a r £± a_l 1984 , Grimes and Boss 1985, H a r o l d and H a r o l d 1986) and/or i n c r e a s e Ca2 + f l u x i n t o the c e l l (Herth 1978, W e i s e n s e e l and K i c h e r e r 1981, A l Khazzar £± aj. 1984 , Foder £± a_l 1984 , I r v i n g j_t a l 1984, Kauss and Rausch 1984, Lehtonen 1984, S k i b s t e d et a l 1984, Grimes and Boss 1985). T h e r e f o r e , when A23187 i s added t o c u l t u r e s , presumably the e x t r a - and i n t r a c e l l u l a r Ca2+ become e q u i l i b r a t e d , thus s i g n i f i c a n t l y r a i s i n g i n t r a c e l l u l a r Ca2+ c o n c e n t r a t i o n s . Herth (1978) showed t h a t i n L i l i u m p o l l e n t u b e s , d e p o l a r i z e d growth i s due t o the d e r e a l i z a t i o n of Ca2 + from a p i c a l growth s i t e s . Lehtonen (1984) a l s o showed 50 t h a t the d i s r u p t i o n of Ca2+ i n f l u x through the plasma membrane i n M i c r a s t e r i a s by A23187 l e a d s t o d i s o r i e n t e d growth due to d e l o c a l i z e d e x o c y t o s i s of d i c t y o s o m e - d e r i v e d v e s i c l e s , c o n t a i n i n g w a l l p r e c u r s o r s , which r e s u l t s i n m o r p h o l o g i c a l a b n o r m a l i t i e s . These o b s e r v a t i o n s concur w i t h p r e s e n t f i n d i n g s t h a t i n V a u c h e r i a . m o r p h o l o g i c a l a b n o r m a l i t i e s produced by A23187 t r e a t m e n t s appear as b u d - l i k e p r o t r u s i o n s w i t h most of the CTC f l u o r e s c e n c e l o c a l i z e d i n them ( F i g . 65) . M o r p h o l o g i c a l a b n o r m a l i t i e s c o u l d a l s o r e s u l t from ionophore-dependent v a r i a t i o n s i n Ca2 + g r a d i e n t s and t h e i r i m p l i c a t i o n s f o r the s t a b i l i t y of the c y t o s k e l e t o n . However, i t has been found t h a t m i c r o t u b u l e i n t e g r i t y i s u n a f f e c t e d i n v i v o by the presence of A23187 (Lonergan, 1984). On the o t h e r hand, i t has been r e p o r t e d t h a t A23187 can d i s r u p t o x i d a t i v e p h o s p h o r y l a t i o n (Peed and L a r d y , 1972). The c a l c i u m i o n ophore A23187 was a l s o s a i d t o p r i m a r i l y a f f e c t w a l l f o r m a t i o n i n p o l l e n tubes ( F e i s s and H e r t h , 1979a) and the caulonema of Funar i a (Schmeidel and Schnepf, 1980). T h e r e f o r e , whether these phenomena are d i r e c t l y induced by C a 2 + - r e l a t e d p e r t u r b a t i o n s of c y t o s k e l e t a l p r o t e i n s and e x o c y t o s i s or o t h e r causes cannot be d e t e r m i n e d at p r e s e n t . To a s c e r t a i n the importance of these v a r i o u s 51 phenomena and the r o l e Ca2+ p l a y s i n them, i t would be of i n t e r e s t t o lower the i n t r a c e l l u l a r Ca2+ l e v e l . To perform t h i s type of experiment i n v i v o , i t would be n e c e s s a r y t o reduce the Ca2+ c o n c e n t r a t i o n i n the growth medium t o a v a l u e below t h a t of f r e e i n t r a c e l l u l a r Ca2+ and then lower i t s i n t r a c e l l u l a r c o n c e n t r a t i o n by i n t r o d u c i n g an ionophore t h a t e q u i l i b r a t e s e x t r a - and i n t r a c e l l u l a r Ca2+. The problem i s t h a t the s i m p l e a c t i o n of l o w e r i n g Ca2+ c o n c e n t r a t i o n s i n the growth medium, t o a v a l u e most p r o b a b l y s t i l l above t h a t of f r e e i n t r a c e l l u l a r Ca2+, r e s u l t s i n the complete c e s s a t i o n of growth. Another p o s s i b i l i t y i s t o determine whether or not c a l m o d u l i n - r e g u l a t e d phenomena, such as c y t o s k e l e t a l s t a b i l i t y , i s i n v o l v e d i n the complex p r o c e s s of a p l a n o s p o r e g e r m i n a t i o n . C a l m o d u l i n i s known t o p a r t i c i p a t e i n the c a l c i u m r e g u l a t i o n of p r o t e i n p h o s p h o r y l a t i o n , of some en z y m a t i c a c t i v i t i e s , c a l c i u m f l u x a c r o s s c e l l membranes, m i c r o t u b u l e assembly and c e l l shape maintenance ( S k i b s t e d eJt &1 1984 , Grimes and Boss 1985 , Marine" 1985 , Smedley and S t a n i s s t r e e t 1985). P h e n o t h i a z i n e compounds, such as TFP, are known to b i n d t o the c a l c i u m - a c t i v a t e d form of c a l m o d u l i n ; hence a c t i n g as c a l m o d u l i n a n t a g o n i s t s ( B a r - S a g i and Proves 1983, H o r w i t z £i a_l 1981). TFP i s r e p o r t e d t o i n h i b i t wound 52 h e a l i n g i n Xenopus embryos (Smedley and S t a n i s s t r e e t , 1 9 8 5), i n h i b i t growth i n p o l l e n tubes of T r a d e s c a n t i a  v i r a i n i a n a ( P i c t o n and S t e e r , 1985), Pyrus communis,. J u g l a n s r e g j a and P r u n u s d u l c j s ( P o l i t o , 1 9 8 5), and t o i n h i b i t f u s i o n of Daucus c a r o t a p r o t o p l a s t s (Grimes and Boss, 1985). In human e r y t h r o c y t e s , TFP p r e v e n t s the t r a n s p o r t of Ca2+ i o n s through Ca2+ c h a n n e l s i n the plasma membrane by b i n d i n g t o the t r a n s p o r t enzyme and t o c a l m o d u l i n i t s e l f (Foder £i a l 1984 , S k i b s t e d sX a_l 1 9 8 4). TFP i s a l s o known t o i n h i b i t Ca2+ i n f l u x i n r o o t s of L e p i d i u m s a t i v u m (Buckhout, 1984) and o t h e r p l a n t s (Grimes and Boss 1985 , P i c t o n and S t e e r 1985 , P o l i t o 1 985). T h e r e f o r e , c a l m o d u l i n p l a y s an i m p o r t a n t r o l e i n g o v e r n i n g i n t r a c e l l u l a r Ca2+ c o n c e n t r a t i o n s ( P i c t o n and S t e e r , 1982). S i n c e n e i t h e r g e r m i n a t i o n nor growth o c c u r r e d i n V a u c h e r i a i n the presence of TFP, i t seems C a 2 + i n f l u x may have been i n h i b i t e d by the t r e a t m e n t . The absence of CTC f l u o r e s c e n c e i n T F P - t r e a t e d m a t e r i a l shows t h a t the t r e a t m e n t p o s s i b l y d i s t u r b s the s t o r a g e of i n t r a -c e l l u l a r Ca2+ as w e l l . T h i s i s not s u r p r i s i n g g i v e n the f a c t t h a t the c o n t r o l of c y t o p l a s m i c Ca2+ i n p l a n t as w e l l as i n animal c e l l s i s , at l e a s t p a r t i a l l y , dependent on the c a l m o d u l i n r e g u l a t e d a c c u m u l a t i o n of Ca2+ i n c e l l u l a r o r g a n e l l e s such as m i t o c h o n d r i a and 53 endoplasmic r e t i c u l u m (Marme, 1985). S c h l i w a e_t a l (1981) r e p o r t e d t h a t c a l m o d u l i n i n h i b i t o r s can c o m p l e t e l y i n h i b i t Ca2+-induced m i c r o t u b u l e d e p o l y m e r i z a t i o n i n l y s e d animal c e l l s . These f i n d i n g s t o g e t h e r w i t h the a v a i l a b l e e v i d e n c e s u g g e s t i n g a r o l e f o r Ca2+-calmodulin r e g u l a t i o n of m i c r o t u b u l e d e p o l y m e r i z a t i o n i n v i v o ( K e i t h si a l 1983, S c h l i w a £ i a l 1981) c o u l d e x p l a i n growth c e s s a t i o n i n T F P - t r e a t e d V a u c h e r i a due t o i n t e r f e r e n c e w i t h the p r e f e r e n t i a l arrangement of c y t o p l a s m i c m i c r o t u b u l e s o b s e r v e d d u r i n g a p l a n o s p o r e g e r m i n a t i o n ( F i t c h and O l i v e i r a , 1986b). The p o s s i b i l i t y t h a t c a l m o d u l i n may be i n v o l v e d i n the r e g u l a t i o n of m i c r o t u b u l e - m i c r o t u b u l e and p o s s i b l y m i c r o t u b u l e - o r g a n e l l e i n t e r a c t i o n s r a t h e r than m i c r o t u b u l e s t a b i l i t y has a l s o been advanced (De Mey £i a l , 1980). D i s r u p t i o n s i n these systems c o u l d l e a d t o an impairment i n the t r a n s p o r t of G o l g i - d e r i v e d v e s i c l e s c o n t a i n i n g c e l l w a l l p r e c u r s o r s t o the c e l l s u r f a c e ( F i t c h and O l i v e i r a , 1986b). Papahadjopoulos (1978) proposed t h a t c a l c i u m p a r t i c i p a t e s i n the r e g u l a t i o n of the p h y s i c a l s t r u c t u r e of the membrane b i l a y e r and thus c o n t r o l s the f u s i o n of membranes. T h i s would a l s o l e a d t o the d i s r u p t i o n of the e x o c y t o s i s of m a t e r i a l s n e c e s s a r y f o r c e l l w a l l e x p a n s i o n ( F i t c h and O l i v e i r a , 54 1986b) and hence t o growth c e s s a t i o n i n T F P - t r e a t e d m a t e r i a l . TFP-induced d i s t u r b a n c e on Ca2+ g r a d i e n t s can, t h e r e f o r e , a f f e c t i n a v a r i e t y of ways the o r g a n i z a t i o n of the a p i c a l c y t o p l a s m of Vaucher i a f i l a m e n t s ; thus s t o p p i n g growth and g e r m i n a t i o n . CONCLUSION 55 The i m p l i c a t i o n s of the u l t r a s t r u c t u r a l and o n t o g e n e t i c d i f f e r e n c e s between a p l a n o s p o r o g e n e s i s ( a p l a n o s p o r e s ) and z o o s p o r o g e n e s i s (zoospores) s h o u l d not be o v e r l o o k e d . The presence of a complete c e l l w a l l and c e n t r a l v a c u o l e and the maintenance of a w a l l - p r o d u c i n g a p p a r a t u s throughout a p l a n o s p o r o g e n e s i s ( F i t c h and O l i v e i r a , 1986a) appear t o p r e p a r e the a p l a n o s p o r e f o r immediate g e r m i n a t i o n and r a p i d growth upon r e l e a s e . These u l t r a s t r u c t u r a l and p h y s i o l o g i c a l a d a p t a t i o n s make the a p l a n o s p o r e a s u i t a b l e v e c t o r f o r a s e x u a l r e p r o d u c t i v e s u c c e s s i n some Vaucher i a s p e c i e s . J u d g i n g by the e x t e n s i v e and p e r s i s t e n t growth of y. l o n g i c a u l i s v a r . m a c o u n i i i n c o a s t a l s a l t marshes of the P a c i f i c N o r t h w e s t , d i s p e r s a l and growth v i a a p l a n o s p o r e s appears c r i t i c a l t o the c o n t i n u e d presence and e x p a n s i o n of t h i s a l g a . The c o m b i n a t i o n of i n c r e a s i n g t u r g o r p r e s s u r e ( F i t c h and O l i v e i r a , 1986b), a c i d i f i c a t i o n and weakening of the a p i c a l c e l l w a l l (Burns £ i a l » 1984), Ca2+ r e d i s t r i b u t i o n and i t s e f f e c t s on the s t a b i l i z a t i o n of the c y t o s k e l e t a l network ( P i c t o n and S t e e r , 1982) and e x o c y t o s i s of v e s i c l e s at the growth s i t e s ( J a f f e £ i a l 1975, H e r t h 1978, P i c t o n and S t e e r 1982, van A d e l s b e r g and A l - A w q a i t 1986) a r e i m p o r t a n t a s p e c t s of the complex mechanism c o n t r o l l i n g p o l a r i z e d g e r m i n a t i o n and growth 56 i n V a u c h e r i a a p l a n o s p o r e s . F u r t h e r s t u d i e s a re r e q u i r e d t o c o n f i r m the l i g h t m i c r o s c o p i c o b s e r v a t i o n s of Ca2+ l o c a l i z a t i o n i n the g e r m i n a t i n g p r o t r u s i o n s and f i l a m e n t t i p s of V a u c h e r i a . T h i s work s h o u l d be extended t o i n c l u d e o t h e r known Ca2+ a n t a g o n i s t s i n order t o get a b e t t e r u n d e r s t a n d i n g of the r o l e s of e x t r a - and i n t r a c e l l u l a r Ca2+ i n these phenomena. The use of m i c r o t u b u l e and m i c r o f i l a m e n t i n h i b i t o r s , such as c o l c h i c i n e and c y t o c h a l a s i n B, and f l u o r e s c e n c e m i c r o s c o p i c s t u d i e s u s i n g monoclonal a n t i b o d i e s t o l o c a l i z e c y t o s k e l e t a l elements i n c o n j u n c t i o n w i t h CTC-dependent l o c a l i z a t i o n of Ca2+ s h o u l d h e l p e l u c i d a t e the r o l e of the c y t o s k e l e t o n and the i n f l u e n c e of Ca2+ i n the g e r m i n a t i o n and growth of V a u c h e r i a a p l a n o s p o r e s . Of p a r t i c u l a r importance would be measuring a c t u a l v a r i a t i o n s i n c e l l u l a r Ca2+ c o n c e n t r a t i o n s by the use of f l u o r e s c e n c e compounds such as Quin 2. C y t o c h e m i c a l l o c a l i z a t i o n and X-ray m i c r o a n a l y s i s may a l s o prove v a l u a b l e t e c h n i q u e s i n f u r t h e r u n d e r s t a n d i n g the r o l e of Ca2+ i n g e r m i n a t i o n and t i p - o r i e n t e d growth. KEY FOE FIGURES 57 A = a p l a n o s p o r e AV = a u t o p h a g i c v e s i c l e AW = a p l a n o s p o r e w a l l ASW = aplanosporangium w a l l B = b a c t e r i u m Ch = c h l o r o p l a s t Cry = c r y s t a l l i n e i n c l u s i o n CW = c e l l w a l l D = dictyosome DV = d i g e s t i v e v a c u o l e ER = endoplasmic r e t i c u l u m L = l i p i d body M = m i t o c h o n d r i o n Mb m i c r o b o d y - l i k e o r g a n e l l e N = n u c l e u s Nu - n u c l e o l u s P = py r e n o i d PM = plasma membrane RER = rough endoplasmic r e t i c u l u m Th = t h y l a k o i d V = f i b r i l l a r m a t e r i a l - c o n t a i n i n g v e s i c l e Vac = v a c u o l e VF = v e g e t a t i v e f i l a m e n t * = paramural f i b r i l l a r m a t e r i a l 58 F i g . 1. A p l a n o s p o r o g e n e s i s i n V.. l o n g i c a u l i s v a r . m a c o u n i i induced by the a d d i t i o n of f r e s h medium. F i g . 2. The i n i t i a t i o n of a p l a n o s p o r o g e n e s i s i s marked by s w e l l i n g a t the t i p of the v e g e t a t i v e f i l a m e n t . F i g . 3. I n i t i a t i o n of s e p t a t i o n of the a p l a n o s p o r e by the i n f u r r o w i n g i n n e r w a l l ( a r r o w h e a d s ) . F i g . 4. Co m p l e t i o n of s e p t a t i o n l e a d i n g t o the i n d i v i d u a l i z a t i o n of an a p l a n o s p o r e from the v e g e t a t i v e f i l a m e n t (arrowhead). F i g . 5. Mature a p l a n o s p o r e w i t h i n the aplanosporangium p r i o r t o i t s r e l e a s e . Note the a p l a n o s p o r a n g i a l w a l l (arrowhead). F i g . 6. D e t a i l of the o r g a n e l l e s seen i n the t i p of a v e g e t a t i v e f i l a m e n t d u r i n g a p l a n o s p o r o g e n e s i s . F i g . 7. D e t a i l of a m i t o c h o n d r i o n - e n d o p l a s m i c r e t i c u l u m -dictoysome a s s o c i a t i o n . A l s o shown i n c l o s e r e l a t i o n s h i p t o these o r g a n e l l e s i s a microbody-l i k e o r g a n e l l e . 59 F i g . 8. A p l a n o s p o r o g e n e s i s ; e a r l y s t a g e . Note the s u b a p i c a l p o s i t i o n of the c e n t r a l v a c u o l e . C r y s t a l l i n e i n c l u s i o n s are observed w i t h i n the v a c u o l e . Densely packed c h l o r o p l a s t s occupy the a p i c a l c y t o p l a s m . F i g . 9. A u t o p h a g i c d i g e s t i o n of c y t o p l a s m i c remnants w i t h i n the v a c u o l e . F i g . 10 . D e t a i l of a c r y s t a l l i n e i n c l u s i o n observed w i t h i n the v a c u o l e ( F i g . 8, c r y ) . F i g . 11 . A c c u m u l a t i o n of f i b r i l l a r m a t e r i a l i n the paramural space (*) of the t i p of the v e g e t a t i v e f i l a m e n t at an e a r l y s tage of a p l a n o s p o r o g e n e s i s (see F i g . 6 a l s o ) . Note the s i n g l e c e l l w a l l . 60 F i g . 12. L o n g i t u d i n a l s e c t i o n of the t i p of a f i l a m e n t u ndergoing a p l a n o s p o r o g e n e s i s . Note the i n f u r r o w i n g of the i n n e r w a l l (arrowheads) and the h i g h l y r e t i c u l a t e d morphology of the c y t o p l a s m of the d e v e l o p i n g a p l a n o s p o r e . F i g . 13. S e p t a t i o n of the a p l a n o s p o r e from the v e g e t a t i v e f i l a m e n t by i n f u r r o w i n g of the i n n e r w a l l ( s m a l l a r r o w h e a d s ) . The w a l l s of the a p l a n o s p o r e ( s m a l l arrowheads) and the aplanosporangium ( l a r g e arrowheads) become c l e a r l y d i s t i n g u i s h a b l e at t h i s s t a g e . F i g . 14. S e p t a t i o n i s n e a r l y complete w i t h only a s m a l l c h annel of c y t o p l a s m i c c o n t i n u i t y r e m a i n i n g (arrowhead) . Note the r e - f o r m a t i o n of the c e n t r a l v a c u o l e and the r e g u l a r arrangement of c h l o r o p l a s t s i n the p e r i p h e r a l c y t o p l a s m . F i g . 15. F i n a l s t a g e i n the i n d i v i d u a l i z a t i o n of the a p l a n o s p o r e from the v e g e t a t i v e f i l a m e n t . 60A 61 F i g . 16. The f i n a l s t a g e of a p l a n o s p o r o g e n e s i s showing the i n d i v i d u a l i z e d w a l l s of the a p l a n o s p o r e (AW) and aplanosporangium (ASW). M i t o c h o n d r i o n - e n d o p l a s m i c r e t i c u l u m - d i c t y o s o m e a s s o c i a t i o n s are observed throughout the c y t o p l a s m ( a r r o w h e a d s ) . F i g . 17. D e t a i l of the w a l l s of the a p l a n o s p o r e (AW) and the aplanosporangium (ASW). Note the f i b r i l l a r m a t e r i a l (*) between the w a l l s . F i g . 18. C h l o r o p l a s t showing the l a r g e p y r e n o i d p e n e t r a t e d by t h y l a k o i d s . F i g . 19. Mature a p l a n o s p o r e w i t h i n an aplanosporangium. Large numbers of c h l o r o p l a s t s are p e r i p h e r a l l y a r r a n g e d and a new c e n t r a l v a c u o l e i s c o a l e s c i n g from the s m a l l e r v a c u o l e s . The a p l a n o s p o r e w a l l (AW) and aplsnoaporangium w a l l (ASW) a r e e a s i l y d i s t i n g u i s h a b l e as w e l l . F i g . 20. S c a n n i n g e l e c t r o n m i c r o g r a p h of a mature a p l a n o s p o r e w i t h i n an aplanosporangium. I s o l a t i o n of the a p l a n o s p o r e from the v e g e t a t i v e f i l a m e n t i s c l e a r l y shown. 62 F i g . 21 Schematic r e p r e s e n t a t i o n of the most i m p o r t a n t events i n a p l a n o s p o r o g e n e s i s i n V.. l o n g i c a u l i s v a r . p a c o u n i (A) V e g e t a t i v e f i l a m e n t p r i o r t o a p l a n o s p o r o g e n e s i s . AZ = a p i c a l zone; SZ = s u b a p i c a l zone; ZoV = zone of v a c u o l a t i o n . (B) E a r l y a p l a n o s p o r o g e n e s i s . Note the s w e l l i n g of the t i p , the a c c u m u l a t i o n of o r g a n e l l e s w i t h i n the t i p and the d i s p l a c e m e n t of the c e n t r a l v a c o u l e from the t i p . (C) I n f u r r o w i n g of the i n n e r w a l l at the neck of the s w o l l e n v e g e t a t i v e f i l a m e n t t i p . The new c e n t r a l v a c u o l e b e g i n s t o c o a l e s c e and r e f o r m amidst the r e t i c u l a t e d c y t o p l a s m and p e r i p h e r a l l y s i t u a t e d c h l o r o p l a s t s . (D) Mature aplanosporangium c o n t a i n i n g a s i n g l e m u l t i n u c l e a t e d a p l a n o s p o r e . S e p t a t i o n from the v e g e t a t i v e f i l a m e n t i s complete; the c e n t r a l v a c u o l e c o n t i n u e s t o expand. 62A 63 F i g . 22. Four v e g e t a t i v e f i l a m e n t s are seen emerging from a s i n g l e a p l a n o s p o r e . F i g . 23. A p l a n o s p o r e body emptied of i t s c o n t e n t s a few days f o l l o w i n g g e r m i n a t i o n . F i g . 24. Mature a p l a n o s p o r e j u s t a f t e r emergence from an aplanosporangium. A node ( l a r g e arrowhead) i n d i c a t e s p r e v i o u s a p l a n o s p o r e p r o d u c t i o n . T h i s f i l a m e n t has grown through an aplanosporangium case ( s m a l l arrowhead). F i g . 25. S e c t i o n through a mature a p l a n o s p o r e s h o r t l y a f t e r r e l e a s e . C h l o r o p l a s t s a r e p e r i p h e r a l l y a r r a n g e d . F i g . 26. In s i t u g e r m i n a t i o n of an a p l a n o s p o r e . Two g e r m i n a t i n g f i l a m e n t s are seen ( s m a l l arrowheads) a l o n g w i t h the aplanosporangium w a l l c a s i n g ( l a r g e a rrowhead). F i g . 27. G e r m i n a t i o n of the a p l a n o s p o r e i s i n d i c a t e d by a p r o t r u s i o n of the c e l l w a l l ( a r r o w h e a d s ) . F i g . 28. S e c t i o n through a g e r m i n a t i n g a p l a n o s p o r e which has produced two f i l a m e n t s . 64 F i g . 29. C e l l w a l l of u n i f o r m t h i c k n e s s and p e r i p h e r a l l y a r r a n g e d c h l o r o p l a s t s c h a r a c t e r i z e the newly r e l e a s e d a p l a n o s p o r e (stage I ) . Note t h a t no s i g n s of r e l e a s e of new w a l l m a t e r i a l i s o b s e r v e d . F i g . 30. D e t a i l of a m i t o c h o n d r i o n - E R - d i c t y c s o m e complex. Two f i b r i l l a r - m a t e r i a l c o n t a i n i n g v e s i c l e s appear at the t r _ a j l S - c i s t e r n a of dictyosomes (arrowheads). F i g . 31. The p r o t r u s i o n which marks the b e g i n n i n g of g e r m i n a t i o n (stage I I ) c o n t a i n s many f i b r i l l a r -m a t e r i a l c o n t a i n i n g v e s i c l e s , n u c l e i , m i t o c h o n d r i a -EF-dictyosome complexes (*) and c h l o r o p l a s t s . F i g . 32. D e t a i l of the paramural space at the t i p of the p r o t r u s i o n . R e l e a s e d m a t e r i a l s accumulate i n t h i s r e g i o n and seem to be i n the p r o c e s s of b e i n g i n c o r p o r a t e d i n t o the w a l l . F i g . 33. E x o c y t o s i s of the c o n t e n t s of a s i n g l e f i b r i l l a r -m a t e r i a l c o n t a i n i n g v e s i c l e i n t o the paramural space. 64A 65 F i g . 34. S m a l l bundle of m i c r o t u b u l e s i n the c y t o p l a s m of an an a p l a n o s p o r e p r i o r t o g e r m i n a t i o n (arrowheads). Note t h a t these m i c r o t u b u l e bundles a re not p o s i t i o n e d p a r a l l e l t o the plasma membrane. F i g . 35. Bundle c o n t a i n i n g numerous m i c r o t u b u l e s showing p r e f e r e n t i a l o r i e n t a t i o n p a r a l l e l t o the plasma membrane of a g e r m i n a t i n g a p l a n o s p o r e (arrowheads). Note the p r o x i m i t y of t h i s bundle to one of the n u c l e i . F i g . 36. C l u s t e r s of e n d o p h y t i c b a c t e r i a a d j a c e n t t o a n u c l e u s . No obvi o u s m o r p h o l o g i c a l damage due t o the presence of the b a c t e r i a i s ob s e r v e d . F i g . 37. A v a c u o l e w i t h f o u r p a r t i a l l y d i g e s t e d b a c t e r i a . F i g . 38. S i n g l e b a c t e r i u m embedded i n the c e l l w a l l of an a p l a n o s p o r e . Note the c l e a r r e g i o n s u r r o u n d i n g the b a c t e r i u m (*), i n d i c a t i v e of the d i g e s t i o n of the a p l a n o s p o r e w a l l m a t e r i a l s . 66 F i g . 39 Volume d e n s i t y of major a p l a n o s p o r e compartments d u r i n g g e r m i n a t i o n (data from l i g h t m i c r o s c o p y s e c t i o n s ) . 67 F i g . 40 Volume d e n s i t y changes o c c u r r i n g d u r i n g a p l a n o s p o r e g e r m i n a t i o n i n c y t o p l a s m i c compartments (data from e l e c t r o n m i c r o s c o p y s e c t i o n s ) . FIGURE 40 II HI GERMINATION STAGES cn 3> 68 F i g u r e 41. Graph showing the e f f e c t of CTC c o n c e n t r a t i o n on the t o t a l f i l a m e n t l e n g t h at 6 hours and 24 hours a f t e r g e r m i n a t i o n . FIGURE 41 FILAMENT LENGTH VS. TIME 5000 -| 69 F i g u r e 42. Graph showing the e f f e c t of CTC c o n c e n t r a t i o n on the growth r a t e of f i l a m e n t s at 2, 4 and 6 hours of g e r m i n a t i o n . FIGURE 42 GROWTH RATE VS. TIME 300 n TIME [HRS] 70 Figure 43. Graph showing the area of the germinating filament reoccupied by CTC fluorescence. CTC F L U O R E S C E N C E [% OF TERMINAL 2 0 0 UM OF FILAMENT] VOZ 71 F i g u r e 44. Graph showing the d i s t r i b u t i o n and i n t e n s i t y of CTC f l u o r e s c e n c e a l o n g the 200 pm t e r m i n a l p o r t i o n of the g e r m i n a t i n g f i l a m e n t s . FLUORESCENCE INTENSITY [REL. UNITS] I V X CO NJ CD x 1 o 70 TO VJ-00 CO S3 I VIZ L i g h t m i c r o g r a p h of f i l a m e n t s grown f o r 24 hours i n CTC-free medium. L i g h t m i c r o g r a p h of f i l a m e n t s grown f o r 24 hours i n 10~ 4M CTC. Morphology and b r a n c h i n g p a t t e r n s are s i m i l a r t o those of the c o n t r o l (compare w i t h F i g . 4 5 ) . L i g h t m i c r o g r a p h of a g e r m i n a t i n g a p l a n o s p o r e . Mote the r e g i o n s of low o p t i c a l d e n s i t y c h a r a c t e r i s t i c of the g e r m i n a t i o n s i t e s (ar rowheads). CTC f l u o r e s c e n c e of the a p l a n o s p o r e d e p i c t e d from F i g . 47. The most i n t e n s e f l u o r e s c e n c e i s l o c a l i z e d at the s i t e s of low o p t i c a l d e n s i t y . L i g h t m i c r o g r a p h of an a p l a n o s p o r e exposed t o the C a 2 + - i n s e n s i t i v e probe OTC. No f l u o r e s c e n c e i s d e t e c t e d w i t h t h i s t r e a t m e n t . 73 F i g u r e 50. T i p - l o c a l i z e d f l u o r e s c e n c e i n a f i l a m e n t exposed t o 10~ 4M CTC two h r s a f t e r the i n i t i a t i o n of g e r m i n a t i o n . F i g u r e 51. M i c r o g r a p h of a f i l a m e n t exposed t o 10 _5M CTC d u r i n g the t r a n s i t i o n p e r i o d from 0-2 to 2-4 hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n . CTC f l u o r e s c e n c e i s l e s s l o c a l i z e d . F i g u r e 52. Ap l a n o s p o r e s h o r t l y a f t e r the i n i t i a t i o n of g e r m i n a t i o n . CTC i s s h a r p l y d e l i m i t e d t o the g e r m i n a t i o n s i t e . F i g u r e 53. S h a r p l y - d e l i m i t e d CTC f l u o r e s c e n c e i s shown l o c a l i z e d at the t i p of a f i l a m e n t 4 hours a f t e r g e r m i n a t i o n . T h i s p a t t e r n i s s i m i l a r t o t h a t o b s e r v e d d u r i n g the f i r s t two hours of g e r m i n a t i o n (compare w i t h F i g s 50 and 5 2 ) . F i g u r e 54. A more d i f f u s e p a t t e r n of CTC f l u o r e s c e n c e i s seen e x t e n d i n g b a s i p e t a l l y from the t i p i n f i l a m e n t s between two t o f o u r hours a f t e r the i n i t i a t i o n of g e r m i n a t i o n . F i g u r e 55 . The p a t t e r n of CTC f l u o r e s c e n c e shown here i n a f r e s h l y c o l l e c t e d v e g e t a t i v e f i l a m e n t . 73A 74 F i g u r e 56. Graph showing the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of EGTA on the growth r a t e of newly g e r m i n a t e d V a u c h e r i a f i l a m e n t s . F i g u r e 57. Graph showing the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of c a l c i u m ionophore A23187 on the growth r a t e of newly germinated V a u c h e r i a f i l a m e n t s . F i g u r e 58. Graph showing the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of the c a l m o d u l i n a n t a g o n i s t TFP on the growth r a t e of newly germinated V a u c h e r i a f i l a m e n t s . F i g u r e 59. Graph comparing the r e s u l t s of the e f f e c t s of a l l t h r e e compounds (EGTA, A23187 and TFP) on the growth r a t e of newly germinated V a u c h e r i a f i l a m e n t s . RGURE 56 EGTA GROWTH RATES CONCENTRATION M FIGURE 68 TFP GROWTH RATES CONCENTRATION (Mj RGURE 57 A23187 G R O W T H RATES CONCENTRATION [M RGURE 59 GROWTH RATES VS. CONCENTRATION CONCENTRATION M M i c r o g r a p h of a g e r m i n a t i n g f i l a m e n t from an ap l a n o s p o r e i n c u b a t e d i n u n t r e a t e d medium. Same specimen as i n F i g . 60, 10 minutes a f t e r i n c u b a t i o n w i t h 10-4^ CTC. Mi c r o g r a p h of a g e r m i n a t i n g f i l a m e n t from an ap l a n o s p o r e i n c u b a t e d i n 10~4M EGTA. Same specimen as i n F i g . 62, 10 minutes a f t e r i n c u b a t i o n w i t h 10"4M CTC. F l u o r e s c e n c e i s l e s s i n t e n s e and more d i f f u s e than i n the u n t r e a t e d m a t e r i a l (compare w i t h F i g . 6 1 ) . Mi c r o g r a p h of g e r m i n a t i n g f i l a m e n t s from an ap l a n o s p o r e i n c u b a t e d i n 1 0 _ 4 M A23187. Note the d i s o r i e n t e d growth p a t t e r n , a p i c a l s w e l l i n g s , b u d - l i k e p r o t r u s i o n s and r e g i o n s low o p t i c a l d e n s i t y ( a r r o w h e a d s ) . M i c r o g r a p h of g e r m i n a t i n g f i l a m e n t s from an ap l a n o s p o r e i n c u b a t e d i n 10~4M A23187, f o l l o w e d by 10-4M CTC. Note the b r i g h t f l u o r e s c e n c e at the s w o l l e n t i p (arrowhead). 7 5 A 76 F i g u r e 66. M i c r o g r a p h of g e r m i n a t i n g f i l a m e n t s from a p l a n o s p o r e s i n c u b a t e d i n 10~5M A23187. I r r e g u l a r f i l a m e n t d i a m e t e r s , s w o l l e n a p i c e s and p r o t r u s i o n s a r e s t i l l p r e s e n t . F i g u r e 67. M i c r o g r a p h of g e r m i n a t i n g f i l a m e n t s from a p l a n o s p o r e s i n c u b a t e d i n 10~6M A23187. The o n l y a b n o r m a l i t y observed i s f i l a m e n t d i a m e t e r s of i r r e g u l a r w i d t h . F i g u r e 68. M i c r o g r a p h of g e r m i n a t i n g f i l a m e n t s from a p l a n o s p o r e s i n c u b a t e d i n 1% DMSO. No m o r p h o l o g i c a l a b n o r m a l i t i e s are seen. F i g u r e 69. M i c r o g r a p h of a s i n g l e a p l a n o s p o r e i n c u b a t e d i n 10-5M TFP, 10 minutes a f t e r 10~4M CTC a p p l i c a t i o n . No s i g n s of f l u o r e s c e n c e i s d e t e c t e d a f t e r i n c u b a t i o n w i t h CTC. LITERATURE CITED 77 A g h a j a n i a n , J.G. 1979. 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